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Sample records for iii secretion signals

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

    SciTech Connect

    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, after 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.

  2. SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli

    PubMed Central

    Deng, Wanyin; Yu, Hong B.; Li, Yuling

    2015-01-01

    ABSTRACT The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. IMPORTANCE Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into

  3. RNA Type III Secretion Signals that require Hfq

    SciTech Connect

    Niemann, George; Brown, Roslyn N.; Mushamiri, Ivy T.; Nguyen, Nhu T.; Taiwo, Rukayat; Stufkens, Afke; Smith, Richard D.; Adkins, Joshua N.; McDermott, Jason E.; Heffron, Fred

    2013-05-01

    effector proteins from the bacterium to a host cell; however, the secretion signal is poorly defined. Effector N-termini are thought to contain the signal, but they lack homology, possess no identifiable motif, and adopt intrinsically disordered structures. We identified a panel of RNA secretion signals that facilitated reporter translocation into host cells via a mechanism dependent upon the RNA chaperone Hfq. Each of these signals was localized to an RNA leader sequence preceding the translational start codon. To obtain this panel of RNA signals, we fused untranslated leader sequences from 42 different Salmonella effector proteins to the adenylate cyclase reporter (CyaA'), and tested each of them for translocation into J774 macrophages. RNA sequences derived from five effectors, gtgA, cigR, gogB, sseL, and steD were sufficient for CyaA' injection into host cells. The gtgA RNA also directed translocation of the β-lactamase reporter. To determine the mechanism of signal recognition, we identified proteins that bound specifically to the gtgA RNA. One of the unique proteins identified was Hfq. Translocation of all five UTR fusions was abolished in the Hfq mutant, confirming the importance of Hfq. Our results suggest that Hfq may direct a subset of RNA transcripts to the T3S apparatus for translation and secretion. Signal diversity may explain why the T3S signal has been difficult to define.

  4. SepD/SepL-dependent secretion signals of the type III secretion system translocator proteins in enteropathogenic Escherichia coli.

    PubMed

    Deng, Wanyin; Yu, Hong B; Li, Yuling; Finlay, B Brett

    2015-04-01

    The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by the sepD and sepL mutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals. Many bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to

  5. A Program of Yersinia enterocolitica Type III Secretion Reactions Is Activated by Specific Signals

    PubMed Central

    Lee, Vincent T.; Mazmanian, Sarkis K.; Schneewind, Olaf

    2001-01-01

    Successful establishment of Yersinia infections requires the type III machinery, a protein transporter that injects virulence factors (Yops) into macrophages. It is reported here that the Yersinia type III pathway responds to environmental signals by transporting proteins to distinct locations. Yersinia enterocolitica cells sense an increase in extracellular amino acids (glutamate, glutamine, aspartate, and asparagine) that results in the activation of the type III pathway. Another signal, provided by serum proteins such as albumin, triggers the secretion of YopD into the extracellular medium. The third signal, a decrease in calcium concentration, appears to be provided by host cells and causes Y. enterocolitica to transport YopE and presumably other virulence factors across the eukaryotic plasma membrane. Mutations in several genes encoding regulatory molecules (lcrG, lcrH, tyeA, yopD, yopN, yscM1, and yscM2) bypass the signal requirement of the type III pathway. Together these results suggest that yersiniae may have evolved distinct secretion reactions in response to environmental signals. PMID:11489848

  6. Secretion of Pseudomonas aeruginosa Type III Cytotoxins is Dependent on Pseudomonas Quinolone Signal Concentration

    PubMed Central

    Singh, G.; Wu, B.; Baek, M.S.; Camargo, A.; Nguyen, A.; Slusher, N.A.; Srinivasan, R.; Wiener-Kronish, J.P.; Lynch, S.V.

    2010-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that can, like other bacterial species, exist in antimicrobial resistant sessile biofilms and as free-swimming, planktonic cells. Specific virulence factors are typically associated with each lifestyle and several two-component response regulators have been shown to reciprocally regulate transition between biofilm-associated chronic, and free-swimming acute infections. Quorum sensing (QS) signal molecules belonging to the las and rhl systems are known to regulate virulence gene expression by P. aeruginosa. However the impact of a recently described family of novel quorum sensing signals produced by the Pseudomonas Quinolone Signal (PQS) biosynthetic pathway, on the transition between these modes of infection is less clear. Using clonal isolates from a patient developing ventilator-associated pneumonia, we demonstrated that clinical observations were mirrored by an in vitro temporal shift in isolate phenotype from a non-secreting, to a Type III cytotoxin secreting (TTSS) phenotype and further, that this phenotypic change was PQS-dependent. While intracellular type III cytotoxin levels were unaffected by PQS concentration, cytotoxin secretion was dependent on this signal molecule. Elevated PQS concentrations were associated with inhibition of cytotoxin secretion coincident with expression of virulence factors such as elastase and pyoverdin. In contrast, low concentrations or the inability to biosynthesize PQS resulted in a reversal of this phenotype. These data suggest that expression of specific P. aeruginosa virulence factors appears to be reciprocally regulated and that an additional level of PQS-dependent posttranslational control, specifically governing type III cytotoxin secretion, exists in this species. PMID:20570614

  7. 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

  8. 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.

  9. Substrate-Activated Conformational Switch on Chaperones Encodes aTargeting Signal in Type III Secretion

    PubMed Central

    Chen, Li; Ai, Xuanjun; Portaliou, Athina G.; Minetti, Conceicao A.S.A.; Remeta, David P.; Economou, Anastassios; Kalodimos, Charalampos G.

    2013-01-01

    SUMMARY Targeting of type III secretion proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone CesAB to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB, but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB–EspA complex and EscN resulted in severe secretion and infection defects. We further show that the targeting and secretion signals are distinct and the two processes are likely regulated by different mechanisms. PMID:23523349

  10. Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system

    PubMed Central

    Okazaki, Shin; Kaneko, Takakazu; Sato, Shusei; Saeki, Kazuhiko

    2013-01-01

    Root–nodule symbiosis between leguminous plants and nitrogen-fixing bacteria (rhizobia) involves molecular communication between the two partners. Key components for the establishment of symbiosis are rhizobium-derived lipochitooligosaccharides (Nod factors; NFs) and their leguminous receptors (NFRs) that initiate nodule development and bacterial entry. Here we demonstrate that the soybean microsymbiont Bradyrhizobium elkanii uses the type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria, to promote symbiosis. Intriguingly, wild-type B. elkanii, but not the T3SS-deficient mutant, was able to form nitrogen-fixing nodules on soybean nfr mutant En1282. Furthermore, even the NF-deficient B. elkanii mutant induced nodules unless T3SS genes were mutated. Transcriptional analysis revealed that expression of the soybean nodulation-specific genes ENOD40 and NIN was increased in the roots of En1282 inoculated with B. elkanii but not with its T3SS mutant, suggesting that T3SS activates host nodulation signaling by bypassing NF recognition. Root-hair curling and infection threads were not observed in the roots of En1282 inoculated with B. elkanii, indicating that T3SS is involved in crack entry or intercellular infection. These findings suggest that B. elkanii has adopted a pathogenic system for activating host symbiosis signaling to promote its infection. PMID:24082124

  11. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function.

    PubMed

    Tomalka, Amanda G; Stopford, Charles M; Lee, Pei-Chung; Rietsch, Arne

    2012-12-01

    Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the Pseudomonas aeruginosa translocator protein PopD as a model to identify its export signals. The N-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. © 2012 Blackwell Publishing Ltd.

  12. A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function

    PubMed Central

    Tomalka, Amanda G.; Stopford, Charles M.; Lee, Pei-Chung; Rietsch, Arne

    2012-01-01

    Summary Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the P. aeruginosa translocator protein PopD as a model to identify its export signals. The amino-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone-binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. PMID:23121689

  13. Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip

    PubMed Central

    Murillo, I.; Martinez-Argudo, I.; Blocker, A. J.

    2016-01-01

    Many Gram-negative bacterial pathogens use type III secretion systems (T3SSs) for virulence. The Shigella T3SS consists of a hollow needle, made of MxiH and protruding from the bacterial surface, anchored in both bacterial membranes by multimeric protein rings. Atop the needle lies the tip complex (TC), formed by IpaD and IpaB. Upon physical contact with eukaryotic host cells, T3S is initiated leading to formation of a pore in the eukaryotic cell membrane, which is made of IpaB and IpaC. Through the needle and pore channels, further bacterial proteins are translocated inside the host cell to meditate its invasion. IpaD and the needle are implicated in transduction of the host cell-sensing signal to the T3S apparatus. Furthermore, the sensing-competent TC seems formed of 4 IpaDs topped by 1 IpaB. However, nothing further is known about the activation process. To investigate IpaB’s role during T3SS activation, we isolated secretion-deregulated IpaB mutants using random mutagenesis and a genetic screen. We found ipaB point mutations in leading to defects in secretion activation, which sometimes diminished pore insertion and host cell invasion. We also demonstrated IpaB communicates intramolecularly and intermolecularly with IpaD and MxiH within the TC because mutations affecting these interactions impair signal transduction. PMID:27277624

  14. Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

    PubMed Central

    Anderson, Jeffrey C.; Wan, Ying; Kim, Young-Mo; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Peck, Scott C.

    2014-01-01

    Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host–pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis. PMID:24753604

  15. Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

    SciTech Connect

    Anderson, Jeffrey C.; Wan, Ying; Kim, Young-Mo; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Peck, Scott C.

    2014-04-21

    Many phytopathogenic bacteria use a type III secretion system (T3SS) to inject defense-suppressing effector proteins into host cells. Genes encoding the T3SS are induced at the start of infection, yet host signals that initiate T3SS gene expression are poorly understood. Here we identify several plant-derived metabolites that induce the T3SS in the bacterial pathogen Pseudomonas syringae pv tomato DC3000. In addition, we report that mkp1 (mapk phosphatase 1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these T3SS-inducing metabolites. Consistent with the observed decrease in these metabolites, T3SS effector delivery by DC3000 was impaired in mkp1. Addition of the bioactive metabolites to the mkp1-DC3000 interaction fully restored T3SS effector delivery and suppressed enhanced resistance in mkp1. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate their virulence program, and reveal a new layer of molecular communication between plants and these pathogenic bacteria.

  16. A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins

    SciTech Connect

    Buchko, Garry W.; Niemann, George; Baker, Erin Shammel; Belov, Mikhail E.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; McDermott, Jason E.

    2010-11-08

    Many pathogenic Gram-negative bacteria use a type III secretion system (T3SS) to deliver effector proteins into the host cell where they reprogram host defenses and facilitate pathogenesis. While it has been determined that the first 20 - 30 N-terminal residues usually contain the ‘secretion signal’ that targets effector proteins for translocation, the molecular basis for recognition of this signal is not understood. Recent machine-learning approaches, such as SVM-based Identification and Evaluation of Virulence Effectors (SIEVE), have improved the ability to identify effector proteins from genomics sequence information. While these methods all suggest that the T3SS secretion signal has a characteristic amino acid composition bias, it is still unclear if the amino acid pattern is important and if there are any unifying structural properties that direct recognition. To address these issues a peptide corresponding to the secretion signal for Salmonella enterica serovar Typhimurium effector SseJ was synthesized (residues 1-30, SseJ) along with scrambled peptides of the same amino acid composition that produced high (SseJ-H) and low (SseJ-L) SIEVE scores. The secretion properties of these three peptides were tested using a secretion signal-CyaA fusion assay and their structures systematically probed using circular dichroism, nuclear magnetic resonance, and ion mobility spectrometry-mass spectrometry. The signal-CyaA fusion assay showed that the native and SseJ-H fusion constructs were secreted into J774 macrophage at similar levels via the SPI-2 secretion pathway while secretion of the SseJ-L fusion construct was substantially retarded, suggesting that the SseJ secretion signal was sequence order dependent. The structural studies showed that the SseJ, SseJ-H, and SseJ-L peptides were intrinsically disordered in aqueous solution with only a small predisposition to adopt nascent helical structure in the presence of the powerful structure stabilizing agent, 1

  17. Sequence-Based Prediction of Type III Secreted Proteins

    PubMed Central

    Arnold, Roland; Brandmaier, Stefan; Kleine, Frederick; Tischler, Patrick; Heinz, Eva; Behrens, Sebastian; Niinikoski, Antti; Mewes, Hans-Werner; Horn, Matthias; Rattei, Thomas

    2009-01-01

    The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of ∼71% and selectivity of ∼85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will

  18. BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages

    PubMed Central

    Kuwae, Asaomi; Momose, Fumitaka; Nagamatsu, Kanna; Suyama, Yasuharu; Abe, Akio

    2016-01-01

    BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200–312 and 400–658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis. PMID:26828590

  19. Impassable YscP substrates and their impact on the Yersinia enterocolitica type III secretion pathway.

    PubMed

    Riordan, Kelly E; Sorg, Joseph A; Berube, Bryan J; Schneewind, Olaf

    2008-09-01

    Yersinia type III machines secrete protein substrates across the bacterial envelope and, following assembly of their secretion needles, transport effector Yops into host cells. According to their destination during type III secretion, early, middle, and late secretion substrates can be distinguished; however, the signals and mechanisms whereby these proteins are recognized and transported by the secretion machine are not understood. Here, we examine several hybrids between secretion substrates and the impassable reporter protein glutathione S-transferase (GST). YscP-GST and YopR-GST blocked type III secretion; however, YscF-, YopD-, YopN-, and LcrV-GST did not. Unlike YopR-GST, which can block type III machines only during their assembly, expression of YscP-GST led to an immediate and complete block of all secretion. The secretion signal of YscP was mapped to its first 10 codons or amino acids; however, YscP(Delta 2-15)-GST, lacking this secretion signal, imposed a partial blockade. YscP-GST copurified with the type III ATPase complex (YscN, YscL, and YscQ) and with YscO, suggesting that the association of specific machine components with the impassable substrate may cause the block in type III secretion.

  20. General secretion signal for the mycobacterial type VII secretion pathway

    PubMed Central

    Daleke, Maria H.; Ummels, Roy; Bawono, Punto; Heringa, Jaap; Vandenbroucke-Grauls, Christina M. J. E.; Luirink, Joen; Bitter, Wilbert

    2012-01-01

    Mycobacterial pathogens use specialized type VII secretion (T7S) systems to transport crucial virulence factors across their unusual cell envelope into infected host cells. These virulence factors lack classical secretion signals and the mechanism of substrate recognition is not well understood. Here we demonstrate that the model T7S substrates PE25/PPE41, which form a heterodimer, are targeted to the T7S pathway ESX-5 by a signal located in the C terminus of PE25. Site-directed mutagenesis of residues within this C terminus resulted in the identification of a highly conserved motif, i.e., YxxxD/E, which is required for secretion. This motif was also essential for the secretion of LipY, another ESX-5 substrate. Pathogenic mycobacteria have several different T7S systems and we identified a PE protein that is secreted by the ESX-1 system, which allowed us to compare substrate recognition of these two T7S systems. Surprisingly, this ESX-1 substrate contained a C-terminal signal functionally equivalent to that of PE25. Exchange of these C-terminal secretion signals between the PE proteins restored secretion, but each PE protein remained secreted via its own ESX secretion system, indicating that an additional signal(s) provides system specificity. Remarkably, the YxxxD/E motif was also present in and required for efficient secretion of the ESX-1 substrates CFP-10 and EspB. Therefore, our data show that the YxxxD/E motif is a general secretion signal that is present in all known mycobacterial T7S substrates or substrate complexes. PMID:22733768

  1. Computational prediction shines light on type III secretion origins

    PubMed Central

    Goldberg, Tatyana; Rost, Burkhard; Bromberg, Yana

    2016-01-01

    Type III secretion system is a key bacterial symbiosis and pathogenicity mechanism responsible for a variety of infectious diseases, ranging from food-borne illnesses to the bubonic plague. In many Gram-negative bacteria, the type III secretion system transports effector proteins into host cells, converting resources to bacterial advantage. Here we introduce a computational method that identifies type III effectors by combining homology-based inference with de novo predictions, reaching up to 3-fold higher performance than existing tools. Our work reveals that signals for recognition and transport of effectors are distributed over the entire protein sequence instead of being confined to the N-terminus, as was previously thought. Our scan of hundreds of prokaryotic genomes identified previously unknown effectors, suggesting that type III secretion may have evolved prior to the archaea/bacteria split. Crucially, our method performs well for short sequence fragments, facilitating evaluation of microbial communities and rapid identification of bacterial pathogenicity – no genome assembly required. pEffect and its data sets are available at http://services.bromberglab.org/peffect. PMID:27713481

  2. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants

    PubMed Central

    Hueck, Christoph J.

    1998-01-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  3. Type III protein secretion systems in bacterial pathogens of animals and plants.

    PubMed

    Hueck, C J

    1998-06-01

    Various gram-negative animal and plant pathogens use a novel, sec-independent protein secretion system as a basic virulence mechanism. It is becoming increasingly clear that these so-called type III secretion systems inject (translocate) proteins into the cytosol of eukaryotic cells, where the translocated proteins facilitate bacterial pathogenesis by specifically interfering with host cell signal transduction and other cellular processes. Accordingly, some type III secretion systems are activated by bacterial contact with host cell surfaces. Individual type III secretion systems direct the secretion and translocation of a variety of unrelated proteins, which account for species-specific pathogenesis phenotypes. In contrast to the secreted virulence factors, most of the 15 to 20 membrane-associated proteins which constitute the type III secretion apparatus are conserved among different pathogens. Most of the inner membrane components of the type III secretion apparatus show additional homologies to flagellar biosynthetic proteins, while a conserved outer membrane factor is similar to secretins from type II and other secretion pathways. Structurally conserved chaperones which specifically bind to individual secreted proteins play an important role in type III protein secretion, apparently by preventing premature interactions of the secreted factors with other proteins. The genes encoding type III secretion systems are clustered, and various pieces of evidence suggest that these systems have been acquired by horizontal genetic transfer during evolution. Expression of type III secretion systems is coordinately regulated in response to host environmental stimuli by networks of transcription factors. This review comprises a comparison of the structure, function, regulation, and impact on host cells of the type III secretion systems in the animal pathogens Yersinia spp., Pseudomonas aeruginosa, Shigella flexneri, Salmonella typhimurium, enteropathogenic Escherichia coli

  4. Derivative of plant phenolic compound inhibits the type III secretion system of Dickeya dadantii via HrpX/HrpY two-component signal transduction and Rsm systems.

    PubMed

    Li, Yan; Hutchins, William; Wu, Xiaogang; Liang, Cuirong; Zhang, Chengfang; Yuan, Xiaochen; Khokhani, Devanshi; Chen, Xin; Che, Yizhou; Wang, Qi; Yang, Ching-Hong

    2015-02-01

    The type III secretion system (T3SS) is a major virulence factor in many Gram-negative bacterial pathogens and represents a particularly appealing target for antimicrobial agents. Previous studies have shown that the plant phenolic compound p-coumaric acid (PCA) plays a role in the inhibition of T3SS expression of the phytopathogen Dickeya dadantii 3937. This study screened a series of derivatives of plant phenolic compounds and identified that trans-4-hydroxycinnamohydroxamic acid (TS103) has an eight-fold higher inhibitory potency than PCA on the T3SS of D. dadantii. The effect of TS103 on regulatory components of the T3SS was further elucidated. Our results suggest that TS103 inhibits HrpY phosphorylation and leads to reduced levels of hrpS and hrpL transcripts. In addition, through a reduction in the RNA levels of the regulatory small RNA RsmB, TS103 also inhibits hrpL at the post-transcriptional level via the rsmB-RsmA regulatory pathway. Finally, TS103 inhibits hrpL transcription and mRNA stability, which leads to reduced expression of HrpL regulon genes, such as hrpA and hrpN. To our knowledge, this is the first inhibitor to affect the T3SS through both the transcriptional and post-transcriptional pathways in the soft-rot phytopathogen D. dadantii 3937. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  5. Yersinia enterocolitica type III secretion: evidence for the ability to transport proteins that are folded prior to secretion.

    PubMed

    Wilharm, Gottfried; Lehmann, Verena; Neumayer, Wibke; Trcek, Janja; Heesemann, Jürgen

    2004-07-12

    Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2-3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs). In this study we have fused YopE secretion signals of various length to the mouse dihydrofolate reductase (DHFR) in order to analyse the DHFR folding state prior to secretion. We could demonstrate that secretion-deficient as well as secretion-competent YopE-DHFR fusions complexed to SycE can be efficiently purified from Yersinia cytosol by affinity chromatography using methotrexate-agarose. This implies the folding of the DHFR fusion moiety despite SycE binding and contradicts the previously presented model of folding inhibition by chaperone binding. Secretion-deficient YopE-DHFR fusions caused severe jamming of the TTSS. This observation contradicts the co-translational secretion model. We present evidence that the Yersinia TTSS is familiar with the processing of transport substrates which are folded prior to secretion. We therefore predict that an unfoldase is involved in type III secretion.

  6. The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion.

    PubMed

    Torruellas, Julie; Jackson, Michael W; Pennock, Jeffry W; Plano, Gregory V

    2005-09-01

    Activation of bacterial virulence-associated type III secretion systems (T3SSs) requires direct contact between a bacterium and a eukaryotic cell. In Yersinia pestis, the cytosolic LcrG protein and a cytosolic YopN-TyeA complex function to block T3S in the presence of extracellular calcium and prior to contact with a eukaryotic cell. The mechanism by which the bacterium senses extracellular calcium and/or cell contact and transmits these signals to the cytosolic compartment is unknown. We report here that YscF, a small protein that polymerizes to form the external needle of the T3SS, is essential for the calcium-dependent regulation of T3S. Alanine-scanning mutagenesis was used to identify YscF mutants that secrete virulence proteins in the presence and absence of calcium and prior to contact with a eukaryotic cell. Interestingly, one of the YscF mutants that exhibited constitutive T3S was unable to translocate secreted proteins across the eukaryotic plasma membrane. These data indicate that the YscF needle is a multifunctional structure that participates in virulence protein secretion, in translocation of virulence proteins across eukaryotic membranes and in the cell contact- and calcium-dependent regulation of T3S.

  7. Substrate recognition by the Yersinia type III protein secretion machinery.

    PubMed

    Ramamurthi, Kumaran S; Schneewind, Olaf

    2003-11-01

    Type III secretion is the designation given to those protein secretion pathways, primarily in pathogenic Gram-negative bacteria, whose secretion machinery components share an amino acid sequence homology to components of the flagellar basal body. In Yersinia spp., these secretion machineries inject virulence proteins called Yops into the cytosol of target macrophages in an effort to evade phagocytic killing. To date, a clear mechanism by which Yops are recognized by the type III secretion machinery has not been elucidated. Unlike most, if not all, previously characterized protein sorting pathways, the information that identifies Yops as substrates for secretion seems not to be wholly encoded within the Yop peptide sequence. In fact, it appears that at least some of this information is contained within yop mRNAs. This review summarizes recent observations that have been made in this unusual field and proposes models by which proteins may be initiated into this pathway.

  8. Crystal structure of the Yersinia type III secretion protein YscE

    SciTech Connect

    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.

  9. The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens

    PubMed Central

    Rossier, Ombeline; Wengelnik, Kai; Hahn, Karoline; Bonas, Ulla

    1999-01-01

    Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen Xanthomonas campestris pv. vesicatoria have been suggested to be part of a type III machinery. Here, we report the hrp-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by X. campestris pv. vesicatoria strains that constitutively express hrp genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly hrp-dependent because a mutant carrying a deletion in hrcV, a conserved hrp gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of X. campestris pv. vesicatoria was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in Ralstonia solanacearum, and AvrB, an avirulence protein from Pseudomonas syringae pv. glycinea. Interestingly, X. campestris pv. vesicatoria also secreted YopE, a type III-secreted cytotoxin of the mammalian pathogen Yersinia pseudotuberculosis in a hrp-dependent manner. YerA, a YopE-specific chaperone, was required for YopE stability but not for secretion in X. campestris pv. vesicatoria. Our results demonstrate the functional conservation of the type III system of X. campestris for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals. PMID:10430949

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

    SciTech Connect

    McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.; Oehmen, Christopher S.; Niemann, George; Cambronne, Eric; Sharp, Danna; Adkins, Joshua N.; Samudrala, Ram; Heffron, Fred

    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 on 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.

  11. Cell-secreted signals shape lymphoma identity.

    PubMed

    Gloghini, Annunziata; Bongarzone, Italia

    2015-10-01

    Sequencing data show that both specific genes and a number of signaling pathways are recurrently mutated in various types of lymphoma. DNA sequencing analyses of lymphoma have identified several aberrations that might affect the interaction between malignant cells and the tumor microenvironment. Microenvironmental functions are essential to lymphoma; they provide survival and proliferation signals and license immune evasion. It is plausible that interventions that aim to destroy tumor-microenvironment interactions may improve responses to therapeutics. Accordingly, the identification of extrinsic factors and their downstream intracellular signaling targets has led to much progress in understanding tumor-microenvironment interactions. Lymphoma cells are differently influenced by cells' interactions with components of their microenvironment; these cell extrinsic factors include soluble and immobilized factors, the extracellular matrix, and signals presented by neighboring cells. Soluble factors, which are often cell-secreted autocrine and paracrine factors, comprise a significant fraction of targetable molecules. To begin to understand how intercellular communication is conducted in lymphoma, a first order of study is deciphering the soluble factors secreted by malignant cells and microenvironmental cells. These soluble factors are shed into the interstitial fluid in lymphoma and can be conveniently explored using mass spectrometry. Protein components can be detected and quantified, thus enabling the routine navigation of the soluble part of the microenvironment. Elucidating functional and signaling states affords a new paradigm for understanding cancer biology and devising new therapies. This review summarizes knowledge in this field and discusses the utility of studying tumor-secreted factors.

  12. Translational regulation of Yersinia enterocolitica mRNA encoding a type III secretion substrate.

    PubMed

    Kopaskie, Karyl S; Ligtenberg, Katherine Given; Schneewind, Olaf

    2013-12-06

    Yersinia enterocolitica type III secretion machines transport YopQ and other Yop effectors into host immune cells. YopD and its chaperone LcrH are essential components of the Yersinia type III pathway, enabling effector translocation into host cells. YopD, LcrH, and YscM1 also regulate yop expression post-transcriptionally in response to environmental signals; however, the molecular mechanisms for this regulation and Yop secretion are unknown. We show here that YopD associates with 30 S ribosomal particles in a manner requiring LcrH. When added to ribosomes, YopD, LcrH, and YscM1 block the translation of yopQ mRNA. We propose a model whereby LcrH-dependent association of YopD with 30 S ribosomal particles enables YscM1 to block yopQ translation unless type III machines are induced to secrete the effector.

  13. Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

    PubMed Central

    Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias

    2014-01-01

    The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28 ± 9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant. PMID:25038096

  14. HpaA from Xanthomonas is a regulator of type III secretion.

    PubMed

    Lorenz, Christian; Kirchner, Oliver; Egler, Monique; Stuttmann, Johannes; Bonas, Ulla; Büttner, Daniela

    2008-07-01

    The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject effector proteins into the host cell cytoplasm. Efficient secretion of several effector proteins depends on the cytoplasmic global T3S chaperone HpaB. In this study, we show that HpaB interacts with the virulence factor HpaA, which is secreted by the T3S system and translocated into the plant cell. HpaA promotes secretion of pilus, translocon and effector proteins and therefore appears to be an important control protein of the T3S system. Protein-protein interaction studies and the analysis of HpaA deletion derivatives revealed that the C-terminal protein region, which contains a HpaB binding site, is crucial for the contribution of HpaA to T3S. Secretion of pilus and translocon proteins is not affected when HpaA is expressed as an N-terminal deletion derivative that lacks the secretion and translocation signal. Our data suggest that binding of HpaA to HpaB within the bacterial cell favours secretion of extracellular components of the secretion apparatus. Secretion of HpaA presumably liberates HpaB and thus promotes effector protein secretion after assembly of the T3S apparatus.

  15. Type III Secretion: Building and Operating a Remarkable Nanomachine.

    PubMed

    Portaliou, Athina G; Tsolis, Konstantinos C; Loos, Maria S; Zorzini, Valentina; Economou, Anastassios

    2016-02-01

    The Type III secretion system (T3SS) is a protein export pathway that is widespread in Gram-negative bacteria and delivers effector proteins directly into eukaryotic cells. At its core lie the injectisome (a sophisticated transmembrane secretion apparatus) and a complex network of specialized chaperones that target secretory proteins to the antechamber of the injectisome. The assembly of the system, and the subsequent secretion of proteins through it, undergo fine-tuned, hierarchical regulation. Here, we present the current understanding of the injectisome assembly process, secretion hierarchy, and the role of chaperones. We discuss these events in light of available structural and biochemical dissection and propose future directions essential to revealing mechanistic insight into this fascinating nanomachine.

  16. The Type III Secretion Translocation Pore Senses Host Cell Contact

    PubMed Central

    Armentrout, Erin I.; Rietsch, Arne

    2016-01-01

    Type III secretion systems (T3SS) are nano-syringes used by a wide range of Gram-negative pathogens to promote infection by directly injecting effector proteins into targeted host cells. Translocation of effectors is triggered by host-cell contact and requires assembly of a pore in the host-cell plasma membrane, which consists of two translocator proteins. Our understanding of the translocation pore, how it is assembled in the host cell membrane and its precise role in effector translocation, is extremely limited. Here we use a genetic technique to identify protein-protein contacts between pore-forming translocator proteins, as well as the T3SS needle-tip, that are critical for translocon function. The data help establish the orientation of the translocator proteins in the host cell membrane. Analysis of translocon function in mutants that break these contacts demonstrates that an interaction between the pore-forming translocator PopD and the needle-tip is required for sensing host cell contact. Moreover, tethering PopD at a dimer interface also specifically prevents host-cell sensing, arguing that the translocation pore is actively involved in detecting host cell contact. The work presented here therefore establishes a signal transduction pathway for sensing host cell contact that is initiated by a conformational change in the translocation pore, and is subsequently transmitted to the base of the apparatus via a specific contact between the pore and the T3SS needle-tip. PMID:27022930

  17. Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234.

    PubMed

    Marie, Corinne; Deakin, William J; Viprey, Virginie; Kopciñska, Joanna; Golinowski, Wladyslaw; Krishnan, Hari B; Perret, Xavier; Broughton, William J

    2003-09-01

    The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

  18. Ubiquitin-Yop hybrids as probes for post-translational transport by the Yersinia type III secretion pathway.

    PubMed

    Quenee, Lauriane E; Schneewind, Olaf

    2007-07-01

    Yersinia enterocolitica uses type III secretion to transport Yop proteins into the cytoplasm of host cells. Previous work generated hypotheses for both co- and post-translational transport mechanisms in the Yersinia type III pathway. Here, we used ubiquitin (Ub) and UBP1, the Ub-specific protease, to examine whether Yops can be secreted when synthesized prior to recognition by the type III machinery. Fusion of Ub to the N-terminus of Yops blocked substrate recognition and secretion of hybrids generated with YopE, YopQ or YopR. UBP1 removed Ub from the N-terminus of these hybrids and allowed YopE, YopQ or YopR cleavage products to enter the secretion pathway. Following the release of Ub, Yersinia type III machines also transported the YopE cleavage product into the cytosol of tissue culture cells. Minimal secretion signals were also examined with the Ub/UBP1 system and some, but not all, of these signals promoted type III secretion even after polypeptides had been freed from Ub. These results suggest that recognition and secretion of Yop substrates by the type III machinery can occur by a post-translational mechanism.

