A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

PubMed

Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

2017-12-01

Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Phytase-Based Reporter System for Identification of Functional Secretion Signals in Bifidobacteria

PubMed Central

Osswald, Annika; Westermann, Christina; Sun, Zhongke; Riedel, Christian U.

2015-01-01

Health-promoting effects have been attributed to a number of Bifidobacterium sp. strains. These effects as well as the ability to colonise the host depend on secreted proteins. Moreover, rational design of protein secretion systems bears the potential for the generation of novel probiotic bifidobacteria with improved health-promoting or therapeutic properties. To date, there is only very limited data on secretion signals of bifidobacteria available. Using in silico analysis, we demonstrate that all bifidobacteria encode the major components of Sec-dependent secretion machineries but only B. longum strains harbour Tat protein translocation systems. A reporter plasmid for secretion signals in bifidobacteria was established by fusing the coding sequence of the signal peptide of a sialidase of Bifidobacterium bifidum S17 to the phytase gene appA of E. coli. The recombinant strain showed increased phytase activity in spent culture supernatants and reduced phytase levels in crude extracts compared to the control indicating efficient phytase secretion. The reporter plasmid was used to screen seven predicted signal peptides in B. bifidum S17 and B. longum E18. The tested signal peptides differed substantially in their efficacy to mediate protein secretion in different host strains. An efficient signal peptide was used for expression and secretion of a therapeutically relevant protein in B. bifidum S17. Expression of a secreted cytosine deaminase led to a 100-fold reduced sensitivity of B. bifidum S17 to 5-fluorocytosine compared to the non-secreted cytosine deaminase suggesting efficient conversion of 5-fluorocytosine to the cytotoxic cancer drug 5-fluorouracil by cytosine deaminase occurred outside the bacterial cell. Selection of appropriate signal peptides for defined protein secretion might improve therapeutic efficacy as well as probiotic properties of bifidobacteria. PMID:26086721

Identification of the DotL coupling protein subcomplex of the Legionella Dot/Icm type IV secretion system.

PubMed

Vincent, Carr D; Friedman, Jonathan R; Jeong, Kwang Cheol; Sutherland, Molly C; Vogel, Joseph P

2012-07-01

Legionella pneumophila, the causative agent of Legionnaires' disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella type IV secretion system substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. © 2012 Blackwell Publishing Ltd.

Secretome of obligate intracellular Rickettsia

PubMed Central

Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

2014-01-01

The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

The role of intrinsic disorder and dynamics in the assembly and function of the type II secretion system.

PubMed

Gu, Shuang; Shevchik, Vladimir E; Shaw, Rosie; Pickersgill, Richard W; Garnett, James A

2017-10-01

Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS. The central role of intrinsic disorder-order transitions in these processes may be a particular feature of type II secretion. Copyright © 2017 Elsevier B.V. All rights reserved.

Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

PubMed

Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Dinakaran, Vasudevan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2016-01-01

Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide.

PubMed

Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

2015-01-01

Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%-92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp.

A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78.

PubMed

Chaves, Daniela Fojo Seixas; de Souza, Emanuel Maltempi; Monteiro, Rose Adele; de Oliveira Pedrosa, Fábio

2009-11-02

Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant-bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOEpatents

Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

2016-08-09

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Regulation of Protein Secretion Through Controlled Aggregation in the Endoplasmic Reticulum

NASA Astrophysics Data System (ADS)

Rivera, Victor M.; Wang, Xiurong; Wardwell, Scott; Courage, Nancy L.; Volchuk, Allen; Keenan, Terence; Holt, Dennis A.; Gilman, Michael; Orci, Lelio; Cerasoli, Frank; Rothman, James E.; Clackson, Tim

2000-02-01

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

Secretion of TcpF by the Vibrio cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires an N-Terminal Determinant

PubMed Central

Megli, Christina J.

2013-01-01

Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81–92, 2003). This phenomenon is not unique to V. cholerae; secreted virulence factors that are dependent on the presence of components of the type IV pilus biogenesis apparatus for secretion have been reported with Dichelobacter nodosus (R. M. Kennan, O. P. Dhungyel, R. J. Whittington, J. R. Egerton, and J. I. Rood, J. Bacteriol. 183:4451–4458, 2001) and Francisella tularensis (A. J. Hager et al., Mol. Microbiol. 62:227–237, 2006). Using site-directed mutagenesis, we demonstrated that the secretion of TcpF is dependent on the presence of selected amino acid R groups at position five. We were unable to find other secretion determinants, suggesting that Y5 is the major secretion determinant within TcpF. We also report that proteins secreted in a type IV pilus biogenesis apparatus-dependent manner have a YXS motif within the first 15 amino acids following the Sec cleavage site. The YXS motif is not present in proteins secreted by type II secretion systems, indicating that this is unique to type IV pilus-mediated secretion. Moreover, we show that TcpF interacts with the pilin TcpA, suggesting that these proteins are secreted by the type IV pilus biogenesis system. These data provide a starting point for understanding how type IV pili can mediate secretion of virulence factors important for bacterial pathogenesis. PMID:23564177

Proteins Exported via the PrsD-PrsE Type I Secretion System and the Acidic Exopolysaccharide Are Involved in Biofilm Formation by Rhizobium leguminosarum

PubMed Central

Russo, Daniela M.; Williams, Alan; Edwards, Anne; Posadas, Diana M.; Finnie, Christine; Dankert, Marcelo; Downie, J. Allan; Zorreguieta, Angeles

2006-01-01

The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria. PMID:16740954

Identification of the DotL Coupling Protein Subcomplex of the Legionella Dot/Icm Type IV Secretion System

PubMed Central

Vincent, Carr D.; Friedman, Jonathan R.; Jeong, Kwang Cheol; Sutherland, Molly C.; Vogel, Joseph P.

2012-01-01

Summary Legionella pneumophila, the causative agent of Legionnaires’ disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella T4SS substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. PMID:22694730

Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium.

PubMed

Huber, Robert J

2017-07-01

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3 - cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3 - cells displayed delayed streaming and aggregation, and interestingly, cln3 - cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3 - CM, of which 3 were absent in cln3 - CM. Moreover, 12 proteins that were present in cln3 - CM were absent in WT CM. Label-free quantification identified 42 proteins that were present in significantly higher amounts in cln3 - CM compared to WT, and 3 proteins that were present in significantly reduced amounts. A GO term enrichment analysis showed that a majority of the affected proteins are linked to endocytosis, vesicle-mediated transport, proteolysis, and metabolism. In total, the results of this study indicate that Cln3 functions in both conventional and unconventional protein secretion and that loss of Cln3 results in deregulated secretion during early development. Importantly, this is the first evidence in any system linking CLN3 function to protein secretion. Copyright © 2017 Elsevier Inc. All rights reserved.

A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion

DOE PAGES

Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko

2014-11-06

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less

DBSecSys: A Database of Burkholderia mallei Secretion Systems

DTIC Science & Technology

2014-07-16

toxins and the lipases, as well as non-proteinaceous substrates, e.g., cyclic β- glucans and polysaccharides. 2* - Represents a Sec/Tat-dependent system...divided into three types: 1) the archetypal bacterial proteins exported into the periplasm via the Sec system; 2) trimeric proteins with a single beta ...barrel domain; and 3) pairs of proteins in which one partner carries the beta barrel domain and the other partner is the secreted protein. 6

Analysis of secreted proteins from Aspergillus flavus.

PubMed

Medina, Martha L; Haynes, Paul A; Breci, Linda; Francisco, Wilson A

2005-08-01

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

The importance of connections between the cell wall integrity pathway and the unfolded protein response in filamentous fungi.

PubMed

Malavazi, Iran; Goldman, Gustavo Henrique; Brown, Neil Andrew

2014-11-01

In the external environment, or within a host organism, filamentous fungi experience sudden changes in nutrient availability, osmolality, pH, temperature and the exposure to toxic compounds. The fungal cell wall represents the first line of defense, while also performing essential roles in morphology, development and virulence. A polarized secretion system is paramount for cell wall biosynthesis, filamentous growth, nutrient acquisition and interactions with the environment. The unique ability of filamentous fungi to secrete has resulted in their industrial adoption as fungal cell factories. Protein maturation and secretion commences in the endoplasmic reticulum (ER). The unfolded protein response (UPR) maintains ER functionality during exposure to secretion and cell wall stress. UPR, therefore, influences secretion and cell wall homeostasis, which in turn impacts upon numerous fungal traits important to pathogenesis and biotechnology. Subsequently, this review describes the relevance of the cell wall and UPR systems to filamentous fungal pathogens or industrial microbes and then highlights interconnections between the two systems. Ultimately, the possible biotechnological applications of an enhanced understanding of such regulatory systems in combating fungal disease, or the removal of natural bottlenecks in protein secretion in an industrial setting, are discussed. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

PubMed

Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W

2018-01-01

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle

USDA-ARS?s Scientific Manuscript database

The locus of enterocyte effacement (LEE) encodes a type III secretion system (T3SS) for secreting factors that enable Escherichia coli O157:H7 to produce attaching and effacing lesions (A/E) on epithelial cells. The importance of LEE-encoded proteins in intestinal colonization of cattle is well-stud...

Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae

PubMed Central

Giraldo, Martha C.; Dagdas, Yasin F.; Gupta, Yogesh K.; Mentlak, Thomas A.; Yi, Mihwa; Martinez-Rocha, Ana Lilia; Saitoh, Hiromasa; Terauchi, Ryohei; Talbot, Nicholas J.; Valent, Barbara

2013-01-01

To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion. PMID:23774898

Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus.

PubMed

Medina, Martha L; Kiernan, Urban A; Francisco, Wilson A

2004-03-01

Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.

Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

PubMed

Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

2013-01-01

The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Crystal structure of the Yersinia type III secretion protein YscE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phan, Jason; Austin, Brian P.; Waugh, David S.

2010-12-06

The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.

Identification of genes encoding the type IX secretion system and secreted proteins in Flavobacterium columnare IA-S-4

USDA-ARS?s Scientific Manuscript database

Flavobacterium columnare, a member of the phylum Bacteroidetes, causes columnaris disease in wild and aquaculture-reared freshwater fish. The mechanisms responsible for columnaris disease are not known. Many members of the phylum Bacteroidetes use type IX secretion systems (T9SSs) to secrete enzymes...

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for Chlamydomonas reinhardtii

PubMed Central

de Carvalho, João Carlos Monteiro; Mayfield, Stephen Patrick

2018-01-01

Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga Chlamydomonas reinhardtii, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP) for heterologous protein secretion in C. reinhardtii by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified in silico. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified in silico secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the C. reinhardtii genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications. PMID:29408937

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

PubMed Central

2012-01-01

Summary: Flagellar and translocation-associated type III secretion (T3S) systems are present in most Gram-negative plant- and animal-pathogenic bacteria and are often essential for bacterial motility or pathogenicity. The architectures of the complex membrane-spanning secretion apparatuses of both systems are similar, but they are associated with different extracellular appendages, including the flagellar hook and filament or the needle/pilus structures of translocation-associated T3S systems. The needle/pilus is connected to a bacterial translocon that is inserted into the host plasma membrane and mediates the transkingdom transport of bacterial effector proteins into eukaryotic cells. During the last 3 to 5 years, significant progress has been made in the characterization of membrane-associated core components and extracellular structures of T3S systems. Furthermore, transcriptional and posttranscriptional regulators that control T3S gene expression and substrate specificity have been described. Given the architecture of the T3S system, it is assumed that extracellular components of the secretion apparatus are secreted prior to effector proteins, suggesting that there is a hierarchy in T3S. The aim of this review is to summarize our current knowledge of T3S system components and associated control proteins from both plant- and animal-pathogenic bacteria. PMID:22688814

Escherichia coli type III secretion system 2: a new kind of T3SS?

PubMed

Zhou, Mingxu; Guo, Zhiyan; Duan, Qiangde; Hardwidge, Philip R; Zhu, Guoqiang

2014-03-19

Type III secretion systems (T3SSs) are employed by Gram-negative bacteria to deliver effector proteins into the cytoplasm of infected host cells. Enteropathogenic Escherichia coli use a T3SS to deliver effector proteins that result in the creation of the attaching and effacing lesions. The genome sequence of the Escherichia coli pathotype O157:H7 revealed the existence of a gene cluster encoding components of a second type III secretion system, the E. coli type III secretion system 2 (ETT2). Researchers have revealed that, although ETT2 may not be a functional secretion system in most (or all) strains, it still plays an important role in bacterial virulence. This article summarizes current knowledge regarding the E. coli ETT2, including its genetic characteristics, prevalence, function, association with virulence, and prospects for future work.

Twin-arginine signal peptide of Bacillus subtilis YwbN can direct Tat-dependent secretion of methyl parathion hydrolase.

PubMed

Liu, Ruihua; Zuo, Zhenqiang; Xu, Yingming; Song, Cunjiang; Jiang, Hong; Qiao, Chuanling; Xu, Ping; Zhou, Qixing; Yang, Chao

2014-04-02

The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.

Dramatic secretion of recombinant protein expressed in tobacco cells with a designer glycopeptide tag is highly impacted by medium composition.

PubMed

Zhang, Ningning; Dolan, Maureen; Wu, Di; Phillips, Gregory C; Xu, Jianfeng

2016-12-01

Cell growth medium composition has profound impacts on the O -glycosylation of a "designer" arabinogalactan protein-based module; full glycosylation is essential in directing efficient extracellular secretion of the tagged recombinant protein. Expression of recombinant proteins in plant cells as fusion with a de novo designed hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) tag, termed HypGP engineering technology, resulted in dramatically increased secreted protein yields. This is due to the function of the HypGP tag as a molecular carrier in promoting efficient transport of conjoined proteins into culture media. To optimize the cell culture to achieve the best secreted protein yields, the medium effects on the cell growth and protein secretion were investigated using as a model system the tobacco BY-2 cell expressing enhanced green fluorescence protein (EGFP) fused with a (SP) 32 tag (32 tandem repeats of "Ser-Pro" motif). The (SP) 32 tag was found to undergo two-stage Hyp-O-glycosylation in plant cells with the dramatic secretion of the conjoined EGFP correlating with the triggering of the second-stage glycosylation. The BY-2 cell culture in SH medium generated a high secreted protein yield (125 mg/L) with a low cell biomass accumulation (~7.5 gDW/L). In contrast, very low secreted protein yields (~1.5 mg/L) with a high cell biomass accumulation (13.5 gDW/L) were obtained in MS medium. The macronutrients, specifically, the nitrogen supply greatly impacted the glycosylation of the (SP) 32 tag and subsequent protein secretion. Modified MS medium with reduced nitrogen levels boosted the secreted EGFP yields to 168 mg/L. This study demonstrates the profound impacts of medium composition on the secreted yields of a HypGP-tagged protein, and provides a basis for medium design to achieve the highest productivity of the HypGP engineering technology.

Expression, Extracellular Secretion, and Immunogenicity of the Plasmodium falciparum Sporozoite Surface Protein 2 in Salmonella Vaccine Strains

PubMed Central

Gómez-Duarte, Oscar G.; Pasetti, Marcela F.; Santiago, Araceli; Sztein, Marcelo B.; Hoffman, Stephen L.; Levine, Myron M.

2001-01-01

Deleting transmembrane α-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04). PMID:11160021

Effects of Psidium guajava leaf extract on secretion systems of Gram-negative enteropathogenic bacteria.

PubMed

Nakasone, Noboru; Ogura, Yasunori; Higa, Naomi; Toma, Claudia; Koizumi, Yukiko; Takaesu, Giichi; Suzuki, Toshihiko; Yamashiro, Tetsu

2018-05-23

We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

The YopJ superfamily of type III efforts in plant-associated bacteria

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defense responses or innate immunity. However, some effector proteins may be recognized by...

The intracellular citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ encodes two novel autotransporters

USDA-ARS?s Scientific Manuscript database

Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of ‘Candidatus Liberibacter asiaticus’ (Las), and designated them as Las...

The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" encodes an afp-like prophage possibly used for protein secretion.

PubMed

Penz, Thomas; Horn, Matthias; Schmitz-Esser, Stephan

2010-01-01

The recently sequenced genome of the obligate intracellular amoeba symbiont 'Candidatus Amoebophilus asiaticus' is unique among prokaryotic genomes due to its extremely large fraction of genes encoding proteins harboring eukaryotic domains such as ankyrin-repeats, TPR/SEL1 repeats, leucine-rich repeats, as well as F- and U-box domains, most of which likely serve in the interaction with the amoeba host. Here we provide evidence for the presence of additional proteins which are presumably presented extracellularly and should thus also be important for host cell interaction. Surprisingly, we did not find homologues of any of the well-known protein secretion systems required to translocate effector proteins into the host cell in the A. asiaticus genome, and the type six secretion systems seems to be incomplete. Here we describe the presence of a putative prophage in the A. asiaticus genome, which shows similarity to the antifeeding prophage from the insect pathogen Serratia entomophila. In S. entomophila this system is used to deliver toxins into insect hosts. This putative antifeeding-like prophage might thus represent the missing protein secretion apparatus in A. asiaticus.

The secretory IgA system of lung secretions in chronic obstructive bronchitis: comparison of sputum with secretions obtained during fibreoptic bronchoscopy.

PubMed Central

Wiggins, J; Hill, S L; Stockley, R A

1984-01-01

The constituents of the secretory immunoglobulin A system (dimeric IgA, total secretory component and free secretory component) were measured in sputum sol phase, tracheal aspirates, and bronchoalveolar lavage fluids from 15 patients undergoing fibreoptic bronchoscopy. All of the proteins showed a progressive decrease in concentration from sputum to the bronchoalveolar lavage fluids (2p less than 0.001). Standardisation of samples by means of protein concentration ratios showed that all secretions were generally similar in respect of their secretory IgA profiles, although major differences remained in some individual patients. The between patient variability of the results was generally reduced by the use of protein concentration ratios, allowing closer comparison between subjects. When the secretion albumin concentration was used as a standard, however, it increased the variability of the sputum sol phase IgA components (2p less than 0.01), whereas it decreased the variability of the IgA components in the bronchoalveolar lavage fluid (2p less than 0.05). The role of albumin as a standard protein for assessing the secretory IgA system in lung secretions remains uncertain. PMID:6463931

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

PubMed

Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

2017-10-01

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?

PubMed Central

Kesimer, Mehmet; Kirkham, Sara; Pickles, Raymond J.; Henderson, Ashley G.; Alexis, Neil E.; DeMaria, Genevieve; Knight, David; Thornton, David J.; Sheehan, John K.

2009-01-01

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces “mucus” with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products. PMID:18931053

The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

PubMed

Siggers, Keri A; Lesser, Cammie F

2008-07-17

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.

Prediction of type III secretion signals in genomes of gram-negative bacteria.

PubMed

Löwer, Martin; Schneider, Gisbert

2009-06-15

Pathogenic bacteria infecting both animals as well as plants use various mechanisms to transport virulence factors across their cell membranes and channel these proteins into the infected host cell. The type III secretion system represents such a mechanism. Proteins transported via this pathway ("effector proteins") have to be distinguished from all other proteins that are not exported from the bacterial cell. Although a special targeting signal at the N-terminal end of effector proteins has been proposed in literature its exact characteristics remain unknown. In this study, we demonstrate that the signals encoded in the sequences of type III secretion system effectors can be consistently recognized and predicted by machine learning techniques. Known protein effectors were compiled from the literature and sequence databases, and served as training data for artificial neural networks and support vector machine classifiers. Common sequence features were most pronounced in the first 30 amino acids of the effector sequences. Classification accuracy yielded a cross-validated Matthews correlation of 0.63 and allowed for genome-wide prediction of potential type III secretion system effectors in 705 proteobacterial genomes (12% predicted candidates protein), their chromosomes (11%) and plasmids (13%), as well as 213 Firmicute genomes (7%). We present a signal prediction method together with comprehensive survey of potential type III secretion system effectors extracted from 918 published bacterial genomes. Our study demonstrates that the analyzed signal features are common across a wide range of species, and provides a substantial basis for the identification of exported pathogenic proteins as targets for future therapeutic intervention. The prediction software is publicly accessible from our web server (www.modlab.org).

Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis.

PubMed

Ates, Louis S; Dippenaar, Anzaan; Ummels, Roy; Piersma, Sander R; van der Woude, Aniek D; van der Kuij, Kim; Le Chevalier, Fabien; Mata-Espinosa, Dulce; Barrios-Payán, Jorge; Marquina-Castillo, Brenda; Guapillo, Carolina; Jiménez, Connie R; Pain, Arnab; Houben, Edith N G; Warren, Robin M; Brosch, Roland; Hernández-Pando, Rogelio; Bitter, Wilbert

2018-02-01

Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems 1,2 . The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus 3,4 . However, the role of these proteins remains largely elusive 1 . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion 5 . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the 'modern' Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution 6,7 .

Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

PubMed Central

Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

2011-01-01

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

External Loops at the C Terminus of Erwinia chrysanthemi Pectate Lyase C Are Required for Species-Specific Secretion through the Out Type II Pathway

PubMed Central

Lindeberg, Magdalen; Boyd, Carol M.; Keen, Noel T.; Collmer, Alan

1998-01-01

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pel1. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning. PMID:9515910

IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system.

PubMed

Bardill, J Patrick; Miller, Jennifer L; Vogel, Joseph P

2005-04-01

Legionella pneumophila replicates inside alveolar macrophages and causes an acute, potentially fatal pneumonia called Legionnaires' disease. The ability of this bacterium to grow inside of macrophages is dependent on the presence of a functional dot/icm type IV secretion system (T4SS). Proteins secreted by the Dot/Icm T4SS are presumed to alter the host endocytic pathway, allowing L. pneumophila to establish a replicative niche within the host cell. Here we show that a member of the SidE family of proteins interacts with IcmS and is required for full virulence in the protozoan host Acanthamoeba castellanii. Using immunofluorescence microscopy and adenylate cyclase fusions, we show that SdeA is secreted into host cells by L. pneumophila in an IcmS-dependent manner. The SidE-like proteins are secreted very early during macrophage infection, suggesting that they are important in the initial formation of the replicative phagosome. Secreted SidE family members show a similar localization to other Dot/Icm substrates, specifically, to the poles of the replicative phagosome. This common localization of secreted substrates of the Dot/Icm system may indicate the formation of a multiprotein complex on the cytoplasmic face of the replicative phagosome.

THE SECRETION OF DIGESTIVE ENZYMES AND CAECAL SIZE ARE DETERMINED BY DIETARY PROTEIN IN THE CRICKET Gryllus bimaculatus.

PubMed

Woodring, Joseph; Weidlich, Sandy

2016-11-01

In Gryllus bimaculatus, the size of the caecum decreases in the latter half of each instar to a stable minimal size with a steady minimal rate of digestive enzyme secretion until feeding resumes after ecdysis. The higher the percent protein in the newly ingested food, the faster and larger the caecum grows, and as a consequent the higher the secretion rate of trypsin and amylase. When hard boiled eggs (40% protein) are eaten the caecum is 2× larger, the trypsin secretion is almost 3× greater, and amylase 2.5× greater then when fed the same amount of apples (1.5% protein). Only dietary protein increases amylase secretion, whereas dietary carbohydrates have no effect on amylase secretion. The minimal caecal size and secretion rate must be supported by utilization of hemolymph amino acids, but the growth of the caecum and increasing enzymes secretions after the molt depend upon an amino acid source in the lumen. This simple regulation of digestive enzyme secretion is ideal for animals that must stop feeding in order to molt. This basic control system does not preclude additional regulation mechanisms, such as prandal, which is also indicated for G. bimaculatus, or even paramonal regulation. © 2016 Wiley Periodicals, Inc.

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae

PubMed Central

2014-01-01

Background The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Results Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. Conclusion In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus. PMID:24961398

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae.

PubMed

Liu, Lifang; Feizi, Amir; Österlund, Tobias; Hjort, Carsten; Nielsen, Jens

2014-06-24

The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus.

Pseudomonas syringae pv. Tomato DC3000 Type III secretion effector polymutants reveal an interplay between hopAD1 and AvrPtoB

USDA-ARS?s Scientific Manuscript database

The model pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of plants by injecting a complex repertoire of effector proteins into host cells via the type III secretion system. The model effector AvrPtoB has multiple domains and plant protein interactors i...

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells.

PubMed

Nyman, Tuula A; Lorey, Martina B; Cypryk, Wojciech; Matikainen, Sampsa

2017-05-01

The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses. Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses. Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.

Protein secretion through autotransporter and two-partner pathways.

PubMed

Jacob-Dubuisson, Françoise; Fernandez, Rachel; Coutte, Loic

2004-11-11

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.

Heterologous Protein Secretion in Lactobacilli with Modified pSIP Vectors

PubMed Central

Karlskås, Ingrid Lea; Maudal, Kristina; Axelsson, Lars; Rud, Ida; Eijsink, Vincent G. H.; Mathiesen, Geir

2014-01-01

We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species. PMID:24614815

Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

EPA Science Inventory

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophilia

EPA Science Inventory

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrop...

Unconventional Secretion of Heat Shock Proteins in Cancer

PubMed Central

Santos, Tiago Góss; Martins, Vilma Regina; Hajj, Glaucia Noeli Maroso

2017-01-01

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies. PMID:28468249

A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins.

PubMed

Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

2017-03-28

The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia , require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. IMPORTANCE The type 3 secretion system is essential for injection of virulence factors, which are delivered directly into the cytosol of the host cells for usurping and subverting host processes. Recent studies have shown that these effectors proteins indeed travel inside an "injectisome" conduit through a single step of translocation by connecting the bacterium and host cell cytoplasms. However, all findings are not compatible with this model. For example, both YopH, a protein detected on the surface of Yersinia , and EspC, an autotransporter protein secreted by enteropathogenic E. coli , require a functional T3SS for host cell translocation. Both proteins have an intermediate extracellular step before their T3SS-dependent translocation. Here, we show an alternative delivery mechanism for these extracellularly secreted virulence factors that are then incorporated into the T3SS to enter the cells; this novel mechanism coexists with but diverges from the canonical injection model that involves the passage of the protein inside the injectisome. Copyright © 2017 Tejeda-Dominguez et al.

Comprehensive assessment and performance improvement of effector protein predictors for bacterial secretion systems III, IV and VI.

PubMed

An, Yi; Wang, Jiawei; Li, Chen; Leier, André; Marquez-Lago, Tatiana; Wilksch, Jonathan; Zhang, Yang; Webb, Geoffrey I; Song, Jiangning; Lithgow, Trevor

2018-01-01

Bacterial effector proteins secreted by various protein secretion systems play crucial roles in host-pathogen interactions. In this context, computational tools capable of accurately predicting effector proteins of the various types of bacterial secretion systems are highly desirable. Existing computational approaches use different machine learning (ML) techniques and heterogeneous features derived from protein sequences and/or structural information. These predictors differ not only in terms of the used ML methods but also with respect to the used curated data sets, the features selection and their prediction performance. Here, we provide a comprehensive survey and benchmarking of currently available tools for the prediction of effector proteins of bacterial types III, IV and VI secretion systems (T3SS, T4SS and T6SS, respectively). We review core algorithms, feature selection techniques, tool availability and applicability and evaluate the prediction performance based on carefully curated independent test data sets. In an effort to improve predictive performance, we constructed three ensemble models based on ML algorithms by integrating the output of all individual predictors reviewed. Our benchmarks demonstrate that these ensemble models outperform all the reviewed tools for the prediction of effector proteins of T3SS and T4SS. The webserver of the proposed ensemble methods for T3SS and T4SS effector protein prediction is freely available at http://tbooster.erc.monash.edu/index.jsp. We anticipate that this survey will serve as a useful guide for interested users and that the new ensemble predictors will stimulate research into host-pathogen relationships and inspiration for the development of new bioinformatics tools for predicting effector proteins of T3SS, T4SS and T6SS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

PubMed

Wilson, Marlena M; Anderson, D Eric; Bernstein, Harris D

2015-01-01

Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM) and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

Secretion of small proteins is species-specific within Aspergillus sp.

PubMed

Valette, Nicolas; Benoit-Gelber, Isabelle; Falco, Marcos Di; Wiebenga, Ad; de Vries, Ronald P; Gelhaye, Eric; Morel-Rouhier, Mélanie

2017-03-01

Small secreted proteins (SSP) have been defined as proteins containing a signal peptide and a sequence of less than 300 amino acids. In this analysis, we have compared the secretion pattern of SSPs among eight aspergilli species in the context of plant biomass degradation and have highlighted putative interesting candidates that could be involved in the degradative process or in the strategies developed by fungi to resist the associated stress that could be due to the toxicity of some aromatic compounds or reactive oxygen species released during degradation. Among these candidates, for example, some stress-related superoxide dismutases or some hydrophobic surface binding proteins (HsbA) are specifically secreted according to the species . Since these latter proteins are able to recruit lytic enzymes to the surface of hydrophobic solid materials and promote their degradation, a synergistic action of HsbA with the degradative system may be considered and need further investigations. These SSPs could have great applications in biotechnology by optimizing the efficiency of the enzymatic systems for biomass degradation. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors, Secretion of Translocators, and Pathogenesis in Fish.

PubMed

Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia

2016-01-01

The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells.

PubMed

Bocsanczy, Ana M; Nissinen, Riitta M; Oh, Chang-Sik; Beer, Steven V

2008-07-01

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

Exocyst Complex Protein Expression in the Human Placenta

PubMed Central

Gonzalez, I.M.; Ackerman, W.E.; Vandre, D.D.; Robinson, J.M.

2014-01-01

Introduction Protein production and secretion are essential to syncytiotrophoblast function and are associated with cytotrophoblast cell fusion and differentiation. Syncytiotrophoblast hormone secretion is a crucial determinant of maternal-fetal health, and can be misregulated in pathological pregnancies. Although, polarized secretion is a key component of placental function, the mechanisms underlying this process are poorly understood. Objective While the octameric exocyst complex is classically regarded as a master regulator of secretion in various mammalian systems, its expression in the placenta remained unexplored. We hypothesized that the syncytiotrophoblast would express all exocyst complex components and effector proteins requisite for vesicle-mediated secretion more abundantly than cytotrophoblasts in tissue specimens. Methods A two-tiered immunobiological approach was utilized to characterize exocyst and ancillary proteins in normal, term human placentas. Exocyst protein expression and localization was documented in tissue homogenates via immunoblotting and immunofluorescence labeling of placental sections. Results The eight exocyst proteins, EXOC1, 2, 3, 4, 5, 6, 7, and 8, were found in the human placenta. In addition, RAB11, an important exocyst complex modulator, was also expressed. Exocyst and Rab protein expression appeared to be regulated during trophoblast differentiation, as the syncytiotrophoblast expressed these proteins with little, if any, expression in cytotrophoblast cells. Additionally, exocyst proteins were localized at or near the syncytiotrophoblast apical membrane, the major site of placental secretion Discussion/Conclusion Our findings highlight exocyst protein expression as novel indicators of trophoblast differentiation. The exocyst’s regulated localization within the syncytiotrophoblast in conjunction with its well known functions suggests a possible role in placental polarized secretion PMID:24856041

MatureP: prediction of secreted proteins with exclusive information from their mature regions.

PubMed

Orfanoudaki, Georgia; Markaki, Maria; Chatzi, Katerina; Tsamardinos, Ioannis; Economou, Anastassios

2017-06-12

More than a third of the cellular proteome is non-cytoplasmic. Most secretory proteins use the Sec system for export and are targeted to membranes using signal peptides and mature domains. To specifically analyze bacterial mature domain features, we developed MatureP, a classifier that predicts secretory sequences through features exclusively computed from their mature domains. MatureP was trained using Just Add Data Bio, an automated machine learning tool. Mature domains are predicted efficiently with ~92% success, as measured by the Area Under the Receiver Operating Characteristic Curve (AUC). Predictions were validated using experimental datasets of mutated secretory proteins. The features selected by MatureP reveal prominent differences in amino acid content between secreted and cytoplasmic proteins. Amino-terminal mature domain sequences have enhanced disorder, more hydroxyl and polar residues and less hydrophobics. Cytoplasmic proteins have prominent amino-terminal hydrophobic stretches and charged regions downstream. Presumably, secretory mature domains comprise a distinct protein class. They balance properties that promote the necessary flexibility required for the maintenance of non-folded states during targeting and secretion with the ability of post-secretion folding. These findings provide novel insight in protein trafficking, sorting and folding mechanisms and may benefit protein secretion biotechnology.

Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells

PubMed Central

Stepanyuk, Galina A.; Xu, Hao; Wu, Chia-Kuei; Markova, Svetlana V.; Lee, John; Vysotski, Eugene S.; Wang, Bi-Cheng

2008-01-01

Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence (λmax = 480 nm). This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, E. coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2–3 mg/L. This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence, indicates that luciferase does not undergo posttranslational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. PMID:18595733

Symbiotic implications of type III protein secretion machinery in Rhizobium.

PubMed

Viprey, V; Del Greco, A; Golinowski, W; Broughton, W J; Perret, X

1998-06-01

The symbiotic plasmid of Rhizobium sp. NGR234 carries a cluster of genes that encodes components of a bacterial type III secretion system (TTSS). In both animal and plant pathogens, the TTSS is an essential component of pathogenicity. Here, we show that secretion of at least two proteins (y4xL and NolX) is controlled by the TTSS of NGR234 and occurs after the induction with flavonoids. Polar mutations in two TTSS genes, rhcN and the nod-box controlled regulator of transcription y4xl, block the secretion of both proteins and strongly affect the ability of NGR234 to nodulate a variety of tropical legumes including Pachyrhizus tuberosus and Tephrosia vogelii.

Warfare between Host Immunity and Bacterial Weapons.

PubMed

Yu, Manda; Lai, Erh-Min

2017-01-11

Bacterial pathogens deploy protein secretion systems to facilitate infection and colonization of their hosts. In this issue of Cell Host & Microbe, Chen et al. (2017) report a new role for a type VI secretion effector in promoting bacterial colonization by preventing inflammasome activation induced by a type III secretion system. Copyright © 2017 Elsevier Inc. All rights reserved.

In-depth characterization of the secretome of mouse CNS cell lines by LC-MS/MS without prefractionation.

PubMed

Woo, Jongmin; Han, Dohyun; Park, Joonho; Kim, Sang Jeong; Kim, Youngsoo

2015-11-01

Microglia, astrocytes, and neurons, which have important functions in the central nervous system (CNS), communicate mutually to generate a signal through secreted proteins or small molecules, but many of which have not been identified. Because establishing a reference for the secreted proteins from CNS cells could be invaluable in examining cell-to-cell communication in the brain, we analyzed the secretome of three murine CNS cell lines without prefractionation by high-resolution mass spectrometry. In this study, 2795 proteins were identified from conditioned media of the three cell lines, and 2125 proteins were annotated as secreted proteins by bioinformatics analysis. Further, approximately 500 secreted proteins were quantifiable as differentially expressed proteins by label-free quantitation. As a result, our secretome references are useful datasets for the future study of neuronal diseases. All MS data have been deposited in the ProteomeXchange with identifier PXD001597 (http://proteomecentral.proteomexchange.org/dataset/PXD001597). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative pan genome analysis of oral Prevotella species implicated in periodontitis.

PubMed

Ibrahim, Maziya; Subramanian, Ahalyaa; Anishetty, Sharmila

2017-09-01

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a "Por_secre_tail" domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.