  19. Inside the Chamber of Secrets of the Type III Secretion System.

    PubMed

    Cascales, Eric

    2017-03-09

    The bacterial type III secretion system is a specialized machine that injects effectors into eukaryotic cells to manipulate the host cell physiology. In this issue of Cell, Hu et al. use cryo-electron tomography to reveal an unprecedented level of details regarding the architecture of this machine and the conformational changes that occur during its assembly.

  20. The Chlamydia trachomatis Type III Secretion Chaperone Slc1 Engages Multiple Early Effectors, Including TepP, a Tyrosine-phosphorylated Protein Required for the Recruitment of CrkI-II to Nascent Inclusions and Innate Immune Signaling

    PubMed Central

    Chen, Yi-Shan; Bastidas, Robert J.; Saka, Hector A.; Carpenter, Victoria K.; Richards, Kristian L.; Plano, Gregory V.; Valdivia, Raphael H.

    2014-01-01

    Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP

  1. The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosine-phosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling.

    PubMed

    Chen, Yi-Shan; Bastidas, Robert J; Saka, Hector A; Carpenter, Victoria K; Richards, Kristian L; Plano, Gregory V; Valdivia, Raphael H

    2014-02-01

    Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient translocation into host cells. In this study, we defined proteins that associate with Slc1 in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry. We identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form large molecular weight complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to EBs at entry sites where it remained associated with nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses. We propose a model wherein TepP acts downstream of TARP

  2. 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.

  3. Type III Secretion in the Melioidosis Pathogen Burkholderia pseudomallei

    PubMed Central

    Vander Broek, Charles W.; Stevens, Joanne M.

    2017-01-01

    Burkholderia pseudomallei is a Gram-negative intracellular pathogen and the causative agent of melioidosis, a severe disease of both humans and animals. Melioidosis is an emerging disease which is predicted to be vastly under-reported. Type III Secretion Systems (T3SSs) are critical virulence factors in Gram negative pathogens of plants and animals. The genome of B. pseudomallei encodes three T3SSs. T3SS-1 and -2, of which little is known, are homologous to Hrp2 secretion systems of the plant pathogens Ralstonia and Xanthomonas. T3SS-3 is better characterized and is homologous to the Inv/Mxi-Spa secretion systems of Salmonella spp. and Shigella flexneri, respectively. Upon entry into the host cell, B. pseudomallei requires T3SS-3 for efficient escape from the endosome. T3SS-3 is also required for full virulence in both hamster and murine models of infection. The regulatory cascade which controls T3SS-3 expression and the secretome of T3SS-3 have been described, as well as the effect of mutations of some of the structural proteins. Yet only a few effector proteins have been functionally characterized to date and very little work has been carried out to understand the hierarchy of assembly, secretion and temporal regulation of T3SS-3. This review aims to frame current knowledge of B. pseudomallei T3SSs in the context of other well characterized model T3SSs, particularly those of Salmonella and Shigella. PMID:28664152

  4. Assembly, structure, function and regulation of type III secretion systems.

    PubMed

    Deng, Wanyin; Marshall, Natalie C; Rowland, Jennifer L; McCoy, James M; Worrall, Liam J; Santos, Andrew S; Strynadka, Natalie C J; Finlay, B Brett

    2017-04-10

    Type III secretion systems (T3SSs) are protein transport nanomachines that are found in Gram-negative bacterial pathogens and symbionts. Resembling molecular syringes, T3SSs form channels that cross the bacterial envelope and the host cell membrane, which enable bacteria to inject numerous effector proteins into the host cell cytoplasm and establish trans-kingdom interactions with diverse hosts. Recent advances in cryo-electron microscopy and integrative imaging have provided unprecedented views of the architecture and structure of T3SSs. Furthermore, genetic and molecular analyses have elucidated the functions of many effectors and key regulators of T3SS assembly and secretion hierarchy, which is the sequential order by which the protein substrates are secreted. As essential virulence factors, T3SSs are attractive targets for vaccines and therapeutics. This Review summarizes our current knowledge of the structure and function of this important protein secretion machinery. A greater understanding of T3SSs should aid mechanism-based drug design and facilitate their manipulation for biotechnological applications.

  5. The Structure and Function of Type III Secretion Systems.

    PubMed

    Notti, Ryan Q; Stebbins, C Erec

    2016-02-01

    Type III secretion systems (T3SSs) afford Gram-negative bacteria an intimate means of altering the biology of their eukaryotic hosts--the direct delivery of effector proteins from the bacterial cytoplasm to that of the eukaryote. This incredible biophysical feat is accomplished by nanosyringe "injectisomes," which form a conduit across the three plasma membranes, peptidoglycan layer, and extracellular space that form a barrier to the direct delivery of proteins from bacterium to host. The focus of this chapter is T3SS function at the structural level; we will summarize the core findings that have shaped our understanding of the structure and function of these systems and highlight recent developments in the field. In turn, we describe the T3SS secretory apparatus, consider its engagement with secretion substrates, and discuss the posttranslational regulation of secretory function. Lastly, we close with a discussion of the future prospects for the interrogation of structure-function relationships in the T3SS.

  6. Type III secretion systems shape up as they ship out.

    PubMed

    Marlovits, Thomas C; Stebbins, C Erec

    2010-02-01

    Virulence associated protein type III secretion systems (T3SSs) are intricately structured organic nanosyringes that achieve the translocation of bacterial proteins from the prokaryotic cytoplasm across three membranes into the host cytosol. The substrates for these systems number in the hundreds, with remarkably diverse biological activities, modulating host cell biology for the benefit of the pathogen. Although there has been tremendous progress on the structure and function of the T3SS substrates, there has been comparatively little progress on the much more highly conserved secretion apparatus itself. This review summarizes recent advances in the field of structural microbiology that have begun to address this shortcoming, finally bringing to bear the power of structural biology to this central virulence system of Gram-negative bacterial pathogens. Copyright 2009 Elsevier Ltd. All rights reserved.

  7. EffectiveDB—updates and novel features for a better annotation of bacterial secreted proteins and Type III, IV, VI secretion systems

    PubMed Central

    Eichinger, Valerie; Nussbaumer, Thomas; Platzer, Alexander; Jehl, Marc-André; Arnold, Roland; Rattei, Thomas

    2016-01-01

    Protein secretion systems play a key role in the interaction of bacteria and hosts. EffectiveDB (http://effectivedb.org) contains pre-calculated predictions of bacterial secreted proteins and of intact secretion systems. Here we describe a major update of the database, which was previously featured in the NAR Database Issue. EffectiveDB bundles various tools to recognize Type III secretion signals, conserved binding sites of Type III chaperones, Type IV secretion peptides, eukaryotic-like domains and subcellular targeting signals in the host. Beyond the analysis of arbitrary protein sequence collections, the new release of EffectiveDB also provides a ‘genome-mode’, in which protein sequences from nearly complete genomes or metagenomic bins can be screened for the presence of three important secretion systems (Type III, IV, VI). EffectiveDB contains pre-calculated predictions for currently 1677 bacterial genomes from the EggNOG 4.0 database and for additional bacterial genomes from NCBI RefSeq. The new, user-friendly and informative web portal offers a submission tool for running the EffectiveDB prediction tools on user-provided data. PMID:26590402

  8. Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.

    PubMed

    Carlsson, Katrin E; Liu, Junfa; Edqvist, Petra J; Francis, Matthew S

    2007-08-01

    Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.

  9. Functional Activation of the Flagellar Type III Secretion Export Apparatus

    PubMed Central

    Phillips, Andrew M.; Calvo, Rebecca A.; Kearns, Daniel B.

    2015-01-01

    Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. PMID:26244495

  10. Yersinia Type III Secretion System Master Regulator LcrF

    PubMed Central

    Schwiesow, Leah; Lam, Hanh

    2015-01-01

    Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

  11. Prevalence of type III secretion system in effective biocontrol pseudomonads.

    PubMed

    Almario, Juliana; Gobbin, Davide; Défago, Geneviève; Moënne-Loccoz, Yvan; Rezzonico, Fabio

    2014-05-01

    Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN(+) biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN(-) biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  12. Type III secretion systems: the bacterial flagellum and the injectisome

    PubMed Central

    Diepold, Andreas; Armitage, Judith P.

    2015-01-01

    The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications. PMID:26370933

  13. Type-III secretion filaments as scaffolds for inorganic nanostructures

    PubMed Central

    Azam, Anum; Tullman-Ercek, Danielle

    2016-01-01

    Nanostructured materials exhibit unique magnetic, electrical and catalytic properties. These characteristics are determined by the chemical composition, size and shape of the nanostructured components, which are challenging to modulate on such small size scales and to interface with living cells. To address this problem, we are using a self-assembling filament protein, PrgI, as a scaffold for bottom-up inorganic nanostructure synthesis. PrgI is a small protein (80 amino acids) that oligomerizes to form the type-III secretion system needle of Salmonella enterica. We demonstrate that purified PrgI monomers also spontaneously self-assemble into long filaments and that high-affinity peptide tags specific for attachment to functionalized particles can be integrated into the N-terminal region of PrgI. The resulting filaments selectively bind to gold, whether the filaments are assembled in vitro, sheared from cells or remain attached to live S. enterica cell membranes. Chemical reduction of the gold-modified PrgI variants results in structures that are several micrometres in length and which incorporate a contiguous gold surface. Mutant strains with genomically incorporated metal-binding tags retain the secretion phenotype. We anticipate that self-assembled, cell-tethered protein/metal filamentous structures have applications in sensing and energy transduction in vivo. PMID:26763334

  14. The Structure and Function of Type III Secretion Systems

    PubMed Central

    Notti, Ryan Q.; Stebbins, C. Erec

    2015-01-01

    ARTICLE SUMMARY Type III secretion systems (T3SS) afford gram-negative bacteria a most intimate means of altering the biology of their eukaryotic hosts — the direct delivery of effector proteins from the bacterial cytoplasm to that of the eukaryote. This incredible biophysical feat is accomplished by nanosyringe “injectisomes,” which form a conduit across the three plasma membranes, peptidoglycan layer and extracellular space that form a barrier to the direct delivery of proteins from bacterium to host. The focus of this chapter is T3SS function at the structural level; we will summarize the core findings that have shaped our understanding of the structure and function of these systems and highlight recent developments in the field. In turn, we describe the T3SS secretory apparatus, consider its engagement with secretion substrates, and discuss the post-translational regulation of secretory function. Lastly, we close with a discussion of the future prospects for the interrogation of structure-function relationships in the T3SS. PMID:26999392

  15. Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

    PubMed Central

    Tardy, F; Homblé, F; Neyt, C; Wattiez, R; Cornelis, G R; Ruysschaert, J M; Cabiaux, V

    1999-01-01

    'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors. PMID:10581252

  16. Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

    PubMed

    Tardy, F; Homblé, F; Neyt, C; Wattiez, R; Cornelis, G R; Ruysschaert, J M; Cabiaux, V

    1999-12-01

    'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

  17. A Bacterial Pathogen uses Distinct Type III Secretion Systems to Alternate between Host Kingdom

    USDA-ARS?s Scientific Manuscript database

    Gram-negative bacterial pathogens of eukaryotes often secrete proteins directly into host cells via a needle-like protein channel called a ‘type III secretion system’ (T3SS). Bacteria that are adapted to either animal or plant hosts use phylogenetically distinct T3SSs for secreting proteins. Here, ...

  18. Translocation of surface-localized effectors in type III secretion

    PubMed Central

    Edgren, Tomas; Wang-Edgren, Helen; Rosqvist, Roland; Fahlgren, Anna; Wolf-Watz, Hans; Fallman, Maria

    2011-01-01

    Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model. PMID:21220342

  19. Role of calcium signaling in epithelial bicarbonate secretion.

    PubMed

    Jung, Jinsei; Lee, Min Goo

    2014-06-01

    Transepithelial bicarbonate secretion plays a key role in the maintenance of fluid and protein secretion from epithelial cells and the protection of the epithelial cell surface from various pathogens. Epithelial bicarbonate secretion is mainly under the control of cAMP and calcium signaling. While the physiological roles and molecular mechanisms of cAMP-induced bicarbonate secretion are relatively well defined, those induced by calcium signaling remain poorly understood in most epithelia. The present review summarizes the current status of knowledge on the role of calcium signaling in epithelial bicarbonate secretion. Specifically, this review introduces how cytosolic calcium signaling can increase bicarbonate secretion by regulating membrane transport proteins and how it synergizes with cAMP-induced mechanisms in epithelial cells. In addition, tissue-specific variations in the pancreas, salivary glands, intestines, bile ducts, and airways are discussed. We hope that the present report will stimulate further research into this important topic. These studies will provide the basis for future medicines for a wide spectrum of epithelial disorders including cystic fibrosis, Sjögren's syndrome, and chronic pancreatitis.

  20. Regulation of the Yersinia type III secretion system: traffic control

    PubMed Central

    Dewoody, Rebecca S.; Merritt, Peter M.; Marketon, Melanie M.

    2013-01-01

    Yersinia species, as well as many other Gram-negative pathogens, use a type III secretion system (T3SS) to translocate effector proteins from the bacterial cytoplasm to the host cytosol. This T3SS resembles a molecular syringe, with a needle-like shaft connected to a basal body structure, which spans the inner and outer bacterial membranes. The basal body of the injectisome shares a high degree of homology with the bacterial flagellum. Extending from the T3SS basal body is the needle, which is a polymer of a single protein, YscF. The distal end of the needle serves as a platform for the assembly of a tip complex composed of LcrV. Though never directly observed, prevailing models assume that LcrV assists in the insertion of the pore-forming proteins YopB and YopD into the host cell membrane. This completes a bridge between the bacterium and host cell to provide a continuous channel through which effectors are delivered. Significant effort has gone into understanding how the T3SS is assembled, how its substrates are recognized and how substrate delivery is controlled. Arguably the latter topic is the least understood; however, recent advances have provided new insight, and therefore, this review will focus primarily on summarizing the current state of knowledge regarding the control of substrate delivery by the T3SS. Specifically, we will discuss the roles of YopK, as well as YopN and YopE, which have long been linked to regulation of translocation. We also propose models whereby the YopK regulator communicates with the basal body of the T3SS to control translocation. PMID:23390616

  1. SecretEPDB: a comprehensive web-based resource for secreted effector proteins of the bacterial types III, IV and VI secretion systems

    PubMed Central

    An, Yi; Wang, Jiawei; Li, Chen; Revote, Jerico; Zhang, Yang; Naderer, Thomas; Hayashida, Morihiro; Akutsu, Tatsuya; Webb, Geoffrey I.; Lithgow, Trevor; Song, Jiangning

    2017-01-01

    Bacteria translocate effector molecules to host cells through highly evolved secretion systems. By definition, the function of these effector proteins is to manipulate host cell biology and the sequence, structural and functional annotations of these effector proteins will provide a better understanding of how bacterial secretion systems promote bacterial survival and virulence. Here we developed a knowledgebase, termed SecretEPDB (Bacterial Secreted Effector Protein DataBase), for effector proteins of type III secretion system (T3SS), type IV secretion system (T4SS) and type VI secretion system (T6SS). SecretEPDB provides enriched annotations of the aforementioned three classes of effector proteins by manually extracting and integrating structural and functional information from currently available databases and the literature. The database is conservative and strictly curated to ensure that every effector protein entry is supported by experimental evidence that demonstrates it is secreted by a T3SS, T4SS or T6SS. The annotations of effector proteins documented in SecretEPDB are provided in terms of protein characteristics, protein function, protein secondary structure, Pfam domains, metabolic pathway and evolutionary details. It is our hope that this integrated knowledgebase will serve as a useful resource for biological investigation and the generation of new hypotheses for research efforts aimed at bacterial secretion systems. PMID:28112271

  2. Dissection of calcium signaling events in exocrine secretion.

    PubMed

    Ambudkar, Indu S

    2011-07-01

    The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca(2+)] ([Ca(2+)](i)) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca(2+)](i) are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca(2+) release, and Ca(2+) entry, as well as those that maintain cellular Ca(2+) homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca(2+)](i) signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca(2+)](i) in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca(2+) signaling mechanisms that determine salivary gland fluid secretion.

  3. SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages

    PubMed Central

    Günster, Regina A.; Matthews, Sophie A.; Holden, David W.

    2017-01-01

    ABSTRACT Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane via the SPI-2 type III secretion system. Previously, it was shown that when expressed ectopically, the effectors SseK1 and SseK3 inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. In this study, we show that ectopically expressed SseK1, SseK2, and SseK3 suppress TNF-α-induced, but not Toll-like receptor 4- or interleukin-induced, NF-κB activation. Inhibition required a DXD motif in SseK1 and SseK3, which is essential for the transfer of N-acetylglucosamine to arginine residues (arginine-GlcNAcylation). During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNF-α-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNF-α-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages, suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain-containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as-yet-unidentified substrates are likely to explain the additive phenotype of a Salmonella strain lacking both SseK1 and SseK3. PMID:28069818

  4. Global impact of Salmonella type III secretion effector SteA on host cells

    SciTech Connect

    Cardenal-Muñoz, Elena Gutiérrez, Gabriel Ramos-Morales, Francisco

    2014-07-11

    Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.

  5. Timing is everything: the regulation of type III secretion.

    PubMed

    Deane, Janet E; Abrusci, Patrizia; Johnson, Steven; Lea, Susan M

    2010-04-01

    Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion.

  6. Microglial Microvesicle Secretion and Intercellular Signaling

    PubMed Central

    Turola, Elena; Furlan, Roberto; Bianco, Fabio; Matteoli, Michela; Verderio, Claudia

    2012-01-01

    Microvesicles (MVs) are released from almost all cell brain types into the microenvironment and are emerging as a novel way of cell-to-cell communication. This review focuses on MVs discharged by microglial cells, the brain resident myeloid cells, which comprise ∼10–12% of brain population. We summarize first evidence indicating that MV shedding is a process activated by the ATP receptor P2X7 and that shed MVs represent a secretory pathway for the inflammatory cytokine IL-β. We then discuss subsequent findings which clarify how IL-1 β can be locally processed and released from MVs into the extracellular environment. In addition, we describe the current understanding about the mechanism of P2X7-dependent MV formation and membrane abscission, which, by involving sphingomyelinase activity and ceramide formation, may share similarities with exosome biogenesis. Finally we report our recent results which show that microglia-derived MVs can stimulate neuronal activity and participate to the propagation of inflammatory signals, and suggest new areas for future investigation. PMID:22661954

  7. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes.

    PubMed

    Nelson, Matthew S; Sadowsky, Michael J

    2015-01-01

    The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses. Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation. Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity.

  8. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes

    PubMed Central

    Nelson, Matthew S.; Sadowsky, Michael J.

    2015-01-01

    The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses. Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation. Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity. PMID:26191069

  9. Notch Signaling Pathway Regulates Progesterone Secretion in Murine Luteal Cells.

    PubMed

    Wang, Jing; Liu, Shuangmei; Peng, Lichao; Dong, Qiming; Bao, Riqiang; Lv, Qiulan; Tang, Min; Hu, Chuan; Li, Gang; Liang, Shangdong; Zhang, Chunping

    2015-10-01

    Notch signaling is an evolutionarily conserved pathway, which involves in various cell life activities. Other studies and our report showed that the Notch signaling plays very important role in follicle development in mammalian ovaries. In luteal cells, Notch ligand, delta-like ligand 4, is involved in normal luteal vasculature. In this study, murine luteal cells were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT). We found that L-658,458 and DAPT treatment decrease basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion. On the contrary, overexpression of intracellular domain of Notch3 increased basal and hCG-stimulated progesterone secretion. Further studies demonstrated that Notch signaling regulated the expression of steroidogenic acute regulatory protein and CYP11A, 2 key enzymes for progesterone synthesis. In conclusion, Notch signaling plays important role in regulating progesterone secretion in murine luteal cells.

  10. How nutritional status signalling coordinates metabolism and lignocellulolytic enzyme secretion.

    PubMed

    Brown, Neil Andrew; Ries, Laure Nicolas Annick; Goldman, Gustavo Henrique

    2014-11-01

    The utilisation of lignocellulosic plant biomass as an abundant, renewable feedstock for green chemistries and biofuel production is inhibited by its recalcitrant nature. In the environment, lignocellulolytic fungi are naturally capable of breaking down plant biomass into utilisable saccharides. Nonetheless, within the industrial context, inefficiencies in the production of lignocellulolytic enzymes impede the implementation of green technologies. One of the primary causes of such inefficiencies is the tight transcriptional control of lignocellulolytic enzymes via carbon catabolite repression. Fungi coordinate metabolism, protein biosynthesis and secretion with cellular energetic status through the detection of intra- and extra-cellular nutritional signals. An enhanced understanding of the signals and signalling pathways involved in regulating the transcription, translation and secretion of lignocellulolytic enzymes is therefore of great biotechnological interest. This comparative review describes how nutrient sensing pathways regulate carbon catabolite repression, metabolism and the utilisation of alternative carbon sources in Saccharomyces cerevisiae and ascomycete fungi.

  11. Control of type III secretion activity and substrate specificity by the cytoplasmic regulator PcrG

    PubMed Central

    Lee, Pei-Chung; Zmina, Stephanie Elizabeth; Stopford, Charles Morgan; Toska, Jonida; Rietsch, Arne

    2014-01-01

    Pathogenic Gram-negative bacteria use syringe-like type III secretion systems (T3SS) to inject effector proteins directly into targeted host cells. Effector secretion is triggered by host cell contact, and before contact is prevented by a set of conserved regulators. How these regulators interface with the T3SS apparatus to control secretion is unclear. We present evidence that the proton motive force (pmf) drives T3SS secretion in Pseudomonas aeruginosa, and that the cytoplasmic regulator PcrG interacts with distinct components of the T3SS apparatus to control two important aspects of effector secretion: (i) It coassembles with a second regulator (Pcr1) on the inner membrane T3SS component PcrD to prevent effectors from accessing the T3SS, and (ii) In conjunction with PscO, it controls protein secretion activity by modulating the ability of T3SS to convert pmf. PMID:24778208

  12. The ttsA gene is required for low-calcium-induced type III secretion of Yop proteins and virulence of Yersinia enterocolitica W22703.

    PubMed

    DeBord, Kristin L; Galanopoulos, Nicholas S; Schneewind, Olaf

    2003-06-01

    Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues. At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals. Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins. To identify such genes, we screened transposon insertion mutants of Y. enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein. ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins. ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes. All of these defects were restored by complementation with plasmid-encoded wild-type ttsA. LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal. Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone. To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice. The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain.

  13. Role of autocleavage in the function of a type III secretion specificity switch protein in Salmonella enterica serovar Typhimurium.

    PubMed

    Monjarás Feria, Julia V; Lefebre, Matthew D; Stierhof, York-Dieter; Galán, Jorge E; Wagner, Samuel

    2015-10-13

    Type III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the "switch protein," a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of the Salmonella enterica serovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage event per se does not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function. Bacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally

  14. Nodulation genes and type III secretion systems in rhizobia

    USDA-ARS?s Scientific Manuscript database

    For establishment of symbiosis, rhizobia and legumes have to communicate. Specific signaling starts with the release of flavonoids by the plant. All rhizobia encode at least one NodD protein, which responds to the presence of specific flavonoids by activation of nodulation genes. In Bradyrhizobium j...

  15. SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli▿

    PubMed Central

    Mellies, Jay L.; Haack, Kenneth R.; Galligan, Derek C.

    2007-01-01

    Genomes of bacterial pathogens contain and coordinately regulate virulence-associated genes in order to cause disease. Enteropathogenic Escherichia coli (EPEC), a major cause of watery diarrhea in infants and a model gram-negative pathogen, expresses a type III secretion system (TTSS) that is encoded by the locus of enterocyte effacement (LEE) and is necessary for causing attaching and effacing intestinal lesions. Effector proteins encoded by the LEE and in cryptic prophage are injected into the host cell cytoplasm by the TTTS apparatus, ultimately leading to diarrhea. The LEE is comprised of multiple polycistronic operons, most of which are controlled by the global, positive regulator Ler. Here we demonstrated that the LEE2 and LEE3 operons also responded to SOS signaling and that this regulation was LexA dependent. As determined by a DNase I protection assay, purified LexA protein bound in vitro to a predicted SOS box located in the divergent, overlapping LEE2/LEE3 promoters. Expression of the lexA1 allele, encoding an uncleavable LexA protein in EPEC, resulted in reduced secretion, particularly in the absence of the Ler regulator. Finally, we obtained evidence that the cryptic phage-located nleA gene encoding an effector molecule is SOS regulated. Thus, we demonstrated, for the first time to our knowledge, that genes encoding components of a TTSS are regulated by the SOS response, and our data might explain how a subset of EPEC effector proteins, encoded in cryptic prophages, are coordinately regulated with the LEE-encoded TTSS necessary for their translocation into host cells. PMID:17237173

  16. Contribution of Bordetella bronchiseptica Type III secretion system to respiratory disease in swine

    USDA-ARS?s Scientific Manuscript database

    Background: The type III secretion system (TTSS) of gram negative bacteria allows injection of effector proteins directly into the cytosol of eukaryotic cells. Previous studies have demonstrated that the B. bronchiseptica TTSS plays a role in the persistent bacterial colonization of the trachea of m...

  17. A bacterial pathogen uses distinct type III secretion systems to alternate between host kingdoms

    USDA-ARS?s Scientific Manuscript database

    Plant and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (Pnss), the causative agent of Stewart’s bacterial wilt and...

  18. Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

    PubMed Central

    Jiang, Guo-Feng; Jiang, Bo-Le; Yang, Mei; Liu, San; Liu, Jiao; Liang, Xiao-Xia; Bai, Xian-Fang; Tang, Dong-Jie; Lu, Guang-Tao; He, Yong-Qiang; Yu, Di-Qiu; Tang, Ji-Liang

    2013-01-01

    It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc. PMID:24516463

  19. Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris.

    PubMed

    Jiang, Guo-Feng; Jiang, Bo-Le; Yang, Mei; Liu, San; Liu, Jiao; Liang, Xiao-Xia; Bai, Xian-Fang; Tang, Dong-Jie; Lu, Guang-Tao; He, Yong-Qiang; Yu, Di-Qiu; Tang, Ji-Liang

    2013-01-01

    It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

  20. Visualization and characterization of individual type III protein secretion machines in live bacteria.

    PubMed

    Zhang, Yongdeng; Lara-Tejero, María; Bewersdorf, Jörg; Galán, Jorge E

    2017-06-06

    Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the Salmonella type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in Salmonella mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.

  1. A common assembly module in injectisome and flagellar type III secretion sorting platforms.

    PubMed

    Notti, Ryan Q; Bhattacharya, Shibani; Lilic, Mirjana; Stebbins, C Erec

    2015-05-21

    Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design.

  2. A common assembly module in injectisome and flagellar type III secretion sorting platforms

    NASA Astrophysics Data System (ADS)

    Notti, Ryan Q.; Bhattacharya, Shibani; Lilic, Mirjana; Stebbins, C. Erec

    2015-05-01

    Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design.

  3. A common assembly module in injectisome and flagellar type III secretion sorting platforms

    PubMed Central

    Notti, Ryan Q.; Bhattacharya, Shibani; Lilic, Mirjana; Stebbins, C. Erec

    2015-01-01

    Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design. PMID:25994170

  4. Bile salt receptor complex activates a pathogenic type III secretion system

    SciTech Connect

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.; Salomon, Dor; Tomchick, Diana R.; Grishin, Nick V.; Orth, Kim

    2016-07-05

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered thatVibrio parahaemolyticusVtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.

  5. Bile salt receptor complex activates a pathogenic type III secretion system

    PubMed Central

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N; Salomon, Dor; Tomchick, Diana R; Grishin, Nick V; Orth, Kim

    2016-01-01

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment. DOI: http://dx.doi.org/10.7554/eLife.15718.001 PMID:27377244

  6. Bile salt receptor complex activates a pathogenic type III secretion system

    DOE PAGES

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.; ...

    2016-07-05

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structuremore » of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.« less

  7. Identification of type III secretion substrates of Chlamydia trachomatis using Yersinia enterocolitica as a heterologous system

    PubMed Central

    2014-01-01

    Background Chlamydia trachomatis is an obligate intracellular human pathogen causing ocular and urogenital infections that are a significant clinical and public health concern. This bacterium uses a type III secretion (T3S) system to manipulate host cells, through the delivery of effector proteins into their cytosol, membranes, and nucleus. In this work, we aimed to find previously unidentified C. trachomatis T3S substrates. Results We first analyzed the genome of C. trachomatis L2/434 strain for genes encoding mostly uncharacterized proteins that did not appear to possess a signal of the general secretory pathway and which had not been previously experimentally shown to be T3S substrates. We selected several genes with these characteristics and analyzed T3S of the encoding proteins using Yersinia enterocolitica as a heterologous system. We identified 23 C. trachomatis proteins whose first 20 amino acids were sufficient to drive T3S of the mature form of β-lactamase TEM-1 by Y. enterocolitica. We found that 10 of these 23 proteins were also type III secreted in their full-length versions by Y. enterocolitica, providing additional support that they are T3S substrates. Seven of these 10 likely T3S substrates of C. trachomatis were delivered by Y. enterocolitica into host cells, further suggesting that they could be effectors. Finally, real-time quantitative PCR analysis of expression of genes encoding the 10 likely T3S substrates of C. trachomatis showed that 9 of them were clearly expressed during infection of host cells. Conclusions Using Y. enterocolitica as a heterologous system, we identified 10 likely T3S substrates of C. trachomatis (CT053, CT105, CT142, CT143, CT144, CT161, CT338, CT429, CT656, and CT849) and could detect translocation into host cells of CT053, CT105, CT142, CT143, CT161, CT338, and CT429. Therefore, we revealed several C. trachomatis proteins that could be effectors subverting host cell processes. PMID:24533538

  8. 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...

  9. Visualization of the type III secretion sorting platform of Shigella flexneri

    PubMed Central

    Hu, Bo; Morado, Dustin R.; Margolin, William; Rohde, John R.; Arizmendi, Olivia; Picking, Wendy L.; Picking, William D.; Liu, Jun

    2015-01-01

    Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella. PMID:25583506

  10. Type III Secretion of ExoU Is Critical during Early Pseudomonas aeruginosa Pneumonia

    PubMed Central

    Howell, Heather A.; Logan, Latania K.; Hauser, Alan R.

    2013-01-01

    ABSTRACT The Pseudomonas aeruginosa type III secretion system has been associated with poor outcomes in both animal models and human patients. Despite a large number of studies exploring the regulation of type III secretion in vitro, little is known about the timing of secretion during mammalian infection. Here we demonstrate that the exoU gene, which encodes the highly cytotoxic type III effector ExoU, is induced early during acute P. aeruginosa pneumonia. Immunofluorescence microscopy indicated that the amount of ExoU protein in the lung also increased over time. The importance of early expression was examined using a strain of P. aeruginosa with inducible production of ExoU. Delays in expression as short as 3 h led to reduced bacterial burdens in the lungs of mice and improved survival. Our results demonstrate that early expression of exoU is critical to bacterial survival during pneumonia and suggest that therapeutic interventions that delay ExoU secretion for even short periods of time may be efficacious. PMID:23481600

  11. Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae.

    PubMed

    Furutani, Ayako; Takaoka, Minako; Sanada, Harumi; Noguchi, Yukari; Oku, Takashi; Tsuno, Kazunori; Ochiai, Hirokazu; Tsuge, Seiji

    2009-01-01

    Many gram-negative bacteria secrete so-called effector proteins via a type III secretion (T3S) system. Through genome screening for genes encoding potential T3S effectors, 60 candidates were selected from rice pathogen Xanthomonas oryzae pv. oryzae MAFF311018 using these criteria: i) homologs of known T3S effectors in plant-pathogenic bacteria, ii) genes with expression regulated by hrp regulatory protein HrpX, or iii) proteins with N-terminal amino acid patterns associated with T3S substrates of Pseudomonas syringae. Of effector candidates tested with the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter for translocation into plant cells, 16 proteins were translocated in a T3S system-dependent manner. Of these 16 proteins, nine were homologs of known effectors in other plant-pathogenic bacteria and seven were not. Most of the effectors were widely conserved in Xanthomonas spp.; however, some were specific to X. oryzae. Interestingly, all these effectors were expressed in an HrpX-dependent manner, suggesting coregulation of effectors and the T3S system. In X. campestris pv. vesicatoria, HpaB and HpaC (HpaP in X. oryzae pv. oryzae) have a central role in recruiting T3S substrates to the secretion apparatus. Secretion of all but one effector was reduced in both HpaB() and HpaP() mutant strains, indicating that HpaB and HpaP are widely involved in efficient secretion of the effectors.

  12. In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

    PubMed

    Hu, Bo; Lara-Tejero, Maria; Kong, Qingke; Galán, Jorge E; Liu, Jun

    2017-03-09

    Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

  13. Calcium and Iron Regulate Swarming and Type III Secretion in Vibrio parahaemolyticus▿ †

    PubMed Central

    Gode-Potratz, Cindy J.; Chodur, Daniel M.; McCarter, Linda L.

    2010-01-01

    Here, we probe the response to calcium during growth on a surface and show that calcium influences the transcriptome and stimulates motility and virulence of Vibrio parahaemolyticus. Swarming (but not swimming) gene expression and motility were enhanced by calcium. Calcium also elevated transcription of one of the organism's two type III secretion systems (T3SS1 but not T3SS2) and heightened cytotoxicity toward host cells in coculture. Calcium stimulation of T3SS gene expression has not been reported before, although low calcium is an inducing signal for the T3SS of many organisms. EGTA was also found to increase T3SS1 gene expression and virulence; however, this was demonstrated to be the consequence of iron rather than calcium chelation. Ectopic expression of exsA, encoding the T3SS1 AraC-type regulator, was used to define the extent of the T3SS1 regulon and verify its coincident induction by calcium and EGTA. To begin to understand the regulatory mechanisms modulating the calcium response, a calcium-repressed, LysR-type transcription factor named CalR was identified and shown to repress swarming and T3SS1 gene expression. Swarming and T3SS1 gene expression were also demonstrated to be linked by LafK, a σ54-dependent regulator of swarming, and additionally connected by a negative-feedback loop on the swarming regulon propagated by ExsA. Thus, calcium and iron, two ions pertinent for a marine organism and pathogen, play a signaling role with global consequences on the regulation of gene sets that are relevant for surface colonization and infection. PMID:20851895

  14. YspM, a newly identified Ysa type III secreted protein of Yersinia enterocolitica.

    PubMed

    Witowski, Sarah E; Walker, Kimberly A; Miller, Virginia L

    2008-11-01

    Yersinia enterocolitica has three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis of Y. enterocolitica and is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersinia secreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression of yspM is regulated by temperature, NaCl concentration, and other known regulators of the ysa system. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain of Y. enterocolitica shows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed in Saccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspM(Y295)) was expressed. Site-directed mutagenesis of the histidine of YspM(Y295) ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).

  15. Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon-associated degradation.

    PubMed

    Losada, Liliana C; Hutcheson, Steven W

    2005-02-01

    The hrp type III secretion system (TTSS) of Pseudomonas syringae translocates effector proteins into the cytoplasm of host cells. Proteolysis of HrpR by Lon has been shown to negatively regulate the hrp TTSS. The inability to bypass Lon-associated effects on the regulatory system by ectopic expression of the known regulators suggested a second site of action for Lon in TTSS-dependent effector secretion. In this study we report that TTSS-dependent effectors are subject to the proteolytic degradation that appears to be rate-limiting to secretion. The half-lives of the effectors AvrPto, AvrRpt2, HopPsyA, HopPsyB1, HopPtoB2, HopPsyV1, HopPtoG and HopPtoM were substantially higher in bacteria lacking Lon. TTSS-dependent secretion of several effectors was enhanced from Lon mutants. A primary role for chaperones appears to be protection of effectors from Lon-associated degradation prior to secretion. When coexpressed with their cognate chaperone, HopPsyB1, HopPsyV1 and HopPtoM were at least 10 times more stable in strains expressing Lon. Distinct Lon-targeting and chaperone-binding domains were identified in HopPtoM. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secreton and modulation of effector secretion.

  16. [Advances in studies of the type III secretion system in Ralstonia solanacearum--A review].

    PubMed

    Zhang, Yong; Li, Muyuan; Luo, Feng

    2015-06-04

    Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating plant diseases worldwide. The syringe-like type III secretion system (T3SS) plays a crucial role in its pathogenicity. R. solanacearum uses the T3SS to inject effector proteins (Type III effectors) into the cytoplasm of host cells, causing diseases in susceptible plants or triggering the hypersensitive response in resistant plants. In this article we review recent advances in studies of R. solanacearum T3SS and highlight their unique features.

  17. Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates.

    PubMed

    Tsou, Lun K; Lara-Tejero, María; RoseFigura, Jordan; Zhang, Zhenrun J; Wang, Yen-Chih; Yount, Jacob S; Lefebre, Matthew; Dossa, Paul D; Kato, Junya; Guan, Fulan; Lam, Wing; Cheng, Yung-Chi; Galán, Jorge E; Hang, Howard C

    2016-02-24

    Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Among the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-1) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin, also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveal a previously unappreciated mechanism of action for anti-infective medicinal plants.

  18. Expression and Quorum Sensing Regulation of Type III Secretion System Genes of Vibrio harveyi during Infection of Gnotobiotic Brine Shrimp.

    PubMed

    Ruwandeepika, H A Darshanee; Karunasagar, Indrani; Bossier, Peter; Defoirdt, Tom

    2015-01-01

    Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells.

  19. Advanced algorithms and architectures for signal processing III

    SciTech Connect

    Luk, F.T.

    1988-01-01

    This book covers proceedings on advanced algorithms and architectures for signal processing III. Topics covered include: Fast QR-based array-processing algorithm; Model for the analysis of fault-tolerant signal processing architecture; Parallel VLSI direction finding algorithm; and Self-calibration techniques for high-resolution array processing.

  20. 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

  1. Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events.

    PubMed

    Gophna, Uri; Ron, Eliora Z; Graur, Dan

    2003-07-17

    Type III secretion systems (TTSS) are unique bacterial mechanisms that mediate elaborate interactions with their hosts. The fact that several of the TTSS proteins are closely related to flagellar export proteins has led to the suggestion that TTSS had evolved from flagella. Here we reconstruct the evolutionary history of four conserved type III secretion proteins and their phylogenetic relationships with flagellar paralogs. Our analysis indicates that the TTSS and the flagellar export mechanism share a common ancestor, but have evolved independently from one another. The suggestion that TTSS genes have evolved from genes encoding flagellar proteins is effectively refuted. A comparison of the species tree, as deduced from 16S rDNA sequences, to the protein phylogenetic trees has led to the identification of several major lateral transfer events involving clusters of TTSS genes. It is hypothesized that horizontal gene transfer has occurred much earlier and more frequently than previously inferred for TTSS genes and is, consequently, a major force shaping the evolution of species that harbor type III secretion systems.

  2. Kinetic Differences and Synergistic Antiviral Effects Between Type I and Type III Interferon Signaling Indicate Pathway Independence

    PubMed Central

    Voigt, Emily A.

    2015-01-01

    The spread of acute respiratory viral infections is controlled by type I and III interferon (IFN) signaling. While the mechanisms of type I IFN signaling have been studied in detail, features that distinguish type III IFN signaling remain poorly understood. Type III IFNs play an essential role in limiting infections of intestinal and respiratory epithelial surfaces; however, type III IFNs have been shown to activate similar genes to type I IFNs, raising the question of how these IFNs differ and their signals interact. We measured the kinetics of type I and III IFN activation, functional stability, and downstream antiviral responses on A549 human lung epithelial cells. Similar kinetics were found for transcriptional upregulation and secretion of type I and III IFNs in response to infection by an RNA virus, peaking at 12 h postinfection, and both protein types had similar stabilities with functional half-lives extending beyond 2 days. Both IFNs activated potent cellular antiviral responses; however, responses to type III IFNs were delayed by 2–6 h relative to type I IFN responses. Combined treatments with type I and III IFNs produced enhanced antiviral effects, and quantitative analysis of these data with a Bliss interaction model provides evidence for independence of type I and III IFN downstream signaling pathways. This novel synergistic interaction has therapeutic implications for treatment of respiratory virus infections. PMID:25938799

  3. Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

    PubMed Central

    Monjarás Feria, Julia V.; Lefebre, Matthew D.; Stierhof, York-Dieter

    2015-01-01

    ABSTRACT Type III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of the Salmonella enterica serovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage event per se does not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function. PMID:26463164

  4. Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

    PubMed Central

    Singer, Hanna M.; Erhardt, Marc; Steiner, Andrew M.; Zhang, Min-Min; Yoshikami, Doju; Bulaj, Grzegorz; Olivera, Baldomero M.; Hughes, Kelly T.

    2012-01-01

    ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. PMID:22647788

  5. Identification of Chromosomal Genes in Yersinia pestis that Influence Type III Secretion and Delivery of Yops into Target Cells

    PubMed Central

    Houppert, Andrew S.; Kwiatkowski, Elizabeth; Glass, Elizabeth M.; DeBord, Kristin L.; Merritt, Peter M.; Schneewind, Olaf; Marketon, Melanie M.

    2012-01-01

    Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague. PMID:22479512

  6. Identification of chromosomal genes in Yersinia pestis that influence type III secretion and delivery of Yops into target cells.

    PubMed

    Houppert, Andrew S; Kwiatkowski, Elizabeth; Glass, Elizabeth M; DeBord, Kristin L; Merritt, Peter M; Schneewind, Olaf; Marketon, Melanie M

    2012-01-01

    Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague.

  7. Design and characterization of a polyamine derivative inhibiting the expression of type III secretion system in Pseudomonas aeruginosa

    PubMed Central

    Wang, Chao; Liu, Xiaoling; Wang, Jing; Zhou, Jianuan; Cui, Zining; Zhang, Lian-Hui

    2016-01-01

    The type III secretion system (TTSS) of Pseudomonas aeruginosa is a key virulence determinant for infection of eukaryotic hosts. Based on the findings that spermidine-mediated host-pathogen signalling is important for activation of type III secretion systems (TTSS), in this study, we designed, synthesized and evaluated a series of polyamine derivatives for their potentials in inhibiting the expression TTSS in P. aeruginosa. In vitro assay of 15 compounds synthesized in this study unveiled stringent structural requirements for TTSS-inhibitory activity. Among them, R101SPM, a conjugate between rhodamine 101 and spermine, showed a potent activity in inhibition of the TTSS gene expression and in attenuation of the TTSS-mediated cytotoxicity on human cells. In vivo analysis demonstrated that R101SPM could rescue mice from the lethal infection by P. aeruginosa. Moreover, genetic analysis showed that the full TTSS-inhibitory activity of R101SPM required a functional spermidine transporter. Taken together, our results present a new class of lead molecules for developing anti-virulence drugs and demonstrate that the spermidine transporter SpuDEGHF of P. aeruginosa is a promising drug target. PMID:27484745

  8. Functional and Computational Analysis of Amino Acid Patterns Predictive of Type III Secretion System Substrates in Pseudomonas syringae

    PubMed Central

    Schechter, Lisa M.; Valenta, Joy C.; Schneider, David J.; Collmer, Alan; Sakk, Eric

    2012-01-01

    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 pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of

  9. Piericidin A1 Blocks Yersinia Ysc Type III Secretion System Needle Assembly

    PubMed Central

    Morgan, Jessica M.; Duncan, Miles C.; Johnson, Kevin S.; Diepold, Andreas; Lam, Hanh; Dupzyk, Allison J.; Martin, Lexi R.; Wong, Weng Ruh; Linington, Roger G.

    2017-01-01

    ABSTRACT The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood

  10. Boreal feather mosses secrete chemical signals to gain nitrogen.

    PubMed

    Bay, Guillaume; Nahar, Nurun; Oubre, Matthieu; Whitehouse, Martin J; Wardle, David A; Zackrisson, Olle; Nilsson, Marie-Charlotte; Rasmussen, Ulla

    2013-10-01

    The mechanistic basis of feather moss-cyanobacteria associations, a main driver of nitrogen (N) input into boreal forests, remains unknown. Here, we studied colonization by Nostoc sp. on two feather mosses that form these associations (Pleurozium schreberi and Hylocomium splendens) and two acrocarpous mosses that do not (Dicranum polysetum and Polytrichum commune). We also determined how N availability and moss reproductive stage affects colonization, and measured N transfer from cyanobacteria to mosses. The ability of mosses to induce differentiation of cyanobacterial hormogonia, and of hormogonia to then colonize mosses and re-establish a functional symbiosis was determined through microcosm experiments, microscopy and acetylene reduction assays. Nitrogen transfer between cyanobacteria and Pleurozium schreberi was monitored by secondary ion mass spectrometry (SIMS). All mosses induced hormogonia differentiation but only feather mosses were subsequently colonized. Colonization on Pleurozium schreberi was enhanced during the moss reproductive phase but impaired by elevated N. Transfer of N from cyanobacteria to their host moss was observed. Our results reveal that feather mosses likely secrete species-specific chemo-attractants when N-limited, which guide cyanobacteria towards them and from which they gain N. We conclude that this signalling is regulated by N demands of mosses, and serves as a control of N input into boreal forests. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  11. RfaL Is Required for Yersinia pestis Type III Secretion and Virulence

    PubMed Central

    Houppert, Andrew S.; Bohman, Lesley; Merritt, Peter M.; Cole, Christopher B.; Caulfield, Adam J.; Lathem, Wyndham W.

    2013-01-01

    Yersinia pestis, the causative agent of plague, uses a type III secretion system (T3SS) to inject cytotoxic Yop proteins directly into the cytosol of mammalian host cells. The T3SS can also be activated in vitro at 37°C in the absence of calcium. The chromosomal gene rfaL (waaL) was recently identified as a virulence factor required for proper function of the T3SS. RfaL functions as a ligase that adds the terminal N-acetylglucosamine to the lipooligosaccharide core of Y. pestis. We previously showed that deletion of rfaL prevents secretion of Yops in vitro. Here we show that the divalent cations calcium, strontium, and magnesium can partially or fully rescue Yop secretion in vitro, indicating that the secretion phenotype of the rfaL mutant may be due to structural changes in the outer membrane and the corresponding feedback inhibition on the T3SS. In support of this, we found that the defect can be overcome by deleting the regulatory gene lcrQ. Consistent with a defective T3SS, the rfaL mutant is less virulent than the wild type. We show here that the virulence defect of the mutant correlates with a decrease in both T3SS gene expression and ability to inject innate immune cells, combined with an increased sensitivity to cationic antimicrobial peptides. PMID:23357388

  12. Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus

    PubMed Central

    Brunner, Matthias J.; Yan, Jun; Franz-Wachtel, Mirita; Schärfe, Charlotta; Grin, Iwan; Galán, Jorge E.; Macek, Boris; Marlovits, Thomas C.; Robinson, Carol V.

    2016-01-01

    Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central “cup” substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation. PMID:27977800

  13. Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation*

    PubMed Central

    Roehrich, A. Dorothea; Bordignon, Enrica; Mode, Selma; Shen, Da-Kang; Liu, Xia; Pain, Maria; Murillo, Isabel; Martinez-Argudo, Isabel; Sessions, Richard B.

    2017-01-01

    Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions. PMID:27974466

  14. Targeting of plant pattern recognition receptor-triggered immunity by bacterial type-III secretion system effectors.

    PubMed

    Macho, Alberto P; Zipfel, Cyril

    2015-02-01

    During infection, microbes are detected by surface-localized pattern recognition receptors (PRRs), leading to an innate immune response that prevents microbial ingress. Therefore, successful pathogens must evade or inhibit PRR-triggered immunity to cause disease. In the past decade, a number of type-III secretion system effector (T3Es) proteins from plant pathogenic bacteria have been shown to suppress this layer of innate immunity. More recently, the detailed mechanisms of action have been defined for several of these effectors. Interestingly, effectors display a wide array of virulence targets, being able to prevent activation of immune receptors and to hijack immune signaling pathways. Besides being a fascinating example of pathogen-host co-evolution, effectors have also emerged as valuable tools to dissect important biological processes in host cells.

  15. [Prevalence of type III secretion system genes in cholera vibrios from different serogroups].

    PubMed

    Eroshenko, G A; Kutyrev, V V; Fadeeva, A V; Shavina, N Iu; Stepanov, A V

    2008-01-01

    Prevalence of vcs genes coding the type III secretion system (T3SS) in cholera vibrios of different serogroups isolated in Russia and neighboring countries was studied for the first time. Virulent strains of O1 and O139 serogroups as well as toxigenic Vibrio cholerae strains of other serogroups contained no T3SS genes. Unlike mentioned strains, 29.2% of atoxigenic non O1/non O139 cholera vibrios isolated from patients in Russia and neighboring countries contained the T3SS genes cluster, which might contribute to the pathogenic properties of these strains.

  16. 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

  17. Cell wall lignin is polymerised by class III secretable plant peroxidases in Norway spruce.

    PubMed

    Fagerstedt, Kurt V; Kukkola, Eija M; Koistinen, Ville V T; Takahashi, Junko; Marjamaa, Kaisa

    2010-02-01

    Class III secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class III plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.

  18. Functional Characterization of Two Type III Secretion Systems of Vibrio parahaemolyticus

    PubMed Central

    Park, Kwon-Sam; Ono, Takahiro; Rokuda, Mitsuhiro; Jang, Myoung-Ho; Okada, Kazuhisa; Iida, Tetsuya; Honda, Takeshi

    2004-01-01

    Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. Recent genome sequencing of the clinical V. parahaemolyticus strain RIMD2210633 identified two sets of genes for the type III secretion system (TTSS), TTSS1 and TTSS2. Here, we constructed a series of mutant strains from RIMD2210633 to determine whether the two putative TTSS apparatus are functional. The cytotoxic activity of mutant strains having a deletion in one of the TTSS1 genes was significantly decreased compared with that of the parent and TTSS2-related mutant strains. In an enterotoxicity assay with the rabbit ileal loop test, intestinal fluid accumulation was diminished by deletion of the TTSS2-related genes while TTSS1-related mutants caused a level of fluid accumulation similar to that of the parent. VopD, a protein encoded in the proximity of the TTSS1 region and a homologue of the Yersinia YopD, was secreted in a TTSS1-dependent manner. In contrast, VopP, which is encoded by a pathogenicity island on chromosome 2 and is homologous to the Yersinia YopP, was secreted via the TTSS2 pathway. These results provide evidence that V. parahaemolyticus TTSSs function as secretion systems and may have a role in the pathogenicity of the organism. This is the first report of functional TTSSs in Vibrio species. The presence of TTSS apparatus gene homologues was demonstrated in other vibrios, such as Vibrio alginolyticus, Vibrio harveyi, and Vibrio tubiashii, suggesting that some other vibrios also contain TTSS and that the TTSS has a role in protein secretion in those organisms during interaction with eukaryotic cells. PMID:15501799

  19. Type III secretion decreases bacterial and host survival following phagocytosis of Yersinia pseudotuberculosis by macrophages.

    PubMed

    Zhang, Yue; Murtha, James; Roberts, Margaret A; Siegel, Richard M; Bliska, James B

    2008-09-01

    Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis, and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV(+) wild-type, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the study of the macrophage response to internalized pYV(+) Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV(+) but not pYV-cured or DeltayopB Y. pseudotuberculosis within phagosomes: only a small fraction of the pYV(+) bacteria internalized replicated by 24 h. In addition, approximately 20% of macrophages infected with wild-type pYV(+) Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH, and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV(+) Y. pseudotuberculosis with chloramphenicol reduced apoptosis, indicating that the de novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a

  20. EphA4 Receptor Forward Signaling Inhibits Glucagon Secretion From α-Cells

    PubMed Central

    Hutchens, Troy

    2015-01-01

    The loss of inhibition of glucagon secretion exacerbates hyperglycemia in type 1 and 2 diabetes. However, the molecular mechanisms that regulate glucagon secretion in unaffected and diabetic states remain relatively unexplained. We present evidence supporting a new model of juxtacrine-mediated regulation of glucagon secretion where neighboring islet cells negatively regulate glucagon secretion through tonic stimulation of α-cell EphA receptors. Primarily through EphA4 receptors, this stimulation correlates with maintenance of a dense F-actin network. In islets, additional stimulation and inhibition of endogenous EphA forward signaling result in inhibition and enhancement, respectively, of glucagon secretion, accompanied by an increase and decrease, respectively, in α-cell F-actin density. Sorted α-cells lack endogenous stimulation of EphA forward signaling from neighboring cells, resulting in enhanced basal glucagon secretion as compared with islets and the elimination of glucose inhibition of glucagon secretion. Restoration of EphA forward signaling in sorted α-cells recapitulates both normal basal glucagon secretion and glucose inhibition of glucagon secretion. Additionally, α-cell–specific EphA4−/− mice exhibit abnormal glucagon dynamics, and EphA4−/− α-cells contain less dense F-actin networks than EphA4+/+ α-cells. This juxtacrine-mediated model provides insight into the functional and dysfunctional regulation of glucagon secretion and opens up new therapeutic strategies for the clinical management of diabetes. PMID:26251403

  1. Complex Function for SicA, a Salmonella enterica Serovar Typhimurium Type III Secretion-Associated Chaperone

    PubMed Central

    Tucker, Stephanie C.; Galán, Jorge E.

    2000-01-01

    Salmonella enterica encodes a type III secretion system within a pathogenicity island located at centisome 63 that is essential for virulence. All type III secretion systems require the function of a family of low-molecular-weight proteins that aid the secretion process by acting as partitioning factors and/or secretion pilots. One such protein is SicA, which is encoded immediately upstream of the type III secreted proteins SipB and SipC. We found that the absence of SicA results in the degradation of both SipB and SipC. Interestingly, in the absence of SipC, SipB was not only stable but also secreted at wild-type levels in a sicA mutant background, indicating that SicA is not required for SipB secretion. We also found that SicA is capable of binding both SipB and SipC. These results are consistent with a SicA role as a partitioning factor for SipB and SipC, thereby preventing their premature association and degradation. We also found that introduction of a sicA null mutation results in the lack of expression of SopE, another type III-secreted protein. Such an effect was shown to be transcriptional. Introduction of a loss-of-function sipC mutation into the sicA mutant background rescued sopE expression. These results indicate that the effect of sicA on sopE expression is indirect and most likely exerted through a regulatory factor(s) partitioned by SicA from SipC. These studies therefore describe a surprisingly complex function for the Salmonella enterica type III secretion-associated chaperone SicA. PMID:10735870

  2. Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-σ factor.

    PubMed

    Ahuja, Umesh; Shokeen, Bhumika; Cheng, Ning; Cho, Yeonjoo; Blum, Charles; Coppola, Giovanni; Miller, Jeff F

    2016-03-01

    The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.

  3. Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-σ factor

    PubMed Central

    Ahuja, Umesh; Shokeen, Bhumika; Cheng, Ning; Cho, Yeonjoo; Blum, Charles; Coppola, Giovanni; Miller, Jeff F.

    2016-01-01

    The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues. PMID:26884180

  4. Plant flavonoids target Pseudomonas syringae pv. tomato DC3000 flagella and type III secretion system.

    PubMed

    Vargas, Paola; Farias, Gabriela A; Nogales, Joaquina; Prada, Harold; Carvajal, Vivian; Barón, Matilde; Rivilla, Rafael; Martín, Marta; Olmedilla, Adela; Gallegos, María-Trinidad

    2013-12-01

    Flavonoids are among the most abundant plant secondary metabolites involved in plant protection against pathogens, but micro-organisms have developed resistance mechanisms to those compounds. We previously demonstrated that the MexAB-OprM efflux pump mediates resistance of Pseudomonas syringae pv. tomato (Pto) DC3000 to flavonoids, facilitating its survival and the colonization of the host. Here, we have shown that tomato plants respond to Pto infection producing flavonoids and other phenolic compounds. The effects of flavonoids on key traits of this model plant-pathogen bacterium have also been investigated observing that they reduce Pto swimming and swarming because of the loss of flagella, and also inhibited the expression and assembly of a functional type III secretion system. Those effects were more severe in a mutant lacking the MexAB-OprM pump. Our results suggest that flavonoids inhibit the function of the GacS/GacA two-component system, causing a depletion of rsmY RNA, therefore affecting the synthesis of two important virulence factors in Pto DC3000, flagella and the type III secretion system. These data provide new insights into the flavonoid role in the molecular dialog between host and pathogen.

  5. Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD*

    PubMed Central

    Chatterjee, Abhishek; Caballero-Franco, Celia; Bakker, Dannika; Totten, Stephanie; Jardim, Armando

    2015-01-01

    Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits. PMID:26324713

  6. YopK regulates the Yersinia pestis type III secretion system from within host cells

    PubMed Central

    Dewoody, Rebecca; Merritt, Peter M.; Houppert, Andrew S.; Marketon, Melanie M.

    2011-01-01

    Summary The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections, yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system. PMID:21205017

  7. The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion

    PubMed Central

    Dale, Colin; Young, Simon A.; Haydon, Daniel T.; Welburn, Susan C.

    2001-01-01

    Sodalis glossinidius is a maternally transmitted secondary endosymbiont residing intracellularly in tissues of the tsetse flies, Glossina spp. In this study, we have used Tn5 mutagenesis and a negative selection procedure to derive a S. glossinidius mutant that is incapable of invading insect cells in vitro and is aposymbiotic when microinjected into tsetse. This mutant strain harbors Tn5 integrated into a chromosomal gene sharing high sequence identity with a type III secretion system invasion gene (invC) previously identified in Salmonella enterica. With the use of degenerate PCR, we have amplified a further six Sodalis inv/spa genes sharing high sequence identity with type III secretion system genes encoded by Salmonella pathogenicity island 1. Phylogenetic reconstructions based on the inv/spa genes of Sodalis and other members of the family Enterobacteriaceae have consistently identified a well-supported clade containing Sodalis and the enteric pathogens Shigella and Salmonella. These results suggest that Sodalis may have evolved from an ancestor with a parasitic intracellular lifestyle, possibly a latter-day entomopathogen. These observations lend credence to a hypothesis suggesting that vertically transmitted mutualistic endosymbionts evolve from horizontally transmitted parasites through a parasitism–mutualism continuum. PMID:11172045

  8. Edwardsiella tarda-Induced Cytotoxicity Depends on Its Type III Secretion System and Flagellin

    PubMed Central

    Xie, Hai-Xia; Lu, Jin-Fang; Rolhion, Nathalie; Holden, David W.; Zhou, Ying

    2014-01-01

    Many Gram-negative bacteria utilize a type III secretion system (T3SS) to translocate virulence proteins into host cells to cause diseases. In responding to infection, macrophages detect some of the translocated proteins to activate caspase-1-mediated cell death, called pyroptosis, and secretion of proinflammatory cytokines to control the infection. Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. In this study, we report that the T3SS of E. tarda facilitates its survival and replication in murine bone marrow-derived macrophages, and E. tarda infection triggers pyroptosis of infected macrophages from mice and fish and increased secretion of the cytokine interleukin 1β in a T3SS-dependent manner. Deletion of the flagellin gene fliC of E. tarda results in decreased cytotoxicity for infected macrophages and does not attenuate its virulence in a fish model of infection, whereas upregulated expression of FliC in the fliC mutant strain reduces its virulence. We propose that the host controls E. tarda infection partially by detecting FliC translocated by the T3SS, whereas the bacteria downregulate the expression of FliC to evade innate immunity. PMID:24891103

  9. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    PubMed Central

    2014-01-01

    Background Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. Results The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Conclusions Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis. PMID:24742141

  10. Hfq negatively regulates type III secretion in EHEC and several other pathogens

    PubMed Central

    Shakhnovich, Elizabeth A.; Davis, Brigid M.; Waldor, Matthew K.

    2009-01-01

    Summary Hfq is a conserved RNA-binding protein that regulates diverse cellular processes through post-transcriptional control of gene expression, often by functioning as a chaperone for regulatory sRNAs. Here, we explored the role of Hfq in enterohaemorrhagic E. coli (EHEC), a group of non-invasive intestinal pathogens. EHEC virulence is dependent on a Type III secretion system encoded in the LEE pathogenicity island. The abundance of transcripts for all 41 LEE genes and more than half of confirmed non-LEE-encoded T3 effectors were elevated in an EHEC hfq deletion mutant. Thus, Hfq promotes coordinated expression of the LEE-encoded T3S apparatus and both LEE- and non-LEE-encoded effectors. Increased transcript levels led to the formation of functional secretion complexes capable of secreting high quantities of effectors into the supernatant. The increase in LEE-derived transcripts and proteins was dependent on Ler, the LEE-encoded transcriptional activator, and the ler transcript appears to be a direct target of Hfq-mediated negative regulation. Finally, we found that Hfq contributes to the negative regulation of T3SSs in several other pathogens, suggesting that Hfq, potentially along with species-specific sRNAs, underlies a common means to prevent unfettered expression of T3SSs. PMID:19703108

  11. Type III secretion system expression in oxygen-limited Pseudomonas aeruginosa cultures is stimulated by isocitrate lyase activity

    PubMed Central

    Chung, Jade C. S.; Rzhepishevska, Olena; Ramstedt, Madeleine; Welch, Martin

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels. PMID:23363478

  12. Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

    PubMed

    Lin, Jian'er; Neo, Shu Hui; Ho, Steven C L; Yeo, Jessna H M; Wang, Tianhua; Zhang, Wei; Bi, Xuezhi; Chao, Sheng-Hao; Yang, Yuansheng

    2017-09-01

    Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Functional relatedness in the Inv/Mxi-Spa type III secretion system family.

    PubMed

    Klein, Jessica A; Dave, Biren M; Raphenya, Amogelang R; McArthur, Andrew G; Knodler, Leigh A

    2017-03-01

    Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic 'tip complex' translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface. © 2016 John Wiley & Sons Ltd.

  14. The Type III Secretion System Effector SptP of Salmonella enterica Serovar Typhi

    PubMed Central

    Johnson, Rebecca; Byrne, Alexander; Berger, Cedric N.; Klemm, Elizabeth; Crepin, Valerie F.; Dougan, Gordon

    2016-01-01

    ABSTRACT Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S. Typhi; direct comparison of the protein sequences revealed that S. Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S. Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S. Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S. Typhi affected its function revealed that S. Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S. Typhi, S. Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S. Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S. Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. IMPORTANCE Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of

  15. Structure of the Yersinia pestis type III secretion chaperone SycH in complex with a stable fragment of YscM2

    SciTech Connect

    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 of 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.

  16. A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp.

    PubMed

    Zhou, Xiaofeng; Hu, Xiufang; Li, Jinyun; Wang, Nian

    2015-02-01

    Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein-protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp.

  17. Bacterial flagella and Type III secretion: case studies in the evolution of complexity.

    PubMed

    Pallen, M J; Gophna, U

    2007-01-01

    Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered 'irreducibly complex' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems ('molecular bricolage'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.