Suppression or activation of immune responses by predicted secreted proteins of the soybean rust pathogen Phakopsora pachyrhizi

USDA-ARS?s Scientific Manuscript database

Rust fungi, such as Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are intimately associated with plant cells. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characte...

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

PubMed

Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

2000-02-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

PubMed Central

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

A FRET-Based DNA Biosensor Tracks OmpR-Dependent Acidification of Salmonella during Macrophage Infection

PubMed Central

Chakraborty, Smarajit; Mizusaki, Hideaki; Kenney, Linda J.

2015-01-01

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed. PMID:25875623

Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes

PubMed Central

Zhu, Yongtao

2013-01-01

The phylum Bacteroidetes is large and diverse, with rapid gliding motility and the ability to digest macromolecules associated with many genera and species. Recently, a novel protein secretion system, the Por secretion system (PorSS), was identified in two members of the phylum, the gliding bacterium Flavobacterium johnsoniae and the nonmotile oral pathogen Porphyromonas gingivalis. The components of the PorSS are not similar in sequence to those of other well-studied bacterial secretion systems. The F. johnsoniae PorSS genes are a subset of the gliding motility genes, suggesting a role for the secretion system in motility. The F. johnsoniae PorSS is needed for assembly of the gliding motility apparatus and for secretion of a chitinase, and the P. gingivalis PorSS is involved in secretion of gingipain protease virulence factors. Comparative analysis of 37 genomes of members of the phylum Bacteroidetes revealed the widespread occurrence of gliding motility genes and PorSS genes. Genes associated with other bacterial protein secretion systems were less common. The results suggest that gliding motility is more common than previously reported. Microscopic observations confirmed that organisms previously described as nonmotile, including Croceibacter atlanticus, “Gramella forsetii,” Paludibacter propionicigenes, Riemerella anatipestifer, and Robiginitalea biformata, exhibit gliding motility. Three genes (gldA, gldF, and gldG) that encode an apparent ATP-binding cassette transporter required for F. johnsoniae gliding were absent from two related gliding bacteria, suggesting that the transporter may not be central to gliding motility. PMID:23123910

A new potential secretion pathway for recombinant proteins in Bacillus subtilis.

PubMed

Wang, Guangqiang; Xia, Yongjun; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Haiqin; Ai, Lianzhong; Chen, Wei

2015-11-10

Secretion of cytoplasmic expressed proteins into growth media has significant advantages. Due to the lack of an outer membrane, Bacillus subtilis is considered as a desirable 'cell factory' for the secretion of recombinant proteins. However, bottlenecks in the classical pathway for the secretion of recombinant proteins limit its use on a wide scale. In this study, we attempted to use four typical non-classically secreted proteins as signals to export three recombinant model proteins to the culture medium. All four non-classically secreted proteins can direct the export of the intrinsically disordered nucleoskeletal-like protein (Nsp). Two of them can guide the secretion of alkaline phosphatase (PhoA). One can lead the secretion of the thermostable β-galactosidase BgaB, which cannot be secreted with the aid of typical Sec-dependent signal peptides. Our results show that the non-classically secreted proteins lead the recombinant proteins to the culture medium, and thus non-classical protein secretion pathways can be exploited as a novel secretion pathway for recombinant proteins.

Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism.

PubMed

Solomonson, Matthew; Setiaputra, Dheva; Makepeace, Karl A T; Lameignere, Emilie; Petrotchenko, Evgeniy V; Conrady, Deborah G; Bergeron, Julien R; Vuckovic, Marija; DiMaio, Frank; Borchers, Christoph H; Yip, Calvin K; Strynadka, Natalie C J

2015-03-03

Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a smallmore » change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.« less

Distribution and diversity of bacterial secretion systems across metagenomic datasets.

PubMed

Barret, Matthieu; Egan, Frank; O'Gara, Fergal

2013-02-01

Bacteria can manipulate their surrounding environment through the secretion of proteins into other living organisms and into the extracellular milieu. In Gram stain negative bacteria this process is mediated by different types of secretion systems from type I through type VI secretion system (T1SS-T6SS). In this study the prevalence of these secretion systems in 312 publicly available microbiomes derived from a wide range of ecosystems was investigated by a gene-centric approach. Our analysis demonstrates that some secretion systems are over-represented in some specific samples. In addition, some T3SS and T6SS phylogenetic clusters were specifically enriched in particular ecological niches, which could indicate specific bacterial adaptation to these environments. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon

DOE PAGES

Lee, Jaemin; Monzingo, Arthur F.; Keatinge-Clay, Adrian T.; ...

2014-12-26

In this paper, the bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. Wemore » isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Finally, possible roles of FlhE, including that of a chaperone, are discussed.« less

Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jaemin; Monzingo, Arthur F.; Keatinge-Clay, Adrian T.

In this paper, the bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. Wemore » isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Finally, possible roles of FlhE, including that of a chaperone, are discussed.« less

The structure of Serratia marcescens Lip, a membrane-bound component of the type VI secretion system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, Vincenzo A.; Shepherd, Sharon M.; English, Grant

2011-12-01

The high-resolution crystal structure of S. marcescens Lip reveals a new member of the transthyretin family of proteins. Lip, a core component of the type VI secretion apparatus, is localized to the outer membrane and is positioned to interact with other proteins forming this complex system. Lip is a membrane-bound lipoprotein and a core component of the type VI secretion system found in Gram-negative bacteria. The structure of a Lip construct (residues 29–176) from Serratia marcescens (SmLip) has been determined at 1.92 Å resolution. Experimental phases were derived using a single-wavelength anomalous dispersion approach on a sample cocrystallized with iodide.more » The membrane localization of the native protein was confirmed. The structure is that of the globular domain lacking only the lipoprotein signal peptide and the lipidated N-terminus of the mature protein. The protein fold is dominated by an eight-stranded β-sandwich and identifies SmLip as a new member of the transthyretin family of proteins. Transthyretin and the only other member of the family fold, 5-hydroxyisourate hydrolase, form homotetramers important for their function. The asymmetric unit of SmLip is a tetramer with 222 symmetry, but the assembly is distinct from that previously noted for the transthyretin protein family. However, structural comparisons and bacterial two-hybrid data suggest that the SmLip tetramer is not relevant to its role as a core component of the type VI secretion system, but rather reflects a propensity for SmLip to participate in protein–protein interactions. A relatively low level of sequence conservation amongst Lip homologues is noted and is restricted to parts of the structure that might be involved in interactions with physiological partners.« less

Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans.

PubMed

Valverde, José R; Gullón, Sonia; Mellado, Rafael P

2018-06-14

Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and variability analysis predicts a large potential for protein overproduction. This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.

Secretion profiles of fungi as potential tools for metal ecotoxicity assessment: a study of enzymatic system in Trametes versicolor.

PubMed

Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

2011-01-01

The relationship between the expression of extracellular enzymatic system and a metal stress is scarce in fungi, hence limiting the possible use of secretion profiles as tools for metal ecotoxicity assessment. In the present study, we investigated the effect of Zn, Cu, Pb and Cd, tested alone or in equimolar cocktail, on the secretion profiles at enzymatic and protein levels in Trametesversicolor. For that purpose, extracellular hydrolases (acid phosphatase, β-glucosidase, β-galactosidase and N-acetyl-β-glucosaminidase) and ligninolytic oxidases (laccase, Mn-peroxidase) were monitored in liquid cultures. Fungal secretome was analyzed by electrophoresis and laccase secretion was characterized by western-blot and mass spectrometry analyses. Our results showed that all hydrolase activities were inhibited by the metals tested alone or in cocktail, whereas oxidase activities were specifically stimulated by Cu, Cd and metal cocktail. At protein level, metal exposure modified the electrophoretic profiles of fungal secretome and affected the diversity of secreted proteins. Two laccase isoenzymes, LacA and LacB, identified by mass spectrometry were differentially glycosylated according to the metal exposure. The amount of secreted LacA and LacB was strongly correlated with the stimulation of laccase activity by Cu, Cd and metal cocktail. These modifications of extracellular enzymatic system suggest that fungal oxidases could be used as biomarkers of metal exposure. Copyright © 2010 Elsevier Ltd. All rights reserved.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE

PubMed Central

Treerat, Puthayalai; Alwis, Priyangi; D’Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J.; Prescott, Mark; Adler, Ben; Boyce, John D.

2015-01-01

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness. PMID:26624293

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

PubMed

Treerat, Puthayalai; Alwis, Priyangi; D'Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

2015-01-01

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family†

PubMed Central

Meyer, Damien; Cunnac, Sébastien; Guéneron, Mareva; Declercq, Céline; Van Gijsegem, Frédérique; Lauber, Emmanuelle; Boucher, Christian; Arlat, Matthieu

2006-01-01

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum. PMID:16788199

Self-Chaperoning of the Type III Secretion System needle tip proteins IpaD and BipD

PubMed Central

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E.; Birket, Susan; Field, Terry; Picking, William D.; Blocker, Ariel; Galyov, Edouard E.; Picking, Wendy L.; Lea, Susan M.

2007-01-01

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. PMID:17077085

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.

PubMed

Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E; Birket, Susan; Field, Terry; Picking, William D; Blocker, Ariel J; Galyov, Edouard E; Picking, Wendy L; Lea, Susan M

2007-02-09

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.

Construction of a reporter system to study Burkholderia mallei type III secretion and identification of the BopA effector protein function in intracellular survival.

PubMed

Whitlock, Gregory C; Estes, D Mark; Young, Glenn M; Young, Briana; Torres, Alfredo G

2008-12-01

Burkholderia mallei, the aetiological agent of glanders disease, is a Gram-negative facultative intracellular bacterium. Despite numerous studies, the detailed mechanism of its pathogenesis is almost unknown. The presence of a type III secretion system (TTSS) is one of the known mechanisms associated with virulence. An intact TTSS indicates that B. mallei is able to secrete proteins in response to different environmental conditions, which could play an important role in pathogenesis. Therefore, characterization of the TTSS and identification of the secreted proteins associated with bacterial pathogenesis could provide crucial information for the development of a candidate vaccine. In the current study, we used an enzymatic reporter system to establish some of the conditions enabling TTS. Construction of the TTSS bopA mutant revealed that BopA is important for B. mallei invasion and intracellular survival. Overall, our study elucidates how BopA can aid in the optimization of TTS and defines the function of TTS effectors in bacterial intracellular survival and invasion.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less

Structure of a Type-1 Secretion System ABC Transporter.

PubMed

Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

2017-03-07

Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

PubMed Central

Chen, Chen; Gao, George F.

2012-01-01

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans. PMID:23056296

The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme.

PubMed

Ligtenberg, Antoon J M; Brand, Henk S; van den Keijbus, Petra A M; Veerman, Enno C I

2015-11-01

Saliva secretion is regulated by the autonomic nervous system. Parasympathic stimuli increase the secretion of water and mucin MUC5B, whereas sympathetic stimuli such as physical exercise increase the secretion of amylase and other proteins. In the present study we investigated the effect of physical exercise, as a sympathetic stimulus, on salivary flow rate and output of MUC5B, amylase, lysozyme and total protein. Unstimulated whole saliva was collected before exercise (1), after 10 min exercise with moderate intensity by running with a heart rate around 130 beats per minute (2), followed by 10 min exercise with high intensity by running to exhaustion (3) and after 30 min recovery (4). Salivary flow rate, protein and MUC5B concentration, and amylase and lysozyme activity were determined. Saliva protein composition was analysed using SDS-PAGE and immunoblotting. Salivary flow rate, protein and lysozyme secretion increased after exercise with moderate intensity and increased further after exercise with high intensity (p<0.01). Amylase and MUC5B increased after exercise with moderate intensity (p<0.0001), but did not differ significantly between moderate and high exercise intensity. SDS-PAGE analysis and immunoblotting showed that, especially after exercise with high intensity, the concentrations of several other salivary proteins, including MUC7, albumin, and extra-parotid glycoprotein, also increased. Exercise may not only lead to the anticipated increase in amylase and protein secretion, but also to an increase in salivary flow rate and MUC5B secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Je Min, E-mail: jemin@knu.ac.kr; Department of Horticultural Science, Kyungpook National University, Daegu; Lee, Sang-Jik

Highlights: • Yeast secretion trap (YST) is a valuable tool for mining secretome. • A total of 80 secreted proteins are newly identified via YST in pepper fruits. • The secreted proteins are differentially regulated during pepper development and ripening. • Transient GFP-fusion assay and in planta secretion trap can effectively validate the secretion of proteins. - Abstract: Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function inmore » fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.« less

Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication

PubMed Central

Wegrzyn, Jill L.; Bark, Steven J.; Funkelstein, Lydiane; Mosier, Charles; Yap, Angel; Kazemi-Esfarjani, Parasa; La Spada, Albert; Sigurdson, Christina; O’Connor, Daniel T.; Hook, Vivian

2010-01-01

Regulated secretion of neurotransmitters and neurohumoural factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 distinct proteins in the soluble fraction and 384 distinct membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease. PMID:20695487

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain.

PubMed

Burkhart, Annette; Andresen, Thomas Lars; Aigner, Achim; Thomsen, Louiza Bohn; Moos, Torben

2017-07-01

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood-brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000™ was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

DOE PAGES

Fan, Yao; Tan, Kemin; Chhor, Gekleng; ...

2015-07-03

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

Functional cloning of Vibrio parahaemolyticus type III secretion system 1 in Escherichia coli K-12 strain as a molecular syringe.

PubMed

Akeda, Yukihiro; Kimura, Tomomi; Yamasaki, Aiko; Kodama, Toshio; Iida, Tetsuya; Honda, Takeshi; Oishi, Kazunori

2012-10-19

The type III secretion system (T3SS) of gram-negative bacteria involves dedicated protein translocation machinery that directly injects proteins into target cells. Pathogenic bacteria already benefit from this unique system. The successful functional cloning of this useful tool into non-pathogenic bacteria would help establish novel clinical and basic biotechnology strategies in areas such as vaccine administration, the development of screening systems for anti-T3SS drugs and the target-specific delivery of bioactive compounds. In this study, we successfully cloned the Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic Escherichia coli K-12 strain. Assays performed here revealed that the T3SS1 cloned into the E. coli K-12 strain has the ability to translocate V. parahaemolyticus T3SS1 secreted proteins. Importantly, we also observed this system to allow the E. coli K-12 strain to inject foreign protein, as well as the V. parahaemolyticus T3SS effector, into cultured cells. These results demonstrate a prospective useful tool with experimental and therapeutic applications. Copyright © 2012 Elsevier Inc. All rights reserved.

Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

PubMed Central

Dolezal, Pavel; Aili, Margareta; Tong, Janette; Jiang, Jhih-Hang; Marobbio, Carlo M.; Lee, Sau fung; Schuelein, Ralf; Belluzzo, Simon; Binova, Eva; Mousnier, Aurelie; Frankel, Gad; Giannuzzi, Giulia; Palmieri, Ferdinando; Gabriel, Kipros; Naderer, Thomas; Hartland, Elizabeth L.; Lithgow, Trevor

2012-01-01

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms. PMID:22241989

Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives.

PubMed

Lakowitz, Antonia; Godard, Thibault; Biedendieck, Rebekka; Krull, Rainer

2018-05-01

Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production. Copyright © 2017. Published by Elsevier B.V.

LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion.

PubMed

Wulff-Strobel, Christine R; Williams, Andrew W; Straley, Susan C

2002-01-01

LcrQ is a regulatory protein unique to Yersinia. Previous study in Yersinia pseudotuberculosis and Yersinia enterocolitica prompted the model in which LcrQ negatively regulates the expression of a set of virulence proteins called Yops, and its secretion upon activation of the Yop secretion (Ysc) type III secretion system permits full induction of Yops expression. In this study, we tested the hypothesis that LcrQ's effects on Yops expression might be indirect. Excess LcrQ was found to exert an inhibitory effect specifically at the level of Yops secretion, independent of production, and a normal inner Ysc gate protein LcrG was required for this activity. However, overexpression of LcrQ did not prevent YopH secretion, suggesting that LcrQ's effects at the Ysc discriminate among the Yops. We tested this idea by determining the effects of deletion or overexpression of LcrQ, YopH and their common chaperone SycH on early Yop secretion through the Ysc. Together, our findings indicated that LcrQ is not a negative regulator directly, but it acts in partnership with SycH at the Ysc gate to control the entry of a set of Ysc secretion substrates. A hierarchy of YopH secretion before YopE appears to be imposed by SycH in conjunction with both LcrQ and YopH. LcrQ and SycH in addition influenced the deployment of LcrV, a component of the Yops delivery mechanism. Accordingly, LcrQ appears to be a central player in determining the substrate specificity of the Ysc.

Discovery of the type VII ESX-1 secretion needle?

PubMed

Ates, Louis S; Brosch, Roland

2017-01-01

Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C. © 2016 John Wiley & Sons Ltd.

Secretome analysis of diarrhea-inducing strains of Escherichia coli

PubMed Central

Nirujogi, Raja Sekhar; Muthusamy, Babylakshmi; Kim, Min-Sik; Sathe, Gajanan J.; Lakshmi, P.T.V.; Kovbasnjuk, Olga N.; Prasad, T.S. Keshava; Wade, Mary; Jabbour, Rabih E.

2017-01-01

Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by Gram-negative bacteria. Enterohemorrhagic Escherichia coli, O157:H7, is the major pathogen often causing outbreaks. However, studies have reported that the significant outbreaks caused by non-O157:H7 E. coli strains, also known as “Big-Six” serogroup strains, are increasing. There is no systematic study describing differential secreted proteins from these non-O157:H7 E. coli strains. In this study, we carried out MS-based differential secretome analysis using tandem mass tags labeling strategy of non-O157:H7 E. coli strains, O103, O111, O121, O145, O26, and O45. We identified 1241 proteins, of which 565 proteins were predicted to be secreted. We also found that 68 proteins were enriched in type III secretion system and several of them were differentially expressed across the strains. Additionally, we identified several strain-specific secreted proteins that could be used for developing potential markers for the identification and strain-level differentiation. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli Big-Six serogroup and the several of these strain-specific secreted proteins can be further studied to develop potential markers for identification and strain-level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and detection and identification of bio threats in biodefense. PMID:28070933

Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27's Phosphorylation Status, and Is Mediated by Exosome Liberation

PubMed Central

Klinkmann, Gerd; Diesing, Karoline; Koensgen, Dominique; Burchardt, Martin

2017-01-01

The heat shock protein HSP27 has been correlated in ovarian cancer (OC) patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker. PMID:28325957

Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27's Phosphorylation Status, and Is Mediated by Exosome Liberation.

PubMed

Stope, Matthias B; Klinkmann, Gerd; Diesing, Karoline; Koensgen, Dominique; Burchardt, Martin; Mustea, Alexander

2017-01-01

The heat shock protein HSP27 has been correlated in ovarian cancer (OC) patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker.

Porphyromonas gingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility

PubMed Central

Nakayama, K

2015-01-01

Porphyromonas gingivalis is a gram-negative, non-motile, anaerobic bacterium implicated as a major pathogen in periodontal disease. P. gingivalis grows as black-pigmented colonies on blood agar, and many bacteriologists have shown interest in this property. Studies of colonial pigmentation have revealed a number of important findings, including an association with the highly active extracellular and surface proteinases called gingipains that are found in P. gingivalis. The Por secretion system, a novel type IX secretion system (T9SS), has been implicated in gingipain secretion in studies using non-pigmented mutants. In addition, many potent virulence proteins, including the metallocarboxypeptidase CPG70, 35 kDa hemin-binding protein HBP35, peptidylarginine deiminase PAD and Lys-specific serine endopeptidase PepK, are secreted through the T9SS. These findings have not been limited to P. gingivalis but have been extended to other bacteria belonging to the phylum Bacteroidetes. Many Bacteroidetes species possess the T9SS, which is associated with gliding motility for some of these bacteria. PMID:25546073

Secretory carrier membrane protein 5 is an autophagy inhibitor that promotes the secretion of α-synuclein via exosome.

PubMed

Yang, Yi; Qin, Meiling; Bao, Puhua; Xu, Wangchao; Xu, Jin

2017-01-01

Autophagy-lysosomal pathway is a cellular protective system to remove aggregated proteins and damaged organelles. Meanwhile, exosome secretion has emerged as a mode to selectively clear the neurotoxic proteins, such as α-synuclein. Mounting evidence suggests that these two cellular processes are coordinated to facilitate the clearance of toxic cellular waste; however the regulators for the transition between these two processes are unclear. Here we show that SCAMP5, a secretory carrier membrane protein significantly induced in the brains of Huntington's disease patients, is quickly and transiently induced by protein stress and autophagic stimulation, and is regulated by the master autophagy transcriptional regulator TFEB. Ironically, SCAMP5 inhibits autophagy flux by blocking the fusion of autophagosomes and lysosomes. Although autophagy is blocked, SCAMP5 does not cause significant protein aggregation in cells. Instead, it promotes the Golgi fragmentation and stimulates the unconventional secretion of the co-localizing α-synuclein via exosome as an exosome component. Therefore, we have identified SCAMP5 as a novel coordinator of autophagy and exosome secretion, which is induced upon protein stress to channel the efficient clearance of toxic proteins via the exosomes rather than autophagy-lysosomal pathway.

A Novel ESAT-6 Secretion System-Secreted Protein EsxX of Community-Associated Staphylococcus aureus Lineage ST398 Contributes to Immune Evasion and Virulence.

PubMed

Dai, Yingxin; Wang, Yanan; Liu, Qian; Gao, Qianqian; Lu, Huiying; Meng, Hongwei; Qin, Juanxiu; Hu, Mo; Li, Min

2017-01-01

The ESAT-6 secretion system (ESS) has been reported to contribute to the virulence and pathogenicity of several Staphylococcus aureus strains such as USA300 and Newman. However, the role of the ESS in community-associated S. aureus (CA-SA) lineage ST398 in China is not well understood. By comparing the ess locus of ST398 with the published S. aureus sequence in the NCBI database, we found one gene in the ess locus encoding a novel WXG superfamily protein that is highly conserved only in ST398. LC-MS/MS and Western blot analysis revealed that this protein is a novel secreted protein controlled by the ST398 ESS, and we named the protein EsxX. Although EsxX was not under the control of the accessory gene regulator like many other virulence factors and had no influence on several phenotypes of ST398, such as growth, hemolysis, and biofilm formation, it showed important impacts on immune evasion and virulence in ST398. An esxX deletion mutant led to significantly reduced resistance to neutrophil killing and decreased virulence in murine skin and blood infection models, indicating its essential contribution to the evasion of innate host defense and virulence to support the pathogenesis of ST398 infections. The function of this novel secreted protein EsxX might help us better understand the role of the ESS in the virulence and epidemic success of the CA-SA lineage ST398.

Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes.

PubMed

Adam, Philip R; Barta, Michael L; Dickenson, Nicholas E

2017-01-01

In vitro characterization of type III secretion system (T3SS) translocator proteins has proven challenging due to complex purification schemes and their hydrophobic nature that often requires detergents to provide protein solubility and stability. Here, we provide experimental details for several techniques that overcome these hurdles, allowing for the direct characterization of the Shigella translocator protein IpaB with respect to phospholipid membrane interaction. The techniques specifically discussed in this chapter include membrane interaction/liposome flotation, liposome sensitive fluorescence quenching, and protein-mediated liposome disruption assays. These assays have provided valuable insight into the role of IpaB in T3SS-mediated phospholipid membrane interactions by Shigella and should readily extend to other members of this important class of proteins.

Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samudrala, Ram; Heffron, Fred; McDermott, Jason E.

2009-04-24

The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates, effector proteins, are not. We have used a machine learning approach to identify new secreted effectors. The method integrates evolutionary measures, such as the pattern of homologs in a range of other organisms, and sequence-based features, such as G+C content, amino acid composition and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from Salmonella typhimurium and validated on a corresponding set of effectors from Pseudomonas syringae, aftermore » eliminating effectors with detectable sequence similarity. The method was able to identify all of the known effectors in P. syringae with a specificity of 84% and sensitivity of 82%. The reciprocal validation, training on P. syringae and validating on S. typhimurium, gave similar results with a specificity of 86% when the sensitivity level was 87%. These results show that type III effectors in disparate organisms share common features. We found that maximal performance is attained by including an N-terminal sequence of only 30 residues, which agrees with previous studies indicating that this region contains the secretion signal. We then used the method to define the most important residues in this putative secretion signal. Finally, we present novel predictions of secreted effectors in S. typhimurium, some of which have been experimentally validated, and apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis. This approach is a novel and effective way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal.« less

Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

PubMed

Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

2016-03-01

The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

Construction of recombinant Lactobacillus casei efficiently surface displayed and secreted porcine parvovirus VP2 protein and comparison of the immune responses induced by oral immunization.

PubMed

Yigang, X U; Yijing, L I

2008-05-01

Lactobacillus casei ATCC 393 was selected as a bacterial carrier for the development of mucosal vaccine against porcine parvovirus (PPV) infection. The PPV major structural polypeptide VP2 was used as the model parvovirus antigen. Two inducible expression systems, namely pPG611.1 of the cell-surface expression system and pPG612.1 of the secretion expression system based on the xylose operon promoter were used to express the VP2 protein. The immunogenicity of recombinant strains producing VP2 protein in two cellular locations, cell-surface exposed and secreted, was compared to each other by immunizing mice through the intragastric administration. The two types of constructs were able to induce strong specific immune responses against VP2 via intragastric administration and maximum titres of IgA and IgG were attained on days 46 post oral immunization, while the highest antibody levels were obtained with the strain producing the VP2 protein in extracellular milieu. The induced antibodies demonstrated neutralizing effects on PPV infection.

Type VI Secretion System in Pseudomonas aeruginosa

PubMed Central

Hachani, Abderrahman; Lossi, Nadine S.; Hamilton, Alexander; Jones, Cerith; Bleves, Sophie; Albesa-Jové, David; Filloux, Alain

2011-01-01

Pseudomonas aeruginosa is a Gram-negative bacterium causing chronic infections in cystic fibrosis patients. Such infections are associated with an active type VI secretion system (T6SS), which consists of about 15 conserved components, including the AAA+ ATPase, ClpV. The T6SS secretes two categories of proteins, VgrG and Hcp. Hcp is structurally similar to a phage tail tube component, whereas VgrG proteins show similarity to the puncturing device at the tip of the phage tube. In P. aeruginosa, three T6SSs are known. The expression of H1-T6SS genes is controlled by the RetS sensor. Here, 10 vgrG genes were identified in the PAO1 genome, among which three are co-regulated with H1-T6SS, namely vgrG1a/b/c. Whereas VgrG1a and VgrG1c were secreted in a ClpV1-dependent manner, secretion of VgrG1b was ClpV1-independent. We show that VgrG1a and VgrG1c form multimers, which confirmed the VgrG model predicting trimers similar to the tail spike. We demonstrate that Hcp1 secretion requires either VgrG1a or VgrG1c, which may act independently to puncture the bacterial envelope and give Hcp1 access to the surface. VgrG1b is not required for Hcp1 secretion. Thus, VgrG1b does not require H1-T6SS for secretion nor does H1-T6SS require VgrG1b for its function. Finally, we show that VgrG proteins are required for secretion of a genuine H1-T6SS substrate, Tse3. Our results demonstrate that VgrG proteins are not only secreted components but are essential for secretion of other T6SS substrates. Overall, we emphasize variability in behavior of three P. aeruginosa VgrGs, suggesting that, although very similar, distinct VgrGs achieve specific functions. PMID:21325275

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems*

PubMed Central

Durand, Eric; Zoued, Abdelrahim; Spinelli, Silvia; Watson, Paul J. H.; Aschtgen, Marie-Stéphanie; Journet, Laure; Cambillau, Christian; Cascales, Eric

2012-01-01

The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families. PMID:22371492

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex*

PubMed Central

Vincent, Maxence S.; Canestrari, Mickaël J.; Leone, Philippe; Stathopulos, Julien; Ize, Bérengère; Zoued, Abdelrahim; Cambillau, Christian; Kellenberger, Christine; Roussel, Alain

2017-01-01

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts. PMID:28057754

Extracellular proteome analysis of Leptospira interrogans serovar Lai.

PubMed

Zeng, Lingbing; Zhang, Yunyi; Zhu, Yongzhang; Yin, Haidi; Zhuang, Xuran; Zhu, Weinan; Guo, Xiaokui; Qin, Jinhong

2013-10-01

Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System*

PubMed Central

Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H.; Wolf-Watz, Magnus

2017-01-01

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria. PMID:28039361

The role of TcdB and TccC subunits in secretion of the Photorhabdus Tcd toxin complex.

PubMed

Yang, Guowei; Waterfield, Nicholas R

2013-01-01

The Toxin Complex (TC) is a large multi-subunit toxin encoded by a range of bacterial pathogens. The best-characterized examples are from the insect pathogens Photorhabdus, Xenorhabdus and Yersinia. They consist of three large protein subunits, designated A, B and C that assemble in a 5∶1∶1 stoichiometry. Oral toxicity to a range of insects means that some have the potential to be developed as pest control technology. The three subunit proteins do not encode any recognisable export sequences and as such little progress has been made in understanding their secretion. We have developed heterologous TC production and secretion models in E. coli and used them to ascribe functions to different domains of the crucial B+C sub-complex. We have determined that the B and C subunits use a secretion mechanism that is either encoded by the proteins themselves or employ an as yet undefined system common to laboratory strains of E. coli. We demonstrate that both the N-terminal domains of the B and C subunits are required for secretion of the whole complex. We propose a model whereby the N-terminus of the C-subunit toxin exports the B+C sub-complex across the inner membrane while that of the B-subunit allows passage across the outer membrane. We also demonstrate that even in the absence of the B-subunit, that the C-subunit can also facilitate secretion of the larger A-subunit. The recognition of this novel export system is likely to be of importance to future protein secretion studies. Finally, the identification of homologues of B and C subunits in diverse bacterial pathogens, including Burkholderia and Pseudomonas, suggests that these toxins are likely to be important in a range of different hosts, including man.

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

PubMed Central

Vicente, Rebeca L.; Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

2016-01-01

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application. PMID:27977736

The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism.

PubMed

Rossello, Jessica; Lima, Analía; Gil, Magdalena; Rodríguez Duarte, Jorge; Correa, Agustín; Carvalho, Paulo C; Kierbel, Arlinet; Durán, Rosario

2017-08-31

The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

Functional type 1 secretion system involved in Legionella pneumophila virulence.

PubMed

Fuche, Fabien; Vianney, Anne; Andrea, Claire; Doublet, Patricia; Gilbert, Christophe

2015-02-01

Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Functional Type 1 Secretion System Involved in Legionella pneumophila Virulence

PubMed Central

Fuche, Fabien; Vianney, Anne; Andrea, Claire; Doublet, Patricia

2014-01-01

Legionella pneumophila is a Gram-negative pathogen found mainly in water, either in a free-living form or within infected protozoans, where it replicates. This bacterium can also infect humans by inhalation of contaminated aerosols, causing a severe form of pneumonia called legionellosis or Legionnaires' disease. The involvement of type II and IV secretion systems in the virulence of L. pneumophila is now well documented. Despite bioinformatic studies showing that a type I secretion system (T1SS) could be present in this pathogen, the functionality of this system based on the LssB, LssD, and TolC proteins has never been established. Here, we report the demonstration of the functionality of the T1SS, as well as its role in the infectious cycle of L. pneumophila. Using deletion mutants and fusion proteins, we demonstrated that the repeats-in-toxin protein RtxA is secreted through an LssB-LssD-TolC-dependent mechanism. Moreover, fluorescence monitoring and confocal microscopy showed that this T1SS is required for entry into the host cell, although it seems dispensable to the intracellular cycle. Together, these results underline the active participation of L. pneumophila, via its T1SS, in its internalization into host cells. PMID:25422301

Chromobacterium violaceum: important insights for virulence and biotechnological potential by exoproteomic studies.

PubMed

Ciprandi, Alessandra; da Silva, Wanderson Marques; Santos, Agenor Valadares; de Castro Pimenta, Adriano Monteiro; Carepo, Marta Sofia Peixe; Schneider, Maria Paula Cruz; Azevedo, Vasco; Silva, Artur

2013-07-01

Chromobacterium violaceum is a beta-proteobacterium with high biotechnological potential, found in tropical environments. This bacterium causes opportunistic infections in both humans and animals, that can spread throughout several tissues, quickly leading to the death of the host. Genomic studies identified potential mechanisms of pathogenicity but no further studies were done to confirm the expression of these systems. In this study 36 unique protein entries were identified in databank from a two-dimensional profile of C. violaceum secreted proteins. Chromobacterium violaceum exoproteomic preliminary studies confirmed the production of proteins identified as virulence factors (such as a collagenase, flagellum proteins, metallopeptidases, and toxins), allowing us to better understand its pathogenicity mechanisms. Biotechnologically interesting proteins (such as chitinase and chitosanase) were also identified among the secreted proteins, as well as proteins involved in the transport and capture of amino acids, carbohydrates, and oxidative stress protection. Overall, the secreted proteins identified provide us important insights on pathogenicity mechanisms, biotechnological potential, and environment adaptation of C. violaceum.

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

PubMed

Wanka, Franziska; Arentshorst, Mark; Cairns, Timothy C; Jørgensen, Thomas; Ram, Arthur F J; Meyer, Vera

2016-08-20

The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency. In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A. niger continuous cultivation of industrially relevant products. Firstly, promoters of genes encoding either an antifungal protein (Panafp) or putative hydrophobin (PhfbD) were confirmed as active throughout retentostat culture by assessing mRNA and protein levels using a luciferase (mluc) reporter system. This demonstrated the anafp promoter mediates a high but temporally variable expression profile, whereas the hfbD promoter mediates a semi-constant, moderate-to-high protein expression during retentostat culture. In order to assess whether these promoters were suitable to produce heterologous proteins during retentostat cultivation, the secreted antifungal protein (AFP) from Aspergillus giganteus, which has many potential biotechnological applications, was expressed in A. niger during retentostat cultivation. Additionally, this assay was used to concomitantly validate that native secretion signals encoded in anafp and hfbD genes can be harnessed for secretion of heterologous proteins. Afp mRNA and protein abundance were comparable to luciferase measurements throughout retentostat cultivation, validating the use of Panafp and PhfbD for perfusion cultivation. Finally, a gene encoding the highly commercially relevant thermal hysteresis protein (THP) was expressed in this system, which did not yield detectable protein. Both hfbD and anafp promoters are suitable for production of useful products in A. niger during perfusion cultivation. These findings provide a platform for further optimisations for high production of heterologous proteins with industrial relevance.

Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody

PubMed Central

Korotkov, Konstantin V.; Pardon, Els

2009-01-01

Summary Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a “nanobody”, the VHH domain of a “heavy-chain” camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains with the second and third subdomains exhibiting the KH-fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonB-dependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible β-strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by β-strand complementation. PMID:19217396

Proteomic Identification of Novel Secreted Antibacterial Toxins of the Serratia marcescens Type VI Secretion System*

PubMed Central

Fritsch, Maximilian J.; Trunk, Katharina; Diniz, Juliana Alcoforado; Guo, Manman; Trost, Matthias; Coulthurst, Sarah J.

2013-01-01

It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species. PMID:23842002

Transporter-mediated biofuel secretion.