  18. Structure and Function of the Type III Secretion System of Pseudomonas aeruginosa

    PubMed Central

    Galle, Marlies; Carpentier, Isabelle; Beyaert, Rudi

    2012-01-01

    Pseudomonas aeruginosa is a dangerous pathogen particularly because it harbors multiple virulence factors. It causes several types of infection, including dermatitis, endocarditis, and infections of the urinary tract, eye, ear, bone, joints and, of particular interest, the respiratory tract. Patients with cystic fibrosis, who are extremely susceptible to Pseudomonas infections, have a bad prognosis and high mortality. An important virulence factor of P. aeruginosa, shared with many other gram-negative bacteria, is the type III secretion system, a hollow molecular needle that transfers effector toxins directly from the bacterium into the host cell cytosol. This complex macromolecular machine works in a highly regulated manner and can manipulate the host cell in many different ways. Here we review the current knowledge of the structure of the P. aeruginosa T3SS, as well as its function and recognition by the immune system. Furthermore, we describe recent progress in the development and use of therapeutic agents targeting the T3SS. PMID:23305368

  19. The type III secretion system as a source of novel antibacterial drug targets.

    PubMed

    Kline, Toni; Felise, Heather B; Sanowar, Sarah; Miller, Samuel I

    2012-03-01

    Type III Secretion Systems (T3SSs) are highly organized multi-protein nanomachines which translocate effector proteins from the bacterial cytosol directly into host cells. These systems are required for the pathogenesis of a wide array of Gram-negative bacterial pathogens, and thus have attracted attention as potential antibacterial drug targets. A decade of research has enabled the identification of natural products, conventional small molecule drug-like structures, and proteins that inhibit T3SSs. The mechanism(s) of action and molecular target(s) of the majority of these inhibitors remain to be determined. At the same time, structural biology methods are providing an increasingly detailed picture of the functional arrangement of the T3SS component proteins. The confluence of these two research areas may ultimately identify non-classical drug targets and facilitate the development of novel therapeutics.

  20. Benzylidene Acylhydrazides Inhibit Chlamydial Growth in a Type III Secretion- and Iron Chelation-Independent Manner

    PubMed Central

    Bao, Xiaofeng; Gylfe, Åsa; Sturdevant, Gail L.; Gong, Zheng; Xu, Shuang; Caldwell, Harlan D.; Elofsson, Mikael

    2014-01-01

    Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value. PMID:24914180

  1. Expression patterns of Wnt signaling component, secreted frizzled‑related protein 3 in astrocytoma and glioblastoma.

    PubMed

    Pećina-Šlaus, Nives; Kafka, Anja; Varošanec, Ana Maria; Marković, Leon; Krsnik, Željka; Njirić, Niko; Mrak, Goran

    2016-05-01

    Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the

  2. Expression patterns of Wnt signaling component, secreted frizzled-related protein 3 in astrocytoma and glioblastoma

    PubMed Central

    PEĆINA-ŠLAUS, NIVES; KAFKA, ANJA; VAROŠANEC, ANA MARIA; MARKOVIĆ, LEON; KRSNIK, ŽELJKA; NJIRIĆ, NIKO; MRAK, GORAN

    2016-01-01

    Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the cytoplasm an

  3. Presence of genes for type III secretion system 2 in Vibrio mimicus strains

    PubMed Central

    2010-01-01

    Background Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear. Results We therefore examined the distribution of the genes for T3SS2 in vibrios other than V. parahaemolyticus by using a PCR assay targeting both T3SS2α and T3SS2β genes. Among the 32 Vibrio species tested in our study, several T3SS2-related genes were detected in three species, V. cholerae, V. mimicus and V. hollisae, and most of the essential genes for type III secretion were present in T3SS2-positive V. cholerae and V. mimicus strains. Moreover, both V. mimicus strains possessing T3SS2α and T3SS2β were identified. The gene organization of the T3SS2 gene clusters in V. mimicus strains was fundamentally similar to that of V. parahaemolyticus and V. cholerae in both T3SS2α- and T3SS2β-possessing strains. Conclusions This study is the first reported evidence of the presence of T3SS2 gene clusters in V. mimicus strains. This finding thus provides a new insight into the pathogenicity of the V. mimicus species. PMID:21110901

  4. Structural analysis of a specialized type III secretion system peptidoglycan-cleaving enzyme.

    PubMed

    Burkinshaw, Brianne J; Deng, Wanyin; Lameignère, Emilie; Wasney, Gregory A; Zhu, Haizhong; Worrall, Liam J; Finlay, B Brett; Strynadka, Natalie C J

    2015-04-17

    The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or "effector" proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the β-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis.

  5. Computational Analysis and Binding Site Identification of Type III Secretion System ATPase from Pseudomonas aeruginosa.

    PubMed

    Dash, Raju; Hosen, S M Zahid; Sultana, Tasniha; Junaid, Md; Majumder, Mohuya; Ishat, Ismat Ara; Uddin, Mir Muhammad Nasir

    2016-12-01

    In many gram-negative bacteria, the type III secretion system (T3SS), as a virulence factor, is an attractive target for developing novel antibacterial. Regarding this, in our study, we aimed to identify the putative drug target for Pseudomonas aeruginosa, considering ATPase enzyme involved in the type III secretion system. Selective protein sequence of P. aeruginosa involved in the T3SS was retrieved from NCBI databases, and its homologues were subjected to phylogenetic analysis. Its association in T3SS was analyzed via STRING, and the 3D structure was determined by means of homology modeling followed by intensive optimization and validation. The binding site was predicted by 3DLigandSite and examined through molecular docking simulation by Autodock Vina with salicylidene acylhydrazide class of virulence-blocking compounds. PROCHECK analysis showed that 96.7 % of the residues were in the most favored regions, 1.9 % were in the additional allowed region, and 1.4 % were in the generously allowed region of the Ramachandran plot. The refined model yielded ERRAT scores of 88.124 and Verify3D value of 0.2, which indicates that the environmental profile of the model is good. The best binding affinity was observed by ME0055 compound, and ALA160, ALA161, GlY162, GLY163, GLY164, GLY165, SER166, THR167, TYR338, and PRO339 residues were found to be having complementary in the ligand-binding site. However, these findings should be further confirmed by wet lab studies for design a targeted therapeutic agent.

  6. Broadly Protective Shigella Vaccine Based on Type III Secretion Apparatus Proteins

    PubMed Central

    Martinez-Becerra, Francisco J.; Kissmann, Julian M.; Diaz-McNair, Jovita; Choudhari, Shyamal P.; Quick, Amy M.; Mellado-Sanchez, Gabriela; Clements, John D.

    2012-01-01

    Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine. PMID:22202122

  7. Structural Analysis of a Specialized Type III Secretion System Peptidoglycan-cleaving Enzyme*

    PubMed Central

    Burkinshaw, Brianne J.; Deng, Wanyin; Lameignère, Emilie; Wasney, Gregory A.; Zhu, Haizhong; Worrall, Liam J.; Finlay, B. Brett; Strynadka, Natalie C.J.

    2015-01-01

    The Gram-negative bacterium enteropathogenic Escherichia coli uses a syringe-like type III secretion system (T3SS) to inject virulence or “effector” proteins into the cytoplasm of host intestinal epithelial cells. To assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetylglucosamine disaccharides cross-linked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from enteropathogenic E. coli. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg white lysozyme. Most notably, the β-hairpin region resembles that of lysozyme and contains an aspartate that aligns with lysozyme Asp-52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families to which the conserved T3SS enzymes had been presumed to belong. Mutation of the EtgA catalytic glutamate, Glu-42, conserved across lytic transglycosylases and hen egg white lysozyme, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis. PMID:25678709

  8. Potassium transport of Salmonella is important for type III secretion and pathogenesis

    PubMed Central

    Liu, Yehao; Ho, Katharina Kim; Su, Jing; Gong, Hao; Chang, Alexander C.

    2013-01-01

    Intracellular cations are essential for the physiology of all living organisms including bacteria. Cations such as potassium ion (K+), sodium ion (Na+) and proton (H+) are involved in nearly all aspects of bacterial growth and survival. K+ is the most abundant cation and its homeostasis in Escherichia coli and Salmonella is regulated by three major K+ transporters: high affinity transporter Kdp and low affinity transporters Kup and Trk. Previous studies have demonstrated the roles of cations and cation transport in the physiology of Escherichia coli; their roles in the virulence and physiology of pathogenic bacteria are not well characterized. We have previously reported that the Salmonella K+ transporter Trk is important for the secretion of effector proteins of the type III secretion system (TTSS) of Salmonella pathogenicity island 1 (SPI-1). Here we further explore the role of Salmonella cation transport in virulence in vitro and pathogenesis in animal models. Impairment of K+ transport through deletion of K+ transporters or exposure to the chemical modulators of cation transport, gramicidin and valinomycin, results in a severe defect in the TTSS of SPI-1, and this defect in the TTSS was not due to a failure to regulate intrabacterial pH or ATP. Our results also show that K+ transporters are critical to the pathogenesis of Salmonella in mice and chicks and are involved in multiple growth and virulence characteristics in vitro, including protein secretion, motility and invasion of epithelial cells. These results suggest that cation transport of the pathogenic bacterium Salmonella, especially K+ transport, contributes to its virulence in addition to previously characterized roles in maintaining homeostasis of bacteria. PMID:23728623

  9. Potassium transport of Salmonella is important for type III secretion and pathogenesis.

    PubMed

    Liu, Yehao; Ho, Katharina Kim; Su, Jing; Gong, Hao; Chang, Alexander C; Lu, Sangwei

    2013-08-01

    Intracellular cations are essential for the physiology of all living organisms including bacteria. Cations such as potassium ion (K(+)), sodium ion (Na(+)) and proton (H(+)) are involved in nearly all aspects of bacterial growth and survival. K(+) is the most abundant cation and its homeostasis in Escherichia coli and Salmonella is regulated by three major K(+) transporters: high affinity transporter Kdp and low affinity transporters Kup and Trk. Previous studies have demonstrated the roles of cations and cation transport in the physiology of Escherichia coli; their roles in the virulence and physiology of pathogenic bacteria are not well characterized. We have previously reported that the Salmonella K(+) transporter Trk is important for the secretion of effector proteins of the type III secretion system (TTSS) of Salmonella pathogenicity island 1 (SPI-1). Here we further explore the role of Salmonella cation transport in virulence in vitro and pathogenesis in animal models. Impairment of K(+) transport through deletion of K(+) transporters or exposure to the chemical modulators of cation transport, gramicidin and valinomycin, results in a severe defect in the TTSS of SPI-1, and this defect in the TTSS was not due to a failure to regulate intrabacterial pH or ATP. Our results also show that K(+) transporters are critical to the pathogenesis of Salmonella in mice and chicks and are involved in multiple growth and virulence characteristics in vitro, including protein secretion, motility and invasion of epithelial cells. These results suggest that cation transport of the pathogenic bacterium Salmonella, especially K(+) transport, contributes to its virulence in addition to previously characterized roles in maintaining homeostasis of bacteria.

  10. The Type III Secretion System Effector SptP of Salmonella enterica Serovar Typhi.

    PubMed

    Johnson, Rebecca; Byrne, Alexander; Berger, Cedric N; Klemm, Elizabeth; Crepin, Valerie F; Dougan, Gordon; Frankel, Gad

    2017-02-15

    Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector.

  11. Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae.

    PubMed

    Matz, Carsten; Moreno, Ana Maria; Alhede, Morten; Manefield, Mike; Hauser, Alan R; Givskov, Michael; Kjelleberg, Staffan

    2008-08-01

    Bacteria and protozoa coexist in a wide range of biofilm communities of natural, technical and medical importance. Generally, this interaction is characterized by the extensive grazing activity of protozoa on bacterial prey populations. We hypothesized that the close spatial coexistence in biofilms should allow opportunistic pathogenic bacteria to utilize their eukaryote-targeting arsenal to attack and exploit protozoan host cells. Studying cocultures of the environmental pathogen Pseudomonas aeruginosa and the amoeba Acanthamoeba castellanii, we found that P. aeruginosa rapidly colonized and killed biofilm-associated amoebae by a quorum-sensing independent mechanism. Analysis of the amoeba-induced transcriptome indicated the involvement of the P. aeruginosa type III secretion system (T3SS) in this interaction. A comparison of mutants with specific defects in the T3SS demonstrated the use of the secretion apparatus and the effectors ExoU, ExoS and ExoT in the killing process, of which ExoU had the greatest impact. T3SS-mediated virulence towards A. castellanii was found to be controlled by the global regulators RpoN and RpoS and through modulation of cAMP and alginate biosynthesis. Our findings suggest that conserved virulence pathways and specifically the T3SS play a central role in bacteria-protozoa interactions in biofilms and may be instrumental for the environmental persistence and evolution of opportunistic bacterial pathogens.

  12. The Salmonella Type III Secretion System Inner Rod Protein PrgJ Is Partially Folded*

    PubMed Central

    Zhong, Dalian; Lefebre, Matthew; Kaur, Kawaljit; McDowell, Melanie A.; Gdowski, Courtney; Jo, Sunhwan; Wang, Yu; Benedict, Stephen H.; Lea, Susan M.; Galan, Jorge E.; De Guzman, Roberto N.

    2012-01-01

    The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65–82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded. PMID:22654099

  13. Modulation of Type III Secretion System in Pseudomonas aeruginosa: Involvement of the PA4857 Gene Product

    PubMed Central

    Zhu, Miao; Zhao, Jingru; Kang, Huaping; Kong, Weina; Zhao, Yuanyu; Wu, Min; Liang, Haihua

    2016-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes serious acute or chronic infections in humans. Acute infections typically involve the type III secretion systems (T3SSs) and bacterial motility, whereas chronic infections are often associated with biofilm formation and the type VI secretion system. To identify new genes required for pathogenesis, a transposon mutagenesis library was constructed and the gene PA4857, named tspR, was found to modulate T3SS gene expression. Deletion of P. aeruginosa tspR reduced the virulence in a mouse acute lung infection model and diminished cytotoxicity. Suppression of T3SS gene expression in the tspR mutant resulted from compromised translation of the T3SS master regulator ExsA. TspR negatively regulated two small RNAs, RsmY and RsmZ, which control RsmA. Our data demonstrated that defects in T3SS expression and biofilm formation in retS mutant could be partially restored by overexpression of tspR. Taken together, our results demonstrated that the newly identified retS-tspR pathway is coordinated with the retS-gacS system, which regulates the genes associated with acute and chronic infections and controls the lifestyle choice of P. aeruginosa. PMID:26858696

  14. 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.

  15. Signalling satiety and starvation to β-Cell insulin secretion.

    PubMed

    Holness, Mark J; Hegazy, Sharif; Sugden, Mary C

    2011-09-01

    The impact of bariatric surgery on insulin sensitivity and glucose tolerance has refocused interest in the role of gut-derived factors in the regulation of insulin secretion and action. The incretins, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) are released from endocrine cells in the small intestinal mucosa primarily in response to oral nutrient ingestion. They have various effects, including augmentation of glucose-stimulated insulin secretion (GSIS), actions that promote the cellular assimilation and storage of dietary glucose and lipid as liver and skeletal muscle glycogen and adipocyte triacylglycerol (TAG) respectively. Similarly, increased delivery of fatty acids (FA) acutely augments GSIS, and the resultant enhancement of GSIS facilitates FA storage as adipocyte TAG. Leptin secretion from white adipocytes curbs appetite to limit dietary nutrient intake and adipocyte TAG storage and, potentially, GSIS, thereby curtailing insulin-dependent TAG storage. On fasting, GSIS is curbed, an effect the mechanism of which is even now incompletely understood, but which may reflect augmented β-cell FA oxidation. The orexigen ghrelin, systemic concentrations of which increase with fasting, exerts enigmatic effects on GSIS, in that acylated ghrelin and unacylated ghrelin exert opposing effects on GSIS, whereas acylated ghrelin and unacylated ghrelin share protective effects on islet survival. This review will build on these emerging studies to evaluate the roles of the incretins, leptin, lipids and acylated and unacylated ghrelin in modulating islet function and survival during feasting and fasting.

  16. Spatial signal correlation from an III-nitride synaptic device

    NASA Astrophysics Data System (ADS)

    Zhang, Shuai; Zhu, Bingcheng; Shi, Zheng; Yuan, Jialei; Jiang, Yuan; Shen, Xiangfei; Cai, Wei; Yang, Yongchao; Wang, Yongjin

    2017-10-01

    The mechanism by which the external environment affects the internal nervous system is investigated via the spatial correlation of an III-nitride synaptic device, which combines in-plane and out-of-plane illumination. The InGaN/GaN multiple-quantum-well collector (MQW-collector) demonstrates a simultaneous light emission and light detection mode due to the unique property of the MQW-diode. The MQW-collector absorbs the internal incoming light and the external illumination at the same time to generate an integration of the excitatory postsynaptic voltages (EPSVs). Signal cognition can be distinctly decoded from the integrated EPSVs because the signal differences are maintained, which is in good agreement with the simulation results. These results suggest that the nervous system can simultaneously amplify the EPSV amplitude and achieve signal cognition by spatial EPSV summation, which can be further optimized to explore the connections between the internal nervous system and the external environment.

  17. Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion

    PubMed Central

    Sanchez-Villamil, Javier; Tapia-Pastrana, Gabriela; Navarro-Garcia, Fernando

    2016-01-01

    cytokine regulation; and (iii) the intracellular bacteria that induce the highest pathways activation and cytokines secretion by using different activation mechanisms. This study provides a comprehensive analysis of how the different pathogenesis schemes of E. coli pathotypes manipulate inflammatory signaling pathways, which leads to a specific proinflammatory cytokine secretion in a cell model infection that reproduce the hallmarks of infection of each pathotype. PMID:27774437

  18. Secret Key Agreement by Soft-Decision of Signals in Gaussian Maurer's Model

    NASA Astrophysics Data System (ADS)

    Naito, Masashi; Watanabe, Shun; Matsumoto, Ryutaroh; Uyematsu, Tomohiko

    We consider the problem of secret key agreement in Gaussian Maurer's Model. In Gaussian Maurer's model, legitimate receivers, Alice and Bob, and a wire-tapper, Eve, receive signals randomly generated by a satellite through three independent memoryless Gaussian channels respectively. Then Alice and Bob generate a common secret key from their received signals. In this model, we propose a protocol for generating a common secret key by using the result of soft-decision of Alice and Bob's received signals. Then, we calculate a lower bound on the secret key rate in our proposed protocol. As a result of comparison with the protocol that only uses hard-decision, we found that the higher rate is obtained by using our protocol.

  19. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    PubMed Central

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  20. IscR Is Essential for Yersinia pseudotuberculosis Type III Secretion and Virulence

    PubMed Central

    Miller, Halie K.; Kwuan, Laura; Schwiesow, Leah; Bernick, David L.; Mettert, Erin; Ramirez, Hector A.; Ragle, James M.; Chan, Patricia P.; Kiley, Patricia J.; Lowe, Todd M.; Auerbuch, Victoria

    2014-01-01

    Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF. PMID:24945271

  1. 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.

  2. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum

    PubMed Central

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  3. EphA4 Receptor Forward Signaling Inhibits Glucagon Secretion From α-Cells.

    PubMed

    Hutchens, Troy; Piston, David W

    2015-11-01

    The loss of inhibition of glucagon secretion exacerbates hyperglycemia in type 1 and 2 diabetes. However, the molecular mechanisms that regulate glucagon secretion in unaffected and diabetic states remain relatively unexplained. We present evidence supporting a new model of juxtacrine-mediated regulation of glucagon secretion where neighboring islet cells negatively regulate glucagon secretion through tonic stimulation of α-cell EphA receptors. Primarily through EphA4 receptors, this stimulation correlates with maintenance of a dense F-actin network. In islets, additional stimulation and inhibition of endogenous EphA forward signaling result in inhibition and enhancement, respectively, of glucagon secretion, accompanied by an increase and decrease, respectively, in α-cell F-actin density. Sorted α-cells lack endogenous stimulation of EphA forward signaling from neighboring cells, resulting in enhanced basal glucagon secretion as compared with islets and the elimination of glucose inhibition of glucagon secretion. Restoration of EphA forward signaling in sorted α-cells recapitulates both normal basal glucagon secretion and glucose inhibition of glucagon secretion. Additionally, α-cell-specific EphA4(-/-) mice exhibit abnormal glucagon dynamics, and EphA4(-/-) α-cells contain less dense F-actin networks than EphA4(+/+) α-cells. This juxtacrine-mediated model provides insight into the functional and dysfunctional regulation of glucagon secretion and opens up new therapeutic strategies for the clinical management of diabetes. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  4. Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

    PubMed Central

    Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

    2010-01-01

    Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

  5. Ca²⁺ signaling and regulation of fluid secretion in salivary gland acinar cells.

    PubMed

    Ambudkar, Indu S

    2014-06-01

    Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca(2+) signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca(2+) is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca(2+)] ([Ca(2+)]i) triggered by IP3-induced release of Ca(2+) from ER via the IP3R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP3Rs determine the site of initiation and the pattern of [Ca(2+)]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+)]i and fluid secretion. This Ca(2+) influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca(2+) entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca(2+) signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca(2+) signal can be ascribed to the polarized arrangement of the Ca(2+) channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca(2+) signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca(2+) signals in the regulation of fluid secretion.

  6. Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

    SciTech Connect

    Kjaerulff, Soren

    2005-10-28

    In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal did not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.

  7. Optimal secretion of alkali-tolerant xylanase in Bacillus subtilis by signal peptide screening.

    PubMed

    Zhang, Weiwei; Yang, Mingming; Yang, Yuedong; Zhan, Jian; Zhou, Yaoqi; Zhao, Xin

    2016-10-01

    Xylanases are industrially important enzymes for xylan digestion. We experimentally screened over 114 Sec and 24 Tat pathway signal peptides, with two different promoters, for optimal production of an alkaline active xylanase (XynBYG) from Bacillus pumilus BYG in a Bacillus subtilis host. Though both promoters yielded highly consistent secretion levels (0.97 Pearson correlation coefficient), the Sec pathway was found to be more efficient than the Tat pathway for XynBYG secretion. Furthermore, the optimal signal peptide (phoB) for XynBYG secretion was found to be different from the optimal peptides for cutinase and esterase reported in previous studies. A partial least squares regression analysis further identified several statistically important variables: helical properties, amino acid composition bias, and the discrimination score in Signal P. These variables explain the observed 23 % variance in the secretion yield of XynBYG by the different signal peptides. The results also suggest that the helical propensity of a signal peptide plays a significant role in the beta-rich xylanase, but not in the helix-rich cutinase, suggesting a coupling of the conformations between the signal peptide and its cargo protein for optimal secretion.

  8. Cell signalling in insulin secretion: the molecular targets of ATP, cAMP and sulfonylurea.

    PubMed

    Seino, S

    2012-08-01

    Clarification of the molecular mechanisms of insulin secretion is crucial for understanding the pathogenesis and pathophysiology of diabetes and for development of novel therapeutic strategies for the disease. Insulin secretion is regulated by various intracellular signals generated by nutrients and hormonal and neural inputs. In addition, a variety of glucose-lowering drugs including sulfonylureas, glinide-derivatives, and incretin-related drugs such as dipeptidyl peptidase IV (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists are used for glycaemic control by targeting beta cell signalling for improved insulin secretion. There has been a remarkable increase in our understanding of the basis of beta cell signalling over the past two decades following the application of molecular biology, gene technology, electrophysiology and bioimaging to beta cell research. This review discusses cell signalling in insulin secretion, focusing on the molecular targets of ATP, cAMP and sulfonylurea, an essential metabolic signal in glucose-induced insulin secretion (GIIS), a critical signal in the potentiation of GIIS, and the commonly used glucose-lowering drug, respectively.

  9. Low molecular weight plant extract induces metabolic changes and the secretion of extracellular enzymes, but has a negative effect on the expression of the type-III secretion system in Xanthomonas campestris pv. campestris.

    PubMed

    Watt, Tony Francis; Vucur, Mihael; Baumgarth, Birgit; Watt, Steven Alexander; Niehaus, Karsten

    2009-03-10

    Xanthomonas campestris pathovar campestris (Xcc) is a plant pathogenic bacterium and as such has to adapt to a variety of environments. During the course of disease, Xcc colonizes the surface of its host, infects the xylem in the early stages, and develops a fully saprophytic life-style, aided by secreted degradative enzymes, in the late stages. To get some insight into this complex regulation, Xcc was cultivated in the presence of low molecular weight host plant extract (<10 kDa). From this experiments it could be observed, that malate and sucrose are taken up preferably in such an environment. Furthermore, it was demonstrated, that the plant extract has a negative effect on the gene expression of the hrp-gene cluster, although the activator hrpG was induced. Also, the secretion of degradative enzymes was shown to be upregulated. These observations indicate, that a low molecular weight plant extract (<10 kDa) is a sufficient signal to regulate metabolic pathways and the secretion of enzymes relevant for the development of virulence in Xanthomonas, but has a negative effect on the expression of genes involved in type-III secretion.

  10. Die another day: molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins

    USDA-ARS?s Scientific Manuscript database

    Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host ...

  11. 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...

  12. Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1

    PubMed Central

    Gong, Siqi; Lei, Lei; Chang, Xiaotong; Belland, Robert; Zhong, Guangming

    2011-01-01

    Using antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N′-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis. PMID:21233161

  13. Genetic analysis of the Salmonella enterica type III secretion-associated ATPase InvC defines discrete functional domains.

    PubMed

    Akeda, Yukihiro; Galán, Jorge E

    2004-04-01

    An essential component of all type III secretion systems is a highly conserved ATPase that shares significant amino acid sequence similarity to the beta subunit of the F(0)F(1) ATPases and is thought to provide the energy for the secretion process. We have performed a genetic and functional analysis of InvC, the ATPase associated with the Salmonella enterica type III secretion system encoded within its pathogenicity island 1. Through a mutagenesis analysis, we have identified amino acid residues that are essential for specific activities of InvC, such as nucleotide hydrolysis and membrane binding. This has allowed us to define discrete domains of InvC that are specifically associated with different essential activities of this protein.

  14. Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach.

    PubMed

    Zilkenat, Susann; Franz-Wachtel, Mirita; Stierhof, York-Dieter; Galán, Jorge E; Macek, Boris; Wagner, Samuel

    2016-05-01

    Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes. Here, we determined the stoichiometry of a type III secretion system of Salmonella enterica serovar Typhimurium using two complementary protocols of gentle complex purification combined with peptide concatenated standard and synthetic stable isotope-labeled peptide-based mass spectrometry. Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems, termed the needle complex, is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. Structural analyses have revealed the stoichiometry of the components of the base, but the stoichiometry of the essential hydrophobic export apparatus components and of the inner rod protein remain unknown. Here, we provide evidence that the export apparatus of type III secretion systems contains five SpaP, one SpaQ, one SpaR, and one SpaS. We confirmed that the previously suggested stoichiometry of nine InvA is valid for assembled needle complexes and describe a loose association of InvA with other needle complex components that may reflect its function. Furthermore, we present evidence that not more than six PrgJ form the inner rod of the needle complex. Providing this structural information will facilitate efforts to obtain an atomic view of type III secretion systems and foster our understanding of the function of these and related flagellar machines. Given that other virulence

  15. Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach*

    PubMed Central

    Zilkenat, Susann; Franz-Wachtel, Mirita; Stierhof, York-Dieter; Galán, Jorge E.; Macek, Boris; Wagner, Samuel

    2016-01-01

    Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes. Here, we determined the stoichiometry of a type III secretion system of Salmonella enterica serovar Typhimurium using two complementary protocols of gentle complex purification combined with peptide concatenated standard and synthetic stable isotope-labeled peptide-based mass spectrometry. Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems, termed the needle complex, is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. Structural analyses have revealed the stoichiometry of the components of the base, but the stoichiometry of the essential hydrophobic export apparatus components and of the inner rod protein remain unknown. Here, we provide evidence that the export apparatus of type III secretion systems contains five SpaP, one SpaQ, one SpaR, and one SpaS. We confirmed that the previously suggested stoichiometry of nine InvA is valid for assembled needle complexes and describe a loose association of InvA with other needle complex components that may reflect its function. Furthermore, we present evidence that not more than six PrgJ form the inner rod of the needle complex. Providing this structural information will facilitate efforts to obtain an atomic view of type III secretion systems and foster our understanding of the function of these and related flagellar machines. Given that other virulence

  16. Links between type III secretion and extracytoplasmic stress responses in Yersinia

    PubMed Central

    Flores-Kim, Josué; Darwin, Andrew J.

    2012-01-01

    The cell envelope of pathogenic bacteria is a barrier against host environmental conditions and immunity molecules, as well as the site where many virulence factors are assembled. Extracytoplasmic stress responses (ESRs) have evolved to help maintain its integrity in conditions where it might be compromised. These ESRs also have important links to the production of envelope-associated virulence systems by the bacteria themselves. One such virulence factor is the type III secretion system (T3SS), the first example of which was provided by the pathogenic Yersinia. This article reviews the reported links between four different ESRs and T3SS function in Yersinia. Components of three of these ESRs affect the function and/or regulation of two different T3SSs. The response regulator of the Rcs ESR is involved in positive regulation of the Ysa-Ysp T3SS found in the highly pathogenic 1B biogroup of Y. enterocolitica. Conversely, the response regulator of the Y. pseudotuberculosis Cpx ESR can down-regulate production of the Ysc-Yop T3SS, and at least one other envelope virulence factor (invasin), by direct repression. Also in Y. pseudotuberculosis, there is some evidence suggesting that an intact RpoE ESR might be important for normal Yersinia outer proteins (Yop) production and secretion. Besides these regulatory links between ESRs and T3SSs, perhaps the most striking connection between T3SS function and an ESR is that between the phage shock protein (Psp) and Ysc-Yop systems of Y. enterocolitica. The Psp response does not affect the regulation or function of the Ysc-Yop system. Instead, Ysc-Yop T3SS production induces the Psp system, which then mitigates T3SS-induced envelope stress. Consequently, the Y. enterocolitica Psp system is essential when the Ysc-Yop T3SS is produced. PMID:23087910

  17. Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

    PubMed Central

    Xu, Xuefang; McAteer, Sean P.; Tree, Jai J.; Shaw, Darren J.; Wolfson, Eliza B. K.; Beatson, Scott A.; Roe, Andrew J.; Allison, Lesley J.; Chase-Topping, Margo E.; Mahajan, Arvind; Tozzoli, Rosangela; Woolhouse, Mark E. J.; Morabito, Stefano; Gally, David L.

    2012-01-01

    Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins. PMID:22615557

  18. Suppression of hedgehog signaling regulates hepatic stellate cell activation and collagen secretion.

    PubMed

    Li, Tao; Leng, Xi-Sheng; Zhu, Ji-Ye; Wang, Gang

    2015-01-01

    Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.

  19. Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles.

    PubMed

    Chen, Xiaotong; Choudhari, Shyamal P; Kumar, Prashant; Toth, Ronald T; Kim, Jae Hyun; Van Roosmalen, Maarten L; Leenhouts, Kees; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2015-12-01

    Diarrhea caused by Shigella, Salmonella, and Yersinia is an important public health problem, but development of safe and effective vaccines against such diseases is challenging. A new antigen delivery platform called bacterium-like particles (BLPs) was explored as a means for delivering protective antigens from the type III secretion systems (T3SS) of these pathogens. BLPs are peptidoglycan skeletons derived from Lactococcus lactis that are safe for newborns and can carry multiple antigens. Hydrophobic T3SS translocator proteins were fused to a peptidoglycan anchor (PA) for BLP attachment. The proteins and protein-BLP complexes associated with BLPs were characterized and the resulting data used to create three-index empirical phase diagrams (EPDs). On the basis of these EPDs, IpaB (Shigella) and SipB (Salmonella) behave distinctly from YopB (Yersinia) under different environmental stresses. Adding the PA domain appears to enhance the stability of both the PA and translocator proteins, which was confirmed using differential scanning calorimetry, and although the particles dominated the spectroscopic signals in the protein-loaded BLPs, structural changes in the proteins were still detected. The protein-BLPs were most stable near neutral pH, but these proteins' hydrophobicity made them sensitive to environmental stresses. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  20. BEAN 2.0: an integrated web resource for the identification and functional analysis of type III secreted effectors.