PubMed

Doshi, Rupak; Nguyen, Tuan; Chang, Geoffrey

2013-05-07

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as "plug-and-play" biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance.

Transporter-mediated biofuel secretion

PubMed Central

Doshi, Rupak; Nguyen, Tuan; Chang, Geoffrey

2013-01-01

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as “plug-and-play” biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance. PMID:23613592

Recombinant protein secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a novel signal peptide.

PubMed

Cheng, Yali; Avis, Tyler J; Bolduc, Sébastien; Zhao, Yingyi; Anguenot, Raphaël; Neveu, Bertrand; Labbé, Caroline; Belzile, François; Bélanger, Richard R

2008-12-01

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srikannathasan, Velupillai; English, Grant; Bui, Nhat Khai

Crystal structures of type VI secretion system-associated immunity proteins, a peptidoglycan endopeptidase and a complex of the endopeptidase and its cognate immunity protein are reported together with assays of endopeptidase activity and functional assessment. Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-d-glutamic acid and l-meso-diaminopimelic acid with differentmore » specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure–activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1–Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2–Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector–immunity protein interactions.« less

DsbA Plays a Critical and Multifaceted Role in the Production of Secreted Virulence Factors by the Phytopathogen Erwinia carotovora subsp. atroseptica*S⃞

PubMed Central

Coulthurst, Sarah J.; Lilley, Kathryn S.; Hedley, Peter E.; Liu, Hui; Toth, Ian K.; Salmond, George P. C.

2008-01-01

Erwinia carotovora subsp. atroseptica is an enterobacterial phytopathogen causing economically significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the type II (Out) secretion system. DsbA catalyzes the introduction of disulfide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type E. carotovora subsp. atroseptica SCRI1043, and dsbA and out mutants, was analyzed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted, and novel candidate virulence factors. Further characterization of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorumsensing signal, and virulence were absent or substantially reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multifaceted role in the pathogenesis of E. carotovora subsp. atroseptica. PMID:18562317

Reconstruction of the bifidobacterial pan-secretome reveals the network of extracellular interactions between bifidobacteria and the infant gut.

PubMed

Lugli, Gabriele Andrea; Mancino, Walter; Milani, Christian; Duranti, Sabrina; Turroni, Francesca; van Sinderen, Douwe; Ventura, Marco

2018-06-08

The repertoire of secreted proteins decoded by a microorganism represents proteins released from or associated with the cell's surface. In gut commensals, such as bifidobacteria, these proteins are perceived to be functionally relevant as they regulate the interaction with the gut environment. In the current study, we have screened the predicted proteome of over 300 bifidobacterial strains amongst the currently recognized bifidobacterial species to generate a comprehensive database encompassing bifidobacterial extracellular proteins. A glycobiome analysis of this predicted bifidobacterial secretome revealed that a correlation exists between particular bifidobacterial species and their capability to hydrolyze HMOs and intestinal glyconjugates such as mucin. Furthermore, exploration of metatranscriptomic datasets of the infant gut microbiota allowed the evaluation of the expression of bifidobacterial genes encoding extracellular proteins, represented by ABC transporter substrate-binding proteins and glycoside hydrolases enzymes involved in the degradation of human milk oligosaccharides and mucin. Overall, this study provides insights into how bifidobacteria interact with their natural yet highly complex environment, the infant gut. Importance The ecological success of bifidobacteria relies on the activity of extracellular proteins that are involved in the metabolism of nutrients and the interaction with the environment. To date, information on secreted proteins encoded by bifidobacteria are incomplete and just related to few species. In this study, we reconstructed the bifidobacterial pan-secretome, revealing extracellular proteins that modulate the interaction of bifidobacteria with their natural environment. Furthermore, a survey of secretion system between bifidobacterial genomes allowed the identification of a conserved Sec-dependent secretion machinery in all the analyzed genomes and the Tat protein translocation system in the chromosomes of 23 strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium aesculapii . Copyright © 2018 American Society for Microbiology.

Campylobacter fetus subspecies contain conserved type IV secretion systems on multiple genomic islands and plasmids

USDA-ARS?s Scientific Manuscript database

The features contributing to the differences in pathogenicity of the C. fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode type IV secretion system (T4SS) and fic-domain (filamentation induced by cyclic AMP) proteins. In the genomes of ...

Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases.

PubMed

Pietrocola, Giampiero; Nobile, Giulia; Rindi, Simonetta; Speziale, Pietro

2017-01-01

Neutrophils, complement system and skin collectively represent the main elements of the innate immune system, the first line of defense of the host against many common microorganisms. Bacterial pathogens have evolved strategies to counteract all these defense activities. Specifically, Staphylococcus aureus , a major human pathogen, secretes a variety of immune evasion molecules including proteases, which cleave components of the innate immune system or disrupt the integrity of extracellular matrix and intercellular connections of tissues. Additionally, S. aureus secretes proteins that can activate host zymogens which, in turn, target specific defense components. Secreted proteins can also inhibit the anti-bacterial function of neutrophils or complement system proteases, potentiating S. aureus chances of survival. Here, we review the current understanding of these proteases and modulators of host proteases in the functioning of innate immunity and describe the importance of these mechanisms in the pathology of staphylococcal diseases.

Staphylococcus aureus Manipulates Innate Immunity through Own and Host-Expressed Proteases

PubMed Central

Pietrocola, Giampiero; Nobile, Giulia; Rindi, Simonetta; Speziale, Pietro

2017-01-01

Neutrophils, complement system and skin collectively represent the main elements of the innate immune system, the first line of defense of the host against many common microorganisms. Bacterial pathogens have evolved strategies to counteract all these defense activities. Specifically, Staphylococcus aureus, a major human pathogen, secretes a variety of immune evasion molecules including proteases, which cleave components of the innate immune system or disrupt the integrity of extracellular matrix and intercellular connections of tissues. Additionally, S. aureus secretes proteins that can activate host zymogens which, in turn, target specific defense components. Secreted proteins can also inhibit the anti-bacterial function of neutrophils or complement system proteases, potentiating S. aureus chances of survival. Here, we review the current understanding of these proteases and modulators of host proteases in the functioning of innate immunity and describe the importance of these mechanisms in the pathology of staphylococcal diseases. PMID:28529927

Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscUCC

PubMed Central

Frost, Stefan; Ho, Oanh; Login, Frédéric H.; Weise, Christoph F.; Wolf-Watz, Hans; Wolf-Watz, Magnus

2012-01-01

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms. PMID:23185318

Autoproteolysis and intramolecular dissociation of Yersinia YscU precedes secretion of its C-terminal polypeptide YscU(CC).

PubMed

Frost, Stefan; Ho, Oanh; Login, Frédéric H; Weise, Christoph F; Wolf-Watz, Hans; Wolf-Watz, Magnus

2012-01-01

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC). Here we show that depletion of calcium induces intramolecular dissociation of YscU(CC) from YscU followed by secretion of the YscU(CC) polypeptide. Thus, YscU(CC) behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU(CC)in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU(CC) dissociation for Yop secretion. We propose that YscU(CC) orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

A Repulsive Electrostatic Mechanism for Protein Export through the Type III Secretion Apparatus

PubMed Central

Rathinavelan, Thenmalarchelvi; Zhang, Lingling; Picking, Wendy L.; Weis, David D.; De Guzman, Roberto N.; Im, Wonpil

2010-01-01

Abstract Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus, which is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. The trajectories reveal a screwlike rotation motion during the export of nativelike helix-turn-helix conformations. Interestingly, the channel interior with excessive electronegative potential creates an energy barrier for MxiH to enter the channel, whereas the same may facilitate the ejection of the effectors into host cells. Structurally known basal regions and ATPase underneath the basal region also have electronegative interiors. Effector proteins also have considerable electronegative potential patches on their surfaces. From these observations, we propose a repulsive electrostatic mechanism for protein translocation through the type III secretion apparatus. Based on this mechanism, the ATPase activity and/or proton motive force could be used to energize the protein translocation through these nanomachines. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported. PMID:20141759

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal*

PubMed Central

Login, Frédéric H.; Wolf-Watz, Hans

2015-01-01

All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca2+-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the “classical” N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. PMID:26338709

The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function

PubMed Central

Lasica, Anna M.; Ksiazek, Miroslaw; Madej, Mariusz; Potempa, Jan

2017-01-01

Protein secretion systems are vital for prokaryotic life, as they enable bacteria to acquire nutrients, communicate with other species, defend against biological and chemical agents, and facilitate disease through the delivery of virulence factors. In this review, we will focus on the recently discovered type IX secretion system (T9SS), a complex translocon found only in some species of the Bacteroidetes phylum. T9SS plays two roles, depending on the lifestyle of the bacteria. It provides either a means of movement (called gliding motility) for peace-loving environmental bacteria or a weapon for pathogens. The best-studied members of these two groups are Flavobacterium johnsoniae, a commensal microorganism often found in water and soil, and Porphyromonas gingivalis, a human oral pathogen that is a major causative agent of periodontitis. In P. gingivalis and some other periodontopathogens, T9SS translocates proteins, especially virulence factors, across the outer membrane (OM). Proteins destined for secretion bear a conserved C-terminal domain (CTD) that directs the cargo to the OM translocon. At least 18 proteins are involved in this still enigmatic process, with some engaged in the post-translational modification of T9SS cargo proteins. Upon translocation across the OM, the CTD is removed by a protease with sortase-like activity and an anionic LPS is attached to the newly formed C-terminus. As a result, a cargo protein could be secreted into the extracellular milieu or covalently attached to the bacterial surface. T9SS is regulated by a two-component system; however, the precise environmental signal that triggers it has not been identified. Exploring unknown systems contributing to bacterial virulence is exciting, as it may eventually lead to new therapeutic strategies. During the past decade, the major components of T9SS were identified, as well as hints suggesting the possible mechanism of action. In addition, the list of characterized cargo proteins is constantly growing. The actual structure of the translocon, situated in the OM of bacteria, remains the least explored area; however, new technical approaches and increasing scientific attention have resulted in a growing body of data. Therefore, we present a compact up-to-date review of this topic. PMID:28603700

Flk prevents premature secretion of the anti-σ factor FlgM into the periplasm

PubMed Central

Aldridge, Phillip; Karlinsey, Joyce E.; Becker, Eric; Chevance, Fabienne F.V.; Hughes, Kelly T.

2012-01-01

Summary The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P- and l-ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti-σ28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic-facing protein anchored to the inner membrane by a single, C-terminal transmembrane-spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti-σ28 factor FlgM was increased in flk mutant strains. Using FlgM–β-lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar-associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes. PMID:16629666

TRPM channels phosphorylation as a potential bridge between old signals and novel regulatory mechanisms of insulin secretion.

PubMed

Diaz-Garcia, Carlos Manlio; Sanchez-Soto, Carmen; Hiriart, Marcia

2013-03-01

Transient receptor potential channels, especially the members of the melastatin family (TRPM), participate in insulin secretion. Some of them are substrates for protein kinases, which are involved in several neurotransmitter, incretin and hormonal signaling cascades in β cells. The functional relationships between protein kinases and TRPM channels in systems of heterologous expression and native tissues rise issues about novel regulation pathways of pancreatic β-cell excitability. The aim of the present work is to review the evidences about phosphorylation of TRPM channels in β cells and to discuss the perspectives on insulin secretion.

HrcU and HrpP are pathogenicity factors in the fire blight pathogen Erwinia amylovora required for the type III secretion of DspA/E.

PubMed

McNally, R Ryan; Zeng, Quan; Sundin, George W

2016-05-20

Many Gram-negative bacterial pathogens mediate host-microbe interactions via utilization of the type III secretion (T3S) system. The T3S system is a complex molecular machine consisting of more than 20 proteins. Collectively, these proteins translocate effectors across extracellular space and into the host cytoplasm. Successful translocation requires timely synthesis and allocation of both structural and secreted T3S proteins. Based on amino acid conservation in animal pathogenic bacteria, HrcU and HrpP were examined for their roles in regulation of T3S hierarchy. Both HrcU and HrpP were shown to be required for disease development in an immature pear infection model and respective mutants were unable to induce a hypersensitive response in tobacco. Using in vitro western blot analyses, both proteins were also shown to be required for the secretion of DspA/E, a type 3 effector and an important pathogenicity factor. Via yeast-two hybridization (Y2H), HrpP and HrcU were revealed to exhibit protein-protein binding. Finally, all HrcU and HrpP phenotypes identified were shown to be dependent on a conserved amino acid motif in the cytoplasmic tail of HrcU. Collectively, these data demonstrate roles for HrcU and HrpP in regulating T3S and represent the first attempt in understanding T3S heirarchy in E. amylovora.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Yao; Tan, Kemin; Chhor, Gekleng

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity Switch Protein in the Yersinia Type III Secretion System.

PubMed

Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H; Wolf-Watz, Magnus

2017-02-24

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia , the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscU C binds to the inner rod protein YscI with a dissociation constant ( K d ) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

An integrated microfluidic chip system for single-cell secretion profiling of rare circulating tumor cells.

PubMed

Deng, Yuliang; Zhang, Yu; Sun, Shuai; Wang, Zhihua; Wang, Minjiao; Yu, Beiqin; Czajkowsky, Daniel M; Liu, Bingya; Li, Yan; Wei, Wei; Shi, Qihui

2014-12-16

Genetic and transcriptional profiling, as well as surface marker identification of single circulating tumor cells (CTCs) have been demonstrated. However, quantitatively profiling of functional proteins at single CTC resolution has not yet been achieved, owing to the limited purity of the isolated CTC populations and a lack of single-cell proteomic approaches to handle and analyze rare CTCs. Here, we develop an integrated microfluidic system specifically designed for streamlining isolation, purification and single-cell secretomic profiling of CTCs from whole blood. Key to this platform is the use of photocleavable ssDNA-encoded antibody conjugates to enable a highly purified CTC population with <75 'contaminated' blood cells. An enhanced poly-L-lysine barcode pattern is created on the single-cell barcode chip for efficient capture rare CTC cells in microchambers for subsequent secreted protein profiling. This system was extensively evaluated and optimized with EpCAM-positive HCT116 cells seeded into whole blood. Patient blood samples were employed to assess the utility of the system for isolation, purification and single-cell secretion profiling of CTCs. The CTCs present in patient blood samples exhibit highly heterogeneous secretion profile of IL-8 and VEGF. The numbers of secreting CTCs are found not in accordance with CTC enumeration based on immunostaining in the parallel experiments.

An Integrated Microfluidic Chip System for Single-Cell Secretion Profiling of Rare Circulating Tumor Cells

PubMed Central

Deng, Yuliang; Zhang, Yu; Sun, Shuai; Wang, Zhihua; Wang, Minjiao; Yu, Beiqin; Czajkowsky, Daniel M.; Liu, Bingya; Li, Yan; Wei, Wei; Shi, Qihui

2014-01-01

Genetic and transcriptional profiling, as well as surface marker identification of single circulating tumor cells (CTCs) have been demonstrated. However, quantitatively profiling of functional proteins at single CTC resolution has not yet been achieved, owing to the limited purity of the isolated CTC populations and a lack of single-cell proteomic approaches to handle and analyze rare CTCs. Here, we develop an integrated microfluidic system specifically designed for streamlining isolation, purification and single-cell secretomic profiling of CTCs from whole blood. Key to this platform is the use of photocleavable ssDNA-encoded antibody conjugates to enable a highly purified CTC population with <75 ‘contaminated' blood cells. An enhanced poly-L-lysine barcode pattern is created on the single-cell barcode chip for efficient capture rare CTC cells in microchambers for subsequent secreted protein profiling. This system was extensively evaluated and optimized with EpCAM-positive HCT116 cells seeded into whole blood. Patient blood samples were employed to assess the utility of the system for isolation, purification and single-cell secretion profiling of CTCs. The CTCs present in patient blood samples exhibit highly heterogeneous secretion profile of IL-8 and VEGF. The numbers of secreting CTCs are found not in accordance with CTC enumeration based on immunostaining in the parallel experiments. PMID:25511131

Secretory expression of Lentinula edodes intracellular laccase by yeast high-cell-density system: sub-milligram production of difficult-to-express secretory protein.

PubMed

Kurose, Takeshi; Saito, Yuta; Kimata, Koichi; Nakagawa, Yuko; Yano, Akira; Ito, Keisuke; Kawarasaki, Yasuaki

2014-06-01

While a number of heterologous expression systems have been reported for extracellular laccases, there are few for the intracellular counterparts. The Lentinula edodes intracellular laccase Lcc4 is an industrially potential enzyme with its unique substrate specificity. The heterologous production of the intracellular laccase, however, had been difficult because of its expression-dependent toxicity. We previously demonstrated that recombinant yeast cells synthesized and, interestingly, secreted Lcc4 only when they were suspended to an inducing medium in a high cell-density (J. Biosci. Bioeng., 113, 154-159, 2012). The high cell-density system was versatile and applicable to other difficult-to-express secretory proteins. Nevertheless, the system's great dependence on aeration, which was a practical obstacle to scale-up production of the enzyme and some other proteins, left the secretion pathway and enzymatic properties of the Lcc4 uncharacterized. In this report, we demonstrate a successful production of Lcc4 by applying a jar-fermentor to the high cell-density system. The elevated yield (0.6 mg L(-1)) due to the sufficient aeration allowed us to prepare and purify the enzyme to homogeneity. The enzyme had been secreted as a hyper-glycosylated protein, resulting in smear band-formations in SDS-PAGE. The amino acid sequencing analysis suggested that the N-terminal 17 residues had been recognized as a secretion signal. The recombinant enzyme showed similar enzymatic properties to the naturally occurring Lcc4. The characteristics of the scale-upped expression system, which includes helpful information for the potential users, have also been described. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

PubMed Central

Lasica, Anna M.; Goulas, Theodoros; Mizgalska, Danuta; Zhou, Xiaoyan; de Diego, Iñaki; Ksiazek, Mirosław; Madej, Mariusz; Guo, Yonghua; Guevara, Tibisay; Nowak, Magdalena; Potempa, Barbara; Goel, Apoorv; Sztukowska, Maryta; Prabhakar, Apurva T.; Bzowska, Monika; Widziolek, Magdalena; Thøgersen, Ida B.; Enghild, Jan J.; Simonian, Mary; Kulczyk, Arkadiusz W.; Nguyen, Ky-Anh; Potempa, Jan; Gomis-Rüth, F. Xavier

2016-01-01

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity. PMID:27883039

Deoxycholate-Enhanced Shigella Virulence Is Regulated by a Rare π-Helix in the Type Three Secretion System Tip Protein IpaD.

PubMed

Bernard, Abram R; Jessop, T Carson; Kumar, Prashant; Dickenson, Nicholas E

2017-12-12

Type three secretion systems (T3SS) are specialized nanomachines that support infection by injecting bacterial proteins directly into host cells. The Shigella T3SS has uniquely evolved to sense environmental levels of the bile salt deoxycholate (DOC) and upregulate virulence in response to DOC. In this study, we describe a rare i + 5 hydrogen bonding secondary structure element (π-helix) within the type three secretion system tip protein IpaD that plays a critical role in DOC-enhanced virulence. Specifically, engineered mutations within the π-helix altered the pathogen's response to DOC, with one mutant construct in particular exhibiting an unprecedented reduction in virulence following DOC exposure. Fluorescence polarization binding assays showed that these altered DOC responses are not the result of differences in affinity between IpaD and DOC, but rather differences in the DOC-dependent T3SS tip maturation resulting from binding of IpaD to translocator/effector protein IpaB. Together, these findings begin to uncover the complex mechanism of DOC-enhanced Shigella virulence while identifying an uncommon structural element that may provide a much needed target for non-antibiotic treatment of Shigella infection.

The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

PubMed Central

Vincent, Maxence S.; Durand, Eric; Cascales, Eric

2016-01-01

The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins

PubMed Central

Maccani, Andreas; Landes, Nils; Stadlmayr, Gerhard; Maresch, Daniel; Leitner, Christian; Maurer, Michael; Gasser, Brigitte; Ernst, Wolfgang; Kunert, Renate; Mattanovich, Diethard

2014-01-01

Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins. PMID:24390926

Genome Sequence and Analysis of the Soil Cellulolytic ActinomyceteThermobifida fusca

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lykidis, Athanasios; Mavromatis, Konstantinos; Ivanova, Natalia

Thermobifida fusca is a moderately thermophilic soilbacterium that belongs to Actinobacteria. 3 It is a major degrader ofplant cell walls and has been used as a model organism for the study of 4secreted, thermostable cellulases. The complete genome sequence showedthat T. fusca has a 5 single circular chromosome of 3642249 bp predictedto encode 3117 proteins and 65 RNA6 species with a coding densityof 85percent. Genome analysis revealed the existence of 29 putative 7glycoside hydrolases in addition to the previously identified cellulasesand xylanases. The 8 glycosyl hydrolases include enzymes predicted toexhibit mainly dextran/starch and xylan 9 degrading functions. T. fuscapossesses twomore » protein secretion systems: the sec general secretion 10system and the twin-arginine translocation system. Several of thesecreted cellulases have 11 sequence signatures indicating theirsecretion may be mediated by the twin-arginine12 translocation system. T.fusca has extensive transport systems for import of carbohydrates 13coupled to transcriptional regulators controlling the expression of thetransporters and14 glycosylhydrolases. In addition to providing anoverview of the physiology of a soil 15 actinomycete, this study presentsinsights on the transcriptional regulation and secretion of16 cellulaseswhich may facilitate the industrial exploitation of thesesystems.« less

Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species.

PubMed

Vivek-Ananth, R P; Mohanraj, Karthikeyan; Vandanashree, Muralidharan; Jhingran, Anupam; Craig, James P; Samal, Areejit

2018-04-26

Aspergillus fumigatus and multiple other Aspergillus species cause a wide range of lung infections, collectively termed aspergillosis. Aspergilli are ubiquitous in environment with healthy immune systems routinely eliminating inhaled conidia, however, Aspergilli can become an opportunistic pathogen in immune-compromised patients. The aspergillosis mortality rate and emergence of drug-resistance reveals an urgent need to identify novel targets. Secreted and cell membrane proteins play a critical role in fungal-host interactions and pathogenesis. Using a computational pipeline integrating data from high-throughput experiments and bioinformatic predictions, we have identified secreted and cell membrane proteins in ten Aspergillus species known to cause aspergillosis. Small secreted and effector-like proteins similar to agents of fungal-plant pathogenesis were also identified within each secretome. A comparison with humans revealed that at least 70% of Aspergillus secretomes have no sequence similarity with the human proteome. An analysis of antigenic qualities of Aspergillus proteins revealed that the secretome is significantly more antigenic than cell membrane proteins or the complete proteome. Finally, overlaying an expression dataset, four A. fumigatus proteins upregulated during infection and with available structures, were found to be structurally similar to known drug target proteins in other organisms, and were able to dock in silico with the respective drug.

Salmonella-secreted Virulence Factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heffron, Fred; Niemann, George; Yoon, Hyunjin

In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellentmore » reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.« less

Secretome Analysis of Vibrio cholerae Type VI Secretion System Reveals a New Effector-Immunity Pair

PubMed Central

Altindis, Emrah; Dong, Tao; Catalano, Christy

2015-01-01

ABSTRACT The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. PMID:25759499

The Virulence Plasmid of Yersinia, an Antihost Genome

PubMed Central

Cornelis, Guy R.; Boland, Anne; Boyd, Aoife P.; Geuijen, Cecile; Iriarte, Maite; Neyt, Cécile; Sory, Marie-Paule; Stainier, Isabelle

1998-01-01

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds. PMID:9841674

Functional and computational analysis of amino acid patterns predictive of type III secretion system substrates in Pseudomonas syringae

USDA-ARS?s Scientific Manuscript database

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant path...

The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation.

PubMed

Ninio, Shira; Zuckman-Cholon, Deborah M; Cambronne, Eric D; Roy, Craig R

2005-02-01

The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.

NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages.

PubMed

Saad, Maged M; Kobayashi, Hajime; Marie, Corinne; Brown, Ian R; Mansfield, John W; Broughton, William J; Deakin, William J

2005-02-01

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.

NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

PubMed Central

Saad, Maged M.; Kobayashi, Hajime; Marie, Corinne; Brown, Ian R.; Mansfield, John W.; Broughton, William J.; Deakin, William J.

2005-01-01

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGRΩrhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB. PMID:15659692

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions.

PubMed

Ploss, Tina N; Reilman, Ewoud; Monteferrante, Carmine G; Denham, Emma L; Piersma, Sjouke; Lingner, Anja; Vehmaanperä, Jari; Lorenz, Patrick; van Dijl, Jan Maarten

2016-03-29

Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

NASA Astrophysics Data System (ADS)

Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

2017-10-01

In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

PubMed Central

Zhou, Xiaohui; Addwebi, Tarek; Davis, Margaret A.; Orfe, Lisa; Call, Douglas R.; Guard, Jean; Besser, Thomas E.

2011-01-01

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. PMID:21292746

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

PubMed

He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-02-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.

Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

2012-04-01

Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meshcheryakov, Vladimir A.; Kitao, Akio; Core Research for Evolutionary Science and Technology, Tokyo 113-0032

2013-05-01

Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, themore » crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.« less

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments[OPEN

PubMed Central

2017-01-01

Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. PMID:28619883

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Prediction of Body Fluids where Proteins are Secreted into Based on Protein Interaction Network

PubMed Central

Hu, Le-Le; Huang, Tao; Cai, Yu-Dong; Chou, Kuo-Chen

2011-01-01

Determining the body fluids where secreted proteins can be secreted into is important for protein function annotation and disease biomarker discovery. In this study, we developed a network-based method to predict which kind of body fluids human proteins can be secreted into. For a newly constructed benchmark dataset that consists of 529 human-secreted proteins, the prediction accuracy for the most possible body fluid location predicted by our method via the jackknife test was 79.02%, significantly higher than the success rate by a random guess (29.36%). The likelihood that the predicted body fluids of the first four orders contain all the true body fluids where the proteins can be secreted into is 62.94%. Our method was further demonstrated with two independent datasets: one contains 57 proteins that can be secreted into blood; while the other contains 61 proteins that can be secreted into plasma/serum and were possible biomarkers associated with various cancers. For the 57 proteins in first dataset, 55 were correctly predicted as blood-secrete proteins. For the 61 proteins in the second dataset, 58 were predicted to be most possible in plasma/serum. These encouraging results indicate that the network-based prediction method is quite promising. It is anticipated that the method will benefit the relevant areas for both basic research and drug development. PMID:21829572

High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

PubMed

Savijoki, K; Kahala, M; Palva, A

1997-02-28

A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

Genomic analysis of the type VI secretion systems in Pseudomonas spp.: novel clusters and putative effectors uncovered.

PubMed

Barret, Matthieu; Egan, Frank; Fargier, Emilie; Morrissey, John P; O'Gara, Fergal

2011-06-01

Bacteria encode multiple protein secretion systems that are crucial for interaction with the environment and with hosts. In recent years, attention has focused on type VI secretion systems (T6SSs), which are specialized transporters widely encoded in Proteobacteria. The myriad of processes associated with these secretion systems could be explained by subclasses of T6SS, each involved in specialized functions. To assess diversity and predict function associated with different T6SSs, comparative genomic analysis of 34 Pseudomonas genomes was performed. This identified 70 T6SSs, with at least one locus in every strain, except for Pseudomonas stutzeri A1501. By comparing 11 core genes of the T6SS, it was possible to identify five main Pseudomonas phylogenetic clusters, with strains typically carrying T6SSs from more than one clade. In addition, most strains encode additional vgrG and hcp genes, which encode extracellular structural components of the secretion apparatus. Using a combination of phylogenetic and meta-analysis of transcriptome datasets it was possible to associate specific subsets of VgrG and Hcp proteins with each Pseudomonas T6SS clade. Moreover, a closer examination of the genomic context of vgrG genes in multiple strains highlights a number of additional genes associated with these regions. It is proposed that these genes may play a role in secretion or alternatively could be new T6S effectors.

The Legionella pneumophila global regulatory protein LetA affects DotA and Mip.

PubMed

Shi, Chunwei; Forsbach-Birk, Vera; Marre, Reinhard; McNealy, Tamara L

2006-02-01

Several genes have been identified in Legionella pneumophila which are necessary for its virulence properties. These genes include the dot/icm type IV secretion system (T4SS), mip and letA. Genes of the dot/icm system, in particular dotA, have been found to be essential for intracellular growth. The macrophage infectivity protein (Mip) is also necessary for full virulence of the bacteria. Although these genes are well characterized, the regulation of such virulence factors is not. The LetA transcriptional activator interacts with the global regulator CsrA in controlling the switch from the replicative, non-infectious to the transmissive, highly infectious form of L. pneumophila. Regulation by LetA of the dot/icm genes has also been previously postulated. Here we show that the letA mutation exerts effects not only on DotA but on a substrate of the secretion system, RalF as well. LetA was found to be necessary for full transcriptional expression of the dotA and ralF genes. Although at the transcriptional level dotA was reduced, this did not result in a decrease of DotA protein in whole cell lysates. The letA mutation, however, does result in decreased amounts of the DotA protein found in the membrane and increased amounts in the culture supernatant. Additionally, the letA mutation dramatically decreased the secretion of Mip. This work demonstrates the participation of the global regulatory protein LetA in the regulation of an essential part of the dot/icm T4SS. Also shown is the presence of secreted Mip and a decrease in this secretion in the letA(-) strain. Exactly how LetA is regulating these virulence factors remains to be elucidated but it obviously occurs at both transcriptional and post-transcriptional levels.

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal.

PubMed

Login, Frédéric H; Wolf-Watz, Hans

2015-10-23

All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca(2+)-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

PubMed

Biedendieck, Rebekka

2016-01-01

For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

X‐linked retinoschisis: an update

PubMed Central

Sikkink, Stephen K; Biswas, Susmito; Parry, Neil R A; Stanga, Paulo E; Trump, Dorothy

2007-01-01

X‐linked retinoschisis is the leading cause of macular degeneration in males and leads to splitting within the inner retinal layers leading to visual deterioration. Many missense and protein truncating mutations have now been identified in the causative retinoschisis gene (RS1) which encodes a 224 amino acid secreting retinal protein, retinoschisin. Retinoschisin octamerises is implicated in cell–cell interactions and cell adhesion perhaps by interacting with β2 laminin. Mutations cause loss of retinoschisin function by one of the three mechanisms: by interfering with protein secretion, by preventing its octamerisation or by reducing function in the secreted octamerised protein. The development of retinoschisis mouse models have provided a model system that closely resembles the human disease. Recent reports of RS1 gene transfer to these models and the sustained restoration of some retinal function and morphology suggest gene replacement may be a possible future therapy for patients. PMID:17172462

Further Characterization of a Type III Secretion System (T3SS) and of a New Effector Protein from a Clinical Isolate of Aeromonas Hydrophila - Part I

EPA Science Inventory

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular cha...

Structures of EccB 1 and EccD 1 from the core complex of the mycobacterial ESX-1 type VII secretion system

DOE PAGES

Wagner, Jonathan M.; Chan, Sum; Evans, Timothy J.; ...

2016-02-27

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB 1 and EccD 1. The periplasmic domain of EccB 1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. Themore » repeat domains of EccB 1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD 1 has a ubiquitin-like fold and forms a dimer with a negatively charged groove. In conclusion, these structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.« less

Structures of EccB 1 and EccD 1 from the core complex of the mycobacterial ESX-1 type VII secretion system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, Jonathan M.; Chan, Sum; Evans, Timothy J.

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system. This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB 1 and EccD 1. The periplasmic domain of EccB 1 consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. Themore » repeat domains of EccB 1 are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD 1 has a ubiquitin-like fold and forms a dimer with a negatively charged groove. In conclusion, these structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.« less

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

PubMed Central

Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter

2013-01-01

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400

The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon

PubMed Central

Fernández de Marco, María del Mar; Alejo, Alí; Hudson, Paul; Damon, Inger K.; Alcami, Antonio

2010-01-01

Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.—Fernández de Marco, M. M., Alejo, A., Hudson, P., Damon, I. K., Alcami, A. The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon. PMID:20019241

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system

PubMed Central

Zoltner, Martin; Ng, Wui M.A.V.; Money, Jillian J.; Fyfe, Paul K.; Kneuper, Holger; Palmer, Tracy; Hunter, William N.

2016-01-01

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein. PMID:27130157

Transcriptional Profiling of the Iron Starvation Response in Bordetella pertussis Provides New Insights into Siderophore Utilization and Virulence Gene Expression ▿ §

PubMed Central

Brickman, Timothy J.; Cummings, Craig A.; Liew, Sin-Yee; Relman, David A.; Armstrong, Sandra K.

2011-01-01

Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes. PMID:21742863

Enhancing the Biological Relevance of Secretome-Based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion.

PubMed

Gregori, Josep; Méndez, Olga; Katsila, Theodora; Pujals, Mireia; Salvans, Cándida; Villarreal, Laura; Arribas, Joaquin; Tabernero, Josep; Sánchez, Alex; Villanueva, Josep

2014-07-15

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.

Type 1 Does The Two-Step: Type 1 Secretion Substrates With A Functional Periplasmic Intermediate.

PubMed

Smith, Timothy J; Sondermann, Holger; O'Toole, George A

2018-06-04

Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to secrete an array of biologically diverse proteins - from small < 10 kDa bacteriocins to gigantic adhesins with a mass over 1 MDa. For the last several decades T1SS have been characterized as a one-step translocation strategy whereby the secreted substrate is transported directly into the extracellular environment from the cytoplasm with no periplasmic intermediate. Recent phylogenetic, biochemical, and genetic evidence point to a distinct sub-group of T1SS machinery linked with a bacterial transglutaminase-like cysteine proteinase (BTLCP), which uses a two-step secretion mechanism. BTLCP-linked T1SS transport a class of repeats-in-toxin (RTX) adhesins that are critical for biofilm formation. The prototype of this RTX adhesin group, LapA of Pseudomonas fluorescens Pf0-1, uses a novel N-terminal retention module to anchor the adhesin at the cell surface as a secretion intermediate threaded through the outer membrane-localized, TolC-like protein LapE. This secretion intermediate is post-translationally cleaved by the BTLCP family LapG protein to release LapA from its cognate T1SS pore. Thus, secretion of LapA and related RTX adhesins into the extracellular environment appears to be a T1SS-mediated, two-step process that involves a periplasmic intermediate. In this review, we contrast the T1SS machinery and substrates of the BLTCP-linked two-step secretion process with those of the classical one-step T1SS to better understand the newly recognized and expanded role of this secretion machinery. Copyright © 2018 American Society for Microbiology.

Mechanisms of Disease: the first kiss-a crucial role for kisspeptin-1 and its receptor, G-protein-coupled receptor 54, in puberty and reproduction.

PubMed

Seminara, Stephanie B

2006-06-01

Although the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) is the defining hormonal event of puberty, the physiologic mechanisms that drive secretion of GnRH at the time of sexual maturation have been difficult to identify. After puberty is initiated, the factors that modulate the frequency and amplitude of GnRH secretion in rapidly changing sex-steroid environments (i.e. the female menstrual cycle) also remain unknown. The discovery that, in both humans and mouse models, loss-of-function mutations in the gene that encodes G-protein-coupled receptor 54 result in phenotypes of hypogonadotropic hypogonadism with an absence of pubertal development has unearthed a novel pathway regulating GnRH secretion. Ligands for G-protein-coupled receptor 54 (KiSS-1R), including metastin (derived from the parent compound, kisspeptin-1) and metastin's C-terminal peptide fragments, have been shown to be powerful stimulants for GnRH release in vivo via their stimulation of G-protein-coupled receptor 54. This article reviews the discovery of the GPR54 gene, places it into the appropriate biological context, and explores the data from in vitro and in vivo studies that point to this ligand-receptor system as a major driver of GnRH secretion.

In sílico identification and characterization of putative Dot/Icm secreted virulence effectors in the fish pathogen Piscirickettsia salmonis.

PubMed

Labra, Álvaro; Arredondo-Zelada, Oscar; Flores-Herrera, Patricio; Marshall, Sergio H; Gómez, Fernando A

2016-03-01

Piscirickettsia salmonis seriously affects the Chilean salmon industry. The bacterium is phylogenetically related to Legionella pneumophila and Coxiella burnetii, sharing a Dot/Icm secretion system with them. Although it is well documented that L. pneumophila and C. burnetii secrete different virulence effectors via this Dot/Icm system in order to attenuate host cell responses, to date there have been no reported virulence effectors secreted by the Dot/Icm system of P. salmonis. Using several annotations of P. salmonis genome, here we report an in silico analyses of 4 putative Dot/Icm effectors. Three of them contain ankyrin repeat domains and the typical conserved 3D structures of this protein family. The fourth one is highly similar to one of the Dot/Icm-dependent effectors of L. pneumophila. Additionally, all the potential P. salmonis effectors contain a classical Dot/Icm secretion signal in their C-terminus, consisting of: an E-Block, a hydrophobic residue in -3 or -4 and an electronegative charge. Finally, qPCR analysis demonstrated that these proteins are overexpressed early in infection, perhaps contributing to the generation of a replicative vacuole, a key step in the neutralizing strategy proposed for the Dot/Icm system. In summary, this report identifies four Dot/Icm-dependent effectors in P. salmonis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

PubMed

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-03-21

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems

PubMed Central

Espina, Marianela; Ausar, S. Fernando; Middaugh, C. Russell; Baxter, M. Aaron; Picking, William D.; Picking, Wendy L.

2007-01-01

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are α-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins. PMID:17327391

The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.

PubMed

Login, Frédéric H; Shevchik, Vladimir E

2006-11-03

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln(29) appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.

System Specificity of the TpsB Transporters of Coexpressed Two-Partner Secretion Systems of Neisseria meningitidis

PubMed Central

ur Rahman, Sadeeq

2013-01-01

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure. PMID:23222722

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

PubMed

Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

2016-01-01

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

PubMed Central

He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-01-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged. Images PMID:1992458

Label-free quantitative secretome analysis of Xanthomonas oryzae pv. oryzae highlights the involvement of a novel cysteine protease in its pathogenicity.

PubMed

Wang, Yiming; Gupta, Ravi; Song, Wei; Huh, Hyun-Hye; Lee, So Eui; Wu, Jingni; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kang, Kyu Young; Park, Sang-Ryeol; Kim, Sun Tae

2017-10-03

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases resulting in a huge loss of the total rice productivity. The initial interaction between rice and Xoo takes place in the host apoplast and is mediated primarily by secretion of various proteins from both partners. Yet, such secretory proteins remain to be largely identified and characterized. This study employed a label-free quantitative proteomics approach and identified 404 and 323 Xoo-secreted proteins from in vitro suspension-cultured cells and in planta systems, respectively. Gene Ontology analysis showed their involvement primarily in catalytic, transporter, and ATPase activities. Of a particular interest was a Xoo cysteine protease (XoCP), which showed dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence. Besides, a parallel analysis of in planta rice-secreted proteins resulted in identification of 186 secretory proteins mainly associated with the catalytic, antioxidant, and electron carrier activities. Identified secretory proteins were exploited to shed light on their possible role in the rice-Xoo interaction, and that further deepen our understanding of such interaction. Xanthomonas oryzae pv. oryzae (Xoo), causative agent of bacterial blight disease, results in a huge loss of the total rice productivity. Using a label-free quantitative proteomics approach, we identified 727 Xoo- and 186 rice-secreted proteins. Functional annotation showed Xoo secreted proteins were mainly associated with the catalytic, transporter, and ATPase activities while the rice secreted proteins were mainly associated with the catalytic, antioxidant, and electron carrier activities. A novel Xoo cysteine protease (XoCP) was identified, showing dramatic increase in its protein abundance in planta upon Xoo interaction with a susceptible rice cultivar. Knock-out mutants of XoCP showed reduced pathogenicity on rice, highlighting its potential involvement in Xoo virulence. Copyright © 2017 Elsevier B.V. All rights reserved.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

PubMed Central

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

2016-01-01

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

PubMed

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J

2016-01-12

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234

PubMed Central

Ausmees, Nora; Kobayashi, Hajime; Deakin, William J.; Marie, Corinne; Krishnan, Hari B.; Broughton, William J.; Perret, Xavier

2004-01-01

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect. PMID:15231809

Assembly of viral particles in Xenopus oocytes: pre-surface-antigens regulate secretion of the hepatitis B viral surface envelope particle.

PubMed Central

Standring, D N; Ou, J H; Rutter, W J

1986-01-01

Infection with hepatitis B virus (HBV) is associated with the production of a viral envelope particle that contains membrane lipids, surface antigen (S), and two presurface-antigens (pre-S) comprised of the entire S moiety with approximately 55 (pre-S2) and 174 (pre-S1) additional NH2-terminal amino acids. We show here that Xenopus oocytes injected with synthetic S mRNA assemble and secrete characteristic 22-nm viral envelope particles. In contrast, pre-S1 and pre-S2 antigens are synthesized but not secreted. By coinjecting mRNAs, we found that synthesis of high levels of pre-S proteins specifically inhibits S antigen secretion. On the other hand, high levels of S synthesis can drive the secretion of small amounts of either pre-S antigen. These observations are consistent with a model for viral envelope assembly in which both S and pre-S proteins are incorporated into a multimeric particle, presumably via interactions between the S protein domains, while the pre-S amino-terminal moieties regulate the secretion of this structure. Our results indicate that Xenopus oocytes will provide a powerful system for studying the morphogenesis of simple structures of viral or cellular origin. Images PMID:3467308

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion*

PubMed Central

Hung, Chien-Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

2016-01-01

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. PMID:27477394

Molecular Characterization of a Functional Type VI Secretion System from a Clinical Isolate of Aeromonas hydrophila

PubMed Central

Suarez, Giovanni; Sierra, Johanna C.; Sha, Jian; Wang, Shaofei; Erova, Tatiana E.; Fadl, Amin A.; Foltz, Sheri M.; Horneman, Amy J.; Chopra, Ashok K.

2008-01-01

Our laboratory recently molecularly characterized the type II secretion system (T2SS)- associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type 6 secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens. PMID:18037263

A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi.

PubMed

Swain, Durga Madhab; Yadav, Sunil Kumar; Tyagi, Isha; Kumar, Rahul; Kumar, Rajeev; Ghosh, Srayan; Das, Joyati; Jha, Gopaljee

2017-09-01

Some bacteria can feed on fungi, a phenomenon known as mycophagy. Here we show that a prophage tail-like protein (Bg_9562) is essential for mycophagy in Burkholderia gladioli strain NGJ1. The purified protein causes hyphal disintegration and inhibits growth of several fungal species. Disruption of the Bg_9562 gene abolishes mycophagy. Bg_9562 is a potential effector secreted by a type III secretion system (T3SS) and is translocated into fungal mycelia during confrontation. Heterologous expression of Bg_9562 in another bacterial species, Ralstonia solanacearum, confers mycophagous ability in a T3SS-dependent manner. We propose that the ability to feed on fungi conferred by Bg_9562 may help the bacteria to survive in certain ecological niches. Furthermore, considering its broad-spectrum antifungal activity, the protein may be potentially useful in biotechnological applications to control fungal diseases.Some bacteria can feed on live fungi through unclear mechanisms. Here, the authors show that a T3SS-secreted protein, which is homologous to phage tail proteins, allows a Burkholderia gladioli strain to kill and feed on various fungal species.

a Computational Approach to Explore Protein Translocation Through Type III Secretion Apparatus

NASA Astrophysics Data System (ADS)

Rathinavelan, Thenmalarchelvi; Im, Wonpil

2010-01-01

Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus (TTSA) that is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. Interestingly, the electronegative channel interior creates an energy barrier for MxiH to enter the channel, while the same may facilitate the ejection of the effectors into host cells. Structurally-known basal regions and ATPase underneath the basal region have also such electronegative interior, while effector proteins have considerable electronegative patches on their surfaces. Based on these observations, we propose a repulsive electrostatic mechanism for protein translocation through the TTSA. This mechanism is supported by the suggestion that an ATPase is required for protein translocation through these nanomachines, which may provide the energy to overcome the initial electrostatic energy barrier. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported.

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-03-15

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. Copyright © 2014 Elsevier Inc. All rights reserved.

Develop Systems for Manufacturing 100,000,000 Doses of an Emergency Pharmaceutical (e.g. Vaccine or Monoclonal Antibody) Within 2 Months of Product Identification

DTIC Science & Technology

2006-06-08

protein production. At Genencor, we have developed strains of Aspergillus niger var. awamori, which demonstrate improved secretion of foreign proteins...2000. Characterization of the kexin-like maturase of Aspergillus niger . Appl. Environ. Microbiol. 66:363-368 Jefferis, R., J. Lund, and J. D. Pound...N., Gieswein C., Park M., Wang H. 2004. Characterization of humanized antibodies secreted by Aspergillus niger . Appl Environ Microbiol. 70:2567

High-Yield Secretion of Multiple Client Proteins in Aspergillus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segato, F.; Damasio, A. R. L.; Goncalves, T. A.

2012-07-15

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degradingmore » enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.« less

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.

PubMed

Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang

2017-01-01

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.

Structure of the WipA protein reveals a novel tyrosine protein phosphatase effector from Legionella pneumophila.

PubMed

Pinotsis, Nikos; Waksman, Gabriel

2017-06-02

Legionnaires' disease is a severe form of pneumonia caused by the bacterium Legionella pneumophila. L. pneumophila pathogenicity relies on secretion of more than 300 effector proteins by a type IVb secretion system. Among these Legionella effectors, WipA has been primarily studied because of its dependence on a chaperone complex, IcmSW, for translocation through the secretion system, but its role in pathogenicity has remained unknown. In this study, we present the crystal structure of a large fragment of WipA, WipA435. Surprisingly, this structure revealed a serine/threonine phosphatase fold that unexpectedly targets tyrosine-phosphorylated peptides. The structure also revealed a sequence insertion that folds into an α-helical hairpin, the tip of which adopts a canonical coiled-coil structure. The purified protein was a dimer whose dimer interface involves interactions between the coiled coil of one WipA molecule and the phosphatase domain of another. Given the ubiquity of protein-protein interaction mediated by interactions between coiled-coils, we hypothesize that WipA can thereby transition from a homodimeric state to a heterodimeric state in which the coiled-coil region of WipA is engaged in a protein-protein interaction with a tyrosine-phosphorylated host target. In conclusion, these findings help advance our understanding of the molecular mechanisms of an effector involved in Legionella virulence and may inform approaches to elucidate the function of other effectors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The TolC-like protein HgdD of the cyanobacterium Anabaena sp. PCC 7120 is involved in secondary metabolite export and antibiotic resistance.

PubMed

Hahn, Alexander; Stevanovic, Mara; Mirus, Oliver; Schleiff, Enrico

2012-11-30

The role of TolC has largely been explored in proteobacteria, where it functions as a metabolite and protein exporter. In contrast, little research has been carried out on the function of cyanobacterial homologues, and as a consequence, not much is known about the mechanism of cyanobacterial antibiotic uptake and metabolite secretion in general. It has been suggested that the TolC-like homologue of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, termed heterocyst glycolipid deposition protein D (HgdD), is involved in both protein and lipid secretion. To describe its function in secondary metabolite secretion, we established a system to measure the uptake of antibiotics based on the fluorescent molecule ethidium bromide. We analyzed the rate of porin-dependent metabolite uptake and confirmed the functional relation between detoxification and the action of HgdD. Moreover, we identified two major facilitator superfamily proteins that are involved in this process. It appears that anaOmp85 (Alr2269) is not required for insertion or assembly of HgdD, because an alr2269 mutant does not exhibit a phenotype similar to the hgdD mutant. Thus, we could assign components of the metabolite efflux system and describe parameters of detoxification by Anabaena sp. PCC 7120.

The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon.

PubMed

Fernández de Marco, María del Mar; Alejo, Alí; Hudson, Paul; Damon, Inger K; Alcami, Antonio

2010-05-01

Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahman, Arman; DeCourcey, Joseph; Larbi, Nadia Ben

Highlights: •Knock-down of syntaxin-4 in U266 plasma cells resulted in reduction of IgE secretion. •Knock-down of syntaxin-4 also leads to the accumulation of IgE in the cell. •Immuno-fluorescence staining shows co-localisation of IgE and syntaxin-4 in U266 cells. •Findings suggest a critical requirement for syntaxin-4 in IgE secretion from plasma cells. -- Abstract: The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immunemore » response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.« less

Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall

PubMed Central

De Caroli, Monica; Lenucci, Marcello S.; Manualdi, Francesca; Dalessandro, Giuseppe; De Lorenzo, Giulia; Piro, Gabriella

2015-01-01

The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1–10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal. PMID:26379688

AtlasT4SS: a curated database for type IV secretion systems.

PubMed

Souza, Rangel C; del Rosario Quispe Saji, Guadalupe; Costa, Maiana O C; Netto, Diogo S; Lima, Nicholas C B; Klein, Cecília C; Vasconcelos, Ana Tereza R; Nicolás, Marisa F

2012-08-09

The type IV secretion system (T4SS) can be classified as a large family of macromolecule transporter systems, divided into three recognized sub-families, according to the well-known functions. The major sub-family is the conjugation system, which allows transfer of genetic material, such as a nucleoprotein, via cell contact among bacteria. Also, the conjugation system can transfer genetic material from bacteria to eukaryotic cells; such is the case with the T-DNA transfer of Agrobacterium tumefaciens to host plant cells. The system of effector protein transport constitutes the second sub-family, and the third one corresponds to the DNA uptake/release system. Genome analyses have revealed numerous T4SS in Bacteria and Archaea. The purpose of this work was to organize, classify, and integrate the T4SS data into a single database, called AtlasT4SS - the first public database devoted exclusively to this prokaryotic secretion system. The AtlasT4SS is a manual curated database that describes a large number of proteins related to the type IV secretion system reported so far in Gram-negative and Gram-positive bacteria, as well as in Archaea. The database was created using the RDBMS MySQL and the Catalyst Framework based in the Perl programming language and using the Model-View-Controller (MVC) design pattern for Web. The current version holds a comprehensive collection of 1,617 T4SS proteins from 58 Bacteria (49 Gram-negative and 9 Gram-Positive), one Archaea and 11 plasmids. By applying the bi-directional best hit (BBH) relationship in pairwise genome comparison, it was possible to obtain a core set of 134 clusters of orthologous genes encoding T4SS proteins. In our database we present one way of classifying orthologous groups of T4SSs in a hierarchical classification scheme with three levels. The first level comprises four classes that are based on the organization of genetic determinants, shared homologies, and evolutionary relationships: (i) F-T4SS, (ii) P-T4SS, (iii) I-T4SS, and (iv) GI-T4SS. The second level designates a specific well-known protein families otherwise an uncharacterized protein family. Finally, in the third level, each protein of an ortholog cluster is classified according to its involvement in a specific cellular process. AtlasT4SS database is open access and is available at http://www.t4ss.lncc.br.

Structural Characterization of the Yersinia pestis Type III Secretion System Needle Protein YscF in Complex with Its Heterodimeric Chaperone YscE/YscG

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Ping; Tropea, Joseph E.; Austin, Brian P.

2008-05-03

The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonasmore » aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.« less

Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation.

PubMed Central

Bahrani, F K; Sansonetti, P J; Parsot, C

1997-01-01

The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins. PMID:9316999

Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

Treesearch

Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

2003-01-01

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

PubMed

Liu, J; Wang, Y; Szalay, A A; Escher, A

2000-01-01

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells. Copyright 2000 John Wiley & Sons, Ltd.

Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry.

PubMed

Shinya, Ryoji; Morisaka, Hironobu; Kikuchi, Taisei; Takeuchi, Yuko; Ueda, Mitsuyoshi; Futai, Kazuyoshi

2013-01-01

Since it was first introduced into Asia from North America in the early 20(th) century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine's proteins. These proteins could have been acquired by host-parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.

Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax.

PubMed

Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim

2016-08-01

Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

PubMed

McCarthy, Alex J; Lindsay, Jodi A

2013-10-01

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of novel secreted virulence factors from Xylella fastidiosa using a TRV expression system

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a bacterium that causes leaf scorch diseases of agriculturally important crops including grapevines and almonds. Little is known about virulence factors that are necessary for X. fastidiosa to grow and cause disease in the xylem vessels of a plant host. Any protein secreted by ...

Identification and characterization of secreted proteins in Eimeria tenella

NASA Astrophysics Data System (ADS)

Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

2015-09-01

Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development.

PubMed

Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

2015-01-01

Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm.

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

PubMed

Gupta, Sanjeev K; Shukla, Pratyoosh

2016-12-01

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

Native structure of a type IV secretion system core complex essential for Legionella pathogenesis.

PubMed

Kubori, Tomoko; Koike, Masafumi; Bui, Xuan Thanh; Higaki, Saori; Aizawa, Shin-Ichi; Nagai, Hiroki

2014-08-12

Bacterial type IV secretion systems are evolutionarily related to conjugation systems and play a pivotal role in infection by delivering numerous virulence factors into host cells. Using transmission electron microscopy, we report the native molecular structure of the core complex of the Dot/Icm type IV secretion system encoded by Legionella pneumophila, an intracellular human pathogen. The biochemically isolated core complex, composed of at least five proteins--DotC, DotD, DotF, DotG, and DotH--has a ring-shaped structure. Intriguingly, morphologically distinct premature complexes are formed in the absence of DotG or DotF. Our data suggest that DotG forms a central channel spanning inner and outer membranes. DotF, a component dispensable for type IV secretion, plays a role in efficient embedment of DotG into the functional core complex. These results highlight a common scheme for the biogenesis of transport machinery.

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

PubMed

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

Structural and biochemical characterization of SrcA, a multi-cargo type III secretion chaperone in Salmonella required for pathogenic association with a host.

PubMed

Cooper, Colin A; Zhang, Kun; Andres, Sara N; Fang, Yuan; Kaniuk, Natalia A; Hannemann, Mandy; Brumell, John H; Foster, Leonard J; Junop, Murray S; Coombes, Brian K

2010-02-05

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

PubMed Central

Hoang, Huy-Dung; Maruyama, Jun-ichi

2014-01-01

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. PMID:25362068

Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels.

PubMed

López-Benito, Saray; Sánchez-Sánchez, Julia; Brito, Verónica; Calvo, Laura; Lisa, Silvia; Torres-Valle, María; Palko, Mary E; Vicente-García, Cristina; Fernández-Fernández, Seila; Bolaños, Juan P; Ginés, Silvia; Tessarollo, Lino; Arévalo, Juan C

2018-06-06

BDNF is a growth factor with important roles in the nervous system in both physiological and pathological conditions, but the mechanisms controlling its secretion are not completely understood. Here, we show that ARMS/Kidins220 negatively regulates BDNF secretion in neurons from the CNS and PNS. Downregulation of the ARMS/Kidins220 protein in the adult mouse brain increases regulated BDNF secretion, leading to its accumulation in the striatum. Interestingly, two mouse models of Huntington's disease (HD) showed increased levels of ARMS/Kidins220 in the hippocampus and regulated BDNF secretion deficits. Importantly, reduction of ARMS/Kidins220 in hippocampal slices from HD mice reversed the impaired regulated BDNF release. Moreover, there are increased levels of ARMS/Kidins220 in the hippocampus and PFC of patients with HD. ARMS/Kidins220 regulates Synaptotagmin-IV levels, which has been previously observed to modulate BDNF secretion. These data indicate that ARMS/Kidins220 controls the regulated secretion of BDNF and might play a crucial role in the pathogenesis of HD. SIGNIFICANCE STATEMENT BDNF is an important growth factor that plays a fundamental role in the correct functioning of the CNS. The secretion of BDNF must be properly controlled to exert its functions, but the proteins regulating its release are not completely known. Using neuronal cultures and a new conditional mouse to modulate ARMS/Kidins220 protein, we report that ARMS/Kidins220 negatively regulates BDNF secretion. Moreover, ARMS/Kidins220 is overexpressed in two mouse models of Huntington's disease (HD), causing an impaired regulation of BDNF secretion. Furthermore, ARMS/Kidins220 levels are increased in brain samples from HD patients. Future studies should address whether ARMS/Kidins220 has any function on the pathophysiology of HD. Copyright © 2018 the authors 0270-6474/18/385415-14$15.00/0.

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

Detection of Protein Interactions in T3S Systems Using Yeast Two-Hybrid Analysis.

PubMed

Nilles, Matthew L

2017-01-01

Two-hybrid systems, sometimes termed interaction traps, are genetic systems designed to find and analyze interactions between proteins. The most common systems are yeast based (commonly Saccharomyces cerevisae) and rely on the functional reconstitution of the GAL4 transcriptional activator. Reporter genes, such as the lacZ gene of Escherichia coli (encodes β-galactosidase), are placed under GAL4-dependent transcriptional control to provide quick and reliable detection of protein interactions. In this method the use of a yeast-based two-hybrid system is described to study protein interactions between components of type III secretion systems.

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

PubMed

Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong

2018-05-18

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis.

PubMed

Chen, Po Ting; Shaw, Jei-Fu; Chao, Yun-Peng; David Ho, Tuan-Hua; Yu, Su-May

2010-05-12

Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated T7 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the T7 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase E1 with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.

Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli.

PubMed

Gao, Dongfang; Wang, Shengjun; Li, Haoran; Yu, Huili; Qi, Qingsheng

2015-04-10

The Gram-negative bacterium Escherichia coli has been widely used as a cell factory for the production of proteins and specialty chemicals because it is the best characterized host with many available expression and regulation systems. However, recombinant proteins produced in Escherichia coli are generally intracellular and often found in the form of inclusion bodies. Extracellular production of proteins is advantageous compared with intracellular production because extracellular proteins can be purified more easily and can avoid protease attack, which results in higher product quality. In this study, we found a catalytic domain of a cellulase (Cel-CD) and its N-terminus can be employed as carriers for extracellular production of recombinant proteins. In this report, we identified the catalytic domain of a cellulase (Cel-CD) from Bacillus sp. that can be secreted into the medium from recombinant E. coli BL21 (DE3) in large quantities without its native signal peptide. By subcellular location analysis, we proved that the secretion was a two-step process and the N-terminal sequence of the full length Cel-CD played a crucial function in secretion. Both the Cel-CD and its N-terminal sequence can serve as carriers for efficient extracellular production of select target proteins. Fusion of heterologous proteins with N20 from Cel-CD can carry the target proteins out of the cells with a concentration from 101 to 691 mg/L in flask cultivation. The extracellular recombinant proteins with a relative high purity. The results suggested that this system has a potential application in plant biomass conversion and industrial production of enzymes and therapeutic proteins.

Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions*

PubMed Central

Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V.; Kwon, Keehwan; Townsend, Katherine; Yu, Chenggang; Yu, Xueping; DeShazer, David; Reifman, Jaques; Wallqvist, Anders

2013-01-01

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes. PMID:23800426

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins

PubMed Central

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-01-01

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common. DOI: http://dx.doi.org/10.7554/eLife.24869.001 PMID:28323618

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

DOE PAGES

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.; ...

2015-06-18

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

Enrichment and Detection of Molecules Secreted by Tumor Cells Using Magnetic Reversed-Phase Particles and LC-MALDI-TOF-MS

PubMed Central

Peter, Jochen F.; Otto, Angela M.; Wolf, Bernhard

2007-01-01

Tumor cells change their genetic expression pattern as they progress to states of increasing malignancy. Investigations at the DNA and RNA level alone cannot provide all the information resulting after the translation and processing of the corresponding proteins, which is one reason for a poor correlation between mRNA and the respective protein abundance. In diagnostics, differentially expressed peptides or proteins are important markers for the early detection of cancer. Unfortunately, tumor cells secrete peptides and proteins in only very low amounts, making mass spectrometric determination very difficult. In this publication, methods have been developed for the effective enrichment and cleanup of substances secreted by cultivated cancer cells. To obviate peptides from fetal calf serum used in cell culture, a serum surrogate was developed, which maintained growth of the cancer cells. After the binding of substances from cell-culture supernatants to custom-made magnetic reversed-phase particles, the substances were eluted and separated by capillary high-performance liquid chromatography. Fractions were spotted directly on a MALDI target, and MALDI-TOF mass spectrometric data acquisition was performed in automatic mode. This technology was used to detect substances secreted by two mammary carcinoma cell lines differing in their malignancy (MCF-7, MDA-MB231). Unequivocal differences in the peptide secretion patterns were observed. In conclusion, this system allows the sensitive investigation of peptides secreted by cancer cells in culture and provides a valuable tool for the investigation of cancer cells in different states of malignancy. PMID:18166672

X-ray Crystal Structures of the Type IVb Secretion System DotB ATPases.

PubMed

Prevost, Marie S; Waksman, Gabriel

2018-05-17

Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilises a type IVb secretion (T4bS) system termed "dot/icm" to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS-like N-terminal domain coupled to a RecA-like C-terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and β, with an αβαβαβ configuration. The existence of α and β conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααβααβ configuration. The two conformers co-exist regardless of the nucleotide-bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug-design to develop more specific antibiotics to tackle Legionnaire's disease. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.

Computational prediction of type III and IV secreted effectors in Gram-negative bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.

2011-01-01

In this review, we provide an overview of the methods employed by four recent papers that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. The results of the studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVEserver (http://www.biopilot.org). Finally, we assess the accuracy of the three type III effector prediction methods onmore » a small set of proteins not known prior to the development of these tools that we have recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif, and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.« less

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

PubMed Central

Ng, Daphne T. W.

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts. PMID:23124224

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

PubMed

Ng, Daphne T W; Sarkar, Casim A

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

PubMed

Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J; Phu, My L; Lebrilla, Carlito B; Dandekar, Abhaya M; Rodriguez, Raymond L; Nandi, Somen; McDonald, Karen A

2017-01-04

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 ( CMG2 ) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus ( CaMV ) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N -glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N -glycans consists of complex type structures in both protein samples. The most abundant (>50%) N -glycan structure was GlcNAc₂(Xy l )Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N -glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting.

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

PubMed

Moua, Pachai S; Gonzalez, Alfonso; Oshiro, Kristin T; Tam, Vivian; Li, Zhiguo Harry; Chang, Jennifer; Leung, Wilson; Yon, Amy; Thor, Der; Venkatram, Sri; Franz, Andreas H; Risser, Douglas D; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

2016-08-01

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris. Copyright © 2016 Elsevier Inc. All rights reserved.

Solution Structure of Homology Region (HR) Domain of Type II Secretion System*

PubMed Central

Gu, Shuang; Kelly, Geoff; Wang, Xiaohui; Frenkiel, Tom; Shevchik, Vladimir E.; Pickersgill, Richard W.

2012-01-01

The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a β-sandwich-like fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the type II secretion system have a structural or sequence ortholog within the type IV pili system. The HR domain is shown to interact with the N0 domain of the secretin. The importance of this interaction is explored in the context of the functional secretion system. PMID:22253442

Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family

PubMed Central

Heinz, Eva; Stubenrauch, Christopher J.; Grinter, Rhys; Croft, Nathan P.; Purcell, Anthony W.; Strugnell, Richard A.; Dougan, Gordon; Lithgow, Trevor

2016-01-01

The bacterial cell surface proteins intimin and invasin are virulence factors that share a common domain structure and bind selectively to host cell receptors in the course of bacterial pathogenesis. The β-barrel domains of intimin and invasin show significant sequence and structural similarities. Conversely, a variety of proteins with sometimes limited sequence similarity have also been annotated as “intimin-like” and “invasin” in genome datasets, while other recent work on apparently unrelated virulence-associated proteins ultimately revealed similarities to intimin and invasin. Here we characterize the sequence and structural relationships across this complex protein family. Surprisingly, intimins and invasins represent a very small minority of the sequence diversity in what has been previously the “intimin/invasin protein family”. Analysis of the assembly pathway for expression of the classic intimin, EaeA, and a characteristic example of the most prevalent members of the group, FdeC, revealed a dependence on the translocation and assembly module as a common feature for both these proteins. While the majority of the sequences in the grouping are most similar to FdeC, a further and widespread group is two-partner secretion systems that use the β-barrel domain as the delivery device for secretion of a variety of virulence factors. This comprehensive analysis supports the adoption of the “inverse autotransporter protein family” as the most accurate nomenclature for the family and, in turn, has important consequences for our overall understanding of the Type V secretion systems of bacterial pathogens. PMID:27190006

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella.

PubMed

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L; Picking, William D; Middaugh, C Russell

2006-03-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.

Analysis of the ZAR1 immune complex reveals determinants for immunity and molecular interactions

USDA-ARS?s Scientific Manuscript database

Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary function of most T3SEs is to supp...

Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.

PubMed

Chahal, Sabreen; Wei, Peter; Moua, Pachai; Park, Sung Pil James; Kwon, Janet; Patel, Arth; Vu, Anthony T; Catolico, Jason A; Tsai, Yu Fang Tina; Shaheen, Nadia; Chu, Tiffany T; Tam, Vivian; Khan, Zill-E-Huma; Joo, Hyun Henry; Xue, Liang; Lin-Cereghino, Joan; Tsai, Jerry W; Lin-Cereghino, Geoff P

2017-01-20

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell Density Control of Staphylococcal Virulence Mediated by an Octapeptide Pheromone

NASA Astrophysics Data System (ADS)

Ji, Guangyong; Beavis, Ronald C.; Novick, Richard P.

1995-12-01

Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

PubMed

Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

2007-02-01

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

The dimer formed by the periplasmic domain of EpsL from the Type 2 Secretion System of Vibrio parahaemolyticus

PubMed Central

Abendroth, Jan; Kreger, Allison C.; Hol, Wim G. J.

2010-01-01

The Type 2 Secretion System (T2SS), occurring in many Gram-negative bacteria, is responsible for the transport of a diversity of proteins from the periplasm across the outer membrane into the extracellular space. In Vibrio cholerae, the T2SS secretes several unrelated proteins including the major virulence factor cholera toxin. The T2SS consists of three subassemblies, one of which is the Inner Membrane Complex which contains multiple copies of five proteins, including the bitopic membrane protein EpsL. Here we report the 2.3 Å resolution crystal structure of the periplasmic domain of EpsL (peri-EpsL) from V. parahaemolyticus, which is 56 % identical in sequence to its homolog in V. cholerae. The domain adopts a circular permutation of the “common” ferredoxin fold with two contiguous sub-domains. Remarkably, this permutation has so far only been observed once before: in the periplasmic domain of EpsM (peri-EpsM), another T2SS protein which interacts with EpsL. These two domains are 18 % identical in sequence which may indicate a common evolutionary origin. Both peri-EpsL and peri-EpsM form dimers, but the organization of the subunits in these dimers appears to be entirely different. We have previously shown that the cytoplasmic domain of EpsL is also dimeric and forms a heterotetramer with the first domain of the “secretion ATPase” EpsE. The latter enzyme is most likely hexameric. The possible consequences of the combination of the different symmetries of EpsE and EpsL for the architecture of the T2SS are discussed. PMID:19646531

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

PubMed Central

Moodley, Yoshan; Uhr, Markus; Stamer, Christiana; Vauterin, Marc; Suerbaum, Sebastian; Achtman, Mark

2010-01-01

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI–carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown. PMID:20808891

A global overview of the genetic and functional diversity in the Helicobacter pylori cag pathogenicity island.

PubMed

Olbermann, Patrick; Josenhans, Christine; Moodley, Yoshan; Uhr, Markus; Stamer, Christiana; Vauterin, Marc; Suerbaum, Sebastian; Achtman, Mark; Linz, Bodo

2010-08-19

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI-carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown.

The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna.

PubMed

Tyson, Jessica Y; Vargas, Paloma; Cianciotto, Nicholas P

2014-12-01

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts. © 2014 The Authors.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain

PubMed Central

de Diego, Iñaki; Ksiazek, Miroslaw; Mizgalska, Danuta; Koneru, Lahari; Golik, Przemyslaw; Szmigielski, Borys; Nowak, Magdalena; Nowakowska, Zuzanna; Potempa, Barbara; Houston, John A.; Enghild, Jan J.; Thøgersen, Ida B.; Gao, Jinlong; Kwan, Ann H.; Trewhella, Jill; Dubin, Grzegorz; Gomis-Rüth, F. Xavier; Nguyen, Ky-Anh; Potempa, Jan

2016-01-01

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway. PMID:27005013

Expression of and secretion through the Aeromonas salmonicida type III secretion system.

PubMed

Ebanks, Roger O; Knickle, Leah C; Goguen, Michel; Boyd, Jessica M; Pinto, Devanand M; Reith, Michael; Ross, Neil W

2006-05-01

Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 degrees C, but not at its more natural growth temperature of 17 degrees C. More modest increases in expression occur at 24 degrees C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 degrees C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 degrees C in salt concentrations ranging from 0.19 to 0.38 M NaCl. It is also shown that growth at 28 degrees C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.

Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ.

PubMed

Jeong, Kwang Cheol; Sexton, Jessica A; Vogel, Joseph P

2015-03-01

Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.

Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry

PubMed Central

Shinya, Ryoji; Takeuchi, Yuko; Ueda, Mitsuyoshi; Futai, Kazuyoshi

2013-01-01

Since it was first introduced into Asia from North America in the early 20th century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine’s proteins. These proteins could have been acquired by host–parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes. PMID:23805310

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell–cell interaction

PubMed Central

Kwak, Minsuk; Mu, Luye; Lu, Yao; Chen, Jonathan J.; Brower, Kara; Fan, Rong

2013-01-01

Secreted proteins including cytokines, chemokines, and growth factors represent important functional regulators mediating a range of cellular behavior and cell–cell paracrine/autocrine signaling, e.g., in the immunological system (Rothenberg, 2007), tumor microenvironment (Hanahan and Weinberg, 2011), or stem cell niche (Gnecchi etal., 2008). Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically identical cell population can give rise to diverse phenotypic differences (Niepel etal., 2009). Non-genetic heterogeneity is also emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies (Cohen etal., 2008; Gascoigne and Taylor, 2008), but can hardly assessed using conventional approaches that do not examine cellular phenotype at the functional level. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer. PMID:23390614

Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shrestha, Manisha; Xiao, Yi; Robinson, Howard

Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domainmore » of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Lastly, additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.« less

Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa

DOE PAGES

Shrestha, Manisha; Xiao, Yi; Robinson, Howard; ...

2015-08-28

Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domainmore » of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Lastly, additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.« less

Reduced active transcellular intestinal oxalate secretion contributes to the pathogenesis of obesity-associated hyperoxaluria.