    PubMed

    Dong, Xiaobao; Lu, Xiaotian; Zhang, Ziding

    2015-01-01

    Gram-negative pathogenic bacteria inject type III secreted effectors (T3SEs) into host cells to sabotage their immune signaling networks. Because T3SEs constitute a meeting-point of pathogen virulence and host defense, they are of keen interest to host-pathogen interaction research community. To accelerate the identification and functional understanding of T3SEs, we present BEAN 2.0 as an integrated web resource to predict, analyse and store T3SEs. BEAN 2.0 includes three major components. First, it provides an accurate T3SE predictor based on a hybrid approach. Using independent testing data, we show that BEAN 2.0 achieves a sensitivity of 86.05% and a specificity of 100%. Second, it integrates a set of online sequence analysis tools. Users can further perform functional analysis of putative T3SEs in a seamless way, such as subcellular location prediction, functional domain scan and disorder region annotation. Third, it compiles a database covering 1215 experimentally verified T3SEs and constructs two T3SE-related networks that can be used to explore the relationships among T3SEs. Taken together, by presenting a one-stop T3SE bioinformatics resource, we hope BEAN 2.0 can promote comprehensive understanding of the function and evolution of T3SEs.

  1. Expression hierarchy in the Yersinia type III secretion system established through YopD recognition of RNA.

    PubMed

    Chen, Yuqing; Anderson, Deborah M

    2011-05-01

    The Yersinia type III secretion system (T3SS) is environmentally responsive to enable its rapid induction upon contact with host cells and is necessary for Yersiniae to establish a replicative niche and cause disease. YopD, a translocator protein, represses the expression of T3SS genes until signalled by environmental cues, a mechanism known as the low calcium response. In this work, we investigated recognition of target genes by Yersinia pestis YopD. Expression of all genes of the T3SS was induced in a yopD mutant, though not to the same degree, with effector Yops most affected. Two, short AU-rich sequence elements up- and downstream of start codons of target genes were necessary but not sufficient for YopD mediated repression. Purified YopD-LcrH bound specifically to target RNAs in vitro with different relative affinities, with effector Yops having greater affinity. Together, the data suggest YopD binds to T3SS transcripts where it may prevent ribosome binding causing accelerated mRNA degradation. This regulatory mechanism may ensure an expression hierarchy during the low calcium response as low affinity YopD targets such as chaperones would be translated prior to high affinity targets such as effector Yops allowing the bacteria another layer of control over Yop translocation during infection. © 2011 Blackwell Publishing Ltd.

  2. 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

    2015-10-12

    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.

  3. Preprotein mature domains contain translocase targeting signals that are essential for secretion.

    PubMed

    Chatzi, Katerina E; Sardis, Marios Frantzeskos; Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G; Karamanou, Spyridoula; Economou, Anastassios

    2017-05-01

    Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. © 2017 Chatzi et al.

  4. Resolvin D1 Increases Mucin Secretion in Cultured Rat Conjunctival Goblet Cells via Multiple Signaling Pathways

    PubMed Central

    Lippestad, Marit; Hodges, Robin R.; Utheim, Tor P.; Serhan, Charles N.; Dartt, Darlene A.

    2017-01-01

    Purpose Goblet cells in the conjunctiva secrete mucin into the tear film protecting the ocular surface. The proresolution mediator resolvin D1 (RvD1) regulates mucin secretion to maintain homeostasis during physiological conditions and in addition, actively terminates inflammation. We determined the signaling mechanisms used by RvD1 in cultured rat conjunctival goblet cells to increase intracellular [Ca2+] ([Ca2+]i) and induce glycoconjugate secretion. Methods Increase in [Ca2+]i were measured using fura 2/AM and glycoconjugate secretion determined using an enzyme-linked lectin assay with the lectin Ulex Europaeus Agglutinin 1. Signaling pathways activated by RvD1 were studied after goblet cells were pretreated with signaling pathway inhibitors before stimulation with RvD1. The results were compared with results when goblet cells were stimulated with RvD1 alone and percent inhibition calculated. Results The increase in [Ca2+]i stimulated by RvD1 was blocked by inhibitors to phospholipases (PL-) -D, -C, -A2, protein kinase C (PKC), extracellular signal-regulated kinases (ERK)1/2 and Ca2+/calmodulin-dependent kinase (Ca2+/CamK). Glycoconjugate secretion was significantly inhibited by PLD, -C, -A2, ERK1/2 and Ca2+/CamK, but not PKC. Conclusions We conclude that RvD1 increases glycoconjugate secretion from goblet cells via multiple signaling pathways including PLC, PLD, and PLA2, as well as their signaling components ERK1/2 and Ca2+/CamK to preserve the mucous layer and maintain homeostasis by protecting the eye from desiccating stress, allergens, and pathogens. PMID:28892824

  5. Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides.

    PubMed

    Fernandez-Brando, Romina J; Yamaguchi, Nao; Tahoun, Amin; McAteer, Sean P; Gillespie, Trudi; Wang, Dai; Argyle, Sally A; Palermo, Marina S; Gally, David L

    2015-11-02

    A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS. Copyright © 2015 Fernandez-Brando et al.

  6. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    NASA Astrophysics Data System (ADS)

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-12-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

  7. Hyperinvasiveness of Salmonella enterica serovar Choleraesuis linked to hyperexpression of type III secretion systems in vitro

    PubMed Central

    Huang, Kuan-Yeh; Wang, Yi-Hsin; Chien, Kun-Yi; Janapatla, Rajendra Prasad; Chiu, Cheng-Hsun

    2016-01-01

    Salmonella enterica serovars Choleraesuis and Typhimurium are among the non-typhoid Salmonella serovars that are important zoonotic pathogens. In clinical observation, S. Typhimurium typically causes diarrheal diseases; however, S. Choleraesuis shows high predilection to cause bacteremia. The mechanism why S. Choleraesuis is more invasive to humans remains unknown. In this study, we compared the S. Typhimurium LT2 and S. Choleraesuis SC-B67 proteomes through stable isotope labeling of amino acid in cell culture (SILAC). In SILAC, the expression of many virulence proteins in two type III secretion systems (T3SSs) were significantly higher in S. Choleraesuis than in S. Typhimurium. Similar differences were also found at the transcriptional level. Compared to S. Typhimurium, S. Choleraesuis showed a higher penetration level to Caco-2 (>100-fold) and MDCK (>10-fold) monolayers. In mice after oral challenge, the invasion of spleen and liver was also higher in S. Choleraesuis than in S. Typhimurium. The transcription of hilD in S. Choleraesuis was increased in physiological (1 mM) or high (10 mM) concentrations of Mg2+, but not in low (8 μM) concentration. We conclude that S. Choleraesuis showed hyperinvasiveness in cellular as well as mouse models due to hyperexpression of T3SS genes. PMID:27886215

  8. Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides

    PubMed Central

    Fernandez-Brando, Romina J.; Yamaguchi, Nao; Tahoun, Amin; McAteer, Sean P.; Gillespie, Trudi; Wang, Dai; Argyle, Sally A.; Palermo, Marina S.

    2015-01-01

    A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS. PMID:26525795

  9. Erwinia amylovora modifies phenolic profiles of susceptible and resistant apple through its type III secretion system.

    PubMed

    Pontais, Isabelle; Treutter, Dieter; Paulin, Jean-Pierre; Brisset, Marie-Noëlle

    2008-03-01

    Fire blight is a disease affecting Maloideae caused by the necrogenic bacterium Erwinia amylovora, which requires the type III protein secretion system (TTSS) for pathogenicity. Profiles of methanol-extractable leaf phenolics of two apple (Malus x domestica) genotypes with contrasting susceptibility to this disease were analyzed by HPLC after infection. Some qualitative differences were recorded between the constitutive compositions of the two genotypes but in both of them dihydrochalcones accounted for more than 90% of total phenolics. Principal component analysis separated leaves inoculated with a virulent wild-type strain from those inoculated with a non-pathogenic TTSS-defective mutant or with water. The changes in levels of the various groups of phenolics in response to the virulent bacterium were similar between the two genotypes, with a significant decrease of dihydrochalcones and a significant increase of hydroxycinnamate derivatives. Differences between genotypes were, however, recorded in amplitude and kinetic of variation in these groups. Occurrence of oxidation and polymerization reactions is proposed, based on the browning process of infected tissues, but whether some by-products act in defense as toxic compounds remain to be tested. Among direct antibacterial constitutive compounds present in apple leaves, the dihydrochalcone phloretin only was found at levels close to lethal concentrations in both genotypes. However, E. amylovora exhibited the ability to stabilize this compound at sublethal levels even in the resistant apple, rejecting the hypothesis of its involvement in the resistance of this genotype.

  10. A bacterial type III secretion-based protein delivery tool for broad applications in cell biology

    PubMed Central

    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.

    2015-01-01

    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. PMID:26598622

  11. Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains▿

    PubMed Central

    Kim, Hye-Sook; Ma, Bing; Perna, Nicole T.; Charkowski, Amy O.

    2009-01-01

    Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers. PMID:19411432

  12. Phylogeny and virulence of naturally occurring type III secretion system-deficient Pectobacterium strains.

    PubMed

    Kim, Hye-Sook; Ma, Bing; Perna, Nicole T; Charkowski, Amy O

    2009-07-01

    Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.

  13. Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos.

    PubMed

    Brannon, Mark K; Davis, J Muse; Mathias, Jonathan R; Hall, Chris J; Emerson, Julia C; Crosier, Philip S; Huttenlocher, Anna; Ramakrishnan, Lalita; Moskowitz, Samuel M

    2009-05-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function. © 2009 Blackwell Publishing Ltd.

  14. Oligoribonuclease is required for the type III secretion system and pathogenesis of Pseudomonas aeruginosa.

    PubMed

    Chen, Gukui; Zhao, Qiang; Zhu, Feng; Chen, Ronghao; Jin, Yongxin; Liu, Chang; Pan, Xiaolei; Jin, Shouguang; Wu, Weihui; Cheng, Zhihui

    2016-01-01

    Oligoribonuclease (Orn) is a 3' to 5' exonuclease that degrades nanoRNAs, which can serve as primers for transcription initiation at a significant fraction of promoters. One of Orn's substrates, pGpG inhibits the enzymatic activity of EAL-domain containing phosphodiesterases (PDEs), thereby increasing intracellular cyclic-di-GMP (c-di-GMP) level. Here, we found that an orn mutant of Pseudomonas aeruginosa displayed reduced cytotoxicity, which was mainly due to deficient type III secretion system (T3SS). Given the importance of T3SS in pathogenicity, we examined the bacterial virulence in a mouse acute pneumonia model and found that the Δorn mutant was highly attenuated compared to the wild type PA14 strain. Overexpression of an EAL domain-containing PDE reduced the c-di-GMP level as well as biofilm formation in the Δorn mutant. However, no effect was observed on the expression of T3SS genes, suggesting that increased c-di-GMP level is not the solely cause of defective T3SS in the Δorn mutant. Overall, our results demonstrated an essential role of Orn in the expression of T3SS as well as pathogenesis of P. aeruginosa. Copyright © 2016 Elsevier GmbH. All rights reserved.

  15. The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system.

    PubMed

    Anderson, Gregory G; Yahr, Timothy L; Lovewell, Rustin R; O'Toole, George A

    2010-03-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes life-long pneumonia in individuals with cystic fibrosis (CF). These long-term infections are maintained by bacterial biofilm formation in the CF lung. We have recently developed a model of P. aeruginosa biofilm formation on cultured CF airway epithelial cells. Using this model, we discovered that mutation of a putative magnesium transporter gene, called mgtE, led to increased cytotoxicity of P. aeruginosa toward epithelial cells. This altered toxicity appeared to be dependent upon expression of the type III secretion system (T3SS). In this study, we found that mutation of mgtE results in increased T3SS gene transcription. Through epistasis analyses, we discovered that MgtE influences the ExsE-ExsC-ExsD-ExsA gene regulatory system of T3SS by either directly or indirectly inhibiting ExsA activity. While variations in calcium levels modulate T3SS gene expression in P. aeruginosa, we found that addition of exogenous magnesium did not inhibit T3SS activity. Furthermore, mgtE variants that were defective for magnesium transport could still complement the cytotoxicity effect. Thus, the magnesium transport function of MgtE does not fully explain the regulatory effects of MgtE on cytotoxicity. Overall, our results indicate that MgtE modulates expression of T3SS genes.

  16. Molecular Models for the Core Components of the Flagellar Type-III Secretion Complex

    PubMed Central

    Matthews-Palmer, Teige R. S.; Beeby, Morgan

    2016-01-01

    We show that by using a combination of computational methods, consistent three-dimensional molecular models can be proposed for the core proteins of the type-III secretion system. We employed a variety of approaches to reconcile disparate, and sometimes inconsistent, data sources into a coherent picture that for most of the proteins indicated a unique solution to the constraints. The range of difficulty spanned from the trivial (FliQ) to the difficult (FlhA and FliP). The uncertainties encountered with FlhA were largely the result of the greater number of helix packing possibilities allowed in a large protein, however, for FliP, there remains an uncertainty in how to reconcile the large displacement predicted between its two main helical hairpins and their ability to sit together happily across the bacterial membrane. As there is still no high resolution structural information on any of these proteins, we hope our predicted models may be of some use in aiding the interpretation of electron microscope images and in rationalising mutation data and experiments. PMID:27855178

  17. Benzylidene acylhydrazides inhibit chlamydial growth in a type III secretion- and iron chelation-independent manner.

    PubMed

    Bao, Xiaofeng; Gylfe, Asa; Sturdevant, Gail L; Gong, Zheng; Xu, Shuang; Caldwell, Harlan D; Elofsson, Mikael; Fan, Huizhou

    2014-08-15

    Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  18. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation

    PubMed Central

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-01-01

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  19. The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system.

    PubMed

    Lefebre, Matthew D; Galán, Jorge E

    2014-01-14

    Type III secretion machines are essential for the biology of many bacteria that are pathogenic or symbiotic for animals, plants, or insects. They exert their function by delivering bacterial effector proteins into target eukaryotic cells. The core component of these machines is the needle complex, a multiprotein structure that spans the bacterial envelope and serves as a conduit for proteins that transit this secretion pathway. The needle complex is composed of a multiring base embedded in the bacterial envelope and a filament-like structure, the needle, that projects from the bacterial surface and is linked to the base by the inner rod. Assembly of the needle complex proceeds in a step-wise fashion that is initiated by the assembly of the base and is followed by the export of the building subunits for the needle and inner rod substructures. Once assembled, the needle complex reprograms its specificity and becomes competent for the secretion of effector proteins. Here through genetic, biochemical, and electron microscopy analyses of the Salmonella inner rod protein subunit PrgJ we present evidence that the assembly of the inner rod dictates the timing of substrate switching and needle length. Furthermore, the identification of mutations in PrgJ that specifically alter the hierarchy of protein secretion provides additional support for a complex role of the inner rod substructure in type III secretion.

  20. A dominant-negative needle mutant blocks type III secretion of early but not late substrates in Yersinia.

    PubMed

    Davis, Alison J; Díaz, Dennise A De Jesús; Mecsas, Joan

    2010-04-01

    Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to deliver effectors into host cells. A key component of the T3SS is the needle, which is a hollow tube on the bacterial surface through which effectors are secreted, composed of the YscF protein. To study needle assembly, we performed a screen for dominant-negative yscF alleles that prevented effector secretion in the presence of wild-type (WT) YscF. One allele, yscF-L54V, prevents WT YscF secretion and needle assembly, although purified YscF-L54V polymerizes in vitro. YscF-L54V binds to its chaperones YscE and YscG, and the YscF-L54V-EG complex targets to the T3SS ATPase, YscN. We propose that YscF-L54V stalls at a binding site in the needle assembly pathway following its release from the chaperones, which blocks the secretion of WT YscF and other early substrates required for building a needle. Interestingly, YscF-L54V does not affect the activity of pre-assembled actively secreting machines, indicating that a factor and/or binding site required for YscF secretion is absent from T3SS machines already engaged in effector secretion. Thus, substrate switching may involve the removal of an early substrate-specific binding site as a mechanism to exclude early substrates from Yop-secreting machines.

  1. A dominant-negative needle mutant blocks type III secretion of early but not late substrates in Yersinia

    PubMed Central

    Davis, Alison J.; De Jesús Díaz, Dennise A.; Mecsas, Joan

    2010-01-01

    Summary Yersinia pseudotuberculosis uses a Type III Secretion System (T3SS) to deliver effectors into host cells. A key component of the T3SS is the needle, which is a hollow tube on the bacterial surface through which effectors are secreted, composed of the YscF protein. To study needle assembly, we performed a screen for dominant-negative yscF alleles that prevented effector secretion in the presence of wild-type (WT) YscF. One allele, yscF-L54V, prevents WT YscF secretion and needle assembly, although purified YscF-L54V polymerizes in vitro. YscF-L54V binds to its chaperones YscE and YscG, and the YscF-L54V-EG complex targets to the T3SS ATPase, YscN. We propose that YscF-L54V stalls at a binding site in the needle assembly pathway following its release from the chaperones, which blocks the secretion of WT YscF and other early substrates required for building a needle. Interestingly, YscF-L54V does not affect the activity of pre-assembled actively secreting machines, indicating that a factor and/or binding site required for YscF secretion is absent from T3SS machines already engaged in effector secretion. Thus, substrate switching may involve the removal of an early substrate-specific binding site as a mechanism to exclude early substrates from Yop-secreting machines. PMID:20199604

  2. Identification and Characterization of Putative Translocated Effector Proteins of the Edwardsiella ictaluri Type III Secretion System

    PubMed Central

    Dubytska, Lidiya P.; Rogge, Matthew L.

    2016-01-01

    ABSTRACT Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000

  3. The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

    PubMed Central

    Sha, Jian; Pillai, Lakshmi; Fadl, Amin A.; Galindo, Cristi L.; Erova, Tatiana E.; Chopra, Ashok K.

    2005-01-01

    Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first

  4. From ingestion to colonization: the influence of the host environment on regulation of the LEE encoded type III secretion system in enterohaemorrhagic Escherichia coli

    PubMed Central

    Connolly, James P. R.; Finlay, B. Brett; Roe, Andrew J.

    2015-01-01

    Enterohaemorrhagic Escherichia coli (EHEC) binds to host tissue and intimately attaches to intestinal cells using a dedicated type III secretion system (T3SS). This complex multi-protein organelle is encoded within a large pathogenicity island called the locus of enterocyte effacement (LEE), which is subject to extensive regulatory control. Over the past 15 years we have gained a wealth of knowledge concerning how the LEE is regulated transcriptionally by specific, global and phage encoded regulators. More recently, significant advances have been made in our understanding of how specific signals, including host or microbiota derived metabolic products and various nutrient sources, can affect how the LEE-encoded T3SS is regulated. In this review we discuss regulation of the LEE, focusing on how these physiologically relevant signals are sensed and how they affect the expression of this major virulence factor. The implications for understanding the disease process by specific regulatory mechanisms are also discussed. PMID:26097473

  5. Cross-Talk between the Aeromonas hydrophila Type III Secretion System and Lateral Flagella System

    PubMed Central

    Zhao, Yu-Hang; Shaw, Jonathan G.

    2016-01-01

    Aeromonas hydrophila is responsible for aeromonad septicaemia in fish, and gastroenteritis and wound infections in humans. The type III secretion system (T3SS) is utilized by aeromonads to inject protein effectors directly into host cells. One of the major genetic regulators of the T3SS in several bacterial species is the AraC-like protein ExsA. Previous studies have suggested a link between T3SS regulation and lateral flagella expression. The aim of this study was to determine the genetic regulation of the T3SS and its potential interaction with the lateral flagella system in A. hydrophila. To investigate the genes encoding the T3SS regulatory components exsA, exsD, exsC, and exsE were mutated and the activities of the T3SS promoters were measured in wild type and mutant backgrounds demonstrating a regulatory network. The Exs proteins were shown to interact with each other by BACTH assay and Far-Western Blot. The findings suggested a regulatory cascade in which ExsE was bound to the chaperone protein ExsC. When ExsC was free it sequestered the anti-activator ExsD thus stopping the inhibition of the T3SS master regulator ExsA allowing T3SS expression. The T3SS regulatory components were also shown to affect the expression of the lateral flagella system. The activities of the lateral flagella promoters were shown to be repressed by the absence of ExsD and ExsE, suggesting that the T3SS master regulator ExsA was a negative regulator of the lateral flagella system. PMID:27656180

  6. Structural Basis of Chaperone Recognition of Type III Secretion System Minor Translocator Proteins*

    PubMed Central

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-01-01

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the “translocators”) are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development. PMID:20385547

  7. Structural basis of chaperone recognition of type III secretion system minor translocator proteins.

    PubMed

    Job, Viviana; Matteï, Pierre-Jean; Lemaire, David; Attree, Ina; Dessen, Andréa

    2010-07-23

    The type III secretion system (T3SS) is a complex nanomachine employed by many Gram-negative pathogens, including the nosocomial agent Pseudomonas aeruginosa, to inject toxins directly into the cytoplasm of eukaryotic cells. A key component of all T3SS is the translocon, a proteinaceous channel that is inserted into the target membrane, which allows passage of toxins into target cells. In most bacterial species, two distinct membrane proteins (the "translocators") are involved in translocon formation, whereas in the bacterial cytoplasm, however, they remain associated to a common chaperone. To date, the strategy employed by a single chaperone to recognize two distinct translocators is unknown. Here, we report the crystal structure of a complex between the Pseudomonas translocator chaperone PcrH and a short region from the minor translocator PopD. PcrH displays a 7-helical tetratricopeptide repeat fold that harbors the PopD peptide within its concave region, originally believed to be involved in recognition of the major translocator, PopB. Point mutations introduced into the PcrH-interacting region of PopD impede translocator-chaperone recognition in vitro and lead to impairment of bacterial cytotoxicity toward macrophages in vivo. These results indicate that T3SS translocator chaperones form binary complexes with their partner molecules, and the stability of their interaction regions must be strictly maintained to guarantee bacterial infectivity. The PcrH-PopD complex displays homologs among a number of pathogenic strains and could represent a novel, potential target for antibiotic development.

  8. Role of host cell polarity and leading edge properties in Pseudomonas type III secretion

    PubMed Central

    Bridge, Dacie R.; Novotny, Matthew J.; Moore, Elizabeth R.; Olson, Joan C.

    2010-01-01

    Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration. PMID:19910414

  9. Immunodominant regions of a Chlamydia trachomatis type III secretion effector protein, Tarp.

    PubMed

    Wang, Jie; Zhang, Yingqian; Yu, Ping; Zhong, Guangming

    2010-09-01

    We have previously shown that individuals infected with Chlamydia trachomatis can develop a robust antibody response to a Chlamydia type III secretion effector protein called Tarp and that immunization with Tarp induces protection against challenge infection in mice. The current study aimed to map the immunodominant regions of the Tarp protein by expressing 11 fragments of Tarp as glutathione S-transferase (GST) fusion proteins and detecting the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms. A major immunodominant region was strongly recognized by all antibodies. This region covers amino acids 152 to 302, consisting of three repeats (amino acids 152 to 201, 202 to 251, and 252 to 302). Each of the repeats contains multiple tyrosine residues that are phosphorylated by host cell kinases when Tarp is injected into host cells. Several other minor immunodominant regions were also identified, including those comprising amino acids 1 to 156, 310 to 431, and 582 to 682 (recognized by antisera from both humans and rabbits), that comprising amino acids 425 to 581 (recognized only by human antisera), and that comprising amino acids 683 to 847 (preferentially recognized by rabbit antisera). This immunodominance was also confirmed by the observations that six out of the nine monoclonal antibodies (MAbs) bound to the major immunodominant region and that the other three each bound to one of the minor fragments, comprising amino acids 1 to 119, 120 to 151, and 310 to 431. The antigenicity analyses have provided important information for further understanding the structure and function of Tarp.

  10. The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus.

    PubMed

    Zhao, Yue; Yang, Jieling; Shi, Jianjin; Gong, Yi-Nan; Lu, Qiuhe; Xu, Hao; Liu, Liping; Shao, Feng

    2011-09-14

    Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity. © 2011 Macmillan Publishers Limited. All rights reserved

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

    SciTech Connect

    Meshcheryakov, Vladimir A.; Kitao, Akio; Matsunami, Hideyuki; Samatey, Fadel A.

    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, the 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.

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

    SciTech Connect

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

    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. We 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.

  13. Chlamydia pneumoniae CopD Translocator Protein Plays a Critical Role in Type III Secretion (T3S) and Infection

    PubMed Central

    Bulir, David C.; Waltho, Daniel A.; Stone, Christopher B.; Mwawasi, Kenneth A.; Nelson, Jordan C.; Mahony, James B.

    2014-01-01

    Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120–125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae. PMID:24959658

  14. Chlamydia pneumoniae CopD translocator protein plays a critical role in type III secretion (T3S) and infection.

    PubMed

    Bulir, David C; Waltho, Daniel A; Stone, Christopher B; Mwawasi, Kenneth A; Nelson, Jordan C; Mahony, James B

    2014-01-01

    Pathogenic Gram-negative bacteria use type III secretion (T3S) to inject effector proteins into the host cell to create appropriate conditions for infection and intracellular replication. Chlamydia spp. are believed to use T3S to infect their host cell, and the translocator proteins are an essential component of this system. Chlamydia pneumoniae contains genes encoding two sets of translocator proteins; CopB and CopD, and CopB2 and CopD2. In this study, we identified novel interactions between CopD and three type III secretion proteins; namely, CopN, CdsN, and CdsF. We identified a CopD putative chaperone binding motif, PxLxxP, within the N-terminal region (CopD amino acids 120-125), which was necessary for interaction with its putative chaperone LcrH_1. Using size exclusion chromatography, we showed that CopD and LcrH_1 formed higher order structures in solution with CopD and LcrH_1 binding in a ratio of 1∶1, which is unique for T3SS translocator proteins. Lastly, we showed that antibodies to CopD reduced C. pneumoniae infectivity by >95%. Collectively, this data suggests that CopD plays a critical role in pathogenesis and likely functions as a hydrophobic translocator of the type III secretion system in Chlamydia pneumoniae.

  15. Aip regulates cAMP signalling and GH secretion in GH3 cells.

    PubMed

    Formosa, R; Xuereb-Anastasi, A; Vassallo, J

    2013-08-01

    Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been linked to predisposition to pituitary adenomas. However, the mechanism by which this occurs remains unknown. AIP interacts with a number of interesting proteins, including members of the cAMP signalling pathway that has been shown to be consistently altered in pituitary tumours. The functional role of Aip was investigated using both over-expression and knock down of Aip in GH3 cells. cAMP signalling and its downstream effectors, including GH secretion, were then investigated. cAMP signalling was analysed using cAMP assays, cAMP-response element-promoter luciferase reporter assays, real-time PCR and finally secreted GH quantification. Over-expression of wild-type (WT)-Aip reduced forskolin-induced cAMP signalling at the total cAMP level, luciferase reporter activity and target gene expression, when compared with empty vector and the non-functional R304X mutant. Additionally, GH secretion was reduced in WT-Aip over-expressing GH3 cells treated with forskolin. Knock down of endogenous Aip resulted in increased cAMP signalling but a decrease in GH secretion was also noted. Inhibition of phosphodiesterase activity using general and selective inhibitors did not completely ablate the effect of Aip on forskolin-augmented cAMP signalling. A mechanism by which Aip acts as a tumour suppressor, by maintaining a low cAMP signalling and concentration, is suggested. Mutations of Aip render the protein incapable of such activity. This effect appears not to be mediated by the AIP-PDE interaction, suggesting the involvement of other interacting partners in mediating this outcome.

  16. Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

    PubMed

    Shibasaki, T; Takahashi, T; Takahashi, H; Seino, S

    2014-09-01

    Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic β-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²⁺ , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in β-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic β-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.

  17. Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

    PubMed

    Sharma, Shailendra; Sharma, Shiveta; Hirabuchi, Akiko; Yoshida, Kentaro; Fujisaki, Koki; Ito, Akiko; Uemura, Aiko; Terauchi, Ryohei; Kamoun, Sophien; Sohn, Kee Hoon; Jones, Jonathan D G; Saitoh, Hiromasa

    2013-05-01

    Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  18. Engineering NK Cells Modified With an EGFRvIII-specific Chimeric Antigen Receptor to Overexpress CXCR4 Improves Immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma.

    PubMed

    Müller, Nadja; Michen, Susanne; Tietze, Stefanie; Töpfer, Katrin; Schulte, Alexander; Lamszus, Katrin; Schmitz, Marc; Schackert, Gabriele; Pastan, Ira; Temme, Achim

    2015-06-01

    Natural killer (NK) cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK cell-resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. On the basis of the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an epidermal growth factor variant III (EGFRvIII)-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII glioblastoma cells in vitro and to established subcutaneous U87-MG tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared with NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared with the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor-engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

  19. Use of a Novel Report Protein to Study the Secretion Signal of Flagellin in Bacillus subtilis.

    PubMed

    Wang, Guangqiang; Xia, Yongjun; Xiong, Zhiqiang; Zhang, Hui; Ai, Lianzhong

    2016-08-01

    Flagellin (also called Hag) is the main component of bacterial flagellum and is transported across the cytoplasmic membrane by flagellar secretion apparatus. Because flagella play an essential role in the pathogenesis of numerous pathogens, the flagellins of Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Campylobacter jejuni, and Vibrio cholerae have been intensively studied; however, very few studies have focused on the flagellin of Bacillus subtilis, which is considered to be a model organism with which to study the secretion of bacteria and is used on an industrial scale for the secretion of proteins. The signal of B. subtilis flagellin is still debated. This study was performed to seek the export signals of flagellin from B. subtilis. The naturally nonsecretory, intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was used as the reporter protein. Our results demonstrate that the export signal is contained within the first 50 amino acids of B. subtilis flagellin. Nsp is easily degraded inside the cell and can be exported into culture medium with the aid of the signal of flagellin. This method provides a new potential strategy for the expression of proteins with high proteolytic susceptibility via fusion to export signals.

  20. The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion.

    PubMed

    Shimizu, Takeshi; Ichimura, Kimitoshi; Noda, Masatoshi

    2015-12-07

    Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection.

  1. The Erwinia amylovora PhoPQ system is involved in resistance to antimicrobial peptide and suppresses gene expression of two novel type III secretion systems.

    PubMed

    Nakka, Sridevi; Qi, Mingsheng; Zhao, Youfu

    2010-10-20

    The PhoPQ system is a pleiotropic two-component signal transduction system that controls many pathogenic properties in several mammalian and plant pathogens. Three different cues have been demonstrated to activate the PhoPQ system including a mild acidic pH, antimicrobial peptides, and low Mg(2+). In this study, our results showed that phoPQ mutants were more resistant to strong acidic conditions (pH 4.5 or 5) than that of the wild-type (WT) strain, suggesting that this system in Erwinia amylovora may negatively regulate acid resistance gene expression. Furthermore, the PhoPQ system negatively regulated gene expression of two novel type III secretion systems in E. amylovora. These results are in contrast to those reported for the PhoPQ system in Salmonella and Xanthomonas, where it positively regulates type III secretion system and acid resistance. In addition, survival of phoPQ mutants was about 10-fold lower than that of WT when treated with cecropin A at pH 5.5, suggesting that the PhoPQ system renders the pathogen more resistant to cecropin A.