PubMed

Amin, Ruhul; Asplin, John; Jung, Daniel; Bashir, Mohamed; Alshaikh, Altayeb; Ratakonda, Sireesha; Sharma, Sapna; Jeon, Sohee; Granja, Ignacio; Matern, Dietrich; Hassan, Hatim

2018-05-01

Most kidney stones are composed of calcium oxalate, and minor changes in urine oxalate affect the stone risk. Obesity is a risk factor for kidney stones and a positive correlation of unknown etiology between increased body size, and elevated urinary oxalate excretion has been reported. Here, we used obese ob/ob (ob) mice to elucidate the pathogenesis of obesity-associated hyperoxaluria. These ob mice have significant hyperoxaluria (3.3-fold) compared with control mice, which is not due to overeating as shown by pair-feeding studies. Dietary oxalate removal greatly ameliorated this hyperoxaluria, confirming that it is largely enteric in origin. Transporter SLC26A6 (A6) plays an essential role in active transcellular intestinal oxalate secretion, and ob mice have significantly reduced jejunal A6 mRNA (- 80%) and total protein (- 62%) expression. While net oxalate secretion was observed in control jejunal tissues mounted in Ussing chambers, net absorption was seen in ob tissues, due to significantly reduced secretion. We hypothesized that the obesity-associated increase in intestinal and systemic inflammation, as reflected by elevated proinflammatory cytokines, suppresses A6-mediated intestinal oxalate secretion and contributes to obesity-associated hyperoxaluria. Indeed, proinflammatory cytokines (elevated in ob mice) significantly decreased intestinal oxalate transport in vitro by reducing A6 mRNA and total protein expression. Proinflammatory cytokines also significantly reduced active mouse jejunal oxalate secretion, converting oxalate transport from net secretion in vehicle-treated tissues to net absorption in proinflammatory cytokines-treated tissues. Thus, reduced active intestinal oxalate secretion, likely secondary to local and systemic inflammation, contributes to the pathogenesis of obesity-associated hyperoxaluria. Hence, proinflammatory cytokines represent potential therapeutic targets. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Development of exosome surface display technology in living human cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu

Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated themore » successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.« less

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

PubMed

Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Cancer-cells on a chip for label-free optic detection of secreted molecules

NASA Astrophysics Data System (ADS)

Berthuy, Ophélie I.; Blum, Loïc. J.; Marquette, Christophe A.

2015-05-01

To unravel cell complexity, living-cell chips have been developed that allow delivery of experimental stimuli but also measurement of the resulting cellular responses. We have been developing a new concept for multiplexed detection of biomolecules secreted by different cancer cells. In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. For that purpose, antibodies and different cell lines were spotted using a piezoelectric spotter. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and β-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip. There is no doubt that our chip will, in a near future, be applied to more multiplexed and complex biological secretion systems for which kinetic data are at the moment not reachable using standard cellular biology tools.

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer.

PubMed

Pera, Edgar M; Kim, James I; Martinez, Sarah L; Brechner, Mariel; Li, Su Yu; Wessely, Oliver; De Robertis, E M

2002-08-01

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.

Gene and transcript abundances of bacterial type III secretion systems from the rumen microbiome are correlated with methane yield in sheep.

PubMed

Kamke, Janine; Soni, Priya; Li, Yang; Ganesh, Siva; Kelly, William J; Leahy, Sinead C; Shi, Weibing; Froula, Jeff; Rubin, Edward M; Attwood, Graeme T

2017-08-08

Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH 4 /kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. This is the first report of bacterial type III secretion system genes being associated with high methane emissions in ruminants, and identifies these secretions systems as potential new targets for methane mitigation research. The effects of S. dextrinosolvens on the growth of rumen methanogens in co-cultures indicate that bacteria-methanogen interactions are important modulators of methane production in ruminant animals.

Ectopic expression of Capsicum-specific cell wall protein Capsicum annuum senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in Nicotiana benthamiana.

PubMed

Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-04-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase.

PubMed

Walker, Michael J; Rylett, Caroline M; Keen, Jeff N; Audsley, Neil; Sajid, Mohammed; Shirras, Alan D; Isaac, R Elwyn

2006-05-02

In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a gamma-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. gamma-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG gamma-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong gamma-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. We have applied biochemical approaches, not used previously, to characterise prominent D. melanogaster accessory gland products. Of the thirteen accessory gland secreted proteins reported in this study, six were represented in a D. simulans male accessory gland EST library that was biased for male-specific genes. Therefore, the present study has identified seven new secreted accessory gland proteins, including GGT-1, which was not recognised previously as a secreted accessory gland product.

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

PubMed Central

Darboe, Numukunda; Kenjale, Roma; Picking, Wendy L.; Picking, William D.; Middaugh, C. Russell

2006-01-01

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular “needle” that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37°C and MxiH having a value near 42°C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (ΔG°0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein–protein interactions. PMID:16501225

A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes.

PubMed

Palomäki, Jaana; Sund, Jukka; Vippola, Minnamari; Kinaret, Pia; Greco, Dario; Savolainen, Kai; Puustinen, Anne; Alenius, Harri

2015-01-01

Certain types of carbon nanotubes (CNT) can evoke inflammation, fibrosis and mesothelioma in vivo, raising concerns about their potential health effects. It has been recently postulated that NLRP3 inflammasome activation is important in the CNT-induced toxicity. However, more comprehensive studies of the protein secretion induced by CNT can provide new information about their possible pathogenic mechanisms. Here, we studied protein secretion from human macrophages with a proteomic approach in an unbiased way. Human monocyte-derived macrophages (MDM) were exposed to tangled or rigid, long multi-walled CNT (MWCNT) or crocidolite asbestos for 6 h. The growth media was concentrated and secreted proteins were analyzed using 2D-DIGE and DeCyder software. Subsequently, significantly up- or down-regulated protein spots were in-gel digested and identified with an LC-MS/MS approach. Bioinformatics analysis was performed to reveal the different patterns of protein secretion induced by these materials. The results show that both long rigid MWCNT and asbestos elicited ample and highly similar protein secretion. In contrast, exposure to long tangled MWCNT induced weaker protein secretion with a more distinct profile. Secretion of lysosomal proteins followed the exposure to all materials, suggesting lysosomal damage. However, only long rigid MWCNT was associated with apoptosis. This analysis suggests that the CNT toxicity in human MDM is mediated via vigorous secretion of inflammation-related proteins and apoptosis. This study provides new insights into the mechanisms of toxicity of high aspect ratio nanomaterials and indicates that not all types of CNT are as hazardous as asbestos fibers.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

PubMed

Brown, I R; Mansfield, J W; Taira, S; Roine, E; Romantschuk, M

2001-03-01

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Translocation of Helicobacter pylori CagA into Gastric Epithelial Cells by Type IV Secretion

NASA Astrophysics Data System (ADS)

Odenbreit, Stefan; Püls, Jürgen; Sedlmaier, Bettina; Gerland, Elke; Fischer, Wolfgang; Haas, Rainer

2000-02-01

The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA+) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.

The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10

PubMed Central

Ates, Louis S.; van der Woude, Aniek D.; Bestebroer, Jovanka; van Stempvoort, Gunny; Musters, René J. P.; Garcia-Vallejo, Juan J.; Picavet, Daisy I.; van de Weerd, Robert; Maletta, Massimiliano; Kuijl, Coenraad P.; van der Wel, Nicole N.; Bitter, Wilbert

2016-01-01

Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation. PMID:27280885

Purification of proteins from baculovirus-infected insect cells.

PubMed

O'Shaughnessy, Luke; Doyle, Sean

2011-01-01

Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

Targeting bacterial secretion systems: benefits of disarmament in the microcosm.

PubMed

Baron, Christian; Coombes, Brian

2007-03-01

Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections.

Structure of the Minor Pseudopilin EpsH From the Type 2 Secretion System of Vibrio Cholerae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanez, M.E.; Korotkov, K.V.; Abendroth, J.

2009-05-28

Many Gram-negative bacteria use the multi-protein type II secretion system (T2SS) to selectively translocate virulence factors from the periplasmic space into the extracellular environment. In Vibrio cholerae the T2SS is called the extracellular protein secretion (Eps) system, which translocates cholera toxin and several enzymes in their folded state across the outer membrane. Five proteins of the T2SS, the pseudopilins, are thought to assemble into a pseudopilus, which may control the outer membrane pore EpsD, and participate in the active export of proteins in a 'piston-like' manner. We report here the 2.0 {angstrom} resolution crystal structure of an N-terminally truncated variantmore » of EpsH, a minor pseudopilin from Vibrio cholerae. While EpsH maintains an N-terminal {alpha}-helix and C-terminal {beta}-sheet consistent with the type 4a pilin fold, structural comparisons reveal major differences between the minor pseudopilin EpsH and the major pseudopilin GspG from Klebsiella oxytoca: EpsH contains a large {beta}-sheet in the variable domain, where GspG contains an {alpha}-helix. Most importantly, EpsH contains at its surface a hydrophobic crevice between its variable and conserved {beta}-sheets, wherein a majority of the conserved residues within the EpsH family are clustered. In a tentative model of a T2SS pseudopilus with EpsH at its tip, the conserved crevice faces away from the helix axis. This conserved surface region may be critical for interacting with other proteins from the T2SS machinery.« less

A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi.

PubMed

Login, Frédéric H; Fries, Markus; Wang, Xiaohui; Pickersgill, Richard W; Shevchik, Vladimir E

2010-05-01

The type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3' subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.

Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii.

PubMed

Brown, Kevin M; Lourido, Sebastian; Sibley, L David

2016-04-29

Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.) © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The cardiokine story unfolds: ischemic stress-induced protein secretion in the heart.

PubMed

Doroudgar, Shirin; Glembotski, Christopher C

2011-04-01

Intercellular communication depends on many factors, including proteins released via the classical or non-classical secretory pathways, many of which must be properly folded to be functional. Owing to their adverse effects on the secretion machinery, stresses such as ischemia can impair the folding of secreted proteins. Paradoxically, cells rely on secreted proteins to mount a response designed to resist stress-induced damage. This review examines this paradox using proteins secreted from the heart, cardiokines, as examples, and focuses on how the ischemic heart maintains or even increases the release of select cardiokines that regulate important cellular processes in the heart, including excitation-contraction coupling, hypertrophic growth, myocardial remodeling and stem cell function, in ways that moderate ischemic damage and enhance cardiac repair. Copyright © 2010 Elsevier Ltd. All rights reserved.

Extracellular secretion of recombinant proteins

DOEpatents

Linger, Jeffrey G.; Darzins, Aldis

2014-07-22

Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

Deletion of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger.

PubMed

Krijgsheld, Pauline; Nitsche, Benjamin M; Post, Harm; Levin, Ana M; Müller, Wally H; Heck, Albert J R; Ram, Arthur F J; Altelaar, A F Maarten; Wösten, Han A B

2013-04-05

Aspergillus niger is a cell factory for the production of enzymes. This fungus secretes proteins in the central part and at the periphery of the colony. The sporulating zone of the colony overlapped with the nonsecreting subperipheral zone, indicating that sporulation inhibits protein secretion. Indeed, strain ΔflbA that is affected early in the sporulation program secreted proteins throughout the colony. In contrast, the ΔbrlA strain that initiates but not completes sporulation did not show altered spatial secretion. The secretome of 5 concentric zones of xylose-grown ΔflbA colonies was assessed by quantitative proteomics. In total 138 proteins with a signal sequence for secretion were identified in the medium of ΔflbA colonies. Of these, 18 proteins had never been reported to be part of the secretome of A. niger, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies. Taken together, inactivation of flbA results in spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain ΔflbA has a thinner cell wall compared to the wild type, enabling efficient release of proteins. These results are of interest to improve A. niger as a cell factory.

Assembly of the Type II Secretion System such as Found in Vibrio cholerae Depends on the Novel Pilotin AspS

PubMed Central

Dunstan, Rhys A.; Heinz, Eva; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Purcell, Anthony W.; Evans, Timothy J.; Praszkier, Judyta; Robins-Browne, Roy M.; Strugnell, Richard A.; Korotkov, Konstantin V.; Lithgow, Trevor

2013-01-01

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS. PMID:23326233

Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins.

PubMed

Walz, Alexander; Mujer, Cesar V; Connolly, Joseph P; Alefantis, Tim; Chafin, Ryan; Dake, Clarissa; Whittington, Jessica; Kumar, Srikanta P; Khan, Akbar S; DelVecchio, Vito G

2007-07-27

The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2-) during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites), lipoprotein signal peptides (13 have SpII sites), and N-terminal membrane helices (9 have transmembrane helices). The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa) of protective antigen (PA) were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and accumulation may be relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins.

Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

PubMed Central

Walz, Alexander; Mujer, Cesar V; Connolly, Joseph P; Alefantis, Tim; Chafin, Ryan; Dake, Clarissa; Whittington, Jessica; Kumar, Srikanta P; Khan, Akbar S; DelVecchio, Vito G

2007-01-01

Background The secretion time course of Bacillus anthracis strain RA3R (pXO1+/pXO2-) during early, mid, and late log phase were investigated under conditions that simulate those encountered in the host. All of the identified proteins were analyzed by different software algorithms to characterize their predicted mode of secretion and cellular localization. In addition, immunogenic proteins were identified using sera from humans with cutaneous anthrax. Results A total of 275 extracellular proteins were identified by a combination of LC MS/MS and MALDI-TOF MS. All of the identified proteins were analyzed by SignalP, SecretomeP, PSORT, LipoP, TMHMM, and PROSITE to characterize their predicted mode of secretion, cellular localization, and protein domains. Fifty-three proteins were predicted by SignalP to harbor the cleavable N-terminal signal peptides and were therefore secreted via the classical Sec pathway. Twenty-three proteins were predicted by SecretomeP for secretion by the alternative Sec pathway characterized by the lack of typical export signal. In contrast to SignalP and SecretomeP predictions, PSORT predicted 171 extracellular proteins, 7 cell wall-associated proteins, and 6 cytoplasmic proteins. Moreover, 51 proteins were predicted by LipoP to contain putative Sec signal peptides (38 have SpI sites), lipoprotein signal peptides (13 have SpII sites), and N-terminal membrane helices (9 have transmembrane helices). The TMHMM algorithm predicted 25 membrane-associated proteins with one to ten transmembrane helices. Immunogenic proteins were also identified using sera from patients who have recovered from anthrax. The charge variants (83 and 63 kDa) of protective antigen (PA) were the most immunodominant secreted antigens, followed by charge variants of enolase and transketolase. Conclusion This is the first description of the time course of protein secretion for the pathogen Bacillus anthracis. Time course studies of protein secretion and accumulation may be relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins. PMID:17662140

Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

PubMed

Freedman, S D; Scheele, G A

1994-03-23

The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

Collagen 18 and agrin are secreted by neural crest cells to remodel their microenvironment and regulate their migration during enteric nervous system development.

PubMed

Nagy, Nandor; Barad, Csilla; Hotta, Ryo; Bhave, Sukhada; Arciero, Emily; Dora, David; Goldstein, Allan M

2018-05-08

The enteric nervous system (ENS) arises from neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the intestinal wall. Many extracellular matrix (ECM) components are present in the embryonic gut, but their role in regulating ENS development is largely unknown. Here, we identify heparan sulfate proteoglycan proteins, including collagen XVIII (Col18) and agrin, as important regulators of enteric neural crest-derived cell (ENCDC) development. In developing avian hindgut, Col18 is expressed at the ENCDC wavefront, while agrin expression occurs later. Both proteins are normally present around enteric ganglia, but are absent in aganglionic gut. Using chick-mouse intestinal chimeras and enteric neurospheres, we show that vagal- and sacral-derived ENCDCs from both species secrete Col18 and agrin. Whereas glia express Col18 and agrin, enteric neurons only express the latter. Functional studies demonstrate that Col18 is permissive whereas agrin is strongly inhibitory to ENCDC migration, consistent with the timing of their expression during ENS development. We conclude that ENCDCs govern their own migration by actively remodeling their microenvironment through secretion of ECM proteins. © 2018. Published by The Company of Biologists Ltd.

Golgi bypass for local delivery of axonal proteins, fact or fiction?

PubMed

González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

2018-04-06

Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

Live cell imaging of phosphoinositide dynamics during Legionella infection.

PubMed

Weber, Stephen; Hilbi, Hubert

2014-01-01

The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.

Autotransporter-based cell surface display in Gram-negative bacteria.

PubMed

Nicolay, Toon; Vanderleyden, Jos; Spaepen, Stijn

2015-02-01

Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

Common and Distinct Capsid and Surface Protein Requirements for Secretion of Complete and Genome-free Hepatitis B Virions.

PubMed

Ning, Xiaojun; Luckenbaugh, Laurie; Liu, Kuancheng; Bruss, Volker; Sureau, Camille; Hu, Jianming

2018-05-09

During the morphogenesis of hepatitis B virus (HBV), an enveloped virus, two types of virions are secreted: (1) a minor population of complete virions containing a mature nucleocapsid with the characteristic, partially double-stranded, relaxed circular DNA genome and (2) a major population containing an empty capsid with no DNA or RNA (empty virions). Secretion of both types of virions requires interactions between the HBV capsid or core protein (HBc) and the viral surface or envelope proteins. We have studied the requirements from both HBc and envelope proteins for empty virion secretion, in comparison with those for secretion of complete virions. Substitutions within the N-terminal domain of HBc that block secretion of DNA-containing virions reduced but did not prevent secretion of empty virions. The HBc C-terminal domain was not essential for empty virion secretion. Among the three viral envelope proteins, the smallest, S, alone was sufficient for empty virion secretion at a basal level. The largest protein, L, essential for complete virion secretion, was not required for, but could stimulate empty virion secretion. Also, substitutions in L that eliminate secretion of complete virions reduced but did not eliminate empty virion secretion. S mutations that block secretion of the hepatitis D virus (HDV), an HBV satellite, did not block secretion of either empty or complete HBV virions. Together, these results indicate that both common and distinct signals on empty capsids vs. mature nucleocapsids interact with the S and L proteins during the formation of complete vs. empty virions. IMPORTANCE Hepatitis B virus (HBV) is a major cause of severe liver diseases including cirrhosis and cancer. In addition to the complete infectious virion particle, which contains an outer envelope layer and an interior capsid that, in turn, encloses a DNA genome, HBV infected cells also secrete non-infectious, incomplete viral particles in large excess over the complete virions. In particular, the empty (or genome-free) virion share with the complete virion the outer envelope and interior capsid but contain no genome. We have carried out a comparative study on the capsid and envelope requirements for the secretion of these two types of virion particles and uncovered both shared and distinct determinants on the capsid and envelope for their secretion. These results provide new information on HBV morphogenesis, and have implications for efforts to develop empty HBV virions as a novel biomarker and a new generation of HBV vaccine. Copyright © 2018 American Society for Microbiology.

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

PubMed

Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

2017-04-24

Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

PubMed Central

Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

2013-01-01

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

The endoplasmic reticulum in plant immunity and cell death

PubMed Central

Eichmann, Ruth; Schäfer, Patrick

2012-01-01

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants. PMID:22936941

The endoplasmic reticulum in plant immunity and cell death.

PubMed

Eichmann, Ruth; Schäfer, Patrick

2012-01-01

The endoplasmic reticulum (ER) is a highly dynamic organelle in eukaryotic cells and a major production site of proteins destined for vacuoles, the plasma membrane, or apoplast in plants. At the ER, these secreted proteins undergo multiple processing steps, which are supervised and conducted by the ER quality control system. Notably, processing of secreted proteins can considerably elevate under stress conditions and exceed ER folding capacities. The resulting accumulation of unfolded proteins is defined as ER stress. The efficiency of cells to re-establish proper ER function is crucial for stress adaptation. Besides delivering proteins directly antagonizing and resolving stress conditions, the ER monitors synthesis of immune receptors. This indicates the significance of the ER for the establishment and function of the plant immune system. Recent studies point out the fragility of the entire system and highlight the ER as initiator of programed cell death (PCD) in plants as was reported for vertebrates. This review summarizes current knowledge on the impact of the ER on immune and PCD signaling. Understanding the integration of stress signals by the ER bears a considerable potential to optimize development and to enhance stress resistance of plants.

Comparative proteome analysis of laboratory grown Brucella abortus 2308 and Brucella melitensis 16M.

PubMed

Eschenbrenner, Michel; Horn, Troy A; Wagner, Mary Ann; Mujer, Cesar V; Miller-Scandle, Tabbi L; DelVecchio, Vito G

2006-07-01

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.

Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system

PubMed Central

2010-01-01

Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei. PMID:20540813

Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B.

PubMed Central

Létoffé, S; Wandersman, C

1992-01-01

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide. Images PMID:1629152

The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1β Secretion.

PubMed

Moriyama, Miyu; Chen, I-Yin; Kawaguchi, Atsushi; Koshiba, Takumi; Nagata, Kyosuke; Takeyama, Haruko; Hasegawa, Hideki; Ichinohe, Takeshi

2016-04-01

Inflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1β (IL-1β) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1β secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1β secretion. However, the precise mechanism by which NS1 inhibits IL-1β secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1β secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1β secretion. Innate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1β secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results will facilitate the development of new anti-inflammatory drugs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1β Secretion

PubMed Central

Moriyama, Miyu; Chen, I-Yin; Kawaguchi, Atsushi; Koshiba, Takumi; Nagata, Kyosuke; Takeyama, Haruko; Hasegawa, Hideki

2016-01-01

ABSTRACT Inflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1β (IL-1β) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1β secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1β secretion. However, the precise mechanism by which NS1 inhibits IL-1β secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1β secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1β secretion. IMPORTANCE Innate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1β secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results will facilitate the development of new anti-inflammatory drugs. PMID:26865721

Genetically encoded photochemical covalent crosslinking within the Hcp-1 self-assembling bacterial secretion machinery.

PubMed

Antonczak, Alicja K; Milholland, Kedric; Tippmann, Eric M

2018-05-01

The target protein, Hcp1, was first described as part of the bacterial Type VI secretion system from Pseudomonas aeruginosa. The protein first self-assembles into a hexamer and then the hexamers further stack into a nanotubular structure. Hcp1 monomers were targeted for mutagenesis with two widely used photoactivatable amino acids: para-benzoyl phenylalanine or para-azidophenylalanine. The ability of these amino acids to form covalent adducts within the Hcp1 self-assembled system was investigated. Multiple residues, putatively of equal distance between the monomer-monomer interface were targeted. The efficiency of each amino acid to covalently link self-assembled hexamers was determined. The results demonstrate the choice and role of genetically encoded tools applied to complicated biological processes such as self-assembly and also suggested some structural dynamics of the Hcp-1 protein not obvious from crystallographic structures.

The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins*

PubMed Central

Goessweiner-Mohr, Nikolaus; Grumet, Lukas; Arends, Karsten; Pavkov-Keller, Tea; Gruber, Christian C.; Gruber, Karl; Birner-Gruenberger, Ruth; Kropec-Huebner, Andrea; Huebner, Johannes; Grohmann, Elisabeth; Keller, Walter

2013-01-01

Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins. PMID:23188825

Type IV Pili in Gram-Positive Bacteria

PubMed Central

Craig, Lisa

2013-01-01

SUMMARY Type IV pili (T4P) are surface-exposed fibers that mediate many functions in bacteria, including locomotion, adherence to host cells, DNA uptake (competence), and protein secretion and that can act as nanowires carrying electric current. T4P are composed of a polymerized protein, pilin, and their assembly apparatuses share protein homologs with type II secretion systems in eubacteria and the flagella of archaea. T4P are found throughout Gram-negative bacterial families and have been studied most extensively in certain model Gram-negative species. Recently, it was discovered that T4P systems are also widespread among Gram-positive species, in particular the clostridia. Since Gram-positive and Gram-negative bacteria have many differences in cell wall architecture and other features, it is remarkable how similar the T4P core proteins are between these organisms, yet there are many key and interesting differences to be found as well. In this review, we compare the two T4P systems and identify and discuss the features they have in common and where they differ to provide a very broad-based view of T4P systems across all eubacterial species. PMID:24006467

Regulation of pulmonary surfactant secretion in the developing lizard, Pogona vitticeps.

PubMed

Sullivan, Lucy C; Orgeig, Sandra; Daniels, Christopher B

2002-11-01

Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar type II cells in the lungs of all air-breathing vertebrates. Pulmonary surfactant functions to reduce the surface tension in the lungs and, therefore, reduce the work of breathing. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones and autonomic neurotransmitters. We have used a co-culture system of embryonic type II cells and lung fibroblasts to investigate the ability of dexamethasone, tri-iodothyronine (T(3)), adrenaline and carbamylcholine (carbachol) to stimulate the cellular secretion of phosphatidylcholine in the bearded dragon (Pogona vitticeps) at day 55 (approx. 92%) of incubation and following hatching. Adrenaline stimulated surfactant secretion both before and after hatching, whereas carbachol stimulated secretion only at day 55. Glucocorticoids and triiodothyronine together stimulated secretion at day 55 but did not after hatching. Therefore, adrenaline, carbachol, dexamethasone and T(3), are all involved in the development of the surfactant system in the bearded dragon. However, the efficacy of the hormones is attenuated during the developmental process. These differences probably relate to the changes in the cellular environment during development and the specific biology of the bearded dragon.

Closely related dermatophyte species produce different patterns of secreted proteins.

PubMed

Giddey, Karin; Favre, Bertrand; Quadroni, Manfredo; Monod, Michel

2007-02-01

Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Various species in this group of fungi show overlapping characteristics. We investigated the possibility that closely related dermatophyte species with different behaviours secrete distinct proteins when grown in the same culture medium. Protein patterns from culture filtrates of several strains of the same species were very similar. In contrast, secreted protein profiles from various species were different, and so a specific signature could be associated with each of the six analysed species. In particular, protein patterns were useful to distinguish Trichophyton tonsurans from Trichophyton equinum, which cannot be differentiated by ribosomal DNA sequencing. The secreted proteases Sub2, Sub6 and Sub7 of the subtilisin family, as well as Mep3 and Mep4 of the fungalisin family were identified. SUB6, SUB7, MEP3 and MEP4 genes were cloned and sequenced. Although the protein sequence of each protease was highly conserved across species, their level of secretion by the various species was not equivalent. These results suggest that a switch of habitat could be related to a differential expression of genes encoding homologous secreted proteins.

Protein adsorption in microengraving immunoassays.

PubMed

Song, Qing

2015-10-16

Microengraving is a novel immunoassay for characterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales  and  determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when  is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 10⁴-10⁵ single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.

Protein Adsorption in Microengraving Immunoassays

PubMed Central

Song, Qing

2015-01-01

Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

Ectopic Expression of Capsicum-Specific Cell Wall Protein Capsicum annuum Senescence-Delaying 1 (CaSD1) Delays Senescence and Induces Trichome Formation in Nicotiana benthamiana

PubMed Central

Seo, Eunyoung; Yeom, Seon-In; Jo, SungHwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil

2012-01-01

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation. PMID:22441673

Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein

PubMed Central

Alcoforado Diniz, Juliana

2015-01-01

ABSTRACT The type VI secretion system (T6SS) is widespread in Gram-negative bacteria and can deliver toxic effector proteins into eukaryotic cells or competitor bacteria. Antibacterial T6SSs are increasingly recognized as key mediators of interbacterial competition and may contribute to the outcome of many polymicrobial infections. Multiple antibacterial effectors can be delivered by these systems, with diverse activities against target cells and distinct modes of secretion. Polymorphic toxins containing Rhs repeat domains represent a recently identified and as-yet poorly characterized class of T6SS-dependent effectors. Previous work had revealed that the potent antibacterial T6SS of the opportunistic pathogen Serratia marcescens promotes intraspecies as well as interspecies competition (S. L. Murdoch, K. Trunk, G. English, M. J. Fritsch, E. Pourkarimi, and S. J. Coulthurst, J Bacteriol 193:6057–6069, 2011, http://dx.doi.org/10.1128/JB.05671-11). In this study, two new Rhs family antibacterial effectors delivered by this T6SS have been identified. One of these was shown to act as a DNase toxin, while the other contains a novel, cytoplasmic-acting toxin domain. Importantly, using S. marcescens, it has been demonstrated for the first time that Rhs proteins, rather than other T6SS-secreted effectors, can be the primary determinant of intraspecies competition. Furthermore, a new family of accessory proteins associated with T6SS effectors has been identified, exemplified by S. marcescens EagR1, which is specifically required for deployment of its associated Rhs effector. Together, these findings provide new insight into how bacteria can use the T6SS to deploy Rhs-family effectors and mediate different types of interbacterial interactions. IMPORTANCE Infectious diseases caused by bacterial pathogens represent a continuing threat to health and economic prosperity. To counter this threat, we must understand how such organisms survive and prosper. The type VI secretion system is a weapon that many pathogens deploy to compete against rival bacterial cells by injecting multiple antibacterial toxins into them. The ability to compete is vital considering that bacteria generally live in mixed communities. We aimed to identify new toxins and understand their deployment and role in interbacterial competition. We describe two new type VI secretion system-delivered toxins of the Rhs class, demonstrate that this class can play a primary role in competition between closely related bacteria, and identify a new accessory factor needed for their delivery. PMID:25939831

MIX and match: mobile T6SS MIX-effectors enhance bacterial fitness

PubMed Central

Salomon, Dor

2016-01-01

ABSTRACT Protein secretion systems that mediate interbacterial competition secret a wide repertoire of antibacterial toxins. A major player in these competitions is the newly discovered bacterial type VI secretion system (T6SS). We recently found that a subset of polymorphic MIX-effectors, which are a widespread class of effectors secreted by T6SSs, are horizontally shared between marine bacteria and are used to diversify their T6SS effector repertoires, thus enhancing their environmental fitness. In this commentary, I expand on the ideas that were introduced in the previous report, and further speculate on the possible mobility of other MIX-effectors. In addition, I discuss the possible role of horizontal gene transfer in the dissemination of MIX-effectors through bacterial genomes, as well as its possible role in diversifying the T6SS effector repertoire. PMID:27066305

Flavobacterium johnsoniae sprB Is Part of an Operon Spanning the Additional Gliding Motility Genes sprC, sprD, and sprF ▿ †

PubMed Central

Rhodes, Ryan G.; Nelson, Shawn S.; Pochiraju, Soumya; McBride, Mark J.

2011-01-01

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces. PMID:21131497

Virulence of Erwinia amylovora, a prevalent apple pathogen: Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses.

PubMed

Holtappels, Michelle; Noben, Jean-Paul; Valcke, Roland

2016-09-01

Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram-negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D-DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression and function of Anoctamin 1/TMEM16A calcium-activated chloride channels in airways of in vivo mouse models for cystic fibrosis research.

PubMed

Hahn, Anne; Salomon, Johanna J; Leitz, Dominik; Feigenbutz, Dennis; Korsch, Lisa; Lisewski, Ina; Schrimpf, Katrin; Millar-Büchner, Pamela; Mall, Marcus A; Frings, Stephan; Möhrlen, Frank

2018-06-02

Physiological processes of vital importance are often safeguarded by compensatory systems that substitute for primary processes in case these are damaged by gene mutation. Ca 2+ -dependent Cl - secretion in airway epithelial cells may provide such a compensatory mechanism for impaired Cl - secretion via cystic fibrosis transmembrane conductance regulator (CFTR) channels in cystic fibrosis (CF). Anoctamin 1 (ANO1) Ca 2+ -gated Cl - channels are known to contribute to calcium-dependent Cl - secretion in tracheal and bronchial epithelia. In the present study, two mouse models of CF were examined to assess a potential protective function of Ca 2+ -dependent Cl - secretion, a CFTR deletion model (cftr -/- ), and a CF pathology model that overexpresses the epithelial Na + channel β-subunit (βENaC), which is encoded by the Scnn1b gene, specifically in airway epithelia (Scnn1b-Tg). The expression levels of ANO1 were examined by mRNA and protein content, and the channel protein distribution between ciliated and non-ciliated epithelial cells was analyzed. Moreover, Ussing chamber experiments were conducted to compare Ca 2+ -dependent Cl - secretion between wild-type animals and the two mouse models. Our results demonstrate that CFTR and ANO1 channels were co-expressed with ENaC in non-ciliated cells of mouse tracheal and bronchial epithelia. Ciliated cells did not express these proteins. Despite co-localization of CFTR and ANO1 in the same cell type, cells in cftr -/- mice displayed no altered expression of ANO1. Similarly, ANO1 expression was unaffected by βENaC overexpression in the Scnn1b-Tg line. These results suggest that the CF-related environment in the two mouse models did not induce ANO1 overexpression as a compensatory system.

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.

PubMed

Veith, Paul D; Glew, Michelle D; Gorasia, Dhana G; Reynolds, Eric C

2017-10-01

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Proteins secreted by the T9SS have an N-terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C-terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C-terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components. © 2017 John Wiley & Sons Ltd.

76 FR 63316 - Prospective Grant of Exclusive License: Secreted Frizzled Related Protein-1 (sFRP-1) and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-12

... Exclusive License: Secreted Frizzled Related Protein-1 (sFRP-1) and derivatives thereof and their Use In... belonging to the patent families having HHS Reference Numbers E-160-1997/0,/1,/2 and/3; E-014-2000/0; and E... Related Protein-1 (sFRP-1). sFRP-1, also known as SARP-2 (Secreted Apoptosis Related Protein-2). The IP...

Determining the relative contribution and hierarchy of qseBC and hha in the regulation of flagellar motility of Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

In a recent study we demonstrated that in comparison to the wild-type enterohemorrhagic Escherichia coli (EHEC) O157:H7, a motility-compromised hha deletion mutant with an up-regulated type III secretion system and increased secretion of adherence proteins showed reduced fecal shedding in cattle. In...

Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton.

PubMed

Veenendaal, Andreas K J; Sundin, Charlotta; Blocker, Ariel J

2009-01-01

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.

Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain

PubMed Central

Garrett, Christopher K.; Broadwell, Lindsey J.; Hayne, Cassandra K.; Neher, Saskia B.

2015-01-01

Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme. PMID:26270534

Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters

PubMed Central

Yordem, Burcu K.; Conte, Sarah S.; Ma, Jian Feng; Yokosho, Kengo; Vasques, Kenneth A.; Gopalsamy, Srinivasa N.; Walker, Elsbeth L.

2011-01-01

Background and Aims Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency. Methods The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography. Key Results Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated. Conclusions PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis. PMID:21831857

Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella.

PubMed

Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C; Dehio, Christoph

2011-02-10

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment.

Parallel Evolution of a Type IV Secretion System in Radiating Lineages of the Host-Restricted Bacterial Pathogen Bartonella

PubMed Central

Engel, Philipp; Salzburger, Walter; Liesch, Marius; Chang, Chao-Chin; Maruyama, Soichi; Lanz, Christa; Calteau, Alexandra; Lajus, Aurélie; Médigue, Claudine; Schuster, Stephan C.; Dehio, Christoph

2011-01-01

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment. PMID:21347280

The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space*

PubMed Central

Perez-Gonzalez, Rocio; Gauthier, Sebastien A.; Kumar, Asok; Levy, Efrat

2012-01-01

In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain. PMID:23129776

Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor

PubMed Central

2010-01-01

Background Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, Lactococcus lactis, and purified from the culture medium. Results The gene segment corresponding to the S. aureus nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An L. lactis subsp cremoris model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure. Conclusions In L. lactis, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD600 of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg. PMID:20492646

Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Hyunjin; Ansong, Charles; Adkins, Joshua N.