  2. Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides.

    PubMed Central

    Chang, C N; Matteucci, M; Perry, L J; Wulf, J J; Chen, C Y; Hitzeman, R A

    1986-01-01

    Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN-alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion. Images PMID:3023906

  3. [Analysis of signal peptides of the secreted proteins in Agrobacterium tumefaciens C58].

    PubMed

    Fan, Cheng-Ming; Li, Cheng-Yun; Zhao, Ming-Fu; He, Yue-Qiu

    2005-08-01

    The 4554 ORFs of Agrobacterium tumefaciens C58 Cereon were used for the prediction of signal peptides by the network tools, such as SignalP3.0, LipoP1.0, TMHMM2.0 and TargetP1.01. Total 203 signal peptides with conserved amino residues are found, among them, 158 are secretary types, 9 are RR-motif types, 28 are SignalPase II types and 8 are bacteriocin-pheromone types. However, only two signal peptides from the secreted proteins, AGR-C-1878p and AGR-C-1880p have the same amino sequences, showing the signal peptides of the strain are highly variable.

  4. Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma.

    PubMed

    Shen, Chan-Juan; Yang, Yu-Xiu; Han, Ethan Q; Cao, Na; Wang, Yun-Fei; Wang, Yi; Zhao, Ying-Ying; Zhao, Li-Ming; Cui, Jian; Gupta, Puja; Wong, Albert J; Han, Shuang-Yin

    2013-05-09

    Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain. A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model. Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII

  5. Inheritance of Pantoea type III secretion systems through both vertical and horizontal transfer.

    PubMed

    Kirzinger, Morgan W B; Butz, Cory J; Stavrinides, John

    2015-12-01

    The type III secretion system (T3SS) is an extracellular apparatus used by many Gram-negative bacteria to deliver effector proteins directly into plant and animal cells, thereby facilitating host-specific association. Strains of the enterobacterial genus, Pantoea, have been isolated from a wide variety of hosts, including plants, insects, and humans, yet it is unclear whether the T3SS may be involved in these associations. In this study, we use comparative genomics and phylogenetic methods to examine the origin and distribution of T3SSs in 35 sequenced environmental and clinical strains of Pantoea. We began our analysis by examining the distribution of the previously characterized plant cell-specific PSI-1 and animal cell-specific PSI-2 of the plant pathogenic Pantoea stewartii subsp. stewartii DC283 (PstDC283), and showed that both had a somewhat limited distribution. Our analysis, however, identified two variants of a unique plant cell-specific T3SS (PSI-1a and PSI-1b) in six Pantoea strains, including a clinical isolate. Our genome analysis of PstDC283 also identified a third T3SS that we named PSI-3, which has a similar genetic content and organization to the Salmonella, animal cell-specific SPI-2 system. Phylogenetic analysis of all three systems suggests that the PSI-1 system has been inherited vertically, whereas the newly identified PSI-1a and PSI-1b systems have been acquired independently from other genera within the Enterobacteriaceae. PSI-2 appears to have been acquired horizontally as far back as the Erwinia/Pantoea common ancestor, with evidence of more recent horizontal acquisition of the PSI-3 system. Our results suggest that Pantoea is a relatively old plant pathogen that has lost and subsequently regained different plant-associated T3SSs. This work has broad implications for understanding the host-associating capacity of Pantoea strains, and reveals the propensity for Pantoea isolates to exchange pathogenicity determinants with human

  6. Evidence for alternative quaternary structure in a bacterial Type III secretion system chaperone

    SciTech Connect

    Barta, Michael L.; Zhang, Lingling; Picking, Wendy L.; Geisbrecht, Brian V.

    2010-10-05

    Type III secretion systems are a common virulence mechanism in many Gram-negative bacterial pathogens. These systems use a nanomachine resembling a molecular needle and syringe to provide an energized conduit for the translocation of effector proteins from the bacterial cytoplasm to the host cell cytoplasm for the benefit of the pathogen. Prior to translocation specialized chaperones maintain proper effector protein conformation. The class II chaperone, Invasion plasmid gene (Ipg) C, stabilizes two pore forming translocator proteins. IpgC exists as a functional dimer to facilitate the mutually exclusive binding of both translocators. In this study, we present the 3.3 {angstrom} crystal structure of an amino-terminally truncated form (residues 10-155, denoted IpgC10-155) of the class II chaperone IpgC from Shigella flexneri. Our structure demonstrates an alternative quaternary arrangement to that previously described for a carboxy-terminally truncated variant of IpgC (IpgC{sup 1-151}). Specifically, we observe a rotationally-symmetric 'head-to-head' dimerization interface that is far more similar to that previously described for SycD from Yersinia enterocolitica than to IpgC1-151. The IpgC structure presented here displays major differences in the amino terminal region, where extended coil-like structures are seen, as opposed to the short, ordered alpha helices and asymmetric dimerization interface seen within IpgC{sup 1-151}. Despite these differences, however, both modes of dimerization support chaperone activity, as judged by a copurification assay with a recombinant form of the translocator protein, IpaB. Conclusions: From primary to quaternary structure, these results presented here suggest that a symmetric dimerization interface is conserved across bacterial class II chaperones. In light of previous data which have described the structure and function of asymmetric dimerization, our results raise the possibility that class II chaperones may transition between

  7. Identification of YsaP, the Pilotin of the Yersinia enterocolitica Ysa Type III Secretion System

    PubMed Central

    Rau, Reina

    2015-01-01

    ABSTRACT Secretins are multimeric outer membrane pore-forming proteins found in complex export systems in Gram-negative bacteria. All type III secretion systems (T3SSs) have a secretin, and one of these is the YsaC secretin of the chromosomally encoded Ysa T3SS of Yersinia enterocolitica. In some cases, pilotin proteins, which are outer membrane lipoproteins, are required for their cognate secretins to multimerize and/or localize to the outer membrane. However, if secretin multimers mislocalize to the inner membrane, this can trigger the protective phage shock protein (Psp) stress response. During a screen for mutations that suppress YsaC toxicity to a psp null strain, we isolated several independent mutations predicted to increase expression of the YE3559 gene within the Ysa pathogenicity island. YE3559, which we have named ysaP, is predicted to encode a small outer membrane lipoprotein, and this location was confirmed by membrane fractionation. Elevated ysaP expression increased the steady-state level of YsaC but made it less toxic to a psp null strain, and it also decreased YsaC-dependent induction of psp gene expression. Subsequent experiments showed that YsaP was not required for YsaC multimerization but was required for the multimers to localize to the outer membrane. Consistent with this, a ysaP null mutation compromised protein export by the Ysa T3SS. All these observations suggest that YsaP is the pilotin for the YsaC secretin. This is only the second pilotin to be characterized for Yersinia and one of only a small number of pilotins described for all bacteria. IMPORTANCE Secretins are essential for the virulence of many bacterial pathogens and also play roles in surface attachment, motility, and competence. This has generated considerable interest in understanding how secretins function. However, their fundamental differences from typical outer membrane proteins have raised various questions about secretins, including how they are assembled into outer

  8. Biofilm Development on Caenorhabditis elegans by Yersinia Is Facilitated by Quorum Sensing-Dependent Repression of Type III Secretion

    PubMed Central

    Atkinson, Steve; Goldstone, Robert J.; Joshua, George W. P.; Chang, Chien-Yi; Patrick, Hannah L.; Cámara, Miguel; Wren, Brendan W.; Williams, Paul

    2011-01-01

    Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS. PMID:21253572

  9. Scc1 (CP0432) and Scc4 (CP0033) function as a type III secretion chaperone for CopN of Chlamydia pneumoniae.

    PubMed

    Silva-Herzog, Eugenia; Joseph, Sabrina S; Avery, Ann K; Coba, Jose A; Wolf, Katerina; Fields, Kenneth A; Plano, Gregory V

    2011-07-01

    The Chlamydia pneumoniae CopN protein is a member of the YopN/TyeA/InvE/MxiC family of secreted proteins that function to regulate the secretion of type III secretion system (T3SS) translocator and effector proteins. In this study, the Scc1 (CP0432) and Scc4 (CP0033) proteins of C. pneumoniae AR-39 were demonstrated to function together as a type III secretion chaperone that binds to an N-terminal region of CopN. The Scc1/Scc4 chaperone promoted the efficient secretion of CopN via a heterologous T3SS, whereas, the Scc3 chaperone, which binds to a C-terminal region of CopN, reduced CopN secretion.

  10. Persistent DNA damage signaling triggers senescence-associated inflammatory cytokine secretion

    PubMed Central

    Rodier, Francis; Coppé, Jean-Philippe; Patil, Christopher K.; Hoeijmakers, Wieteke A. M.; Muñoz, Denise P.; Raza, Saba R.; Freund, Adam; Campeau, Eric; Davalos, Albert R.; Campisi, Judith

    2009-01-01

    Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells1. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments2. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signaling, usually associated with senescence, not after transient DNA damage responses (DDR). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Further, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell cycle checkpoints and DNA repair, a novel and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue. PMID:19597488

  11. Calcium-regulated type III secretion of Yop proteins by an Escherichia coli hha mutant carrying a Yersinia pestis pCD1 virulence plasmid.

    PubMed

    Bartra, Sara Schesser; Jackson, Michael W; Ross, Julia A; Plano, Gregory V

    2006-02-01

    A series of four large deletions that removed a total of ca. 36 kb of DNA from the ca. 70-kb Yersinia pestis pCD1 virulence plasmid were constructed using lambda Red-mediated recombination. Escherichia coli hha deletion mutants carrying the virulence plasmid with the deletions expressed a functional calcium-regulated type III secretion system. The E. coli hha/pCD1 system should facilitate molecular studies of the type III secretion process.

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

    SciTech Connect

    Sun, Ping; Tropea, Joseph E.; Austin, Brian P.; Cherry, Scott; Waugh, David S.

    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 Pseudomonas 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.

  13. Chlamydia trachomatis Slc1 is a type III secretion chaperone that enhances the translocation of its invasion effector substrate TARP.

    PubMed

    Brinkworth, Amanda J; Malcolm, Denise S; Pedrosa, António T; Roguska, Katarzyna; Shahbazian, Sevanna; Graham, James E; Hayward, Richard D; Carabeo, Rey A

    2011-10-01

    Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200-amino-acid residue N-terminal region of TARP (TARP¹⁻²⁰⁰). Slc1 formed homodimers in vitro, as shown in cross-linking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP¹⁻²⁰⁰ complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.

  14. A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System▿

    PubMed Central

    Rodgers, Loren; Mukerjea, Romila; Birtalan, Sara; Friedberg, Devorah; Ghosh, Partho

    2010-01-01

    Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component required for translocation. We sought to test a prediction of this model by identifying a potential association site for the Yersinia pseudotuberculosis chaperone-effector pair SycE-YopE. We identified a set of residues in the YopE Cb region that are required for translocation but are dispensable for expression, SycE binding, secretion into the extrabacterial milieu, and stability in mammalian cells. These residues form a solvent-exposed patch on the surface of the chaperone-bound Cb region, and thus their effect on translocation is consistent with the structure of the chaperone-bound Cb region serving as a signal for translocation. PMID:20382763

  15. Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

    PubMed Central

    Farfán, Mauricio J.; Toro, Cecilia S.; Barry, Eileen M.; Nataro, James P.

    2011-01-01

    We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study we show that ShET-2 secretion into the extracellular space requires the T3SS in S. flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is co-transcribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Though other phenotypes were not different compared to the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells. PMID:21219446

  16. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

    PubMed

    Pechsrichuang, Phornsiri; Songsiriritthigul, Chomphunuch; Haltrich, Dietmar; Roytrakul, Sittiruk; Namvijtr, Peenida; Bonaparte, Napolean; Yamabhai, Montarop

    2016-01-01

    The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.

  17. Secretion of anti-Plasmodium effector proteins from a natural Pantoea agglomerans isolate by using PelB and HlyA secretion signals.

    PubMed

    Bisi, Dawn C; Lampe, David J

    2011-07-01

    The insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodium spp.) in the mosquito host. We hypothesized that Pantoea agglomerans, a bacterial symbiont of Anopheles mosquitoes, could be engineered to express and secrete anti-Plasmodium effector proteins, a strategy termed paratransgenesis. To this end, plasmids that include the pelB or hlyA secretion signals from the genes of related species (pectate lyase from Erwinia carotovora and hemolysin A from Escherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodium effector proteins (SM1, anti-Pbs21, and PLA2) in P. agglomerans and E. coli. P. agglomerans successfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodium activity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

  18. Type III secretion as a generalizable strategy for the production of full-length biopolymer-forming proteins.

    PubMed

    Azam, Anum; Li, Cheng; Metcalf, Kevin J; Tullman-Ercek, Danielle

    2016-11-01

    Biopolymer-forming proteins are integral in the development of customizable biomaterials, but recombinant expression of these proteins is challenging. In particular, biopolymer-forming proteins have repetitive, glycine-rich domains and, like many heterologously expressed proteins, are prone to incomplete translation, aggregation, and proteolytic degradation in the production host. This necessitates tailored purification processes to isolate each full-length protein of interest from the truncated forms as well as other contaminating proteins; owing to the repetitive nature of these proteins, the truncated polypeptides can have very similar chemistry to the full-length form and are difficult to separate from the full-length protein. We hypothesized that bacterial expression and secretion would be a promising alternative option for biomaterials-forming proteins, simplifying isolation of the full-length target protein. By using a selective secretion system, truncated forms of the protein are not secreted and thus are not found in the culture harvest. We show that a synthetically upregulated type III secretion system leads to a general increase in secretion titer for each protein that we tested. Moreover, we observe a substantial enhancement in the homogeneity of full-length forms of pro-resilin, tropo-elastin crosslinking domains, and silk proteins produced in this manner, as compared with proteins purified from the cytosol. Secretion via the type III apparatus limits co-purification of truncated forms of the target protein and increases protein purity without extensive purification steps. Demonstrating the utility of such a system, we introduce several modifications to resilin-based peptides and use an un-optimized, single-column process to purify these proteins. The resulting materials are of sufficiently high quantity and yield for the production of antimicrobial hydrogels with highly reproducible rheological properties. The ease of this process and its

  19. Application of near-infrared spectroscopy to measurement of hemodynamic signals accompanying stimulated saliva secretion.

    PubMed

    Sato, Hiroki; Obata, Akiko N; Moda, Ichiro; Ozaki, Kazutaka; Yasuhara, Takaomi; Yamamoto, Yukari; Kiguchi, Masashi; Maki, Atsushi; Kubota, Kisou; Koizumi, Hideaki

    2011-04-01

    We aim to test the feasibility of using near-infrared spectroscopy (NIRS) for indirect measurement of human saliva secretion in response to taste stimuli for potential application to organoleptic testing. We use an NIRS system to measure extracranial hemodynamics (Hb-signals around the temples) of healthy participants when taste stimuli are taken in their mouths. First, the Hb-signals and volume of expelled saliva (stimulated by distilled-water or sucrose-solution intake) are simultaneously measured and large Hb-signal changes in response to the taste stimuli (Hb-responses) are found. Statistical analysis show that both the Hb response and saliva volume are larger for the sucrose solution than for the distilled water with a significant correlation between them (r = 0.81). The effects of swallowing on the Hb-signals are investigated. Similar Hb responses, differing from the sucrose solution and distilled water, are obtained even though the participants swallow the mouth contents. Finally, functional magnetic resonance imaging is used to identify possible sources of the Hb signals corresponding to salivation. Statistical analysis indicates similar responses in the extracranial regions, mainly around the middle meningeal artery. In conclusion, the identified correlation between extracranial hemodynamics and the saliva volume suggests that NIRS is applicable to the measurement of hemodynamic signals accompanying stimulated saliva secretion.

  20. In beta-cells, mitochondria integrate and generate metabolic signals controlling insulin secretion.

    PubMed

    Maechler, Pierre; Carobbio, Stefania; Rubi, Blanca

    2006-01-01

    Pancreatic beta-cells are unique neuroendocrine cells displaying the peculiar feature of responding to nutrients, principally glucose, as primary stimulus. This requires translation of a metabolic substrate into intracellular messengers recognized by the exocytotic machinery. Central to this signal transduction mechanism, mitochondria integrate and generate metabolic signals, thereby coupling glucose recognition to insulin secretion. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, to the stimulation of insulin exocytosis. This review describes the mitochondrion-dependent pathways of regulated insulin secretion. In particular, importance of cataplerotic and anaplerotic processes is discussed, with special attention to the mitochondrial enzyme glutamate dehydrogenase. Mitochondrial defects, such as mutations and reactive oxygen species production, are presented in the context of beta-cell failure in the course of type 2 diabetes.

  1. Effects of chromium(III) picolinate on cortisol and DHEAs secretion in H295R human adrenocortical cells.

    PubMed

    Kim, Beob G; Adams, Julye M; Jackson, Brian A; Lindemann, Merlin D

    2010-02-01

    Dietary chromium(III) picolinate (CrPic) effects on circulating steroid hormones have been reported in various experimental animals. However, direct effects of CrPic on adrenocortical steroidogenesis are uncertain. Therefore, the objective was to determine the effects of CrPic on cortisol and dehydroepiandrosterone sulfate (DHEAs) secretion from H295R cells. In experiment 1, a 24-h exposure to CrPic (0 to 200 microM) had both linear (p < 0.001) and quadratic (p < 0.001) effects on cortisol secretion from forskolin-stimulated cells with the highest cortisol secretion at 0.1 microM of CrPic and the lowest at 200 microM of CrPic. In experiment 2, a 48-h exposure to CrPic (200 microM) decreased cortisol (p < 0.07) release from forskolin-stimulated cells during a 24-h collection period. In experiment 3, a 48-h exposure to CrPic (100 microM) decreased cortisol (p < 0.05) and DHEAs (p < 0.01) from forskolin-stimulated cells during a 24-h sampling period. In experiment 4, a 24-h exposure to forskolin followed by a 24-h exposure to both forskolin and CrPic (100 and 200 microM) decreased both cortisol and DHEAs secretion (p < 0.01). This study suggests that at high concentrations, CrPic inhibits aspects of steroidogenesis in agonist-stimulated adrenocortical cells.

  2. The Yersinia pestis type III secretion system: expression, assembly and role in the evasion of host defenses.

    PubMed

    Plano, Gregory V; Schesser, Kurt

    2013-12-01

    Yersinia pestis, the etiologic agent of plague, utilizes a type III secretion system (T3SS) to subvert the defenses of its mammalian hosts. T3SSs are complex nanomachines that allow bacterial pathogens to directly inject effector proteins into eukaryotic cells. The Y. pestis T3SS is not expressed during transit through the flea vector, but T3SS gene expression is rapidly thermoinduced upon entry into a mammalian host. Assembly of the T3S apparatus is a highly coordinated process that requires the homo- and hetero-oligomerization over 20 Yersinia secretion (Ysc) proteins, several assembly intermediates and the T3S process to complete the assembly of the rod and external needle structures. The activation of effector secretion is controlled by the YopN/TyeA/SycN/YscB complex, YscF and LcrG in response to extracellular calcium and/or contact with a eukaryotic cell. Cell contact triggers the T3S process including the secretion and assembly of a pore-forming translocon complex that facilitates the translocation of effector proteins, termed Yersinia outer proteins (Yops), across the eukaryotic membrane. Within the host cell, the Yop effector proteins function to inhibit bacterial phagocytosis and to suppress the production of pro-inflammatory cytokines.

  3. Influence of phenolic acids on indole acetic acid production and on the type III secretion system gene transcription in food-associated Pseudomonas fluorescens KM05.

    PubMed

    Myszka, Kamila; Schmidt, Marcin T; Olejnik-Schmidt, Agnieszka K; Leja, Katarzyna; Czaczyk, Katarzyna

    2014-12-01

    The purpose of these investigations was to evaluate the reduction capability of phenolic acids (ferulic, chlorogenic, gallic, and p-coumaric acids) on indole acetic acid synthesis by food-associated Pseudomonas fluorescens KM05. Specific genetic primer for the type III secretion system (TTSS) in P. fluorescens KM05 was designed and the influence of phenolic acids on its expression was investigated. In the work the ferulic and chlorogenic acids at the concentration of 0.02 and 0.04 μg/ml affected on bacterial growth pattern and the signal molecules production. The phenolic acids, that were appreciable effective against P. fluorescens KM05 indole acetic acid production, significantly suppressed TTSS gene.

  4. Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion.

    PubMed

    MacDonald, Michael J; Fahien, Leonard A; Brown, Laura J; Hasan, Noaman M; Buss, Julian D; Kendrick, Mindy A

    2005-01-01

    The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis

  5. Integration of hormonal and nutrient signals that regulate leptin synthesis and secretion

    PubMed Central

    Lee, Mi-Jeong; Fried, Susan K.

    2009-01-01

    This review summarizes recent advances in our understanding of the pre- and posttranscriptional mechanisms that regulate leptin production and secretion in adipocytes. Basal leptin production is proportional to the status of energy stores, i.e., fat cell size, and this is mainly regulated by alterations in leptin mRNA levels. Leptin mRNA levels are regulated by hormones, including glucocorticoids and catecholamines, but little is known about the transcriptional mechanisms involved. Leptin synthesis and secretion is also acutely modulated in response to hormones such as insulin and the availability of metabolic fuels. Acute variations in leptin production over a time course of minutes to hours are mediated at the levels of both translation and secretion. Increases in amino acids and insulin after a meal activate the mammalian target of rapamycin (mTOR) pathway, leading to an increase in specific rates of leptin biosynthesis. Cross-talk among mTOR, PKA, and AMP-activated protein kinase pathways appears to integrate hormonal and nutrient signals that regulate leptin mRNA translation, at least in part through mechanisms involving its 5′- and 3′-untranslated regions. In addition, the rate of leptin secretion from preformed stores in response to hormonal cues is also regulated. Insulin stimulates, and adrenergic agonists inhibit, leptin secretion, and this likely contributes to variations in the magnitude of nutrition-related leptin excursions and oscillations. Overall, the study of leptin production has contributed to a deepening understanding of leptin biology and, more broadly, to our understanding of the cellular and molecular mechanisms by which the adipocyte integrates hormonal and nutrient signals to regulate adipokine production. PMID:19318513

  6. Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli.

    PubMed

    Han, SooJin; Machhi, Shushil; Berge, Mark; Xi, Guoling; Linke, Thomas; Schoner, Ronald

    2017-12-01

    Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against Staphylococcus aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. Indeed, one of the novel signal peptides improved the yield of secreted ATH35L by 3.5-fold in a fed-batch fermentation process and at the same time maintained processing at the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.

  7. Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement

    PubMed Central

    Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R. D.; Heath, James R.

    2016-01-01

    Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell–cell signaling can depend on cell–cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell–cell signaling that induces a directed cell–cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell–cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell–cell distances. We show that glioblastoma cell–cell movement can be described as Brownian motion biased by cell–cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells. PMID:27140641

  8. Epidermal Wnt/β-catenin signaling regulates adipocyte differentiation via secretion of adipogenic factors

    PubMed Central

    Donati, Giacomo; Proserpio, Valentina; Lichtenberger, Beate Maria; Natsuga, Ken; Sinclair, Rodney; Fujiwara, Hironobu; Watt, Fiona M.

    2014-01-01

    It has long been recognized that the hair follicle growth cycle and oscillation in the thickness of the underlying adipocyte layer are synchronized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether the epidermis can regulate adipogenesis. We show that inhibition of epidermal Wnt/β-catenin signaling reduced adipocyte differentiation in developing and adult mouse dermis. Conversely, ectopic activation of epidermal Wnt signaling promoted adipocyte differentiation and hair growth. When the Wnt pathway was activated in the embryonic epidermis, there was a dramatic and premature increase in adipocytes in the absence of hair follicle formation, demonstrating that Wnt activation, rather than mature hair follicles, is required for adipocyte generation. Epidermal and dermal gene expression profiling identified keratinocyte-derived adipogenic factors that are induced by β-catenin activation. Wnt/β-catenin signaling-dependent secreted factors from keratinocytes promoted adipocyte differentiation in vitro, and we identified ligands for the bone morphogenetic protein and insulin pathways as proadipogenic factors. Our results indicate epidermal Wnt/β-catenin as a critical initiator of a signaling cascade that induces adipogenesis and highlight the role of epidermal Wnt signaling in synchronizing adipocyte differentiation with the hair growth cycle. PMID:24706781

  9. Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

    PubMed

    Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R D; Heath, James R

    2016-05-17

    Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell-cell signaling that induces a directed cell-cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell-cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell-cell distances. We show that glioblastoma cell-cell movement can be described as Brownian motion biased by cell-cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

  10. A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species.

    PubMed

    Attallah, Carolina; Etcheverrigaray, Marina; Kratje, Ricardo; Oggero, Marcos

    2017-01-10

    Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.

  11. Novel alpha1-adrenergic receptor signaling pathways: secreted factors and interactions with the extracellular matrix.

    PubMed

    Shi, Ting; Duan, Zhong-Hui; Papay, Robert; Pluskota, Elzbieta; Gaivin, Robert J; de la Motte, Carol A; Plow, Edward F; Perez, Dianne M

    2006-07-01

    alpha1-Adrenergic receptor (alpha1-ARs) subtypes (alpha1A, alpha1B, and alpha1D) regulate multiple signal pathways, such as phospholipase C, protein kinase C (PKC), and mitogen-activated protein kinases. We employed oligonucleotide microarray technology to explore the effects of both short- (1 h) and long-term (18 h) activation of the alpha1A-AR to enable RNA changes to occur downstream of earlier well characterized signaling pathways, promoting novel couplings. Polymerase chain reaction (PCR) studies confirmed that PKC was a critical regulator of alpha1A-AR-mediated gene expression, and secreted interleukin (IL)-6 also contributed to gene expression alterations. We next focused on two novel signaling pathways that might be mediated through alpha1A-AR stimulation because of the clustering of gene expression changes for cell adhesion/motility (syndecan-4 and tenascin-C) and hyaluronan (HA) signaling. We confirmed that alpha1-ARs induced adhesion in three cell types to vitronectin, an interaction that was also integrin-, FGF7-, and PKC-dependent. alpha1-AR activation also inhibited cell migration, which was integrin- and PKC-independent but still required secretion of FGF7. alpha1-AR activation also increased the expression and deposition of HA, a glycosaminoglycan, which displayed two distinct structures: pericellular coats and long cable structures, as well as increasing expression of the HA receptor, CD44. Long cable structures of HA can bind leukocytes, which this suggests that alpha1-ARs may be involved in proinflammatory responses. Our results indicate alpha1-ARs induce the secretion of factors that interact with the extracellular matrix to regulate cell adhesion, motility and proinflammatory responses through novel signaling pathways.

  12. Caspase-1 activation in macrophages infected with Yersinia pestis KIM requires the type III secretion system effector YopJ.

    PubMed

    Lilo, Sarit; Zheng, Ying; Bliska, James B

    2008-09-01

    Pathogenic Yersinia species utilize a type III secretion system (T3SS) to translocate effectors called Yersinia outer proteins (Yops) into infected host cells. Previous studies demonstrated a role for effector Yops in the inhibition of caspase-1-mediated cell death and secretion of interleukin-1beta (IL-1beta) in naïve macrophages infected with Yersinia enterocolitica. Naïve murine macrophages were infected with a panel of different Yersinia pestis and Yersinia pseudotuberculosis strains to determine whether Yops of these species inhibit caspase-1 activation. Cell death was measured by release of lactate dehydrogenase (LDH), and enzyme-linked immunosorbent assay for secreted IL-1beta was used to measure caspase-1 activation. Surprisingly, isolates derived from the Y. pestis KIM strain (e.g., KIM5) displayed an unusual ability to activate caspase-1 and kill infected macrophages compared to other Y. pestis and Y. pseudotuberculosis strains tested. Secretion of IL-1beta following KIM5 infection was reduced in caspase-1-deficient macrophages compared to wild-type macrophages. However, release of LDH was not reduced in caspase-1-deficient macrophages, indicating that cell death occurred independently of caspase-1. Analysis of KIM-derived strains defective for production of functional effector or translocator Yops indicated that translocation of catalytically active YopJ into macrophages was required for caspase-1 activation and cell death. Release of LDH and secretion of IL-1beta were not reduced when actin polymerization was inhibited in KIM5-infected macrophages, indicating that extracellular bacteria translocating YopJ could trigger cell death and caspase-1 activation. This study uncovered a novel role for YopJ in the activation of caspase-1 in macrophages.

  13. IpaD Localizes to the Tip of the Type III Secretion System Needle of Shigella flexneri

    PubMed Central

    Espina, Marianela; Olive, Andrew J.; Kenjale, Roma; Moore, David S.; Ausar, S. Fernando; Kaminski, Robert W.; Oaks, Edwin V.; Middaugh, C. Russell; Picking, William D.; Picking, Wendy L.

    2006-01-01

    Shigella flexneri, the causative agent of shigellosis, is a gram-negative bacterial pathogen that initiates infection by invading cells within the colonic epithelium. Contact with host cell surfaces induces a rapid burst of protein secretion via the Shigella type III secretion system (TTSS). The first proteins secreted are IpaD, IpaB, and IpaC, with IpaB and IpaC being inserted into the host cell membrane to form a pore for translocating late effectors into the target cell cytoplasm. The resulting pathogen-host cross talk results in localized actin polymerization, membrane ruffling, and, ultimately, pathogen entry. IpaD is essential for host cell invasion, but its role in this process is just now coming to light. IpaD is a multifunctional protein that controls the secretion and presentation of IpaB and IpaC at the pathogen-host interface. We show here that antibodies recognizing the surface-exposed N terminus of IpaD neutralize Shigella's ability to promote pore formation in erythrocyte membranes. We further show that MxiH and IpaD colocalize on the bacterial surface. When TTSS needles were sheared from the Shigella surface, IpaD was found at only the needle tips. Consistent with this, IpaD localized to the exposed tips of needles that were still attached to the bacterium. Molecular analyses then showed that the IpaD C terminus is required for this surface localization and function. Furthermore, mutations that prevent IpaD surface localization also eliminate all IpaD-related functions. Thus, this study demonstrates that IpaD localizes to the TTSA needle tip, where it functions to control the secretion and proper insertion of translocators into host cell membranes. PMID:16861624

  14. Supramolecular Structure and Functional Analysis of the Type III Secretion System in Pseudomonas fluorescens 2P24

    PubMed Central

    Liu, Ping; Zhang, Wei; Zhang, Li-Qun; Liu, Xingzhong; Wei, Hai-Lei

    2016-01-01

    The type III secretion system (T3SS) of plant and animal bacterial pathogens directs the secretion and injection of proteins into host cells. Some homologous genes of T3SS were found also in non-pathogenic bacteria, but the organization of its machinery and basic function are still unknown. In this study, we identified a T3SS gene cluster from the plant growth-promoting Pseudomonas fluorescens 2P24 and isolated the corresponding T3SS apparatus. The T3SS gene cluster of strain 2P24 is similar organizationally to that of pathogenic P. syringae, except that it lacks the regulator hrpR and the hrpK1 and hrpH genes, which are involved in translocation of proteins. Electron microscopy revealed that the T3SS supramolecular structure of strain 2P24 was comprised of two distinctive substructures: a long extracellular, filamentous pilus, and a membrane-embedded base. We show that strain 2P24 deploys a harpin homolog protein, RspZ1, to elicit a hypersensitive response when infiltrated into Nicotiana tabacum cv. xanthi leaves with protein that is partially purified, and by complementing the hrpZ1 mutation of pHIR11. The T3SS of strain 2P24 retained ability to secrete effectors, whereas its effector translocation activity appeared to be excessively lost. Mutation of the rscC gene from 2P24 T3SS abolished the secretion of effectors, but the general biocontrol properties were unaffected. Remarkably, strain 2P24 induced functional MAMP-triggered immunity that included a burst of reactive oxygen species, strong suppression of challenge cell death, and disease expansion, while it was not associated with the secretion functional T3SS. PMID:26779224

  15. A type III effector antagonises death receptor signalling during bacterial gut infection

    PubMed Central

    Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Wong, Tania; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare VL; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O’Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

    2013-01-01

    Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. PMID:24025841

  16. Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae

    PubMed Central

    Waite, Christopher; Schumacher, Jörg; Jovanovic, Milija; Bennett, Mark

    2017-01-01

    ABSTRACT   The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL. In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses. PMID:28119474

  17. 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

  18. EscA is a crucial component of the type III secretion system of enteropathogenic Escherichia coli.

    PubMed

    Sal-Man, Neta; Biemans-Oldehinkel, Esther; Sharon, David; Croxen, Matthew A; Scholz, Roland; Foster, Leonard J; Finlay, B Brett

    2012-06-01

    The virulence of many Gram-negative pathogens is associated with type III secretion systems (T3SSs), which deliver virulence effector proteins into the cytoplasm of host cells. Components of enteropathogenic Escherichia coli (EPEC) T3SS are encoded within the locus of enterocyte effacement (LEE). While most LEE-encoded T3SS proteins in EPEC have assigned names and functions, a few of them remain poorly characterized. Here, we studied a small LEE-encoded protein, Orf15, that shows no homology to other T3SS/flagellar proteins and is only present in attaching and effacing pathogens, including enterohemorrhagic E. coli and Citrobacter rodentium. Our findings demonstrated that it is essential for type III secretion (T3S) and that it is localized to the periplasm and associated with the inner membrane. Membrane association was driven by the N-terminal 19 amino acid residues, which were also shown to be essential for T3S. Consistent with its localization, Orf15 was found to interact with the EPEC T3SS outer membrane ring component, EscC, which was previously shown to be embedded within the outer membrane and protruding into the periplasmic space. Interestingly, we found that the predicted coiled-coil structure of Orf15 is critical for the protein's function. Overall, our findings suggest that Orf15 is a structural protein that contributes to the structural integrity of the T3S complex, and therefore we propose to rename it EscA.