We have previously shown that the regulators SpvR, FruR, IHF, PhoP/PhoQ, SsrA/SsrB, SlyA, Hnr, RpoE, SmpB, CsrA, RpoS, Crp, OmpR/EnvZ, and Hfq are essential for Salmonella Typhimurium virulence in mice. Here we use quantitative LC-MS-based proteomics profiling of in-frame deletion mutants of these 14 regulators to identify proteins that are coordinately regulated by these virulence regulators and are thus presumably novel factors contributing to Salmonella pathogenesis. Putative candidate proteins from proteomics analysis were determined, which exhibited similar abundance profiles to those of Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS) proteins. A subset of 5 proteins including STM0082, STM1548,more » PdgL, STM1633, and STM3595 was selected for further analysis. All 5 proteins were expressed inside macrophage cells and STM0082 (SrfN) was secreted into host cytoplasm. Furthermore, deletion of STM0082 attenuated virulence in mice when administered intraperitoneally as determined by competitive index. srfN transcription was positively regulated by SsrAB, however, secretion was independent of SPI-2 TTSS as well as SPI-1 TTSS and flagella. Proteins including PagK and STM2585A, which are positively regulated by PhoP/PhoQ, have sec signal peptides as predicted for SrfN and were secreted into macrophage cytoplasm regardless of SPI-2 TTSS. Isolation of outer membrane vesicles (OMVs) revealed the presence of SrfN, PagK, and STM2585A inside vesicle compartments. This result is the first case showing delivery of virulence effectors via OMVs in S. Typhimurium. Moreover, Hfq regulation of SrfN translation suggests that small non-coding RNAs may be responsible for regulating effector protein expression.« less

Global Secretome Characterization of Herpes Simplex Virus 1-Infected Human Primary Macrophages

PubMed Central

Miettinen, Juho J.; Matikainen, Sampsa

2012-01-01

Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-β) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-β-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-β priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1β was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense. PMID:22973042

Computational Identification and Comparative Analysis of Secreted and Transmembrane Proteins in Six Burkholderia Species.

PubMed

Nguyen, Thao Thi; Lee, Hyun-Hee; Park, Jungwook; Park, Inmyoung; Seo, Young-Su

2017-04-01

As a step towards discovering novel pathogenesis-related proteins, we performed a genome scale computational identification and characterization of secreted and transmembrane (TM) proteins, which are mainly responsible for bacteria-host interactions and interactions with other bacteria, in the genomes of six representative Burkholderia species. The species comprised plant pathogens ( B. glumae BGR1, B. gladioli BSR3), human pathogens ( B. pseudomallei K96243, B. cepacia LO6), and plant-growth promoting endophytes ( Burkholderia sp. KJ006, B. phytofirmans PsJN). The proportions of putative classically secreted proteins (CSPs) and TM proteins among the species were relatively high, up to approximately 20%. Lower proportions of putative type 3 non-classically secreted proteins (T3NCSPs) (~10%) and unclassified non-classically secreted proteins (NCSPs) (~5%) were observed. The numbers of TM proteins among the three clusters (plant pathogens, human pathogens, and endophytes) were different, while the distribution of these proteins according to the number of TM domains was conserved in which TM proteins possessing 1, 2, 4, or 12 TM domains were the dominant groups in all species. In addition, we observed conservation in the protein size distribution of the secreted protein groups among the species. There were species-specific differences in the functional characteristics of these proteins in the various groups of CSPs, T3NCSPs, and unclassified NCSPs. Furthermore, we assigned the complete sets of the conserved and unique NCSP candidates of the collected Burkholderia species using sequence similarity searching. This study could provide new insights into the relationship among plant-pathogenic, human-pathogenic, and endophytic bacteria.

The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background.

PubMed

Zhang, Hui; Wang, Shuang; Zhang, Xiang Xiang; Ji, Wei; Song, Fuping; Zhao, Yue; Li, Jie

2016-04-28

The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion. The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain. Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

Membrane Fusion Proteins of Type I Secretion System and Tripartite Efflux Pumps Share a Binding Motif for TolC in Gram-Negative Bacteria

PubMed Central

Yoon, Bo-Young; Song, Saemee; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed. PMID:22792337

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

PubMed

Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

2017-01-01

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease. Copyright © 2016 American Society for Microbiology.

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS.

PubMed

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R; Changelian, Armen; Laws, Edward R; Santagata, Sandro; Agar, Nathalie Y R; Van Berkel, Gary J

2015-08-01

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections, using a fully automated droplet-based liquid-microjunction surface-sampling-HPLC-ESI-MS-MS system for spatially resolved sampling, HPLC separation, and mass spectrometric detection. Excellent correlation was found between the protein distribution data obtained with this method and data obtained with matrix-assisted laser desorption/ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland. AVP was most abundant in the posterior pituitary gland region (neurohypophysis), and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH-secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH-secreting adenomas and in normal anterior adenohypophysis compared with non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis, as expected. This work reveals that a fully automated droplet-based liquid-microjunction surface-sampling system coupled to HPLC-ESI-MS-MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, including AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity, and specificity of this method support the potential of this basic technology, with further advancement, for assisting surgical decision-making. Graphical Abstract Mass spectrometry based profiling of hormones in human pituitary gland and tumor thin tissue sections.

The N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD

PubMed Central

Dickenson, Nicholas E.; Arizmendi, Olivia; Patil, Mrinalini K.; Toth, Ronald T.; Middaugh, C. Russell; Picking, William D.; Picking, Wendy L.

2014-01-01

The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri, providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is comprised of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g. deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. While the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study due to the hydrophobic nature of the IpaB and IpaC translocator proteins. Here we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11–27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation. PMID:24236510

Hcp Family Proteins Secreted via the Type VI Secretion System Coordinately Regulate Escherichia coli K1 Interaction with Human Brain Microvascular Endothelial Cells

PubMed Central

Zhou, Yan; Tao, Jing; Yu, Hao; Ni, Jinjing; Zeng, Lingbing; Teng, Qihui; Kim, Kwang Sik; Zhao, Guo-Ping

2012-01-01

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. Based on sequence analysis, we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis. PMID:22184413

Hcp family proteins secreted via the type VI secretion system coordinately regulate Escherichia coli K1 interaction with human brain microvascular endothelial cells.

PubMed

Zhou, Yan; Tao, Jing; Yu, Hao; Ni, Jinjing; Zeng, Lingbing; Teng, Qihui; Kim, Kwang Sik; Zhao, Guo-Ping; Guo, Xiaokui; Yao, Yufeng

2012-03-01

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several gram-negative bacteria. Based on sequence analysis, we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

Low-density lipoproteins modulate endothelial cells to secrete endothelin-1 in a polarized pattern: a study using a culture model system simulating arterial intima.

PubMed

Unoki, H; Fan, J; Watanabe, T

1999-01-01

We investigated the structural and functional properties of human umbilical vein endothelial cells (HUVECs) cultured on a two-chamber culture model system using an amnion membrane. Compared to HUVECs cultured on a plastic dish, HUVECs cultured on the model system exhibited several features similar to those of in vivo vessels, including formation of the intercellular junctional devices and expression of tight junction-associated protein ZO-1 and adherence junction-associated protein alpha-catenin. Furthermore, we found that HUVECs had a property of polar secretion of endothelin-1 (ET-1). About 90% of the total amount of synthesized ET-1 was found in the lower well, designated as the basal side. When HUVECs were incubated with either native low-density lipoproteins (nLDLs) or oxidized LDLs (oxLDLs) at a concentration of 100 microgram/ml, ET-1 secretion was significantly increased, dependent on the cell side (apical vs basal) on which the nLDLs or oxLDLs were loaded. When the LDLs were loaded on the apical side, the secretion of ET-1 from HUVECs on the apical side was increased by 48% (nLDL) and 61% (oxLDL), whereas it was accompanied by a concomitant decrease of ET-1 on the basal side (45% by nLDLs and 38% by oxLDLs). When loaded on the basal side, however, ET-1 was increased by 23% (nLDLs) and 53% (oxLDLs) on the basal side, with a 26% simultaneous decrease of ET-1 on the opposite side for both nLDLs and oxLDLs. On the contrary, high-density lipoproteins (HDLs) inhibited ET-1 secretion from HUVECs on the opposite side of the well on which HDLs were loaded; there was a 57% decrease on the basal side when HDLs were loaded on the apical side, and a 46% decrease on the apical side when loaded on the basal side. These results indicate that modulation of ET-1 secretion from ECs by lipoproteins is virtually dependent on the place (apical vs basal) where these proteins are present. The finding that nLDLs and oxLDLs enhance ET-1 secretion by ECs in a polarized pattern suggests that ET-1 may be involved in pathophysiological processes such as atherogenesis.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

PubMed

Ittig, Simon J; Schmutz, Christoph; Kasper, Christoph A; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R; Nigg, Erich A; Arrieumerlou, Cécile

2015-11-23

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. © 2015 Ittig et al.

Erwinia amylovora effector protein Eop1 suppresses PAMP-triggered immunity in Malus

USDA-ARS?s Scientific Manuscript database

Erwinia amylovora (Ea) utilizes a type three secretion system (T3SS) to deliver effector proteins into plant host cells. Several Ea effectors have been identified based on their sequence similarity to plant and animal bacterial pathogen effectors; however, the function of the majority of Ea effecto...

Crystallization and preliminary X-ray diffraction analysis of a high-affinity phosphate-binding protein endowed with phosphatase activity from Pseudomonas aeruginosa PAO1

PubMed Central

Djeghader, Ahmed; Gotthard, Guillaume; Suh, Andrew; Gonzalez, Daniel; Scott, Ken; Chabriere, Eric; Elias, Mikael

2013-01-01

In prokaryotes, phosphate starvation induces the expression of numerous phosphate-responsive genes, such as the pst operon including the high-affinity phosphate-binding protein (PBP or pstS) and alkaline phosphatases such as PhoA. This response increases the cellular inorganic phosphate import efficiency. Notably, some Pseudomonas species secrete, via a type-2 secretion system, a phosphate-binding protein dubbed LapA endowed with phosphatase activity. Here, the expression, purification, crystallization and X-ray data collection at 0.87 Å resolution of LapA are described. Combined with biochemical and enzymatic characterization, the structure of this intriguing phosphate-binding protein will help to elucidate the molecular origin of its phosphatase activity and to decipher its putative role in phosphate uptake. PMID:24100568

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

PubMed

Ennouri, Habiba; d'Abzac, Paul; Hakil, Florence; Branchu, Priscilla; Naïtali, Murielle; Lomenech, Anne-Marie; Oueslati, Ridha; Desbrières, Jacques; Sivadon, Pierre; Grimaud, Régis

2017-01-01

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

The targeting of plant cellular systems by injected type III effector proteins.

PubMed

Lewis, Jennifer D; Guttman, David S; Desveaux, Darrell

2009-12-01

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector-host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.

Temporal Profiling and Pulsed SILAC Labeling Identify Novel Secreted Proteins During Ex Vivo Osteoblast Differentiation of Human Stromal Stem Cells*

PubMed Central

Kristensen, Lars P.; Chen, Li; Nielsen, Maria Overbeck; Qanie, Diyako W.; Kratchmarova, Irina; Kassem, Moustapha; Andersen, Jens S.

2012-01-01

It is well established that bone forming cells (osteoblasts) secrete proteins with autocrine, paracrine, and endocrine function. However, the identity and functional role for the majority of these secreted and differentially expressed proteins during the osteoblast (OB) differentiation process, is not fully established. To address these questions, we quantified the temporal dynamics of the human stromal (mesenchymal, skeletal) stem cell (hMSC) secretome during ex vivo OB differentiation using stable isotope labeling by amino acids in cell culture (SILAC). In addition, we employed pulsed SILAC labeling to distinguish genuine secreted proteins from intracellular contaminants. We identified 466 potentially secreted proteins that were quantified at 5 time-points during 14-days ex vivo OB differentiation including 41 proteins known to be involved in OB functions. Among these, 315 proteins exhibited more than 2-fold up or down-regulation. The pulsed SILAC method revealed a strong correlation between the fraction of isotope labeling and the subset of proteins known to be secreted and involved in OB differentiation. We verified SILAC data using qRT-PCR analysis of 9 identified potential novel regulators of OB differentiation. Furthermore, we studied the biological effects of one of these proteins, the hormone stanniocalcin 2 (STC2) and demonstrated its autocrine effects in enhancing osteoblastic differentiation of hMSC. In conclusion, combining complete and pulsed SILAC labeling facilitated the identification of novel factors produced by hMSC with potential role in OB differentiation. Our study demonstrates that the secretome of osteoblastic cells is more complex than previously reported and supports the emerging evidence that osteoblastic cells secrete proteins with endocrine functions and regulate cellular processes beyond bone formation. PMID:22801418

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.

PubMed

Avril, Marion; Hathaway, Marianne J; Cartwright, Megan M; Gose, Severin O; Narum, David L; Smith, Joseph D

2009-06-29

VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant protein secretion from Pichia pastoris and if domain boundaries were applicable across different VAR2CSA alleles. VAR2CSA sequences were bioinformatically analysed to identify the predicted C11 and C12 cysteine residues at the C-termini of DBL domains and revised N- and C-termimal domain boundaries were predicted in VAR2CSA. Multiple construct boundaries were systematically evaluated for protein secretion in P. pastoris and secreted proteins were tested as immunogens. From a total of 42 different VAR2CSA constructs, 15 proteins (36%) were secreted. Longer construct boundaries, including the predicted C11 and C12 cysteine residues, generally improved expression of poorly or non-secreted domains and permitted expression of all six VAR2CSA DBL domains. However, protein secretion was still highly empiric and affected by subtle differences in domain boundaries and allelic variation between VAR2CSA sequences. Eleven of the secreted proteins were used to immunize rabbits. Antibodies reacted with CSA-binding infected erythrocytes, indicating that P. pastoris recombinant proteins possessed native protein epitopes. These findings strengthen emerging data for a revision of DBL domain boundaries in var-encoded proteins and may facilitate pregnancy malaria vaccine development.

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris

PubMed Central

Avril, Marion; Hathaway, Marianne J; Cartwright, Megan M; Gose, Severin O; Narum, David L; Smith, Joseph D

2009-01-01

Background VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant protein secretion from Pichia pastoris and if domain boundaries were applicable across different VAR2CSA alleles. Methods VAR2CSA sequences were bioinformatically analysed to identify the predicted C11 and C12 cysteine residues at the C-termini of DBL domains and revised N- and C-termimal domain boundaries were predicted in VAR2CSA. Multiple construct boundaries were systematically evaluated for protein secretion in P. pastoris and secreted proteins were tested as immunogens. Results From a total of 42 different VAR2CSA constructs, 15 proteins (36%) were secreted. Longer construct boundaries, including the predicted C11 and C12 cysteine residues, generally improved expression of poorly or non-secreted domains and permitted expression of all six VAR2CSA DBL domains. However, protein secretion was still highly empiric and affected by subtle differences in domain boundaries and allelic variation between VAR2CSA sequences. Eleven of the secreted proteins were used to immunize rabbits. Antibodies reacted with CSA-binding infected erythrocytes, indicating that P. pastoris recombinant proteins possessed native protein epitopes. Conclusion These findings strengthen emerging data for a revision of DBL domain boundaries in var-encoded proteins and may facilitate pregnancy malaria vaccine development. PMID:19563628

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

PubMed

Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

2012-02-01

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. Copyright © 2011 Elsevier Inc. All rights reserved.

The Crystal Structure of a Binary Complex of Two Pseudopilins: EpsI And EpsJ From the Type 2 Secretion System of Vibrio Vulnificus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanez, M.E.; Korotkov, K.V.; Abendroth, J.

2009-05-28

Type II secretion systems (T2SS) translocate virulence factors from the periplasmic space of many pathogenic bacteria into the extracellular environment. The T2SS of Vibrio cholerae and related species is called the extracellular protein secretion (Eps) system that consists of a core of multiple copies of 11 different proteins. The pseudopilins, EpsG, EpsH, EpsI, EpsJ and EpsK, are five T2SS proteins that are thought to assemble into a pseudopilus, which is assumed to interact with the outer membrane pore, and may actively participate in the export of proteins. We report here biochemical evidence that the minor pseudopilins EpsI and EpsJ frommore » Vibrio species interact directly with one another. Moreover, the 2.3 {angstrom} resolution crystal structure of a complex of EspI and EpsJ from Vibrio vulnificus represents the first atomic resolution structure of a complex of two different pseudopilin components from the T2SS. Both EpsI and EpsJ appear to be structural extremes within the family of type 4a pilin structures solved to date, with EpsI having the smallest, and EpsJ the largest, 'variable pilin segment' seen thus far. A high degree of sequence conservation in the EpsI:EpsJ interface indicates that this heterodimer occurs in the T2SS of a large number of bacteria. The arrangement of EpsI and EpsJ in the heterodimer would correspond to a right-handed helical character of proteins assembled into a pseudopilus.« less

Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968.

PubMed

Kozak, Natalia A; Buss, Meghan; Lucas, Claressa E; Frace, Michael; Govil, Dhwani; Travis, Tatiana; Olsen-Rasmussen, Melissa; Benson, Robert F; Fields, Barry S

2010-02-01

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

N-glycans in liver-secreted and immunoglogulin-derived protein fractions

PubMed Central

Bekesova, S.; Kosti, O.; Chandler, K.B.; Wu, J.; Madej, H.L.; Brown, K.C.; Simonyan, V.; Goldman, R.

2013-01-01

N-glycosylation of proteins provides a rich source of information on liver disease progression because majority of serum glycoproteins, with the exception of immunoglobulins, are secreted by the liver. In this report, we present results of an optimized workflow for MALDI-TOF analysis of permethylated N-glycans detached from serum proteins and separated into liver secreted and immunoglobulin fractions. We have compared relative intensities of N-glycans in 23 healthy controls and 23 cirrhosis patients. We were able to detect 82 N-glycans associated primarily with liver secreted glycoproteins, 54 N-glycans in the protein G bound fraction and 52 N-glycans in the fraction bound to protein A. The N-glycan composition of the fractions differed substantially, independent of liver disease. The relative abundance of approximately 53% N-glycans in all fractions was significantly altered in the cirrhotic liver. The removal of immunoglobulins allowed detection of an increase in a series of high mannose and hybrid N-glycans associated with the liver secreted protein fraction. PMID:22326963

The Coxiella Burnetii type IVB secretion system (T4BSS) component DotA is released/secreted during infection of host cells and during in vitro growth in a T4BSS-dependent manner.

PubMed

Luedtke, Brandon E; Mahapatra, Saugata; Lutter, Erika I; Shaw, Edward I

2017-06-01

Coxiella burnetii is a Gram-negative intracellular pathogen and is the causative agent of the zoonotic disease Q fever. To cause disease, C. burnetii requires a functional type IVB secretion system (T4BSS) to transfer effector proteins required for the establishment and maintenance of a membrane-bound parasitophorous vacuole (PV) and further modulation of host cell process. However, it is not clear how the T4BSS interacts with the PV membrane since neither a secretion pilus nor an extracellular pore forming apparatus has not been described. To address this, we used the acidified citrate cysteine medium (ACCM) along with cell culture infection and immunological techniques to identify the cellular and extracellular localization of T4BSS components. Interestingly, we found that DotA and IcmX were secreted/released in a T4BSS-dependent manner into the ACCM. Analysis of C. burnetii-infected cell lines revealed that DotA colocalized with the host cell marker CD63 (LAMP3) at the PV membrane. In the absence of bacterial protein synthesis, DotA also became depleted from the PV membrane. These data are the first to identify the release/secretion of C. burnetii T4BSS components during axenic growth and the interaction of a T4BSS component with the PV membrane during infection of host cells. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells.

PubMed

Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A; Schmid, Michael C; Schröder, Gunnar; Vergunst, Annette C; Carena, Ilaria; Dehio, Christoph

2005-01-18

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells.

Yarrowia lipolytica vesicle-mediated protein transport pathways

PubMed Central

Swennen, Dominique; Beckerich, Jean-Marie

2007-01-01

Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii). These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO) encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular transport shows that 40% of Y. lipolytica proteins are closer to animal ones, whereas they are only 13% in the case of S. cerevisiae. Conclusion These results provide further support for the idea, previously noted about the endoplasmic reticulum translocation pathway, that Y. lipolytica is more representative of vesicular secretion of animals and other fungi than is S. cerevisiae. PMID:17997821

The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

PubMed Central

Benjak, Andrej; Uplekar, Swapna; Rougemont, Jacques; Guilhot, Christophe; Malaga, Wladimir; Martín, Carlos; Cole, Stewart T.

2014-01-01

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. PMID:24874799

The Rab11 Effector Protein FIP1 Regulates Adiponectin Trafficking and Secretion

PubMed Central

Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

2013-01-01

Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321

Investment in secreted enzymes during nutrient-limited growth is utility dependent.

PubMed

Cezairliyan, Brent; Ausubel, Frederick M

2017-09-12

Pathogenic bacteria secrete toxins and degradative enzymes that facilitate their growth by liberating nutrients from the environment. To understand bacterial growth under nutrient-limited conditions, we studied resource allocation between cellular and secreted components by the pathogenic bacterium Pseudomonas aeruginosa during growth on a protein substrate that requires extracellular digestion by secreted proteases. We identified a quantitative relationship between the rate of increase of cellular biomass under nutrient-limiting growth conditions and the rate of increase in investment in secreted proteases. Production of secreted proteases is stimulated by secreted signals that convey information about the utility of secreted proteins during nutrient-limited growth. Growth modeling using this relationship recapitulated the observed kinetics of bacterial growth on a protein substrate. The proposed regulatory strategy suggests a rationale for quorum-sensing-dependent stimulation of the production of secreted enzymes whereby investment in secreted enzymes occurs in proportion to the utility they confer. Our model provides a framework that can be applied toward understanding bacterial growth in many environments where growth rate is limited by the availability of nutrients.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

PubMed

Skoczinski, Pia; Volkenborn, Kristina; Fulton, Alexander; Bhadauriya, Anuseema; Nutschel, Christina; Gohlke, Holger; Knapp, Andreas; Jaeger, Karl-Erich

2017-09-25

Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.

PubMed

Jones, J T; Reavy, B; Smant, G; Prior, A E

2004-01-07

We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.

Human Rhinovirus 16 Causes Golgi Apparatus Fragmentation without Blocking Protein Secretion

PubMed Central

Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V.; Walton, Ross; Johnston, Sebastian L.

2014-01-01

ABSTRACT The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins. PMID:25100828

Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria.

PubMed

Majewski, Dorothy D; Worrall, Liam J; Strynadka, Natalie Cj

2018-03-23

The acquisition and evolution of customized and often highly complex secretion systems allows Gram-negative bacteria to efficiently passage large macromolecules across both inner and outer membranes and, in some cases, that of the infected host. Essential to the virulence and ultimate survival of the many pathogenic species that encode them, secretion systems export a wide variety of effector proteins and DNA as well as the downstream extracellular filaments of the secretion apparatus themselves. Although these customized secretion systems differ in their cytosolic and inner membrane components, several commonly rely on the secretin family of giant pores to allow these large substrates to traverse the outer membrane. Recently, several near-atomic resolution cryo-EM secretin structures have unveiled the first insights into the unique structural motifs required for outer membrane localization, assembly, hallmark ultrastable nature, spontaneous membrane insertion, and mechanism of action-including the requisite central gating needed to prevent deleterious passage of periplasmic contents to the extracellular space. Copyright © 2018. Published by Elsevier Ltd.

Single-cell analysis and sorting using droplet-based microfluidics.

PubMed

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2013-05-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

Single-cell analysis and sorting using droplet-based microfluidics

PubMed Central

Mazutis, Linas; Gilbert, John; Ung, W Lloyd; Weitz, David A; Griffiths, Andrew D; Heyman, John A

2014-01-01

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. as an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. the beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ~200 Hz as well as cell enrichment. the microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ~1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d. PMID:23558786

From Cytosol to the Apoplast: The Hygromycin Phosphotransferase (HYG(R)) Model in Arabidopsis.

PubMed

Zhang, Haiyan; Li, Jinjin

2016-01-01

The process by which proteins are secreted via endoplasmic reticulum (ER)/Golgi-independent mechanism is conveniently called unconventional protein secretion. Recent studies have revealed that unconventional protein secretion operates in plants, but little is known about its underlying mechanism and function. This chapter provides methods we have used to analyze unconventional character of hygromycin phosphotransferase (HYG(R)) secretion in plant cells. Following isolation of protoplasts from HYG (R) -GFP-transgenic plants and incubation with brefeldin A (BFA), an inhibitor of conventional secretory pathway, we easily obtain protein extracts from protoplasts and culture medium separately. These proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis with anti-GFP antibodies.

The Role of Prokineticins in the Pathogenesis of Hypogonadotropic Hypogonadism

PubMed Central

Abreu, Ana Paula; Kaiser, Ursula B.; Latronico, Ana Claudia

2010-01-01

The prokineticin system comprises two multifunctional secreted proteins, prokineticin-1 (PROK1) and prokineticin-2 (PROK2), and their cognate G protein-coupled receptors. The prokineticins were originally identified as endogenous regulators of gastrointestinal motility. Currently, these bioactive peptides are involved in a wide spectrum of biological functions, including angiogenesis, neurogenesis, circadian rhythms, nociception, hematopoiesis and immune response. Mice homozygous for null mutations in Prokr2 or Prok2 recapitulate the human phenotype of Kallmann syndrome, exhibiting severe atrophy of the reproductive system and hypoplastic olfactory bulbs. Indeed, the evidence from several naturally inactivating mutations in the PROK2 and PROKR2 genes in patients with Kallmann syndrome and normosmic hypogonadotropic hypogonadism also indicate the essential role of PROK2 in olfactory bulb morphogenesis and GnRH secretion in humans. PMID:20502053

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus.

PubMed

Akeda, Yukihiro; Okayama, Kanna; Kimura, Tomomi; Dryselius, Rikard; Kodama, Toshio; Oishi, Kazunori; Iida, Tetsuya; Honda, Takeshi

2009-07-01

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus. In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30-100 amino acids and an amino terminal secretion signal encompassing the first 5-20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.

Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner.

PubMed

Kroukamp, Heinrich; den Haan, Riaan; la Grange, Daniël C; Sibanda, Ntsako; Foulquié-Moreno, Maria R; Thevelein, Johan M; van Zyl, Willem H

2017-10-01

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

PubMed

Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

2012-01-10

The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Antisense RNA to the first N-glycosylation gene, ALG7, inhibits protein N-glycosylation and secretion by Xenopus oocytes.

PubMed

Kukuruzinska, M A; Apekin, V; Lamkin, M S; Hiltz, A; Rodriguez, A; Lin, C C; Paz, M A; Oppenheim, F G

1994-02-15

N-Glycosylation has been shown to affect the rate of glycoprotein transport through the secretory pathway. In order to identify the critical components in the N-glycosylation pathway that directly influence protein secretion, we have studied the effects of downregulation of the first gene in the dolichol pathway, ALG7, on the synthesis, glycosylation and secretion of native and heterologous proteins by Xenopus laevis oocytes. Our strategy involved the use of ALG7 antisense RNA (asRNA) to lower the effective abundance of the ALG7 protein in oocytes. The results showed that there was an inverse dose-response relationship between ALG7 asRNA and the amount of glycosylated and secreted proteins. These effects were also observed for heterologously expressed rat parotid amylase. Since ALG7 asRNA did not inhibit overall protein synthesis, we conclude that downregulation of ALG7 expression directly lowered protein export.

The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms.

PubMed

Fry, Bryan G; Roelants, Kim; Champagne, Donald E; Scheib, Holger; Tyndall, Joel D A; King, Glenn F; Nevalainen, Timo J; Norman, Janette A; Lewis, Richard J; Norton, Raymond S; Renjifo, Camila; de la Vega, Ricardo C Rodríguez

2009-01-01

Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase/prokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A(2), sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., vampire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, P.; Whitwam, R.E.; Tien, Ming

1996-03-01

A manganese peroxidase (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3{prime} untranslated region of the glucoamylase gene of Asperigillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies. 34 refs., 7 figs., 1 tab.

Elucidating the Mechanism of Gain of Toxic Function from Mutant C1 Inhibitor Proteins in Hereditary Angioedema

DTIC Science & Technology

2016-10-01

this is due, at least in part, to an additional acquired GOTF defect caused by the mutant protein that interferes with the secretion of WT C1INH. Our...overall hypothesis is that mutant C1INH proteins exert a variable GOTF phenotype that inhibit secretion of WT C1INH protein and worsen disease...will assess the mechanisms of the GOTF with a hypothesis that misfolding of mutant C1INH protein in the ER causes impairment of WT C1INH secretion

High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr

DOE PAGES

Reeve, Wayne; van Berkum, Peter; Ardley, Julie; ...

2017-03-04

Bradyrhizobium elkanii USDA 76 T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76 T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76 T are described, together with its genome sequence information and annotation. The 9,484,767 bpmore » high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76 T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76 T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76 T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76 T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76 T to introduce various effector molecules into this host to enable nodulation.« less

High-quality permanent draft genome sequence of the Bradyrhizobium elkanii type strain USDA 76T, isolated from Glycine max (L.) Merr

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reeve, Wayne; van Berkum, Peter; Ardley, Julie

Bradyrhizobium elkanii USDA 76 T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76 T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76 T are described, together with its genome sequence information and annotation. The 9,484,767 bpmore » high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76 T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76 T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76 T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76 T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76 T to introduce various effector molecules into this host to enable nodulation.« less

Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

PubMed

Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

2009-07-01

The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

A Catalog of Proteins Expressed in the AG Secreted Fluid during the Mature Phase of the Chinese Mitten Crabs (Eriocheir sinensis)

PubMed Central

He, Lin; Li, Qing; Liu, Lihua; Wang, Yuanli; Xie, Jing; Yang, Hongdan; Wang, Qun

2015-01-01

The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database (http://www.ncbi.nlm.nih.gov/) and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction. PMID:26305468

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

PubMed Central

Amyot, Whitney M.; deJesus, Dennise

2013-01-01

Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536

The effect of guar gum on carbohydrate-, fat- and protein-stimulated gut hormone secretion: modification of postprandial gastric inhibitory polypeptide and gastrin responses.

PubMed

Morgan, L M; Tredger, J A; Madden, A; Kwasowski, P; Marks, V

1985-05-01

The effect of incorporating guar gum into predominantly single-component meals of carbohydrate, fat or protein on liquid gastric emptying and on the secretion of gastric inhibitory polypeptide (GIP), gastrin and motilin, was studied in healthy human volunteers. Volunteers were given either 80 ml Hycal (carbohydrate meal), 150 g cooked lean minced beef (protein meal) or 200 ml double cream (fat meal) either with or without 5 or 6 g guar gum. Liquid gastric emptying was monitored in the fat and protein meals by taking 1.5 g paracetamol, consumed in water, with the meals and monitoring its appearance in circulation. Postprandial insulin and GIP levels were both significantly reduced by addition of guar gum to the carbohydrate meal. Postprandial GIP secretion was also reduced by addition of guar gum to the protein meal, but protein-stimulated gastrin secretion was enhanced by guar gum. There was a significant negative correlation between peak circulating gastrin levels and the corresponding GIP levels. Postprandial GIP secretion and plasma motilin levels were unaffected by addition of guar gum to the fat meal. 5 and 10 g guar gum/l solutions in water possessed buffering capacities between pH 2.75 and 5.5. Guar gum at 5 g/l caused no detectable change in liquid gastric-emptying time. The observed augmentation of gastrin secretion by guar gum following a protein meal could be due either to the buffering capacity of guar gum or to the attenuation of GIP secretion. It is possible that the chronic use of guar gum could be associated with changes in gastric acid secretion.

Tools for controlling protein interactions with light

PubMed Central

Tucker, Chandra L.; Vrana, Justin D.; Kennedy, Matthew J.

2014-01-01

Genetically-encoded actuators that allow control of protein-protein interactions with light, termed ‘optical dimerizers’, are emerging as new tools for experimental biology. In recent years, numerous new and versatile dimerizer systems have been developed. Here we discuss the design of optical dimerizer experiments, including choice of a dimerizer system, photoexcitation sources, and coordinate use of imaging reporters. We provide detailed protocols for experiments using two dimerization systems we previously developed, CRY2/CIB and UVR8/UVR8, for use controlling transcription, protein localization, and protein secretion with light. Additionally, we provide instructions and software for constructing a pulse-controlled LED light device for use in experiments requiring extended light treatments. PMID:25181301

Identification of the Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the T3SS2-specific effector VopC.

PubMed

Akeda, Yukihiro; Kodama, Toshio; Saito, Kazunobu; Iida, Tetsuya; Oishi, Kazunori; Honda, Takeshi

2011-11-01

The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Protein-restriction diet during the suckling phase programs rat metabolism against obesity and insulin resistance exacerbation induced by a high-fat diet in adulthood.

PubMed

Martins, Isabela Peixoto; de Oliveira, Júlio Cezar; Pavanello, Audrei; Matiusso, Camila Cristina Ianoni; Previate, Carina; Tófolo, Laize Peron; Ribeiro, Tatiane Aparecida; da Silva Franco, Claudinéia Conationi; Miranda, Rosiane Aparecida; Prates, Kelly Valério; Alves, Vander Silva; Francisco, Flávio Andrade; de Moraes, Ana Maria Praxedes; de Freitas Mathias, Paulo Cezar; Malta, Ananda

2018-04-03

Protein restriction during the suckling phase can malprogram rat offspring to a lean phenotype associated with metabolic dysfunctions later in life. We tested whether protein-caloric restriction during lactation can exacerbate the effect of a high-fat (HF) diet at adulthood. To test this hypothesis, we fed lactating Wistar dams with a low-protein (LP; 4% protein) diet during the first 2 weeks of lactation or a normal-protein (NP; 23% protein) diet throughout lactation. Rat offspring from NP and LP mothers received a normal-protein diet until 60 days old. At this time, a batch of animals from both groups was fed an HF (35% fat) diet, while another received an NF (7% fat) diet. Maternal protein-caloric restriction provoked lower body weight and fat pad stores, hypoinsulinemia, glucose intolerance, higher insulin sensitivity, reduced insulin secretion and altered autonomic nervous system (ANS) function in adult rat offspring. At 90 days old, NP rats fed an HF diet in adulthood displayed obesity, impaired glucose homeostasis and altered insulin secretion and ANS activity. Interestingly, the LP/HF group also presented fat pad and body weight gain, altered glucose homeostasis, hyperleptinemia and impaired insulin secretion but at a smaller magnitude than the NP-HF group. In addition, LP/HF rats displayed elevated insulin sensitivity. We concluded that protein-caloric restriction during the first 14 days of life programs the rat metabolism against obesity and insulin resistance exacerbation induced by an obesogenic HF diet. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteomic profiling of the planarian Schmidtea mediterranea and its mucous reveals similarities with human secretions and those predicted for parasitic flatworms.