  19. Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris.

    PubMed

    Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

    2017-02-15

    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.

  20. Characterization of the ysa pathogenicity locus in the chromosome of Yersinia enterocolitica and phylogeny analysis of type III secretion systems.

    PubMed

    Foultier, Boris; Troisfontaines, Paul; Müller, Simone; Opperdoes, Fred R; Cornelis, Guy R

    2002-07-01

    Several Gram negative bacteria use a complex system called "type III secretion system" (TTSS) to engage their host. The archetype of TTSS is the plasmid-encoded "Yop virulon" shared by the three species of pathogenic Yersinia (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica). A second TTSS, called Ysa (for Yersinia secretion apparatus) was recently described in Y. enterocolitica 8081, a strain from serotype O:8. In this study, we describe the ysa locus from A127/90, another strain of serotype O:8, and we extend the sequence to several new genes encoding Ysp proteins which are the substrates of this secretion system, and a putative chaperone SycB. According to the deduced protein sequences, the ysa system from A127/90 is identical to that of 8081. It is different from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. We further demonstrated that the ysa locus is only present in biotype IB strains of Y. enterocolitica. Including this new Ysa system, a phylogenetic analysis of the 26 known TTSSs was carried out, based on the sequence analysis of three conserved proteins. All the TTSSs fall into five different clusters. The phylogenetic tree of these TTSSs is completely different from the evolutionary tree based on 16S RNA, indicating that TTSSs have been distributed by horizontal transfer.

  1. Expression of the Bradyrhizobium japonicum type III secretion system in legume nodules and analysis of the associated tts box promoter.

    PubMed

    Zehner, Susanne; Schober, Grit; Wenzel, Mandy; Lang, Kathrin; Göttfert, Michael

    2008-08-01

    In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.

  2. The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector.

    PubMed

    Correa, Valdir R; Majerczak, Doris R; Ammar, El-Desouky; Merighi, Massimo; Pratt, Richard C; Hogenhout, Saskia A; Coplin, David L; Redinbaugh, Margaret G

    2012-09-01

    Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host.

  3. The Bacterium Pantoea stewartii Uses Two Different Type III Secretion Systems To Colonize Its Plant Host and Insect Vector

    PubMed Central

    Correa, Valdir R.; Majerczak, Doris R.; Ammar, El-Desouky; Merighi, Massimo; Pratt, Richard C.; Hogenhout, Saskia A.; Coplin, David L.

    2012-01-01

    Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host. PMID:22773631

  4. Eukaryotic pathways targeted by the type III secretion system effector protein, BipC, involved in the intracellular lifecycle of Burkholderia pseudomallei

    PubMed Central

    Kang, Wen-Tyng; Vellasamy, Kumutha Malar; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei, the etiological agent for melioidosis, is known to secrete a type III secretion system (TTSS) protein into the host’s internal milieu. One of the TTSS effector protein, BipC, has been shown to play an important role in the B. pseudomallei pathogenesis. To identify the host response profile that was directly or indirectly regulated by this protein, genome-wide transcriptome approach was used to examine the gene expression profiles of infected mice. The transcriptome analysis of the liver and spleen revealed that a total of approximately 1,000 genes were transcriptionally affected by BipC. Genes involved in bacterial invasion, regulation of actin cytoskeleton, and MAPK signalling pathway were over-expressed and may be specifically regulated by BipC in vivo. These results suggest that BipC mainly targets pathways related to the cellular processes which could modulate the cellular trafficking processes. The host transcriptional response exhibited remarkable differences with and without the presence of the BipC protein. Overall, the detailed picture of this study provides new insights that BipC may have evolved to efficiently manipulate host-cell pathways which is crucial in the intracellular lifecycle of B. pseudomallei. PMID:27634329

  5. Genetically Engineered Frameshifted YopN-TyeA Chimeras Influence Type III Secretion System Function in Yersinia pseudotuberculosis

    PubMed Central

    Amer, Ayad A. A.; Costa, Tiago R. D.; Farag, Salah I.; Avican, Ummehan; Forsberg, Åke; Francis, Matthew S.

    2013-01-01

    Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact. PMID

  6. Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis.

    PubMed

    Amer, Ayad A A; Costa, Tiago R D; Farag, Salah I; Avican, Ummehan; Forsberg, Åke; Francis, Matthew S

    2013-01-01

    Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

  7. The Structures of Coiled-Coil Domains from Type III Secretion System Translocators Reveal Homology to Pore-Forming Toxins

    SciTech Connect

    Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.

    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. 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.

  8. Identification of Small-Molecule Inhibitors of Yersinia pestis Type III Secretion System YscN ATPase

    PubMed Central

    Swietnicki, Wieslaw; Carmany, Daniel; Retford, Michael; Guelta, Mark; Dorsey, Russell; Bozue, Joel; Lee, Michael S.; Olson, Mark A.

    2011-01-01

    Yersinia pestis is a Gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS) to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC50 values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species. PMID:21611119

  9. Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation

    PubMed Central

    Koyama, Takashi; Mirth, Christen K.

    2016-01-01

    In Drosophila, the fat body, functionally equivalent to the mammalian liver and adipocytes, plays a central role in regulating systemic growth in response to nutrition. The fat body senses intracellular amino acids through Target of Rapamycin (TOR) signaling, and produces an unidentified humoral factor(s) to regulate insulin-like peptide (ILP) synthesis and/or secretion in the insulin-producing cells. Here, we find that two peptides, Growth-Blocking Peptide (GBP1) and CG11395 (GBP2), are produced in the fat body in response to amino acids and TOR signaling. Reducing the expression of GBP1 and GBP2 (GBPs) specifically in the fat body results in smaller body size due to reduced growth rate. In addition, we found that GBPs stimulate ILP secretion from the insulin-producing cells, either directly or indirectly, thereby increasing insulin and insulin-like growth factor signaling activity throughout the body. Our findings fill an important gap in our understanding of how the fat body transmits nutritional information to the insulin producing cells to control body size. PMID:26928023

  10. HpaP modulates type III effector secretion in Ralstonia solanacearum and harbours a substrate specificity switch domain essential for virulence.

    PubMed

    Lohou, David; Turner, Marie; Lonjon, Fabien; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2014-08-01

    Many pathogenic bacteria have evolved a type III secretion system (T3SS) to successfully invade their host. This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. T3Es are virulence factors that have been shown to interfere with the host's immunity or to provide nutrients from the host to the bacteria. The Gram-negative bacterium Ralstonia solanacearum is a worldwide major crop pest whose virulence strongly relies on the T3SS. In R. solanacearum, transcriptional regulation has been extensively studied. However, very few data are available concerning the role played by type III-associated regulators, such as type III chaperones and T3SS control proteins. Here, we characterized HpaP, a putative type III secretion substrate specificity switch (T3S4) protein of R. solanacearum which is not secreted by the bacterium or translocated in the plant cells. HpaP self-interacts and interacts with the PopP1 T3E. HpaP modulates the secretion of early (HrpY pilin) and late (AvrA and PopP1 T3Es) type III substrates. HpaP is dispensable for the translocation of T3Es into the host cells. Finally, we identified two regions of five amino acids in the T3S4 domain that are essential for efficient PopP1 secretion and for HpaP's role in virulence on tomato and Arabidopsis thaliana, but not required for HpaP-HpaP and HpaP-PopP1 interactions. Taken together, our results indicate that HpaP is a putative R. solanacearum T3S4 protein important for full pathogenicity on several hosts, acting as a helper for PopP1 secretion, and repressing AvrA and HrpY secretion. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  11. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase

    PubMed Central

    Kato, Junya; Lefebre, Matthew

    2015-01-01

    ABSTRACT Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. IMPORTANCE Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. PMID:26170413

  12. Secretion and lysophospholipase D activity of autotaxin by adipocytes are controlled by N-glycosylation and signal peptidase.

    PubMed

    Pradère, Jean Philippe; Tarnus, Evelyne; Grès, Sandra; Valet, Philippe; Saulnier-Blache, Jean Sébastien

    2007-01-01

    Autotaxin (ATX) is a lysophospholipase D involved in synthesis of lysophosphatidic acid (LPA). ATX is secreted by adipocytes and is associated with adipogenesis and obesity-associated diabetes. Here we have studied the mechanisms involved in biosynthesis and secretion of ATX by mouse 3T3-F442A adipocytes. We found that inhibition of N-glycosylation with tunicamycin or by double point deletion of the amino-acids N53 and N410 of ATX inhibit its secretion. In addition, N-glycosidase treatment and point deletion of the amino-acid N410 inhibits the lysophospholipase D activity of ATX. Analysis of the amino-acid sequence of mouse ATX shows the presence of a N-terminal signal peptide. Treatment with the signal peptidase inhibitor globomycin inhibits ATX secretion by adipocytes. Transfection in Cos-7 cells of site-directed deleted ATX shows that ATX secretion is dependent on the hydrophobic core sequence of the signal peptide, not on the putative signal peptidase cleavage site sequence. Analysis of the amino-acid sequence of mouse ATX also reveals the presence of a putative cleavage site by the protein convertase furin. Treatment of adipocytes with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone does not modified secretion or lysophospholipase D activity of ATX. Transfection in Cos-7 cells of site-directed deleted ATX shows that the furin recognition site is not required for secretion or lysophospholipase D activity of ATX. In conclusion, the present work demonstrates the crucial role of N-glycosylation in secretion and activity of ATX. The present work also confirms the crucial role signal peptidase in secretion of ATX by adipocytes.

  13. Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells.

    PubMed

    Ferdaoussi, Mourad; Dai, Xiaoqing; Jensen, Mette V; Wang, Runsheng; Peterson, Brett S; Huang, Chao; Ilkayeva, Olga; Smith, Nancy; Miller, Nathanael; Hajmrle, Catherine; Spigelman, Aliya F; Wright, Robert C; Plummer, Gregory; Suzuki, Kunimasa; Mackay, James P; van de Bunt, Martijn; Gloyn, Anna L; Ryan, Terence E; Norquay, Lisa D; Brosnan, M Julia; Trimmer, Jeff K; Rolph, Timothy P; Kibbey, Richard G; Manning Fox, Jocelyn E; Colmers, William F; Shirihai, Orian S; Neufer, P Darrell; Yeh, Edward T H; Newgard, Christopher B; MacDonald, Patrick E

    2015-10-01

    Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

  14. Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

    PubMed Central

    Ferdaoussi, Mourad; Dai, Xiaoqing; Jensen, Mette V.; Wang, Runsheng; Peterson, Brett S.; Huang, Chao; Ilkayeva, Olga; Smith, Nancy; Miller, Nathanael; Hajmrle, Catherine; Spigelman, Aliya F.; Wright, Robert C.; Plummer, Gregory; Suzuki, Kunimasa; Mackay, James P.; van de Bunt, Martijn; Gloyn, Anna L.; Ryan, Terence E.; Norquay, Lisa D.; Brosnan, M. Julia; Trimmer, Jeff K.; Rolph, Timothy P.; Kibbey, Richard G.; Manning Fox, Jocelyn E.; Colmers, William F.; Shirihai, Orian S.; Neufer, P. Darrell; Yeh, Edward T.H.; Newgard, Christopher B.; MacDonald, Patrick E.

    2015-01-01

    Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D. PMID:26389676

  15. Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties

    PubMed Central

    Vyas, Neha; Walvekar, Ankita; Tate, Dhananjay; Lakshmanan, Vairavan; Bansal, Dhiru; Cicero, Alessandra Lo; Raposo, Graca; Palakodeti, Dasaradhi; Dhawan, Jyotsna

    2014-01-01

    Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3β and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation. PMID:25483805

  16. Vertebrate Hedgehog is secreted on two types of extracellular vesicles with different signaling properties.

    PubMed

    Vyas, Neha; Walvekar, Ankita; Tate, Dhananjay; Lakshmanan, Vairavan; Bansal, Dhiru; Lo Cicero, Alessandra; Raposo, Graca; Palakodeti, Dasaradhi; Dhawan, Jyotsna

    2014-12-08

    Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3β and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation.

  17. Structural Insights into Fibronectin Type III Domain Mediated Signaling

    PubMed Central

    Bencharit, Sompop; Cui, Cai Bin; Siddiqui, Adnan; Howard-Williams, Escher L.; Sondek, John; Zuobi-Hasona, Kheir; Aukhil, Ikramuddin

    2007-01-01

    The alternatively spliced type-III extradomain B (EIIIB) of Fibronectin (FN) is only expressed during embryogenesis, wound healing and tumorigenesis. The biological function of this domain remains unclear. We describe here the first crystal structure of the interface between alternatively-spliced domain EIIIB and its adjacent FN type-III domain 8 (FN B-8). The opened CC′ loop of EIIIB and the rotation and tilt of EIIIB domain allows good access to the FG loop of FN-8 which is normally hindered by the CC′ loop of FN-7. In addition, the AGEGIP sequence of the CC′ loop of EIIIB replaces the NGQQGN sequence of the CC′ loop of FN-7. Finally, the CC” loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilagenous development, wound healing, and tumorigenesis, respectively. PMID:17261313

  18. The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System.

    PubMed

    Walker, Kimberly A; Griggs, Lauren A; Obrist, Markus; Bode, Addys; Summers, R Patrick; Miller, Virginia L

    2016-06-15

    The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes

  19. The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System

    PubMed Central

    Griggs, Lauren A.; Obrist, Markus; Bode, Addys; Summers, R. Patrick; Miller, Virginia L.

    2016-01-01

    ABSTRACT The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes

  20. Temperature-induced protein secretion by Leishmania mexicana modulates macrophage signalling and function.

    PubMed

    Hassani, Kasra; Antoniak, Elisabeth; Jardim, Armando; Olivier, Martin

    2011-05-03

    Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.

  1. Phylogenetic evidence for extensive horizontal gene transfer of type III secretion system genes among enterobacterial plant pathogens.

    PubMed

    Naum, Marianna; Brown, Eric W; Mason-Gamer, Roberta J

    2009-10-01

    This study uses sequences from four genes, which are involved in the formation of the type III secretion apparatus, to determine the role of horizontal gene transfer in the evolution of virulence genes for the enterobacterial plant pathogens. Sequences of Erwinia, Brenneria, Pectobacterium, Dickeya and Pantoea were compared (a) with one another, (b) with sequences of enterobacterial animal pathogens, and (c) with sequences of plant pathogenic gamma and beta proteobacteria, to evaluate probable paths of lateral exchange leading to the current distribution of virulence determinants among these micro-organisms. Phylogenies were reconstructed based on hrcC, hrcR, hrcJ and hrcV gene sequences using parsimony and maximum-likelihood algorithms. Virulence gene phylogenies were also compared with several housekeeping gene loci in order to evaluate patterns of lateral versus vertical acquisition. The resulting phylogenies suggest that multiple horizontal gene transfer events have occurred both within and among the enterobacterial plant pathogens and plant pathogenic gamma and beta proteobacteria. hrcJ sequences are the most similar, exhibiting anywhere from 2 to 50 % variation at the nucleotide level, with the highest degree of variation present between plant and animal pathogen sequences. hrcV sequences are conserved among plant and animal pathogens at the N terminus. The C-terminal domain is conserved only among the enterobacterial plant pathogens, as are the hrcC and hrcR sequences. Additionally, hrcJ and hrcV sequence phylogenies suggest that at least some type III secretion system virulence genes from enterobacterial plant pathogens are related more closely to those of the genus Pseudomonas, a conclusion neither supported nor refuted by hrcC or hrcR.

  2. Functional interaction between type III-secreted protein IncA of Chlamydophila psittaci and human G3BP1.

    PubMed

    Borth, Nicole; Litsche, Katrin; Franke, Claudia; Sachse, Konrad; Saluz, Hans Peter; Hänel, Frank

    2011-01-31

    Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation.

  3. Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum.

    PubMed

    Goodin, Jeremy L; Raab, Ronald W; McKown, Robert L; Coffman, George L; Powell, Bradford S; Enama, Jeff T; Ligon, John A; Andrews, Gerard P

    2005-03-01

    The type III secretion system (YscC) protein of Yersinia pestis plays an essential role in the translocation of Yersinia outer proteins (Yops) into eukaryotic target cells through a type III secretion mechanism. To assess the immunogenicity and potential protective efficacy of YscC against lethal plague challenge, we cloned, overexpressed, and purified YscC using two different bacterial expression and purification systems. The resulting expression plasmids for YscC, pETBlue-2-YscC and pTYB11-YscC, were regulated by robust T7 promoters that were induced with isopropyl-beta-D-thiogalactopyranoside. The intein-fusion pTYB11-YscC system and the six-histidine-tagging pETBlue-2-YscC system were both successful for producing and purifying YscC. The intein-mediated purification system produced about 1mg of soluble YscC per liter of bacterial culture while the YscC-His(6)-tag method resulted in 16mg of insoluble YscC per liter of bacterial culture. Protein identity for purified YscC-His(6) was confirmed by ion trap mass spectrometry. Antisera were produced against both YscC and YscC-His(6). The specific immune response generated in YscC-vaccinated mice was relative to the particular purified protein, YscC or YscC-His(6), which was used for vaccination as determined by Western blot analysis and ELISA. Regardless of the purification method, either form of the YscC protein failed to elicit a protective immune response against lethal plague challenge with either F1 capsule forming Y. pestis CO92 or the isogenic F1(-)Y. pestis C12.

  4. Development of loop-mediated isothermal amplification assays for genotyping of Type III Secretion System in Pseudomonas aeruginosa.

    PubMed

    Shi, H; Chen, Z; Kan, J

    2015-10-01

    Pseudomonas aeruginosa is a well-known environmental bacterium capable of causing a variety of life-threatening human infections, with a Type III Secretion System (T3SS) as the most significant virulence determinant. P. aeruginosa strains exhibit unique T3SS virulence genotypes defined by the presence of either exoS or exoU. In this study, loop-mediated isothermal amplification (LAMP) assays for rapid detection of exoS and exoU in P. aeruginosa have been developed and evaluated. Set of four primers were designed for LAMP-based amplification of exoS and exoU respectively. The LAMP reactions were performed at 63°C for 40 min, with detection limits of 100 fg purified DNA. In 107 river water isolates, exoS and exoU were detected in 10 (9%) and 89 (83%) isolates, respectively, and in 38 soil isolates, they were detected in 7 (18%) and 31 (82%) cases respectively. In conclusion, the LAMP assays are rapid, simple and cost-effective tools for detection of the exoU- and exoS-types of P. aeruginosa strains. This method can be used for the rapid, sensitive and low-cost detection of genes (exoS and exoU) encoding proteins that are part of Type III Secretion System of Pseudomonas aeruginosa. It can serve as an efficient method in outbreak situations or in routine surveillance studies to judge virulence potential and to investigate pathogenesis of P. aeruginosa. © 2015 The Society for Applied Microbiology.

  5. Hereditary Hemochromatosis Predisposes Mice to Yersinia pseudotuberculosis Infection Even in the Absence of the Type III Secretion System

    PubMed Central

    Miller, Halie K.; Schwiesow, Leah; Au-Yeung, Winnie; Auerbuch, Victoria

    2016-01-01

    The iron overload disorder hereditary hemochromatosis (HH) predisposes humans to serious disseminated infection with pathogenic Yersinia as well as several other pathogens. Recently, we showed that the iron-sulfur cluster coordinating transcription factor IscR is required for type III secretion in Y. pseudotuberculosis by direct control of the T3SS master regulator LcrF. In E. coli and Yersinia, IscR levels are predicted to be regulated by iron bioavailability, oxygen tension, and oxidative stress, such that iron depletion should lead to increased IscR levels. To investigate how host iron overload influences Y. pseudotuberculosis virulence and the requirement for the Ysc type III secretion system (T3SS), we utilized two distinct murine models of HH: hemojuvelin knockout mice that mimic severe, early-onset HH as well as mice with the HfeC282Y∕C282Y mutation carried by 10% of people of Northern European descent, associated with adult-onset HH. Hjv−∕− and HfeC282Y∕C282Y transgenic mice displayed enhanced colonization of deep tissues by Y. pseudotuberculosis following oral inoculation, recapitulating enhanced susceptibility of humans with HH to disseminated infection with enteropathogenic Yersinia. Importantly, HH mice orally infected with Y. pseudotuberculosis lacking the T3SS-encoding virulence plasmid, pYV, displayed increased deep tissue colonization relative to wildtype mice. Consistent with previous reports using monocytes from HH vs. healthy donors, macrophages isolated from HfeC282Y∕C282Y mice were defective in Yersinia uptake compared to wildtype macrophages, indicating that the anti-phagocytic property of the Yersinia T3SS plays a less important role in HH animals. These data suggest that Yersinia may rely on distinct virulence factors to cause disease in healthy vs. HH hosts. PMID:27446816

  6. Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

    PubMed Central

    Borth, Nicole; Litsche, Katrin; Franke, Claudia; Sachse, Konrad; Saluz, Hans Peter; Hänel, Frank

    2011-01-01

    Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation. PMID:21304914

  7. The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

    PubMed

    Chaudhury, Sukanya; de Azevedo Souza, Clarice; Plano, Gregory V; De Guzman, Roberto N

    2015-09-25

    The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV. Copyright © 2015. Published by Elsevier Ltd.

  8. Understanding the sequential activation of Type III and Type VI Secretion Systems in Salmonella typhimurium using Boolean modeling

    PubMed Central

    2013-01-01

    Background Three pathogenicity islands, viz. SPI-1 (Salmonella pathogenicity island 1), SPI-2 (Salmonella pathogenicity island 2) and T6SS (Type VI Secretion System), present in the genome of Salmonella typhimurium have been implicated in the virulence of the pathogen. While the regulation of SPI-1 and SPI-2 (both encoding components of the Type III Secretion System - T3SS) are well understood, T6SS regulation is comparatively less studied. Interestingly, inter-connections among the regulatory elements of these three virulence determinants have also been suggested to be essential for successful infection. However, till date, an integrated view of gene regulation involving the regulators of these three secretion systems and their cross-talk is not available. Results In the current study, relevant regulatory information available from literature have been integrated into a single Boolean network, which portrays the dynamics of T3SS (SPI-1 and SPI-2) and T6SS mediated virulence. Some additional regulatory interactions involving a two-component system response regulator YfhA have also been predicted and included in the Boolean network. These predictions are aimed at deciphering the effects of osmolarity on T6SS regulation, an aspect that has been suggested in earlier studies, but the mechanism of which was hitherto unknown. Simulation of the regulatory network was able to recreate in silico the experimentally observed sequential activation of SPI-1, SPI-2 and T6SS. Conclusions The present study integrates relevant gene regulatory data (from literature and our prediction) into a single network, representing the cross-communication between T3SS (SPI-1 and SPI-2) and T6SS. This holistic view of regulatory interactions is expected to improve the current understanding of pathogenesis of S. typhimurium. PMID:24079299

  9. 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 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.

  10. NMR characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa

    PubMed Central

    Chaudhury, Sukanya; Nordhues, Bryce A.; Kaur, Kawaljit; Zhang, Na; De Guzman, Roberto N.

    2017-01-01

    Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS) to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologs are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by CD and NMR spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residues 16–41 and 55–76. NMR backbone dynamics data show that the helices in PcrG have semi-rigid flexibility and they tumble as a single entity with similar backbone dynamics. NMR titrations show that the entire length of PcrG residues from 9–76 is involved in binding to PcrV. Thus the PcrG family of T3SS chaperone proteins is essentially partially folded. PMID:26451841

  11. Angiogenic inhibitors delivered by the type III secretion system of tumor-targeting Salmonella typhimurium safely shrink tumors in mice.

    PubMed

    Shi, Lei; Yu, Bin; Cai, Chun-Hui; Huang, Jian-Dong

    2016-12-01

    Despite of a growing number of bacterial species that apparently exhibit intrinsic tumor-targeting properties, no bacterium is able to inhibit tumor growth completely in the immunocompetent hosts, due to its poor dissemination inside the tumors. Oxygen and inflammatory reaction form two barriers and restrain the spread of the bacteria inside the tumors. Here, we engineered a Salmonella typhimurium strain named ST8 which is safe and has limited ability to spread beyond the anaerobic regions of tumors. When injected systemically to tumor-bearing immunocompetent mice, ST8 accumulated in tumors at levels at least 100-fold greater than parental obligate anaerobic strain ST4. ST8/pSEndo harboring therapeutic plasmids encoding Endostatin fused with a secreted protein SopA could target vasculature at the tumor periphery, can stably maintain and safely deliver a therapeutic vector, release angiogenic inhibitors through a type III secretion system (T3SS) to interfere with the pro-angiogenic action of growth factors in tumors. Mice with murine CT26 colon cancer that had been injected with ST8/pSEndo showed efficient tumor suppression by inducing more severe necrosis and inhibiting blooding vessel density within tumors. Our findings provide a therapeutic platform for indirectly acting therapeutic strategies such as anti-angiogenesis and immune therapy.

  12. Biophysical Characterization of Chlamydia trachomatis CT584 Supports Its Potential Role as a Type III Secretion Needle Tip Protein

    PubMed Central

    Markham, Aaron P.; Jaafar, Zane A.; Kemege, Kyle E.; Middaugh, C. Russell; Hefty, P. Scott

    2014-01-01

    Chlamydia are obligate intracellular bacterial pathogens that cause a variety of diseases. Likemany Gram-negative bacteria, they employ type III secretion systems (T3SS) for invasion, establishing and maintaining their unique intracellular niche, and possibly cellular exit. Computational structure prediction indicated that ORF CT584 is homologous to other T3SS needle tip proteins. Tip proteins have been shown to be localized to the extracellular end of the T3SS needle and play a key role in controlling secretion of effector proteins. We have previously demonstrated that T3SS needle tip proteins from different bacteria share many biophysical characteristics. To support the hypothesis that CT584 is a T3SS needle tip protein, biophysical properties of CT584 were explored as a function of pH and temperature, using spectroscopic techniques. Far-UV circular dichroism, Fourier transform infrared spectroscopy, UV absorbance spectroscopy, ANS extrinsic fluorescence, turbidity, right angle static light scattering, and analytical ultracentrifugation were all employed to monitor the secondary, tertiary, quaternary, and aggregation behavior of this protein. An empirical phase diagram approach is also employed to facilitate such comparisons. These analyses demonstrate that CT584 shares many biophysical characteristics with other T3SS needle tip proteins. These data support the hypothesis that CT584 is a member of the same functional family, although future biologic analyses are required. PMID:19769366

  13. InvB is a type III secretion-associated chaperone for the Salmonella enterica effector protein SopE.

    PubMed

    Lee, Sang Ho; Galán, Jorge E

    2003-12-01

    SopE is a bacteriophage-encoded effector protein of Salmonella enterica serovar Typhimurium that is translocated into the cytosol of eukaryotic cells by a type III secretion system (TTSS) (W.-D. Hardt, H. Urlaub, and J. E. Galán, Proc. Natl. Acad. Sci. USA 95:2574-2579, 1998; M. W. Wood, R. Rosqvist, P. B. Mullan, M. H. Edwards, and E. E. Galyov, Mol. Microbiol. 22:327-338, 1996). In this study, we provide evidence that an unlinked gene carried within the Salmonella pathogenicity island 1 (SPI-1), invB (K. Eichelberg, C. Ginocchio, and J. E. Galán, J. Bacteriol. 176:4501-4510, 1994), is required for the secretion of SopE through the SPI-1 TTSS. Furthermore, far-Western blotting analysis shows that SopE directly interacts with InvB through a domain located at its amino terminus. We conclude that InvB is the TTSS-associated chaperone for SopE.

  14. The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

    PubMed Central

    Intile, Peter J.; Balzer, Grant J.; Wolfgang, Matthew C.