PubMed

Bocchinfuso, Donald G; Taylor, Paul; Ross, Eric; Ignatchenko, Alex; Ignatchenko, Vladimir; Kislinger, Thomas; Pearson, Bret J; Moran, Michael F

2012-09-01

The freshwater planarian Schmidtea mediterranea has been used in research for over 100 years, and is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. Exteriorly, planarians are covered in mucous secretions of unknown composition, implicated in locomotion, predation, innate immunity, and substrate adhesion. Although the planarian genome has been sequenced, it remains mostly unannotated, challenging both genomic and proteomic analyses. The goal of the current study was to annotate the proteome of the whole planarian and its mucous fraction. The S. mediterranea proteome was analyzed via mass spectrometry by using multidimensional protein identification technology with whole-worm tryptic digests. By using a proteogenomics approach, MS data were searched against an in silico translated planarian transcript database, and by using the Swiss-Prot BLAST algorithm to identify proteins similar to planarian queries. A total of 1604 proteins were identified. The mucous subproteome was defined through analysis of a mucous trail fraction and an extract obtained by treating whole worms with the mucolytic agent N-acetylcysteine. Gene Ontology analysis confirmed that the mucous fractions were enriched with secreted proteins. The S. mediterranea proteome is highly similar to that predicted for the trematode Schistosoma mansoni associated with intestinal schistosomiasis, with the mucous subproteome particularly highly conserved. Remarkably, orthologs of 119 planarian mucous proteins are present in human mucosal secretions and tear fluid. We suggest planarians have potential to be a model system for the characterization of mucous protein function and relevant to parasitic flatworm infections and diseases underlined by mucous aberrancies, such as cystic fibrosis, asthma, and other lung diseases.

Protein Biosynthesis and Maturation in the ER.

PubMed

Pedrazzini, Emanuela; Vitale, Alessandro

2018-01-01

The endoplasmic reticulum takes care of the folding, assembly, and quality control of thousands of proteins destined to the different compartments of the endomembrane system, or to be secreted in the apoplast. Here we describe how these early events in the life of all these proteins can be followed biochemically by using velocity or isopycnic ultracentrifugation, metabolic labeling with radioactive amino acids, and immunoprecipitation in various conditions.

A novel marine silk.

PubMed

Kronenberger, Katrin; Dicko, Cedric; Vollrath, Fritz

2012-01-01

The discovery of a novel silk production system in a marine amphipod provides insights into the wider potential of natural silks. The tube-building corophioid amphipod Crassicorophium bonellii produces from its legs fibrous, adhesive underwater threads that combine barnacle cement biology with aspects of spider silk thread extrusion spinning. We characterised the filamentous silk as a mixture of mucopolysaccharides and protein deriving from glands representing two distinct types. The carbohydrate and protein silk secretion is dominated by complex β-sheet structures and a high content of charged amino acid residues. The filamentous secretion product exits the gland through a pore near the tip of the secretory leg after having moved through a duct, which subdivides into several small ductules all terminating in a spindle-shaped chamber. This chamber communicates with the exterior and may be considered the silk reservoir and processing/mixing space, in which the silk is mechanically and potentially chemically altered and becomes fibrous. We assert that further study of this probably independently evolved, marine arthropod silk processing and secretion system can provide not only important insights into the more complex arachnid and insect silks but also into crustacean adhesion cements.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.

Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression hostmore » offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.« less

Structure of the cytoplasmic domain of Yersinia pestis YscD, an essential component of the type III secretion system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

2012-09-17

The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are requiredmore » for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.« less

Secretomes reveal several novel proteins as well as TGF-β1 as the top upstream regulator of metastatic process in breast cancer.

PubMed

Erin, Nuray; Ogan, Nur; Yerlikaya, Azmi

2018-03-20

Metastatic breast cancer is resistant to many conventional treatments and novel therapeutic targets are needed. We previously isolated subsets of 4T1 murine breast cancer cells which metastasized to liver (4TLM), brain (4TBM), and heart (4THM). Among these cells, 4TLM is the most aggressive one, demonstrating mesenchymal phenotype. Here we compared secreted proteins from 4TLM, 4TBM, and 4THM cells and compared with that of hardly metastatic 67NR cells to detect differentially secreted factors involved in organ-specific metastasis. Label-free LC-MS/MS proteomic technique was used to detect the differentially secreted proteins. Eighty-five of over 500 secreted proteins were significantly altered in metastatic breast cancer cells. Differential expression of several proteins such as fibulin-4, Bone Morphogenetic Protein 1, TGF-β1 MMP-3, MMP-9, and Thymic Stromal Lymphopoietin were further verified using ELISA or Western blotting. Many of these identified proteins were also present in human metastatic breast carcinomas. Annexin A1 and A5, laminin beta 1, Neutral alpha-glucosidase AB were commonly found at least in three out of six studies examined here. Ingenuity Pathway Analysis showed that proteins differentially secreted from metastatic cells are involved primarily in carcinogenesis and TGF-β1 is the top upstream regulator in all metastatic cells. Cells metastasized to different organs displayed significant differences in several of secreted proteins. Proteins differentially altered were fibronectin, insulin-like growth factor-binding protein 7, and Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. On the other hand, many exosomal proteins were also common to all metastatic cells, demonstrating involvement of key universal factors in distant metastatic process.

Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein.

PubMed

Wartenberg, Dirk; Lapp, Katrin; Jacobsen, Ilse D; Dahse, Hans-Martin; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

2011-11-01

Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive analysis of secreted proteins of A. fumigatus and a detailed characterization of hemolysin mutants. Copyright © 2011 Elsevier GmbH. All rights reserved.

Small ubiquitin-related modifier is secreted and shows cytokine-like activity.

PubMed

Hosono, Hidetaka; Yokosawa, Hideyoshi

2008-05-01

Small ubiquitin-related modifier (SUMO) is a type I ubiquitin-like protein family member and is covalently attached to various target proteins. Through this post-translational modification, SUMO plays important roles in various cellular events. Here, we show that SUMO is secreted from cultured cells in an endoplasmic reticulum (ER)/Golgi-independent manner and that this secretion occurs without covalent binding to target proteins or chain formation. Overexpression experiments using C-terminally truncated mutants of SUMO revealed that the secretion requires the C-terminal sequence. Recombinant SUMO-3 protein was capable of binding to and promoting the proliferation of cultured cells. Thus, we propose that SUMO functions as a cytokine-like molecule extracellularly.

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate.

PubMed

Thomas, Sabrina; Bakkes, Patrick J; Smits, Sander H J; Schmitt, Lutz

2014-09-01

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca(2+) binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca(2+) is essential for stability as well as correct folding of the whole protein. In the absence of Ca(2+), pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca(2+) addition, indicating that these are not dead-end species and that Ca(2+) drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6-7M urea, relates to Ca(2+) dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca(2+) induced folding is not restricted to the RTX domain. Taken together, Ca(2+) binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-gamma production in suspension and inside microcarriers in protein-free media.

PubMed

Ballez, J S; Mols, J; Burteau, C; Agathos, S N; Schneider, Y J

2004-03-01

We have recently developed a protein-free medium (PFS) able to support the growth of Chinese hamster ovary (CHO) cells in suspension. Upon further supplementation with some plant protein hydrolysates, medium performances reached what could be observed in serum-containing media [Burteau et al. In Vitro Cell. Dev. Biol.-Anim. 39 (2003) 291]. Now, we describe the use of rice and wheat protein hydrolysates, as non-nutritional additives to the culture medium to support productivity and cell growth in suspension or in microcarriers. When CHO-320 cells secreting recombinant interferon-gamma (IFN-gamma) were cultivated in suspension in a bioreactor with our PFS supplemented with wheat hydrolysates, the maximum cell density increased by 25% and the IFN-gamma secretion by 60% compared to the control PFS. A small-scale perfusion system consisting of CHO-320 cells growing on and inside fibrous microcarriers under discontinuous operation was first developed. Under these conditions, rice protein hydrolysates stimulated recombinant IFN-gamma secretion by 30% compared to the control PFS. At the bioreactorscale, similar results were obtained but when compared to shake-flasks studies, nutrients, oxygen or toxic by-products gradients inside the microcarriers seemed to be the main limitation of the system. An increase of the perfusion rate to maintain glucose concentration over 5.5 mM and dissolved oxygen (DO) at 60% was able to stimulate the production of IFN-gamma to a level of 6.6 mug h(-1) g(-1) of microcarriers after 160 h when a cellular density of about 4 x 10(8) cell g(-1) of carriers was reached.

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters.

PubMed

Eom, Gyeong Tae; Lee, Seung Hwan; Oh, Young Hoon; Choi, Ji Eun; Park, Si Jae; Song, Jae Kwang

2014-10-01

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml(-1)) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.

The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms.

PubMed

Lin, Wei-Jye; Salton, Stephen R

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

The Regulated Secretory Pathway and Human Disease: Insights from Gene Variants and Single Nucleotide Polymorphisms

PubMed Central

Lin, Wei-Jye; Salton, Stephen R.

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired. PMID:23964269

Small-Molecule Type III Secretion System Inhibitors Block Assembly of the Shigella Type III Secreton▿ †

PubMed Central

Veenendaal, Andreas K. J.; Sundin, Charlotta; Blocker, Ariel J.

2009-01-01

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds. PMID:18996990

The Legionella IcmSW Complex Directly Interacts with DotL to Mediate Translocation of Adaptor-Dependent Substrates

PubMed Central

Sutherland, Molly C.; Nguyen, Thuy Linh; Tseng, Victor; Vogel, Joseph P.

2012-01-01

Legionella pneumophila is a Gram-negative bacterium that replicates within human alveolar macrophages by evasion of the host endocytic pathway through the formation of a replicative vacuole. Generation of this vacuole is dependent upon the secretion of over 275 effector proteins into the host cell via the Dot/Icm type IVB secretion system (T4SS). The type IV coupling protein (T4CP) subcomplex, consisting of DotL, DotM, DotN, IcmS and IcmW, was recently defined. DotL is proposed to be the T4CP of the L. pneumophila T4SS based on its homology to known T4CPs, which function as inner-membrane receptors for substrates. As a result, DotL is hypothesized to play an integral role(s) in the L. pneumophila T4SS for the engagement and translocation of substrates. To elucidate this role, a genetic approach was taken to screen for dotL mutants that were unable to survive inside host cells. One mutant, dotLY725Stop, did not interact with the type IV adaptor proteins IcmS/IcmW (IcmSW) leading to the identification of an IcmSW-binding domain on DotL. Interestingly, the dotLY725Stop mutant was competent for export of one class of secreted effectors, the IcmSW-independent substrates, but exhibited a specific defect in secretion of IcmSW-dependent substrates. This differential secretion illustrates that DotL requires a direct interaction with the type IV adaptor proteins for the secretion of a major class of substrates. Thus, by identifying a new target for IcmSW, we have discovered that the type IV adaptors perform an additional role in the export of substrates by the L. pneumophila Dot/Icm T4SS. PMID:23028312

Biophysical Characterization of the Type III Secretion Tip Proteins and the Tip Proteins Attached to Bacterium-Like Particles

PubMed Central

Choudhari, Shyamal P.; Chen, Xiaotong; Kim, Jae Hyun; van Roosmalen, Maarten L.; Greenwood, Jamie C.; Joshi, Sangeeta B.; Picking, William D.; Leenhouts, Kees; Middaugh, C. Russell; Picking, Wendy L.

2014-01-01

Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring domain (PA). In this study, the tip proteins IpaD, SipD and LcrV belonging to type three secretion systems of Shigella flexneri, Salmonella enterica and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation. PMID:24916512

Identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity

PubMed Central

Wu, Lindsay E.; Samocha-Bonet, Dorit; Whitworth, P. Tess; Fazakerley, Daniel J.; Turner, Nigel; Biden, Trevor J.; James, David E.; Cantley, James

2014-01-01

A critical feature of obesity is enhanced insulin secretion from pancreatic β-cells, enabling the majority of individuals to maintain glycaemic control despite adiposity and insulin resistance. Surprisingly, the factors coordinating this adaptive β-cell response with adiposity have not been delineated. Here we show that fatty acid binding protein 4 (FABP4/aP2) is an adipokine released from adipocytes under obesogenic conditions, such as hypoxia, to augment insulin secretion. The insulinotropic action of FABP4 was identified using an in vitro system that recapitulates adipocyte to β-cell endocrine signalling, with glucose-stimulated insulin secretion (GSIS) as a functional readout, coupled with quantitative proteomics. Exogenous FABP4 potentiated GSIS in vitro and in vivo, and circulating FABP4 levels correlated with GSIS in humans. Insulin inhibited FABP4 release from adipocytes in vitro, in mice and in humans, consistent with feedback regulation. These data suggest that FABP4 and insulin form an endocrine loop coordinating the β-cell response to obesity. PMID:24944906

The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

PubMed

Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph

2017-01-03

The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

PubMed

Pillai, Viju Vijayan; Weber, Darren M; Phinney, Brett S; Selvaraj, Vimal

2017-01-01

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

The secretory pathway: exploring yeast diversity.

PubMed

Delic, Marizela; Valli, Minoska; Graf, Alexandra B; Pfeffer, Martin; Mattanovich, Diethard; Gasser, Brigitte

2013-11-01

Protein secretion is an essential process for living organisms. In eukaryotes, this encompasses numerous steps mediated by several hundred cellular proteins. The core functions of translocation through the endoplasmic reticulum membrane, primary glycosylation, folding and quality control, and vesicle-mediated secretion are similar from yeasts to higher eukaryotes. However, recent research has revealed significant functional differences between yeasts and mammalian cells, and even among diverse yeast species. This review provides a current overview of the canonical protein secretion pathway in the model yeast Saccharomyces cerevisiae, highlighting differences to mammalian cells as well as currently unresolved questions, and provides a genomic comparison of the S. cerevisiae pathway to seven other yeast species where secretion has been investigated due to their attraction as protein production platforms, or for their relevance as pathogens. The analysis of Candida albicans, Candida glabrata, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Schizosaccharomyces pombe reveals that many - but not all - secretion steps are more redundant in S. cerevisiae due to duplicated genes, while some processes are even absent in this model yeast. Recent research obviates that even where homologous genes are present, small differences in protein sequence and/or differences in the regulation of gene expression may lead to quite different protein secretion phenotypes. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Label-free detection of protein biomolecules secreted from a heart-on-a-chip model for drug cardiotoxicity evaluation

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Zhang, Yu Shrike; Bustamante, Gilbert; Li, Le; Lauderdale, Matthew; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

2018-02-01

Efficient methods for the accurate analysis of drug toxicities are in urgent demand as failures of newly discovered drug candidates due to toxic side effects have resulted in about 30% of clinical attrition. The high failure rate is partly due to current inadequate models to study drug side effects, i.e., common animal models may fail due to its misrepresentation of human physiology. Therefore, much effort has been allocated in the development of organ-on-a-chip models which offer a variety of human organ models mimicking a multitude of human physiological conditions. However, it is extremely challenging to analyze the transient and long-term response of the organ models to drug treatments during drug toxicity tests, as the proteins secreted from the organ-on-a-chip model are minute due to its volumetric size, and current methods for detecting said biomolecules are not suitable for real-time monitoring. As protein biomolecules are being continuously secreted from the human organ model, fluorescence techniques are practically impossible to achieve real-time fluorescence labeling in the dynamically changing environment, thus making a label-free approach highly desirable for the organ-on-achip applications. In this paper, we report the use of a photonic-crystal biosensor integrated with a microfluidic system for sensitive label-free bioassays of secreted protein biomolecules from a heart-on-the-chip model created with cardiomyocytes derived from human induced pluripotent stem cells.

Immunomodulatory molecules are released from the first trimester and term placenta via exosomes

PubMed Central

Kshirsagar, S.K.; Alam, S.M.; Jasti, S.; Hodes, H.; Nauser, T.; Gilliam, M.; Billstrand, C.; Hunt, J.S.; Petroff, M.G.

2012-01-01

The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular colocalization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 hours. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment. PMID:23107341

Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

PubMed Central

2012-01-01

Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins. PMID:22234238

Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

PubMed

Staats, Charley Christian; Junges, Angela; Guedes, Rafael Lucas Muniz; Thompson, Claudia Elizabeth; de Morais, Guilherme Loss; Boldo, Juliano Tomazzoni; de Almeida, Luiz Gonzaga Paula; Andreis, Fábio Carrer; Gerber, Alexandra Lehmkuhl; Sbaraini, Nicolau; da Paixão, Rana Louise de Andrade; Broetto, Leonardo; Landell, Melissa; Santi, Lucélia; Beys-da-Silva, Walter Orlando; Silveira, Carolina Pereira; Serrano, Thaiane Rispoli; de Oliveira, Eder Silva; Kmetzsch, Lívia; Vainstein, Marilene Henning; de Vasconcelos, Ana Tereza Ribeiro; Schrank, Augusto

2014-09-29

Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

The Small GTPase MoSec4 Is Involved in Vegetative Development and Pathogenicity by Regulating the Extracellular Protein Secretion in Magnaporthe oryzae

PubMed Central

Zheng, Huakun; Chen, Simiao; Chen, Xiaofeng; Liu, Shuyan; Dang, Xie; Yang, Chengdong; Giraldo, Martha C.; Oliveira-Garcia, Ely; Zhou, Jie; Wang, Zonghua; Valent, Barbara

2016-01-01

The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in Magnaporthe oryzae. To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in M. oryzae, namely MoSEC4. The ΔMosec4 mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the ΔMosec4 mutant. Further studies showed that deletion of the MoSEC4 gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with a role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus. PMID:27729922

Functions of KLK4 and MMP-20 in dental enamel formation

PubMed Central

Lu, Yuhe; Papagerakis, Petros; Yamakoshi, Yasuo; Hu, Jan C-C.; Bartlett, John D.; Simmer, James P.

2009-01-01

Two proteases are secreted into the enamel matrix of developing teeth. The early protease is enamelysin (MMP-20). The late protease is kallikrein 4 (KLK4). Mutations in MMP20 and KLK4 both cause autosomal recessive amelogenesis imperfecta, a condition featuring soft, porous enamel containing residual protein. MMP-20 is secreted along with enamel proteins by secretory stage ameloblasts. Enamel protein cleavage products accumulate in the space between the crystal ribbons, helping to support them. MMP-20 steadily cleaves accumulated enamel proteins, so their concentration decreases with depth. Kallikrein 4 is secreted by transition and maturation stage ameloblasts. KLK4 aggressively degrades the retained organic matrix following the termination of enamel protein secretion. The principle functions of MMP-20 and KLK4 in dental enamel formation are to facilitate the orderly replacement of organic matrix with mineral, generating an enamel layer that is harder, less porous, and unstained by retained enamel proteins. PMID:18627287

Quantitative visualization of synchronized insulin secretion from 3D-cultured cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Takahiro; Kanamori, Takao; Inouye, Satoshi

Quantitative visualization of synchronized insulin secretion was performed in an isolated rat pancreatic islet and a spheroid of rat pancreatic beta cell line using a method of video-rate bioluminescence imaging. Video-rate images of insulin secretion from 3D-cultured cells were obtained by expressing the fusion protein of insulin and Gaussia luciferase (Insulin-GLase). A subclonal rat INS-1E cell line stably expressing Insulin-GLase, named iGL, was established and a cluster of iGL cells showed oscillatory insulin secretion that was completely synchronized in response to high glucose. Furthermore, we demonstrated the effect of an antidiabetic drug, glibenclamide, on synchronized insulin secretion from 2D- andmore » 3D-cultured iGL cells. The amount of secreted Insulin-GLase from iGL cells was also determined by a luminometer. Thus, our bioluminescence imaging method could generally be used for investigating protein secretion from living 3D-cultured cells. In addition, iGL cell line would be valuable for evaluating antidiabetic drugs. - Highlights: • An imaging method for protein secretion from 3D-cultured cells was established. • The fused protein of insulin to GLase, Insulin-GLase, was used as a reporter. • Synchronous insulin secretion was visualized in rat islets and spheroidal beta cells. • A rat beta cell line stably expressing Insulin-GLase, named iGL, was established. • Effect of an antidiabetic drug on insulin secretion was visualized in iGL cells.« less

Delivery of cytoplasmic and apoplastic effectors from Phytophthora infestans haustoria by distinct secretion pathways.

PubMed

Wang, Shumei; Boevink, Petra C; Welsh, Lydia; Zhang, Ruofang; Whisson, Stephen C; Birch, Paul R J

2017-10-01

The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease. We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection. The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells. Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1

PubMed Central

Salomon, Dor; Klimko, John A.

2014-01-01

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity. PMID:24987102

Influence of Flavonoids on Mechanism of Modulation of Insulin Secretion.

PubMed

Soares, Juliana Mikaelly Dias; Pereira Leal, Ana Ediléia Barbosa; Silva, Juliane Cabral; Almeida, Jackson R G S; de Oliveira, Helinando Pequeno

2017-01-01

The development of alternatives for insulin secretion control in vivo or in vitro represents an important aspect to be investigated. In this direction, natural products have been progressively explored with this aim. In particular, flavonoids are potential candidates to act as insulin secretagogue. To study the influence of flavonoid on overall modulation mechanisms of insulin secretion. The research was conducted in the following databases and platforms: PubMed, Scopus, ISI Web of Knowledge, SciELO, LILACS, and ScienceDirect, and the MeSH terms used for the search were flavonoids, flavones, islets of Langerhans, and insulin-secreting cells. Twelve articles were included and represent the basis of discussion on mechanisms of insulin secretion of flavonoids. Papers in ISI Web of Knowledge were in number of 1, Scopus 44, PubMed 264, ScienceDirect 511, and no papers from LILACS and SciELO databases. According to the literature, the majority of flavonoid subclasses can modulate insulin secretion through several pathways, in an indication that corresponding molecule is a potential candidate for active materials to be applied in the treatment of diabetes. The action of natural products on insulin secretion represents an important investigation topic due to their importance in the diabetes controlIn addition to their typical antioxidant properties, flavonoids contribute to the insulin secretionThe modulation of insulin secretion is induced by flavonoids according to different mechanisms. Abbreviations used: K ATP channels: ATP-sensitive K + channels, GLUT4: Glucose transporter 4, ERK1/2: Extracellular signal-regulated protein kinases 1 and 2, L-VDCCs: L-type voltage-dependent Ca +2 channels, GLUT1: Glucose transporter 1, AMPK: Adenosine monophosphate-activated protein kinase, PTP1B: Protein tyrosine phosphatase 1B, GLUT2: Glucose transporter 2, cAMP: Cyclic adenosine monophosphate, PKA: Protein kinase A, PTK: Protein tyrosine kinase, CaMK II: Ca 2+ /calmodulin-dependent protein kinase II, GSIS: Glucose-stimulated insulin secretion, Insig-1: Insulin-induced gene 1, IRS-2: Insulin receptor substrate 2, PDX-1: Pancreatic and duodenal homeobox 1, SREBP-1c: Sterol regulatory element binding protein-1c, DMC: Dihydroxy-6'-methoxy-3',5'-dimethylchalcone, GLP-1: Glucagon-like peptide-1, GLP-1R: Glucagon-like peptide 1 receptor.

Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1β by human fetal membranes in a two-compartment culture system.

PubMed

Osorio-Caballero, M; Perdigón-Palacio, C; García-López, G; Flores-Herrera, O; Olvera-Sánchez, S; Morales-Méndez, I; Sosa-González, I; Acevedo, J F; Guzmán-Grenfell, A M; Molina-Hernández, A; Díaz, N F; Flores-Herrera, H

2015-03-01

Escherichia coli is recognized as an etiological bacteria associated with chorioamnionitis and the preterm premature rupture of fetal membranes. This pathological condition induces pro-inflammatory cytokines and degradative metalloproteinases, which are considered biological markers secreted in an acute stage of infection. Heat-shock proteins (HSPs) are an important component of the innate immunity response and are found in different pathological conditions. They have not been previously measured in human fetal membranes in response to infectious conditions. We hypothesized that the choriodecidual tissue and amniotic epithelium secreted temporal and differential Hsp-60, Hsp-70, and interleukin (IL)-1β mediated by E. coli infection. Fetal membranes were mounted in a two-compartment culture system and infected with two passes of live E. coli at different doses (10², 10⁴, 10⁵, and 10⁶ colony-forming units (CFU)/mL) and intervals of incubation (3, 6, and 24 h). The culture medium was collected, and Hsp-60, Hsp-70, and IL-1β were assessed using the enzyme-linked immunosorbent assay (ELISA) method. After 3 and 6 h of infection, E. coli induced an increase in Hsp-70 secretion in the choriodecidual tissue. However, after 24 h of incubation, Hsp-70 was downregulated and we observed an increase in IL-1β secretion. By contrast, E. coli induced a lower Hsp-60 secretion in the amnion compared to Hsp-70. Human fetal membranes responded actively to E. coli infection, with an increase in Hsp-70 during the first hours of infection. After 24 h, there was an increase in the liberation of IL-1β. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protein Secretion Is Required for Pregnancy-Associated Plasma Protein-A to Promote Lung Cancer Growth In Vivo

PubMed Central

Pan, Hong; Hanada, Sayaka; Zhao, Jun; Mao, Li; Ma, Mark Zhi-Qing

2012-01-01

Pregnancy-associated plasma protein-A (PAPPA) has been reported to regulate the activity of insulin-like growth factor (IGF) signal pathway through proteolytic degradation of IGF binding proteins (IGFBPs) thereby increasing the local concentration of free IGFs available to receptors. In this study we found that PAPPA is secreted from two out of seven lung cancer cell lines examined. None of immortalized normal bronchial epithelial cells (HBE) tested secrets PAPPA. There is no correlation between expression level and secretion of PAPPA in these cells. A cell line over-expressing PAPPA accompanied with secretion shows no notable changes in proliferation under cell culture conditions in vitro, but displays significantly augmentation of tumor growth in vivo in a xenograft model. In contrast, a cell line over-expressing PAPPA without secretion exhibits reduction of tumor growth both in vitro and in vivo. Down-regulation of PAPPA expression and secretion by RNAi knockdown decreases tumor growth after implanted in vivo. The tumor promoting activity of PAPPA appears to be mediated mainly through augmentation of the IGF signaling pathway as indicated by notable increases in downstream Akt kinase phosphorylation in tumor samples. Our results indicate that PAPPA secretion may play an important role in lung cancer growth and progression. PMID:23152806

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

PubMed

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Yersinia pestis Yop secretion protein F: purification, characterization, and protective efficacy against bubonic plague.

PubMed

Swietnicki, Wieslaw; Powell, Bradford S; Goodin, Jeremy

2005-07-01

Yersinia pestis is a gram-negative human pathogen that uses a type III secretion system to deliver virulence factors into human hosts. The delivery is contact-dependent and it has been proposed that polymerization of Yop secretion protein F (YscF) is used to puncture mammalian cell membranes to facilitate delivery of Yersinia outer protein effectors into host cells. To evaluate the potential immunogenicity and protective efficacy of YscF against Y. pestis, we used a purified recombinant YscF protein as a potential vaccine candidate in a mouse subcutaneous infection model. YscF was expressed and purified from Escherichia coli by immobilized metal-ion affinity chromatography and protein identity was confirmed by ion trap mass spectrometry. The recombinant protein was highly alpha-helical and formed relatively stable aggregates under physiological conditions. The properties were consistent with behavior expected for the native YscF, suggesting that the antigen was properly folded. Ten mice were inoculated subcutaneously, administered booster injections after one month, and challenged with 130 LD(50) of wild type Y. pestis CO92. Six animals in the vaccinated group but none in the control group survived the challenge. The vaccinated animals produced high levels of specific antibodies against YscF as determined by Western blot. The data were statistically significant (P = 0.053 by two-tailed Fisher's test), suggesting that the YscF protein can provide a protective immune response against lethal plague challenge during subcutaneous plague infection.

The dynamics of secretion during sea urchin embryonic skeleton formation.

PubMed

Wilt, Fred H; Killian, Christopher E; Hamilton, Patricia; Croker, Lindsay

2008-05-01

Skeleton formation involves secretion of massive amounts of mineral precursor, usually a calcium salt, and matrix proteins, many of which are deposited on, or even occluded within, the mineral. The cell biological underpinnings of this secretion and subsequent assembly of the biomineralized skeletal element is not well understood. We ask here what is the relationship of the trafficking and secretion of the mineral and matrix within the primary mesenchyme cells of the sea urchin embryo, cells that deposit the endoskeletal spicule. Fluorescent labeling of intracellular calcium deposits show mineral precursors are present in granules visible by light microscopy, from whence they are deposited in the endoskeletal spicule, especially at its tip. In contrast, two different matrix proteins tagged with GFP are present in smaller post-Golgi vesicles only seen by electron microscopy, and the secreted protein are only incorporated into the spicule in the vicinity of the cell of origin. The matrix protein, SpSM30B, is post-translationally modified during secretion, and this processing continues after its incorporation into the spicule. Our findings also indicate that the mineral precursor and two well characterized matrix proteins are trafficked by different cellular routes.

The Dynamics of Secretion during Sea Urchin Embryonic Skeleton Formation

PubMed Central

Wilt, Fred H.; Killian, Christopher E.; Hamilton, Patricia; Croker, Lindsay

2008-01-01

Skeleton formation involves secretion of massive amounts of mineral precursor, usually a calcium salt, and matrix proteins, many of which are deposited on, or even occluded within, the mineral. The cell biological underpinnings of this secretion and subsequent assembly of the biomineralized skeletal element is not well understood. We ask here what is the relationship of the trafficking and secretion of the mineral and matrix within the primary mesenchyme cells of the sea urchin embryo, cells that deposit the endoskeletal spicule. Fluorescent labeling of intracellular calcium deposits show mineral precursors are present in granules visible by light microscopy, from whence they are deposited in the endoskeletal spicule, especially at its tip. In contrast, two different matrix proteins tagged with GFP are present in smaller post-Golgi vesicles only seen by electron microscopy, and the secreted protein are only incorporated into the spicule in the vicinity of the cell of origin. The matrix protein, SpSM30B, is post-translationally modified during secretion, and this processing continues after its incorporation into the spicule. Our findings also indicate that the mineral precursor and two well characterized matrix proteins are trafficked by different cellular routes. PMID:18355808

Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle.

PubMed

Sharma, V K; Sacco, R E; Kunkle, R A; Bearson, S M D; Palmquist, D E

2012-04-01

The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1β was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.

An emerging case for membrane pore formation as a common mechanism for the unconventional secretion of FGF2 and IL-1β.

PubMed

Brough, David; Pelegrin, Pablo; Nickel, Walter

2017-10-01

Extracellular proteins with important signalling roles in processes, such as inflammation and angiogenesis, are known to employ unconventional routes of protein secretion. Although mechanisms of unconventional protein secretion are beginning to emerge, the precise molecular details have remained elusive for the majority of cargo proteins secreted by unconventional means. Recent findings suggest that for two examples of unconventionally secreted proteins, interleukin 1β (IL-1β) and fibroblast growth factor 2 (FGF2), the common molecular principle of pore formation may be shared. Under specific experimental conditions, secretion of IL-1β and FGF2 is triggered by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]-dependent formation of pores across the plasma membrane. However, the underlying mechanisms are different, with FGF2 known to directly interact with PI(4,5)P 2 , whereas in the case of IL-1β secretion, it is proposed that the N-terminal fragment of gasdermin D interacts with PI(4,5)P 2 to form the pore. Thus, although implemented in different ways, these findings suggest that pore formation may be shared by the unconventional secretion mechanisms for FGF2 and IL-1β in at least some cases. In this Opinion article, we discuss the unconventional mechanisms of FGF2 and IL-1β release with a particular emphasis on recent discoveries suggesting the importance of pore formation on the plasma membrane. © 2017. Published by The Company of Biologists Ltd.

A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells

PubMed Central

Schulein, Ralf; Guye, Patrick; Rhomberg, Thomas A.; Schmid, Michael C.; Schröder, Gunnar; Vergunst, Annette C.; Carena, Ilaria; Dehio, Christoph

2005-01-01

Bacterial type IV secretion (T4S) systems mediate the transfer of macromolecular substrates into various target cells, e.g., the conjugative transfer of DNA into bacteria or the transfer of virulence proteins into eukaryotic host cells. The T4S apparatus VirB of the vascular tumor-inducing pathogen Bartonella henselae causes subversion of human endothelial cell (HEC) function. Here we report the identification of multiple protein substrates of VirB, which, upon translocation into HEC, mediate all known VirB-dependent cellular changes. These Bartonella-translocated effector proteins (Beps) A-G are encoded together with the VirB system and the T4S coupling protein VirD4 on a Bartonella-specific pathogenicity island. The Beps display a modular architecture, suggesting an evolution by extensive domain duplication and reshuffling. The C terminus of each Bep harbors at least one copy of the Bep-intracellular delivery domain and a short positively charged tail sequence. This biparte C terminus constitutes a transfer signal that is sufficient to mediate VirB/VirD4-dependent intracellular delivery of reporter protein fusions. The Bep-intracellular delivery domain is also present in conjugative relaxases of bacterial conjugation systems. We exemplarily show that the C terminus of such a conjugative relaxase mediates protein transfer through the Bartonella henselae VirB/VirD4 system into HEC. Conjugative relaxases may thus represent the evolutionary origin of the here defined T4S signal for protein transfer into human cells. PMID:15642951

Redox Proteomics Applied to the Thiol Secretome.

PubMed

Ghezzi, Pietro; Chan, Philippe

2017-03-01

Secreted proteins are important both as signaling molecules and potential biomarkers. Recent Advances: Protein can undergo different types of oxidation, both in physiological conditions or under oxidative stress. Several redox proteomics techniques have been successfully applied to the identification of glutathionylated proteins, an oxidative post-translational modification consisting in the formation of a mixed disulfide between a protein cysteine and glutathione. Redox proteomics has also been used to study other forms of protein oxidation. Because of the highest proportion of free cysteines in the cytosol, redox proteomics of protein thiols has focused, so far, on intracellular proteins. However, plasma proteins, such as transthyretin and albumin, have been described as glutathionylated or cysteinylated. The present review discusses the redox state of protein cysteines in relation to their cellular distribution. We describe the various approaches used to detect secreted glutathionylated proteins, the only thiol modification studied so far in secreted proteins, and the specific problems presented in the study of the secretome. This review focusses on glutathionylated proteins secreted under inflammatory conditions and that may act as soluble mediators (cytokines). Future studies on the redox secretome (including other forms of oxidation) might identify new soluble mediators and biomarkers of oxidative stress. Antioxid. Redox Signal. 26, 299-312.

Detergent Isolation Stabilizes and Activates the Shigella Type III Secretion System Translocator Protein IpaC.

PubMed

Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E

2016-07-01

Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

PubMed

Zhong, Guitao; Liu, Ronghe; Zhuang, Menglong; Wang, Hao

2017-01-01

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata.

PubMed

Hatano, Naoya; Hamada, Tatsuro

2012-08-03

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes. Copyright © 2012 Elsevier B.V. All rights reserved.

The protein composition of the digestive fluid from the venus flytrap sheds light on prey digestion mechanisms.

PubMed

Schulze, Waltraud X; Sanggaard, Kristian W; Kreuzer, Ines; Knudsen, Anders D; Bemm, Felix; Thøgersen, Ida B; Bräutigam, Andrea; Thomsen, Line R; Schliesky, Simon; Dyrlund, Thomas F; Escalante-Perez, Maria; Becker, Dirk; Schultz, Jörg; Karring, Henrik; Weber, Andreas; Højrup, Peter; Hedrich, Rainer; Enghild, Jan J

2012-11-01

The Venus flytrap (Dionaea muscipula) is one of the most well-known carnivorous plants because of its unique ability to capture small animals, usually insects or spiders, through a unique snap-trapping mechanism. The animals are subsequently killed and digested so that the plants can assimilate nutrients, as they grow in mineral-deficient soils. We deep sequenced the cDNA from Dionaea traps to obtain transcript libraries, which were used in the mass spectrometry-based identification of the proteins secreted during digestion. The identified proteins consisted of peroxidases, nucleases, phosphatases, phospholipases, a glucanase, chitinases, and proteolytic enzymes, including four cysteine proteases, two aspartic proteases, and a serine carboxypeptidase. The majority of the most abundant proteins were categorized as pathogenesis-related proteins, suggesting that the plant's digestive system evolved from defense-related processes. This in-depth characterization of a highly specialized secreted fluid from a carnivorous plant provides new information about the plant's prey digestion mechanism and the evolutionary processes driving its defense pathways and nutrient acquisition.

Comprehensive Analysis of Transport Proteins Encoded Within the Genome of Bdellovibrio bacteriovorus

PubMed Central

Barabote, Ravi D.; Rendulic, Snjezana; Schuster, Stephan C.; Saier, Milton H.

2012-01-01

Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the transporter classification database (TCDB). A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least 4 types of inner membrane secretion systems and 5 types for outer membrane secretion are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer membrane transport and motility than does E. coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes were also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses. PMID:17706914

New insights in Trichoderma harzianum antagonism of fungal plant pathogens by secreted protein analysis.

PubMed

Monteiro, Valdirene Neves; do Nascimento Silva, Roberto; Steindorff, Andrei Stecca; Costa, Fabio Teles; Noronha, Eliane Ferreira; Ricart, Carlos André Ornelas; de Sousa, Marcelo Valle; Vainstein, Marilene Henning; Ulhoa, Cirano José

2010-10-01

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, β-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.

Outer membrane components of the Tad (tight adherence) secreton of Aggregatibacter actinomycetemcomitans.

PubMed

Clock, Sarah A; Planet, Paul J; Perez, Brenda A; Figurski, David H

2008-02-01

Prokaryotic secretion relies on proteins that are widely conserved, including NTPases and secretins, and on proteins that are system specific. The Tad secretion system in Aggregatibacter actinomycetemcomitans is dedicated to the assembly and export of Flp pili, which are needed for tight adherence. Consistent with predictions that RcpA forms the multimeric outer membrane secretion channel (secretin) of the Flp pilus biogenesis apparatus, we observed the RcpA protein in multimers that were stable in the presence of detergent and found that rcpA and its closely related homologs form a novel and distinct subfamily within a well-supported gene phylogeny of the entire secretin gene superfamily. We also found that rcpA-like genes were always linked to Aggregatibacter rcpB- or Caulobacter cpaD-like genes. Using antisera, we determined the localization and gross abundances of conserved (RcpA and TadC) and unique (RcpB, RcpC, and TadD) Tad proteins. The three Rcp proteins (RcpA, RcpB, and RcpC) and TadD, a putative lipoprotein, localized to the bacterial outer membrane. RcpA, RcpC, and TadD were also found in the inner membrane, while TadC localized exclusively to the inner membrane. The RcpA secretin was necessary for wild-type abundances of RcpB and RcpC, and TadC was required for normal levels of all three Rcp proteins. TadC abundance defects were observed in rcpA and rcpC mutants. TadD production was essential for wild-type RcpA and RcpB abundances, and RcpA did not multimerize or localize to the outer membrane without the expression of TadD. These data indicate that membrane proteins TadC and TadD may influence the assembly, transport, and/or function of individual outer membrane Rcp proteins.

Genomic and Proteomic Dissection of the Ubiquitous Plant Pathogen, Armillaria mellea: Toward a New Infection Model System

PubMed Central

2013-01-01

Armillaria mellea is a major plant pathogen. Yet, no large-scale “-omics” data are available to enable new studies, and limited experimental models are available to investigate basidiomycete pathogenicity. Here we reveal that the A. mellea genome comprises 58.35 Mb, contains 14473 gene models, of average length 1575 bp (4.72 introns/gene). Tandem mass spectrometry identified 921 mycelial (n = 629 unique) and secreted (n = 183 unique) proteins. Almost 100 mycelial proteins were either species-specific or previously unidentified at the protein level. A number of proteins (n = 111) was detected in both mycelia and culture supernatant extracts. Signal sequence occurrence was 4-fold greater for secreted (50.2%) compared to mycelial (12%) proteins. Analyses revealed a rich reservoir of carbohydrate degrading enzymes, laccases, and lignin peroxidases in the A. mellea proteome, reminiscent of both basidiomycete and ascomycete glycodegradative arsenals. We discovered that A. mellea exhibits a specific killing effect against Candida albicans during coculture. Proteomic investigation of this interaction revealed the unique expression of defensive and potentially offensive A. mellea proteins (n = 30). Overall, our data reveal new insights into the origin of basidiomycete virulence and we present a new model system for further studies aimed at deciphering fungal pathogenic mechanisms. PMID:23656496

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vogel, Lotte K.; Larsen, Jakob E.; Hansen, Martin

2005-05-13

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity ofmore » secretion is determined by conformational sensitive sorting signals.« less

Expression of bovine non-classical major histocompatibility complex class 1 proteins in mouse P815 and human K562 cells

USDA-ARS?s Scientific Manuscript database

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-class...

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells.

PubMed

Phung, Thuy Thi Bich; Sugamata, Ryuichi; Uno, Kazuko; Aratani, Yasuaki; Ozato, Keiko; Kawachi, Shoji; Thanh Nguyen, Liem; Nakayama, Toshinori; Suzuki, Kazuo

2011-12-01

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae

PubMed Central

2011-01-01

Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA), the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (Vir)B/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail. PMID:21489243

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

PubMed Central

Gagic, Dragana; Ciric, Milica; Wen, Wesley X.; Ng, Filomena; Rakonjac, Jasna

2016-01-01

Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to “bait” of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea. PMID:27092113

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

PubMed

Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

2016-03-01

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags.

PubMed

Celińska, Ewelina; Borkowska, Monika; Białas, Wojciech; Korpys, Paulina; Nicaud, Jean-Marc

2018-06-01

Upon expression of a given protein in an expression host, its secretion into the culture medium or cell-surface display is frequently advantageous in both research and industrial contexts. Hence, engineering strategies targeting folding, trafficking, and secretion of the proteins gain considerable interest. Yarrowia lipolytica has emerged as an efficient protein expression platform, repeatedly proved to be a competitive secretor of proteins. Although the key role of signal peptides (SPs) in secretory overexpression of proteins and their direct effect on the final protein titers are widely known, the number of reports on manipulation with SPs in Y. lipolytica is rather scattered. In this study, we assessed the potential of ten different SPs for secretion of two heterologous proteins in Y. lipolytica. Genomic and transcriptomic data mining allowed us to select five novel, previously undescribed SPs for recombinant protein secretion in Y. lipolytica. Their secretory potential was assessed in comparison with known, widely exploited SPs. We took advantage of Golden Gate approach, for construction of expression cassettes, and micro-volume enzymatic assays, for functional screening of large libraries of recombinant strains. Based on the adopted strategy, we identified novel secretory tags, characterized their secretory capacity, indicated the most potent SPs, and suggested a consensus sequence of a potentially robust synthetic SP to expand the molecular toolbox for engineering Y. lipolytica.

Mapping the Interactions of Dengue Virus NS1 Protein with Human Liver Proteins Using a Yeast Two-Hybrid System: Identification of C1q as an Interacting Partner

PubMed Central

Allonso, Diego; Nogueira, Mauricio L.; Mohana-Borges, Ronaldo

2013-01-01

Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection. PMID:23516407

Protein-Linked Glycan Degradation in Infants Fed Human Milk

PubMed Central

Dallas, David C.; Sela, David; Underwood, Mark A.; German, J. Bruce; Lebrilla, Carlito

2014-01-01

Many human milk proteins are glycosylated. Glycosylation is important in protecting bioactive proteins and peptide fragments from digestion. Protein-linked glycans have a variety of functions; however, there is a paucity of information on protein-linked glycan degradation in either the infant or the adult digestive system. Human digestive enzymes can break down dietary disaccharides and starches, but most of the digestive enzymes required for complex protein-linked glycan degradation are absent from both human digestive secretions and the external brush border membrane of the intestinal lining. Indeed, complex carbohydrates remain intact throughout their transit through the stomach and small intestine, and are undegraded by in vitro incubation with either adult pancreatic secretions or intact intestinal brush border membranes. Human gastrointestinal bacteria, however, produce a wide variety of glycosidases with regio- and anomeric specificities matching those of protein-linked glycan structures. These bacteria degrade a wide array of complex carbohydrates including various protein-linked glycans. That bacteria possess glycan degradation capabilities, whereas the human digestive system, perse, does not, suggests that most dietary protein-linked glycan breakdown will be of bacterial origin. In addition to providing a food source for specific bacteria in the colon, protein-linked glycans from human milk may act as decoys for pathogenic bacteria to prevent invasion and infection of the host. The composition of the intestinal microbiome may be particularly important in the most vulnerable humans-the elderly, the immunocompromised, and infants (particularly premature infants). PMID:24533224

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

PubMed

Redhai, Siamak; Hellberg, Josephine E E U; Wainwright, Mark; Perera, Sumeth W; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C I; Wilson, Clive

2016-10-01

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells

PubMed Central

Redhai, Siamak; Hellberg, Josephine E. E. U.; Wainwright, Mark; Perera, Sumeth W.; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C. I.

2016-01-01

Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion. PMID:27727275

Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phan, Jason; Tropea, Joseph E.; Waugh, David S.

2010-11-16

Pathogenic Yersinia species use a type III secretion system to inject cytotoxic effector proteins directly into the cytosol of mammalian cells, where they neutralize the innate immune response by interfering with the signal-transduction pathways that control phagocytosis and inflammation. To be exported efficiently, some effectors must transiently associate with cognate cytoplasmic secretion chaperones. SycH is the chaperone for YopH, a potent eukaryotic-like protein tyrosine phosphatase that is essential for virulence. SycH also binds two negative regulators of type III secretion, YscM1 and YscM2, both of which share significant sequence homology with the chaperone-binding domain of YopH. Here, the structure ofmore » a complex between SycH and a stable fragment of YscM2 that was designed on the basis of limited proteolysis experiments is presented. The overall fold of SycH is very similar to the structures of other homodimeric secretion chaperones that have been determined to date. YscM2 wraps around SycH in an extended fashion, with some secondary but no tertiary structure, assuming a conformation distinct from the globular fold that it is predicted to adopt in the absence of SycH.« less

In vitro and in vivo assessment of oral autologous artificial connective tissue characteristics that influence its performance as a graft.

PubMed

Fontanilla, Marta Raquel; Espinosa, Lady Giovanna

2012-09-01

Several studies have evaluated proteins secreted by fibroblasts comprising skin substitutes, finding that they are secreted in combinations and concentrations that promote wound healing. However, assessment of proteins secreted by oral fibroblasts forming a part of oral substitutes is scarce. In our previous work, collagen type-I scaffolds (CSs) and autologous artificial connective tissue (AACT) were produced and implanted in rabbit oral lesions, evidencing that AACT outperforms CS. The present work determined the secreted factor profile of AACT in the time of grafting as well as that of the AACT embedded in the clot. It also evaluated the proliferation and viability of AACT fibroblasts to establish the dwell time of these cells in the grafted area. Finally, it assessed whether CS, AACT, and clot-embedded AACT increase fibroblast recruitment induced by a fibrin clot, because the cell migratory response has been associated with the wound-healing outcome. We found that some of the factors secreted by AACT fibroblasts are significantly different from those secreted by clot-embedded AACT fibroblasts. Also, that the profile of proteins secreted by AACT fibroblasts and clot-embedded AACT fibroblasts is different from already reported protein secretion profiles of other engineered tissues used in treating oral mucosa wounds. It was also found that AACT fibroblasts are viable when grafted and remain in the treated area for almost 2 weeks, and that the migratory response of fibroblasts to tissue-substitute stimulus is significantly less than the migratory response induced by the clot alone. Overall, data suggest that AACT secretion of proteins is modulated by three-dimensionality and environment factors. This bioactivity and the fact that AACT does not increase fibroblast migration can be held accountable for AACT's good performance as a graft.

The tad locus: postcards from the widespread colonization island.

PubMed

Tomich, Mladen; Planet, Paul J; Figurski, David H

2007-05-01

The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.

Production enhancement of the extracellular lipase LipA in Bacillus subtilis: Effects of expression system and Sec pathway components.

PubMed

Ma, Ran Jing; Wang, Yan Hong; Liu, Lu; Bai, Lei Lei; Ban, Rui

2018-02-01

Lipases are among the most versatile biocatalysts, and are used in a range of industrially relevant bioconversion reactions. However, the production of LipA in recombinant Bacillus subtilis is still limited, due to unresolved issues surrounding the regulation of the expression and secretion systems. In this study, the gene encoding LipA from B. subtilis 168 was expressed in BNA under the control of the P 43 and the P AE promoter. The extracellular lipase activity of the resulting strains BNACL and BNAAL was 7.8 U ml -1 and 12.6 U ml -1 , respectively. To further enhance the expression of LipA, pHP13L was constructed by inserting the P AE -lip into the shuttle vector pHP13, which produced an extracellular lipase activity of 180.5 U ml -1 of BNA/pHP13L. The strain BNAY8 described in Supplement data which lacks eight extracellular proteins was constructed and the deletion a few of the much weaker secreting proteins had no significant effect on the secretion of LipA. Moreover, the four Sec pathway components, secA-prfB, secDF, secYEG, prsA, were individually overexpressed in BNA. The overexpression of secDF and prsA enhanced the production of LipA by 28% and 49%, respectively. Furthermore, the co-overexpression of secDF with prsA improved the extracellular amount of LipA by 59% over that of BNA/pHP13L, reaching 287.8 U ml -1 . It can therefore be said that both regulatory elements and secretion pathway had an impact on the production of secreted LipA. Their optimization and modification is a useful strategy to improve the homologous overproduction of other extracellular proteins in B. subtilis. Copyright © 2017. Published by Elsevier Inc.

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

PubMed Central

2013-01-01

Background Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. Results We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. Conclusions Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. PMID:24252298

Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.

PubMed

Mols, J; Peeters-Joris, C; Wattiez, R; Agathos, S N; Schneider, Y-J

2005-01-01

Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

PubMed

Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

2015-08-25

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

Cystic Fibrosis Heterozygote Resistance to Cholera Toxin in the Cystic Fibrosis Mouse Model

NASA Astrophysics Data System (ADS)

Gabriel, Sherif E.; Brigman, Kristen N.; Koller, Beverly H.; Boucher, Richard C.; Stutts, M. Jackson

1994-10-01

The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model. CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT. Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in response to CT. This correlation between CFTR protein and CT-induced chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

PubMed

Kumita, Janet R; Helmfors, Linda; Williams, Jocy; Luheshi, Leila M; Menzer, Linda; Dumoulin, Mireille; Lomas, David A; Crowther, Damian C; Dobson, Christopher M; Brorsson, Ann-Christin

2012-01-01

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Structure and biophysics of type III secretion in bacteria.

PubMed

Chatterjee, Srirupa; Chaudhury, Sukanya; McShan, Andrew C; Kaur, Kawaljit; De Guzman, Roberto N

2013-04-16

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

PubMed Central

Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

2017-01-01

Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

C1q/TNF-related Protein 4 (CTRP4) Is a Unique Secreted Protein with Two Tandem C1q Domains That Functions in the Hypothalamus to Modulate Food Intake and Body Weight*

PubMed Central

Byerly, Mardi S.; Petersen, Pia S.; Ramamurthy, Santosh; Seldin, Marcus M.; Lei, Xia; Provost, Elayne; Wei, Zhikui; Ronnett, Gabriele V.; Wong, G. William

2014-01-01

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance. PMID:24366864

A microfluidic in-line ELISA for measuring secreted protein under perfusion.

PubMed

Luan, Qiyue; Cahoon, Stacey; Wu, Agnes; Bale, Shyam Sundhar; Yarmush, Martin; Bhushan, Abhinav

2017-11-11

Recent progress in the development of microfluidic microphysiological systems such as 'organs-on-chips' and microfabricated cell culture is geared to simulate organ-level physiology. These tissue models leverage microengineering technologies that provide capabilities of presenting cultured cells with input signals in a more physiologically relevant context such as perfused flow. Proteins that are secreted from cells have important information about the health of the cells. Techniques to quantify cellular proteins include mass spectrometry to ELISA (enzyme-linked immunosorbent assay). Although our capability to perturb the cells in the microphysiological systems with varying inputs is well established, we lack the tools to monitor in-line the cellular responses. User intervention for sample collection and off-site is cumbersome, causes delays in obtaining results, and is especially expensive because of collection, storage, and offline processing of the samples, and in many case, technically impractical to carry out because of limitated sample volumes. To address these shortcomings, we report the development of an ELISA that is carried out in-line under perfusion within a microfluidic device. Using this assay, we measured the albumin secreted from perfused hepatocytes without and under stimulation by IL-6. Since the method is based on a sandwich ELISA, we envision broad application of this technology to not just organs-on-chips but also to characterizing the temporal release and measurement of soluble factors and response to drugs.

The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

PubMed Central

Zhou, Yan; Dong, Na; Hu, Liyan; Shao, Feng

2013-01-01

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells. PMID:23469023

An assay for entry of secreted fungal effectors into plant cells.

PubMed

Lo Presti, Libera; Zechmann, Bernd; Kumlehn, Jochen; Liang, Liang; Lanver, Daniel; Tanaka, Shigeyuki; Bock, Ralph; Kahmann, Regine

2017-01-01

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity.

PubMed

Chen, De-Ju; Xu, Yan-Ming; Zheng, Wei; Huang, Dong-Yang; Wong, Wing-Yan; Tai, William Chi-Shing; Cho, Yong-Yeon; Lau, Andy T Y

2015-09-01

For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS-2B) treated with cadmium chloride (CdCl2 ), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two-dimensional electrophoresis and visualized by silver staining. Differentially-secreted proteins were identified by MALDI-TOF-MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS-2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor-suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress-induced cytotoxicity; and the differentially-secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

PubMed Central

Arranz, S E; Albertali, I E; Cabada, M O

1997-01-01

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium. PMID:9173897

Quantitative proteomic analysis reveals effects of epidermal growth factor receptor (EGFR) on invasion-promoting proteins secreted by glioblastoma cells.

PubMed

Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D

2014-10-01

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

PubMed

Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

2017-09-01

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP) n , wherein n = 10 or 20]. The yields of the (SP) n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP) n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP) n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP) n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

PubMed

Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

2015-11-01

The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. Copyright © 2015 Elsevier GmbH. All rights reserved.

Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion.

PubMed

Wei, Wei; Gao, Yanhui; Wang, Cheng; Zhao, Lijun; Sun, Dianjun

2013-06-01

Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis. Copyright © 2011 Wiley Periodicals, Inc.

The calcium-sensing receptor regulates mammary gland parathyroid hormone–related protein production and calcium transport

PubMed Central

VanHouten, Joshua; Dann, Pamela; McGeoch, Grace; Brown, Edward M.; Krapcho, Karen; Neville, Margaret; Wysolmerski, John J.

2004-01-01

The transfer of calcium from mother to milk during lactation is poorly understood. In this report, we demonstrate that parathyroid hormone–related protein (PTHrP) production and calcium transport in mammary epithelial cells are regulated by extracellular calcium acting through the calcium-sensing receptor (CaR). The CaR becomes expressed on mammary epithelial cells at the transition from pregnancy to lactation. Increasing concentrations of calcium, neomycin, and a calcimimetic compound suppress PTHrP secretion by mammary epithelial cells in vitro, whereas in vivo, systemic hypocalcemia increases PTHrP production, an effect that can be prevented by treatment with a calcimimetic. Hypocalcemia also reduces overall milk production and calcium content, while increasing milk osmolality and protein concentrations. The changes in milk calcium content, milk osmolality, and milk protein concentration were mitigated by calcimimetic infusions. Finally, in a three-dimensional culture system that recapitulates the lactating alveolus, activation of the basolateral CaR increases transcellular calcium transport independent of its effect on PTHrP. We conclude that the lactating mammary gland can sense calcium and adjusts its secretion of calcium, PTHrP, and perhaps water in response to changes in extracellular calcium concentration. We believe this defines a homeostatic system that helps to match milk production to the availability of calcium. PMID:14966569

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

PubMed

Rhimi, Moez; Bermudez-Humaran, Luis G; Huang, Yuan; Boudebbouze, Samira; Gaci, Nadia; Garnier, Alexandrine; Gratadoux, Jean-Jacques; Mkaouar, Héla; Langella, Philippe; Maguin, Emmanuelle

2015-12-21

The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the L-arabinose isomerase has the ability to produce D-tagatose in vivo and conferred an anti-hyperglycemic effect to mice.

The Drosophila cytokine Unpaired 2 regulates physiological homeostasis by remotely controlling Insulin secretion

PubMed Central

Rajan, Akhila; Perrimon, Norbert

2012-01-01

In Drosophila the fat body (FB), a functional analog of the vertebrate adipose tissue, is the 'nutrient sensor' that conveys the nutrient status to the insulin producing cells (IPCs) in the fly brain to release insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. We identify Unpaired2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the ‘fed’ state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, we find that human Leptin, can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin. PMID:23021220

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tykvart, J.; Sacha, P.; Barinka, C.

2012-02-07

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo.more » We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.« less

Construction and Screening of a Lentiviral Secretome Library.

PubMed

Liu, Tao; Jia, Panpan; Ma, Huailei; Reed, Sean A; Luo, Xiaozhou; Larman, H Benjamin; Schultz, Peter G

2017-06-22

Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

NASA Astrophysics Data System (ADS)

Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

2015-10-01

Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

Unconventional Protein Secretion in Animal Cells.

PubMed

Ng, Fanny; Tang, Bor Luen

2016-01-01

All eukaryotic cells secrete a range of proteins in a constitutive or regulated manner through the conventional or canonical exocytic/secretory pathway characterized by vesicular traffic from the endoplasmic reticulum, through the Golgi apparatus, and towards the plasma membrane. However, a number of proteins are secreted in an unconventional manner, which are insensitive to inhibitors of conventional exocytosis and use a route that bypasses the Golgi apparatus. These include cytosolic proteins such as fibroblast growth factor 2 (FGF2) and interleukin-1β (IL-1β), and membrane proteins that are known to also traverse to the plasma membrane by a conventional process of exocytosis, such as α integrin and the cystic fibrosis transmembrane conductor (CFTR). Mechanisms underlying unconventional protein secretion (UPS) are actively being analyzed and deciphered, and these range from an unusual form of plasma membrane translocation to vesicular processes involving the generation of exosomes and other extracellular microvesicles. In this chapter, we provide an overview on what is currently known about UPS in animal cells.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System

PubMed Central

Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.

2016-01-01

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252

Comparative secretome analysis of rat stomach under different nutritional status.

PubMed

Senin, Lucia L; Roca-Rivada, Arturo; Castelao, Cecilia; Alonso, Jana; Folgueira, Cintia; Casanueva, Felipe F; Pardo, Maria; Seoane, Luisa M

2015-02-26

Obesity is a major public health threat for many industrialised countries. Bariatric surgery is the most effective treatment against obesity, suggesting that gut derived signals are crucial for energy balance regulation. Several descriptive studies have proven the presence of gastric endogenous systems that modulate energy homeostasis; however, these systems and the interactions between them are still not well known. In the present study, we show for the first time the comparative 2-DE gastric secretome analysis under different nutritional status. We have identified 38 differently secreted proteins by comparing stomach secretomes from tissue explant cultures of rats under feeding, fasting and re-feeding conditions. Among the proteins identified, glyceraldehyde-3-phosphate dehydrogenase was found to be more abundant in gastric secretome and plasma after re-feeding, and downregulated in obesity. Additionally, two calponin-1 species were decreased in feeding state, and other were modulated by nutritional and metabolic conditions. These and other secreted proteins identified in this work may be considered as potential gastrokines implicated in food intake regulation. The present work has an important impact in the field of obesity, especially in the regulation of body weight maintenance by the stomach. Nowadays, the most effective treatment in the fight against obesity is bariatric surgery, which suggests that stomach derived signals might be crucial for the regulation of the energy homeostasis. However, until now, the knowledge about the gastrokines and its mechanism of action has been poorly elucidated. In the present work, we had updated a previously validated explant secretion model for proteomic studies; this analysis allowed us, for the first time, to study the gastric secretome without interferences from other organs. We had identified 38 differently secreted proteins comparing ex vivo cultured stomachs from rats under feeding, fasting and re-feeding regimes. The results in the present article provide novel targets to study the role of the stomach in body weight and appetite regulation, and suggest new potential therapeutic targets for treating obesity. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of reoxygenation on the hypoxia-induced up-regulation of serine protease inhibitor PAI-1 in head and neck cancer cells.

PubMed

Sprague, Lisa D; Mengele, Karin; Schilling, Daniela; Geurts-Moespot, Anneke; Sweep, Fred C G J; Stadler, Peter; Schmitt, Manfred; Molls, Michael

2006-01-01

In squamous cell carcinoma of the head and neck (SCCHN), hypoxia is considered a crucial physiological modulator for malignant progression, wherebythe plasminogen activation system is involved in overlapping functions such as moulding of the extracellular matrix, cell proliferation and signal transduction. Little is known about the effects of reoxygenation on the plasminogen activation system in SCCHN cells. Three human SCCHN cell lines (BHY, CAL27, FaDu) and a non-transformed human fibroblast cell line (VH7) were exposed to hypoxic (<0.5% O(2)) conditions for up to 72 h and subsequently reoxygenated at normoxic conditions for 24 h. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein concentration and former protein activity were determined by ELISA and complex ELISA, respectively. Reoxygenation induced significant changes in cell-associated and secreted PAI-1 protein compared to the normoxic control. Significant increase in cell-associated and secreted uPA protein after reoxygenation was only observed for some of the cell lines. Determination of uPA-PAI-1 complex formation revealed the release of active protein into the cell supernatant. The beneficial role of reoxygenation during radiation therapy is widely accepted. However, reoxygenation does not seem to counteract the effects induced by hypoxia on the plasminogen activation system. Fatally irradiated reoxygenat- ed tumour cells might still produce sufficient amounts of 'harmful' protein and thus initiate a path for invasion and metastasis for surviving tumour cells.

THE STRUCTURES OF COILED-COIL DOMAINS FROM TYPE THREE SECRETION SYSTEM TRANSLOCATORS REVEAL HOMOLOGY TO PORE-FORMING TOXINS

PubMed Central

Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

2012-01-01

Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SS) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) which is responsible for over one million deaths per year. The Shigella type III secretion apparatus (T3SA) is comprised of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 Å and 2.8 Å limiting resolution, respectively. These newly identified domains are comprised of extended length (114 Å in IpaB and 71 Å in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably colicin Ia. This suggests that these mechanistically-separate and functionally-distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events. PMID:22321794

The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini

2012-03-26

Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC.more » While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.« less

A new class of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in plant parasitic cyst nematodes.

PubMed

Tytgat, Tom; Vanholme, Bartel; De Meutter, Jan; Claeys, Myriam; Couvreur, Marjolein; Vanhoutte, Isabelle; Gheysen, Greetje; Van Criekinge, Wim; Borgonie, Gaetan; Coomans, August; Gheysen, Godelieve

2004-08-01

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells.

PubMed

Zhong, Xiaotian; He, Tao; Prashad, Amar S; Wang, Wenge; Cohen, Justin; Ferguson, Darren; Tam, Amy S; Sousa, Eric; Lin, Laura; Tchistiakova, Lioudmila; Gatto, Scott; D'Antona, Aaron; Luan, Yen-Tung; Ma, Weijun; Zollner, Richard; Zhou, Jing; Arve, Bo; Somers, Will; Kriz, Ronald

2017-04-20

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellular Basis of Secondary Infections and Impaired Desquamation in Certain Inherited Ichthyoses

PubMed Central

Chan, Aegean; Godoy-Gijon, Elena; Nuno-Gonzalez, Almudena; Crumrine, Debra; Hupe, Melanie; Choi, Eung-Ho; Gruber, Robert; Williams, Mary L.; Choate, Keith; Fleckman, Philip H.; Elias, Peter M.

2015-01-01

IMPORTANCE Secondary infections and impaired desquamation complicate certain inherited ichthyoses, but their cellular basis remains unknown. In healthy human epidermis, the antimicrobial peptides cathelicidin (LL-37) and human β-defensin 2 (HBD2), as well as the desquamatory protease kallikrein-related peptidase 7 (KLK7), are delivered to the stratum corneum (SC) interstices by lamellar body (LB) exocytosis. OBJECTIVE To assess whether abnormalities in the LB secretory system could account for increased risk of infections and impaired desquamation in inherited ichthyoses with known abnormalities in LB assembly (Harlequin ichthyosis [HI]), secretion (epidermolytic ichthyosis [EI]), or postsecretory proteolysis (Netherton syndrome [NS]). DESIGN, SETTING, AND PARTICIPANTS Samples from library material were taken from patients with HI, EI, NS, and other ichthyoses, but with a normal LB secretory system, and in healthy controls and were evaluated by electron microscopy and immunohistochemical analysis from July 1, 2010, through March 31, 2013. MAIN OUTCOME AND MEASURES Changes in LB secretion and in the fate of LB-derived enzymes and antimicrobial peptides in ichthyotic patients vs healthy controls. RESULTS In healthy controls and patients with X-linked ichthyosis, neutral lipid storage disease with ichthyosis, and Gaucher disease, LB secretion is normal, and delivery of LB-derived proteins and LL-37 immunostaining persists high into the SC. In contrast, proteins loaded into nascent LBs and their delivery to the SC interstices decrease markedly in patients with HI, paralleled by reduced immunostaining for LL-37, HBD2, and KLK7 in the SC. In patients with EI, the cytoskeletal abnormality impairs the exocytosis of LB contents and thus results in decreased LL-37, HBD2, and KLK7 secretion, causing substantial entombment of these proteins within the corneocyte cytosol. Finally, in patients with NS, although abundant enzyme proteins loaded in parallel with accelerated LB production, LL-37 disappears, whereas KLK7 levels increase markedly in the SC. CONCLUSIONS AND RELEVANCE Together, these results suggest that diverse abnormalities in the LB secretory system account for the increased risk of secondary infections and impaired desquamation in patients with HI, EI, and NS. PMID:25565224

RIC-7 Promotes Neuropeptide Secretion

PubMed Central

Hao, Yingsong; Hu, Zhitao; Sieburth, Derek; Kaplan, Joshua M.

2012-01-01

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion. PMID:22275875

Genetic mapping of secretion and functional determinants of the Vibrio cholerae TcpF colonization factor.

PubMed

Krebs, Shelly J; Kirn, Thomas J; Taylor, Ronald K

2009-06-01

Colonization of the human small intestine by Vibrio cholerae requires the type IV toxin-coregulated pilus (TCP). TcpF, which is encoded within the tcp operon, is secreted from the bacterial cell by the TCP apparatus and is also essential for colonization. Bacteria lacking tcpF are deficient in colonization, and anti-TcpF antibodies are protective in the infant mouse cholera model. In order to elucidate the regions of the protein that are required for secretion through the TCP apparatus and for its function in colonization, random mutagenesis of tcpF was performed. Analysis of these mutants suggests that multiple regions throughout the protein influence extracellular secretion and that determinants near the C terminus are important for the function of TcpF in colonization. The TcpF proteins of certain environmental V. cholerae isolates with 31% to 66% identity to pathogenic V. cholerae TcpF showed higher similarity in regions identified as secretion determinants but diverged in regions found to be important for colonization. These environmental TcpF proteins are secreted from the pathogenic strain; however, they do not mediate colonization in the infant mouse model. Here we provide genetic evidence pointing toward regions of TcpF that influence secretion, as well as regions that play an important role in in vivo colonization.

Somatostatin inhibits cholecystokinin-induced pancreatic protein secretion via cholinergic pathways.

PubMed

Brodish, R J; Kuvshinoff, B W; McFadden, D W; Fink, A S

1995-05-01

Although somatostatin is a potent inhibitor of pancreatic exocrine secretion in vivo, its mechanism of action remains unclear. The influence of extrapancreatic nerves and intrapancreatic cholinergic activity on somatostatin-induced inhibition of pancreatic exocrine secretion was studied in conscious dogs. Chronic pancreatic fistulae were created in six mongrel dogs, and a second group of six dogs also underwent complete pancreatic denervation. The pancreatic responses to graded doses of cholecystokinin (12.5-200 ng/kg/h) and bethanechol (57-916 micrograms/kg/h), both alone and during background infusion of somatostatin-14 (800 pm/kg/h), were determined in all dogs. The cholecystokinin dose-response with a somatostatin-14 background was then repeated with the addition of atropine (10 micrograms/kg/h). In both groups of animals, cholecystokinin elicited a dose-dependent increase in pancreatic protein secretion that was inhibited significantly by somatostatin-14. Regardless of the status of extrapancreatic nerves, atropine further inhibited cholecystokinin-induced protein secretion beyond that evoked by somatostatin-14. In both innervated and denervated animals, cholinergic stimulation with bethanechol elicited a dose-dependent increase in pancreatic protein secretion that was unaffected by somatostatin-14. We conclude that extrapancreatic nerves do not mediate the inhibitory effects of somatostatin-14. Somatostatin-14 appears to inhibit cholecystokinin-induced pancreatic secretion by an intrapancreatic cholinergic mechanism.

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira

PubMed Central

Fouts, Derrick E.; Matthias, Michael A.; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E.; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L.; Haake, David A.; Haft, Daniel H.; Hartskeerl, Rudy; Ko, Albert I.; Levett, Paul N.; Matsunaga, James; Mechaly, Ariel E.; Monk, Jonathan M.; Nascimento, Ana L. T.; Nelson, Karen E.; Palsson, Bernhard; Peacock, Sharon J.; Picardeau, Mathieu; Ricaldi, Jessica N.; Thaipandungpanit, Janjira; Wunder, Elsio A.; Yang, X. Frank; Zhang, Jun-Jie; Vinetz, Joseph M.

2016-01-01

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts. PMID:26890609

What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira.

PubMed

Fouts, Derrick E; Matthias, Michael A; Adhikarla, Haritha; Adler, Ben; Amorim-Santos, Luciane; Berg, Douglas E; Bulach, Dieter; Buschiazzo, Alejandro; Chang, Yung-Fu; Galloway, Renee L; Haake, David A; Haft, Daniel H; Hartskeerl, Rudy; Ko, Albert I; Levett, Paul N; Matsunaga, James; Mechaly, Ariel E; Monk, Jonathan M; Nascimento, Ana L T; Nelson, Karen E; Palsson, Bernhard; Peacock, Sharon J; Picardeau, Mathieu; Ricaldi, Jessica N; Thaipandungpanit, Janjira; Wunder, Elsio A; Yang, X Frank; Zhang, Jun-Jie; Vinetz, Joseph M

2016-02-01

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.