    2015-01-01

    ABSTRACT The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS. IMPORTANCE Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box

  15. Mapping Type IV Secretion Signals on the Primase Encoded by the Broad-Host-Range Plasmid R1162 (RSF1010).

    PubMed

    Meyer, Richard

    2015-10-01

    The plasmid R1162 (RSF1010) encodes a primase essential for its replication. This primase makes up the C-terminal part of MobA, a multifunctional protein with the relaxase as a separate N-terminal domain. The primase is also translated separately as the protein RepB'. Here, we map two signals for type IV secretion onto the recently solved structure of RepB'. One signal is located internally within RepB' and consists of a long α-helix and an adjacent disordered region rich in arginines. The second signal is made up of the same α-helix and a second, arginine-rich region at the C-terminal end of the protein. Successive arginine-to-alanine substitutions revealed that either signal can be utilized by the type IV secretion complex of the plasmid R751. The internal signal also enables conjugal transfer when linked to the relaxase part of MobA. Both signals are similar to those previously identified for type IV secretion substrates in the Vir system of Agrobacterium tumefaciens. Moreover, the C-terminal arginine-rich segment of RepB' has been shown to be secreted by Vir. However, with R751, the signals require MobB, an R1162-encoded accessory protein active in conjugal transfer. The results of two-hybrid assays revealed that MobB interacts, via its membrane-associated domain, with the R751 plasmid coupling protein TraG. In addition, MobB interacts with a region of MobA just outside the RepB' domain. Therefore, MobB is likely an adaptor that is essential for recognition of the primase-associated signals by the R751 secretion machinery. For most plasmids, type IV secretion is an intrinsic part of the mechanism for conjugal transfer. Protein relaxases, bound to the 5' end of the transferring strand, are mobilized into recipient cells by the type IV pathway. In this work, we identify and characterize two signals for secretion in the primase domain of MobA, the relaxase of the IncQ plasmid R1162 (RSF1010). We also show that the adaptor protein MobB is required for engagement

  16. Lytic Activity of LysH5 Endolysin Secreted by Lactococcus lactis Using the Secretion Signal Sequence of Bacteriocin Lcn972

    PubMed Central

    Rodríguez-Rubio, Lorena; Gutiérrez, Dolores; Martínez, Beatriz; Rodríguez, Ana

    2012-01-01

    Bacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded by Staphylococcus aureus phage vB_SauS-phi-IPLA88, was expressed and secreted in Lactococcus lactis using the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts. PMID:22344638

  17. Leukotriene B4 potentiates CpG signaling for enhanced cytokine secretion by human leukocytes.

    PubMed

    Gaudreault, Eric; Gosselin, Jean

    2009-08-15

    TLRs are known to be important in innate host defense against a variety of microbial infections. In particular, TLR9 has been associated with immune defense against different foreign organisms by recognition of unmethylated DNA sequences. In this report, we provide evidence that leukotriene B(4) (LTB(4)) has the capacity to modulate TLR9 expression on human neutrophils. The effect of LTB(4) was found to be specific, because related leukotrienes such as LTC(4) and LTD(4) or neutrophil agonists IL-8 and C5a failed to modulate TLR9 expression in neutrophils. Using fluorochrome-tagged CpG DNA, we observed that LTB(4) treatment also increased TLR9 ligand binding in neutrophils. Moreover, LTB(4) stimulation potentiates CpG-mediated signaling via an endosome-independent mechanism in human neutrophils, leading to enhanced secretion of proinflammatory cytokines. The increase in cytokine secretion by LTB(4) following CpG stimulation of neutrophils was associated with the activation of TGF-beta-activated kinase (TAK-1) as well as p38 and c-Jun (JNK) kinases. In contrast, in PBMC LTB(4) leads to an increase in cytokine secretion following CpG stimulation but via a MyD88- and endosome-dependent mechanism. As observed in neutrophils, PBMC stimulation with LTB(4) in the presence of CpG also results in enhanced TAK-1, p38, and JNK phosphorylation/activation. These data provide new evidence underlying the immunomodulatory properties of LTB(4) leading to antimicrobial defense.

  18. Effects of terbium (III) on signaling molecules in horseradish.

    PubMed

    Wang, Lihong; Zhang, Xuanbo; Zhou, Qing; Huang, Xiaohua

    2015-03-01

    Rare earth elements, especially terbium (Tb), are high-valence heavy metal elements that accumulate in the environment, and they show toxic effects on plants. Signaling molecules regulate many physiological and biochemical processes in plants. How rare earth elements affect signaling molecules remains largely unknown. In the present study, the effects of Tb(3+) on some extracellular and intracellular signaling molecules (gibberellic acid, abscisic acid, auxin, H2O2, and Ca(2+)) in horseradish leaves were investigated by using high-performance liquid chromatography, X-ray energy spectrometry, and transmission electron microscopy, and Tb(3+) was sprayed on the surface of leaves. Tb(3+) treatment decreased the auxin and gibberellic acid contents and increased the abscisic acid content. These changes in the contents of phytohormones (gibberellic acid, abscisic acid, and auxin) triggered excessive production of intracellular H2O2. Consequently, the increase in H2O2 content stimulated the influx of extracellular Ca(2+) and the release of Ca(2+) from Ca(2+) stores, leading to Ca(2+) overload and the resulting inhibition of physiological and biochemical processes. The effects outlined above were more evident with increasing the concentration of Tb(3+) sprayed on horseradish leaves. Our data provide a possible underlying mechanism of Tb(3+) action on plants.

  19. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

    SciTech Connect

    Davis, Jamaine; Wang, Jiawei; Tropea, Joseph E.; Zhang, Di; Dauter, Zbigniew; Waugh, David S.; Wlodawer, Alexander

    2009-01-28

    VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave {alpha}-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 {angstrom} resolution. The shape of the molecule resembles the letter 'V,' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

  20. Chronic intermittent hypoxia disturbs insulin secretion and causes pancreatic injury via the MAPK signaling pathway.

    PubMed

    Wang, Yeying; Hai, Bing; Niu, Xiaoqun; Ai, Li; Cao, Yu; Li, Ran; Li, Yongxia

    2017-06-01

    Obstructive sleep apnea (OSA) is a breathing disorder during sleep, with a most prominent character of chronic intermittent hypoxia (CIH), which induces the generation of reactive oxygen species (ROS) that damages multiple tissues and causes metabolic disorders. In this study, we established a rat model of varying OSA with different grades of CIH (12.5% O2, 10% O2, 7.5% O2, and 5% O2) for 12 weeks, and found that CIH stimulated insulin secretion, reduced the insulin:proinsulin ratio in pancreatic tissue, and caused pancreatic tissue lesions and cell apoptosis in a dose-dependent manner. Moreover, CIH promoted the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and activated mitogen-activated protein kinase (MAPK) family members, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and P38, depending on the O2 concentration. In summary, CIH disturbed insulin secretion, and caused inflammation, lesions, and cell apoptosis in pancreatic tissue via the MAPK signaling pathway, which may be of great significance for clinical treatment of OSA and type 2 diabetes mellitus (T2DM).

  1. Vesicular Trafficking and Signaling for Cytokine and Chemokine Secretion in Mast Cells

    PubMed Central

    Blank, Ulrich; Madera-Salcedo, Iris Karina; Danelli, Luca; Claver, Julien; Tiwari, Neeraj; Sánchez-Miranda, Elizabeth; Vázquez-Victorio, Genaro; Ramírez-Valadez, Karla Alina; Macias-Silva, Marina; González-Espinosa, Claudia

    2014-01-01

    Upon activation mast cells (MCs) secrete numerous inflammatory compounds stored in their cytoplasmic secretory granules by a process called anaphylactic degranulation, which is responsible for type I hypersensitivity responses. Prestored mediators include histamine and MC proteases but also some cytokines and growth factors making them available within minutes for a maximal biological effect. Degranulation is followed by the de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as a vast array of cytokines, chemokines, and growth factors, which are responsible for late phase inflammatory responses. While lipid mediators diffuse freely out of the cell through lipid bilayers, both anaphylactic degranulation and secretion of cytokines, chemokines, and growth factors depends on highly regulated vesicular trafficking steps that occur along the secretory pathway starting with the translocation of proteins to the endoplasmic reticulum. Vesicular trafficking in MCs also intersects with endocytic routes, notably to form specialized cytoplasmic granules called secretory lysosomes. Some of the mediators like histamine reach granules via specific vesicular monoamine transporters directly from the cytoplasm. In this review, we try to summarize the available data on granule biogenesis and signaling events that coordinate the complex steps that lead to the release of the inflammatory mediators from the various vesicular carriers in MCs. PMID:25295038

  2. The cardiokine secreted Frizzled-related protein 3, a modulator of Wnt signalling, in clinical and experimental heart failure.

    PubMed

    Askevold, E T; Aukrust, P; Nymo, S H; Lunde, I G; Kaasbøll, O J; Aakhus, S; Florholmen, G; Ohm, I K; Strand, M E; Attramadal, H; Fiane, A; Dahl, C P; Finsen, A V; Vinge, L E; Christensen, G; Yndestad, A; Gullestad, L; Latini, R; Masson, S; Tavazzi, L; Ueland, T

    2014-06-01

    Experimental studies have shown involvement of Wnt signalling in heart failure (HF). We hypothesized that secreted frizzled-related protein 3 (sFRP3), a modulator of Wnt signalling, is related to the progression of HF. Circulating sFRP3 was measured in 153 HF patients and compared with 25 healthy controls. The association of sFRP3 with mortality was evaluated in 1202 patients (GISSI-HF trial). sFRP3 mRNA expression was assessed in failing human and murine left ventricles (LV), and cellular localization was determined after fractioning of myocardial tissue. In vitro studies were carried out in cardiac fibroblasts subjected to cyclic mechanical stretch. (i) Heart failure patients had significantly raised serum sFRP3 levels compared with controls, (ii) during a median follow-up of 47 months, 315 patients died in the GISSI-HF substudy. In univariable Cox regression, tertiles of baseline sFRP3 concentration were significantly associated with all-cause and cardiovascular mortality. After adjustment for demographic and clinical variables, but not for CRP and NT-proBNP, the associations with mortality remained significant for the third tertile (all-cause, HR 1.45, P = 0.011; cardiovascular, HR 1.66, P = 0.003), (iii) sFRP3 mRNA expression was increased in failing human LV, with a decline following LV assist device therapy. LV from post-MI mice showed an increased sFRP3 mRNA level, particularly in cardiac fibroblasts, and (iv) mechanical stretch enhanced sFRP3 expression and release in myocardial fibroblasts. There is an association between increased sFRP3 expression and adverse outcome in HF, suggesting that the failing myocardium itself contributes to an increase in circulating sFRP3. © 2013 The Association for the Publication of the Journal of Internal Medicine.

  3. 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...

  4. 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...

  5. 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...

  6. Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors

    USDA-ARS?s Scientific Manuscript database

    Many bacterial pathogens of plants and animals disarm and remodel host cells by injecting large repertoires of effectors via the type III secretion system (T3SS). The repertoires of individual strains appear to function as robust systems that can tolerate loss of individual effectors with little or ...

  7. New protein-protein interactions identified for the regulatory and structural components and substrates of the type III Secretion system of the phytopathogen Xanthomonas axonopodis Pathovar citri.

    PubMed

    Alegria, Marcos C; Docena, Cassia; Khater, Leticia; Ramos, Carlos H I; da Silva, Ana C R; Farah, Chuck S

    2004-09-01

    We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.

  8. New Protein-Protein Interactions Identified for the Regulatory and Structural Components and Substrates of the Type III Secretion System of the Phytopathogen Xanthomonas axonopodis Pathovar citri

    PubMed Central

    Alegria, Marcos C.; Docena, Cassia; Khater, Leticia; Ramos, Carlos H. I.; da Silva, Ana C. R.; Farah, Chuck S.

    2004-01-01

    We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity. PMID:15342589

  9. Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N).

    PubMed

    Squires, P E; Amiranoff, B; Dunne, M J

    1994-10-01

    Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1-100 microM) on the intracellular free calcium ion concentration ([Ca2+]i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca2+]i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca2+]i transients. Overall, the pattern of response was concentration related. In general, 0.1-10 microM acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in [Ca2+]i followed by a smaller more sustained increase. Without external Ca2+, 100 microM acetylcholine caused only a transient rise in [Ca2+]i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca2+]i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1-10 microM), verapamil (100 microM) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca2+]i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca2+]i. Ryanodine (50-500 nM) and caffeine (1-20 mM) did not increase basal Ca2+ levels, but the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca2+]i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.

  10. Regulation of Pol III Transcription by Nutrient and Stress Signaling Pathways

    PubMed Central

    Moir, Robyn D.; Willis, Ian M.

    2012-01-01

    Transcription by RNA polymerase III (pol III) is responsible for ~15% of total cellular transcription through the generation of small structured RNAs such as tRNA and 5S RNA. The coordinate synthesis of these molecules with ribosomal protein mRNAs and rRNA couples the production of ribosomes and their tRNA substrates and balances protein synthetic capacity with the growth requirements of the cell. Ribosome biogenesis in general and pol III transcription in particular is known to be regulated by nutrient availability, cell stress and cell cycle stage and is perturbed in pathological states. High throughput proteomic studies have catalogued modifications to pol III subunits, assembly, initiation and accessory factors but most of these modifications have yet to be linked to functional consequences. Here we review our current understanding of the major points of regulation in the pol III transcription apparatus, the targets of regulation and the signaling pathways known to regulate their function. PMID:23165150

  11. Can the 21-cm signal probe Population III and II star formation?

    NASA Astrophysics Data System (ADS)

    Yajima, Hidenobu; Khochfar, Sadegh

    2015-03-01

    Using varying models for the star formation rate (SFR) of Population (Pop) III and II stars at z > 6 we derive the expected redshift history of the global 21-cm signal from the intergalactic medium (IGM). To recover the observed Thomson scattering optical depth of the cosmic microwave background (CMB) requires SFRs at the level of ˜ 10- 3 M⊙ yr- 1 Mpc- 3 at z ˜ 15 from Pop III stars, or ˜ 10- 1 M⊙ yr- 1 Mpc- 3 at z ˜ 7 from Pop II stars. In the case the SFR is dominated by Pop III stars, the IGM quickly heats above the CMB at z ≳ 12 due to heating from supernovae. In addition, Lyα photons from haloes hosting Pop III stars couple the spin temperature to that of the gas, resulting in a deep absorption signal. If the SFR is dominated by Pop II stars, the IGM slowly heats and exceeds the CMB temperature at z ˜ 10. However, the larger and varying fraction of Pop III stars are able to break this degeneracy. We find that the impact of the initial mass function (IMF) of Pop III stars on the 21-cm signal results in an earlier change to a positive signal if the IMF slope is ˜-1.2. Measuring the 21-cm signal at z ≳ 10 with next generation radio telescopes such as the Square Kilometre Array will be able to investigate the contribution from Pop III and Pop II stars to the global SFR.

  12. The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins.

    PubMed

    Chen, Ning; Greenwald, Iva

    2004-02-01

    The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Here, we computationally identify ten genes that encode potential ligands for LIN-12, and show that three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. We also show that transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, we show that DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays.

  13. Specific maceration and induction of PR-3 gene in potato tuber tissue by Pectobacterium carotovorum subsp. atrosepticum type III secretion system mutants.

    PubMed

    Aghabozorgy, Sohrab; Niakan, Mohammad

    2009-12-15

    The exact function of type III secretion system in some phytopathogenes including Pectobacterium carotovorum subsp. atrosepticum (Pca) is not understood and is a matter of debate. The aim of this study were to determine specific effect of type III secretion system on potato tubers and to reveal the connection of this system with potato resistant genes such as PR-3. A Pca hrpW fragment was subcloned into a low-copy-number cloning vector (pZH448). The resulting plasmid (pAS19) was then conjugated into the wild-type and mutant strains of Pca by type III secretion system. The virulence property of different Pca strains was studied and the influence of over expression of hrpW on maceration activity was also investigated. Furthermore, the effect of mentioned mutation on the maceration of carrot-root was evaluated. Finally, using real-time PCR, the copy-number of PR-3 gene in potato tuber tissue was assessed. In conclusion, for type III secretion system mutant strains, in contrast with the wild-type, the maceration amount of potato tuber tissue decreased after over expression of hrpW while inoculation of tubers by mutants, increased this amount. In the case of potato, HrpN and DspE proteins appeared to be avirulent factors. Compared with the wild-type strains, Pca nominated mutants significantly reduced potato PR-3 expression thus, PR-3 expression level in potato tuber tissue in answer to infiltration by Pca, depends on functional type III secretion system in the bacterium.

  14. Enantiomeric NMR signal separation behavior and mechanism of samarium(III) and neodymium(III) complexes with (S,S)-ethylenediamine-N,N'-disuccinate.

    PubMed

    Aizawa, Sen-Ichi; Okano, Masaru; Kidani, Takahiro

    2017-04-12

    Enantiomeric (1) H and (13) C NMR signal separation behaviors of various α-amino acids and DL-tartarate were investigated by using the samarium(III) and neodymium(III) complexes with (S,S)-ethylenediamine-N,N'-disuccinate as chiral shift reagents. A relatively smaller concentration ratio of the lanthanide(III) complex to substrates was suitable for the neodymium(III) complex compared with the samarium(III) one, striking a balance between relatively greater signal separation and broadening. To clarify the difference in the signal separation behavior, the chemical shifts of β-protons for fully bound D- and L-alanine (δb (D) and δb (L)) and their adduct formation constants (Ks) were obtained for both metal complexes. Preference for D-alanine was similarly observed for both complexes, while it was revealed that the difference between the δb (D) and δb (L) values is the significant factor to determine the enantiomeric signal separation. The neodymium(III) and samarium(III) complexes can be used complementarily for higher and smaller concentration ranges of substrates, respectively, because the neodymium(III) complex gives the larger difference between the δb (D) and δb (L) values with greater signal broadening compared to the samarium(III) complex.

  15. Glutamate Acts as a Key Signal Linking Glucose Metabolism to Incretin/cAMP Action to Amplify Insulin Secretion

    PubMed Central

    Gheni, Ghupurjan; Ogura, Masahito; Iwasaki, Masahiro; Yokoi, Norihide; Minami, Kohtaro; Nakayama, Yasumune; Harada, Kazuo; Hastoy, Benoit; Wu, Xichen; Takahashi, Harumi; Kimura, Kazushi; Matsubara, Toshiya; Hoshikawa, Ritsuko; Hatano, Naoya; Sugawara, Kenji; Shibasaki, Tadao; Inagaki, Nobuya; Bamba, Takeshi; Mizoguchi, Akira; Fukusaki, Eiichiro; Rorsman, Patrik; Seino, Susumu

    2014-01-01

    Summary Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion. PMID:25373904

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

    PubMed Central

    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. PMID:26473722

  17. Induction and Relaxation Dynamics of the Regulatory Network Controlling the Type III Secretion System encoded within Salmonella Pathogenicity Island 1

    PubMed Central

    Temme, Karsten; Salis, Howard; Tullman-Ercek, Danielle; Levskaya, Anselm; Hong, Soon-Ho; Voigt, Christopher A.

    2008-01-01

    Summary Bacterial pathogenesis requires the precise spatial and temporal control of gene expression, the dynamics of which are controlled by regulatory networks. A network encoded within Salmonella Pathogenicity Island 1 controls the expression of a type III protein secretion system involved in the invasion of host cells. The dynamics of this network are measured in single cells using promoter-green fluorescent protein (gfp) reporters and flow cytometry. During induction, there is a temporal order of gene expression, with transcriptional inputs turning on first, followed by structure, and effector genes. The promoters show varying stochastic properties, where graded inputs are converted into all-or-none and hybrid responses. The relaxation dynamics are measured by shifting cells from inducing into non-inducing conditions and measuring the fluorescence decay. The gfp expressed from promoters controlling the transcriptional inputs (hilC and hilD) and structural genes (prgH) decay exponentially with a characteristic time of 50–55 minutes. In contrast, the gfp expressed from a promoter controlling the expression of effectors (sicA) persists for 110 ± 9 minutes. This promoter is controlled by a genetic circuit formed by a transcription factor (InvF), chaperone (SicA) and secreted protein (SipC) that regulates effector expression in response to the secretion capacity of the cell. A mathematical model of this circuit demonstrates that the delay is due to a split positive feedback loop. This model is tested in a ΔsicA knockout where sicA is complemented with and without the feedback loop. The delay is eliminated when the feedback loop is deleted. Further, a robustness analysis of the model predicts that the delay time can be tuned by changing the affinity of SicA:InvF multimers to an operator in the sicA promoter. This prediction is used to construct a targeted library, which contains mutants with both longer and shorter delays. This combination of theory and

  18. Kisspeptin Restores Pulsatile LH Secretion in Patients with Neurokinin B Signaling Deficiencies: Physiological, Pathophysiological and Therapeutic Implications

    PubMed Central

    Young, Jacques; George, Jyothis T.; Tello, Javier A.; Francou, Bruno; Bouligand, Jerome; Guiochon-Mantel, Anne; Brailly-Tabard, Sylvie; Anderson, Richard A.; Millar, Robert P.

    2013-01-01

    Pulsatile gonadotropin-releasing hormone (GnRH) is crucial to normal reproductive function and abnormalities in pulse frequency give rise to reproductive dysfunction. Kisspeptin and neurokinin B (NKB), neuropeptides secreted by the same neuronal population in the ventral hypothalamus, have emerged recently as critical central regulators of GnRH and thus gonadotropin secretion. Patients with mutations resulting in loss of signaling by either of these neuroendocrine peptides fail to advance through puberty but the mechanisms mediating this remain unresolved. We report here that continuous kisspeptin infusion restores gonadotropin pulsatility in patients with loss-of-function mutations in NKB (TAC3) or its receptor (TAC3R), indicating that kisspeptin on its own is sufficient to stimulate pulsatile GnRH secretion. Moreover, our findings suggest that NKB action is proximal to kisspeptin in the reproductive neuroendocrine cascade regulating GnRH secretion, and may act as an autocrine modulator of kisspeptin secretion. The ability of continuous kisspeptin infusion to induce pulsatile gonadotropin secretion further indicates that GnRH neurons are able to set up pulsatile secretion in the absence of pulsatile exogenous kisspeptin. PMID:22377698

  19. Role of CD14 and TLR4 in type I, type III collagen expression, synthesis and secretion in LPS-induced normal human skin fibroblasts

    PubMed Central

    Yang, Hongming; Li, Juncong; Wang, Yihe; Hu, Quan

    2015-01-01

    Objectives: The primary aim of this study was to investigate the role of CD14 and TLR4 in type I, type III collagen expression, synthesis and secretion in LPS-induced normal human skin fibroblasts. The secondary aim was to provide theoretical basis for the molecular mechanisms of scar formation induced by LPS. Methods: The normal skin fibroblasts cultured in vitro were randomly divided into four groups: 0.1 μg/mL LPS reference group, CD14 pretreatment + LPS, TLR4 pretreatment + LPS, CD14 and TLR4 pretreatment + LPS. The collagen DNA synthesis was assessed by 3H-proline incorporation method. Real-time Quantitative PCR was used to detect type I, type III collagen mRNA expression. Results: Similar results were revealed for mRNA expression levels. The immunofluorescence staining suggested that type I and type III collagen were expressed in all investigated groups and that the expression was differentially downregulated in groups B, C, D. ELISA demonstrated markedly decreased levels in secreting type I, type III collagens and hydroxyproline in groups B, C, D (P<0.05), and the lowest level was detected in group D (P<0.01). Conclusion: Pretreatment with CD14 or TLR4 alone or their combination can significantly reduce the levels of type I and type III collagen expression, synthesis and secretion, with the most notable reduction detected in case of CD14 and TLR4 combined. We could thus conclude that both CD14 and TLR4 are involved in type I and type III collagen expression, synthesis and secretion in LPS-induced skin fibroblasts. PMID:25932184

  20. Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism*

    PubMed Central

    Rountree, Austin M.; Neal, Adam S.; Lisowski, Mark; Rizzo, Norma; Radtke, Jared; White, Sarah; Luciani, Dan S.; Kim, Francis; Hampe, Christiane S.; Sweet, Ian R.

    2014-01-01

    The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal. PMID:24841202

  1. The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway.

    PubMed

    Sawamoto, K; Okabe, M; Tanimura, T; Mikoshiba, K; Nishida, Y; Okano, H

    1996-08-25

    The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.

  2. Electrochemical monitoring of cellular signal transduction with a secreted alkaline phosphatase reporter system.

    PubMed

    Torisawa, Yu-Suke; Ohara, Noriko; Nagamine, Kuniaki; Kasai, Shigenobu; Yasukawa, Tomoyuki; Shiku, Hitoshi; Matsue, Tomokazu

    2006-11-15

    Electrochemical monitoring of cellular signal transduction under three-dimensional (3-D) cell culture conditions has been demonstrated by combining cell-based microarrays with a secreted alkaline phosphatase (SEAP) reporter system. The cells were genetically engineered to produce SEAP under the control of nuclear factor kappaB (NFkappaB) enhancer elements, and they were embedded with a small volume of a collagen gel matrix on a pyramidal-shaped silicon microstructure. Cellular SEAP expression triggered by NFkappaB activation was assessed by two types of electrochemical systems. First, SEAP expression of a 3-D cell array on a chip was continuously monitored in situ for 2 days by scanning electrochemical microscopy (SECM). Since the SECM-based assay enables the evaluation of cellular respiratory activity, simultaneous measurements of cellular viability and signal transduction were possible. Further, we have developed an electrode-integrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was integrated around the silicon microhole. Two kinds of cells were immobilized on the array of microholes on the same chip for comparative characterization of their SEAP activity. This electrochemical microdevice can be applied to evaluate the SEAP expression activity in multiple cellular microarrays by a high-throughput method.

  3. Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis.

    PubMed

    Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2011-01-01

    Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

  4. Metabolic signaling of insulin secretion by pancreatic β-cell and its derangement in type 2 diabetes.

    PubMed

    Zou, C-Y; Gong, Y; Liang, J

    2014-01-01

    Pancreatic beta-cell is responsible for insulin secretion in response to the availability of nutrients. Type 2 diabetes mellitus (T2D) is the result of pancreatic b-cell failure to supply sufficient amount of insulin accompanied with decreased sensitivity of the body tissues to respond to insulin. The insulin secretion apparatus of beta-cell is uniquely equipped with multiple metabolic and signaling steps that are under rigorous control. The metabolic machinery of beta-cell is designed to sense the fluctuations in blood glucose level and supply insulin accordingly to the needs of body. Besides glucose, amino acids including glutamine and leucine and also fatty acids are known to either stimulate the beta-cell directly or potentiate the glucose stimulated insulin secretion (GSIS) response. Glucose metabolism dependent GSIS is linked with the production of ATP that is needed for K+ATP channel inhibition and influx of calcium, necessary for insulin granule exocytosis. Besides glucose metabolism, amino acid metabolism and lipid metabolism derived metabolites mediate the optimal glucose response of beta-cells to secrete insulin. Metabolites derived from nutrient secretagogues that directly or indirectly participate in the enhancement of GSIS are considered as metabolic coupling factors. In this review, we will discuss the regulation of insulin secretion by b-cell keeping the recent developments in metabolic signaling in focus. The relevant metabolic pathways in pancreatic beta-cell and their role in the control of fuel-stimulated insulin secretion will be reviewed to arrive at a consensus picture with respect to the metabolic signaling of insulin secretion.

  5. Roles of the signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi.

    PubMed Central

    Chang, S C; Su, M H; Lee, Y H

    1997-01-01

    The neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a prepro-Npr precursor form consisting of a secretory signal peptide, a propeptide and the mature metalloprotease. The maturation of Npr occurs extracellularly via an autoproteolytic processing of the secreted pro-Npr. The integrity of the propeptide is essential for the formation of mature active Npr but not for its secretion [Chang, Chang and Lee (1994) J. Biol. Chem. 269, 3548-3554]. In this study we investigated whether the secretion and maturation of Npr require the integrity of its signal peptide region and mature protease domain. Five signal peptide mutants were generated, including the substitution mutations at the positively charged region (mutant IR6LE), the central hydrophobic region (mutants GI19EL and G19N), the boundary of the hydrophobic core-cleavage region (mutant P30L) and at the residues adjacent to the signal peptidase cleavage site (mutant YA33SM). All these lesions delayed the export of Npr to the growth medium and also resulted in a 2-10-fold decrease in Npr export. The most severe effect was noted in mutants GI19EL and P30L. When these signal peptide mutations were fused separately with the propeptide lacking the Npr mature domain, the secretory defect on the propeptide was also observed, and this impairment was again more severely expressed in mutants GI19EL and P30L. Thus the Npr signal peptide seems to have more constraints on the hydrophobic core region and at the proline residue within the boundary of the hydrophobic core-cleavage site. Deletion mutations within the C-terminal mature protease domain that left its active site intact still blocked the proteolytic processing of mutant precursor forms of pro-Npr, although their secretions were unaffected. These results, together with our previous findings, strongly suggest that the signal peptide of Npr plays a pivotal role in the secretion of both Npr and the propeptide, but not in the maturation of Npr. On the

  6. Rhb1 regulates the expression of secreted aspartic protease 2 through the TOR signaling pathway in Candida albicans.

    PubMed

    Chen, Yu-Ting; Lin, Chia-Ying; Tsai, Pei-Wen; Yang, Cheng-Yao; Hsieh, Wen-Ping; Lan, Chung-Yu

    2012-02-01

    Candida albicans is a major fungal pathogen in humans. In C. albicans, secreted aspartyl protease 2 (Sap2) is the most highly expressed secreted aspartic protease in vitro and is a virulence factor. Recent research links the small GTPase Rhb1 to C. albicans target of rapamycin (TOR) signaling in response to nitrogen availability. The results of this study show that Rhb1 is related to cell growth through the control of SAP2 expression when protein is the major nitrogen source. This process involves various components of the TOR signaling pathway, including Tor1 kinase and its downstream effectors. TOR signaling not only controls SAP2 transcription but also affects Sap2 protein levels, possibly through general amino acid control. DNA microarray analysis identifies other target genes downstream of Rhb1 in addition to SAP2. These findings provide new insight into nutrients, Rhb1-TOR signaling, and expression of C. albicans virulence factor.

  7. YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

    PubMed Central

    Amer, Ayad A. A.; Gurung, Jyoti M.; Costa, Tiago R. D.; Ruuth, Kristina; Zavialov, Anton V.; Forsberg, Åke; Francis, Matthew S.

    2016-01-01

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity. PMID:27446813

  8. The type III secretion system of biocontrol Pseudomonas fluorescens KD targets the phytopathogenic Chromista Pythium ultimum and promotes cucumber protection.

    PubMed

    Rezzonico, Fabio; Binder, Christian; Défago, Geneviève; Moënne-Loccoz, Yvan

    2005-09-01

    The type III secretion system (TTSS) is used by Proteobacteria for pathogenic or symbiotic interaction with plant and animal hosts. Recently, TTSS genes thought to originate from the phytopathogen Pseudomonas syringae were evidenced in Pseudomonas fluorescens KD, which protects cucumber from the oomycete Pythium ultimum (kingdom Chromista/Stramenopila). However, it is not known whether the TTSS contributes to plant protection by the bacterium and, if so, whether it targets the plant or the phytopathogen. Inactivation of TTSS gene hrcV following the insertion of an omega cassette strongly reduced the biocontrol activity of the pseudomonad against P. ultimum on cucumber when compared with the wild type, but had no effect on its root-colonization ability. Analysis of a plasmid-based transcriptional hrpJ'-inaZ reporter fusion revealed that expression in strain KD of the operon containing hrcV was strongly stimulated in vitro and in situ by the oomycete and not by the plant. In vitro, both strain KD and its hrcV mutant reduced the activity level of the pectinase polygalacturonase (a key pathogenicity factor) from P. ultimum, but the reduction was much stronger with the wild type. Together, these results show that the target range of bacterial TTSS is not restricted to plants and animals but also can include members of Chromista/Stramenopila, and suggest that virulence genes acquired horizontally from phytopathogenic bacteria were functionally recycled in biocontrol saprophytic Pseudomonas spp., resulting in enhanced plant protection by the latter.

  9. Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

    PubMed

    Bibova, Ilona; Hot, David; Keidel, Kristina; Amman, Fabian; Slupek, Stephanie; Cerny, Ondrej; Gross, Roy; Vecerek, Branislav

    2015-01-01

    Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

  10. Identification of Novel Protein-Protein Interactions of Yersinia pestis Type III Secretion System by Yeast Two Hybrid System

    PubMed Central

    Tang, Liujun; Wang, Jian; Ke, Yuehua; Guo, Zhaobiao; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2013-01-01

    Type III secretion system (T3SS) of the plague bacterium Y. pestis encodes a syringe-like structure consisting of more than 20 proteins, which can inject virulence effectors into host cells to modulate the cellular functions. Here in this report, interactions among the possible components in T3SS of Yersinia pestis were identified using yeast mating technique. A total of 57 genes, including all the pCD1-encoded genes except those involved in plasmid replication and partition, pseudogenes, and the putative transposase genes, were subjected to yeast mating analysis. 21 pairs of interaction proteins were identified, among which 9 pairs had been previously reported and 12 novel pairs were identified in this study. Six of them were tested by GST pull down assay, and interaction pairs of YscG-SycD, YscG-TyeA, YscI-YscF, and YopN-YpCD1.09c were successfully validated, suggesting that these interactions might play potential roles in function of Yersinia T3SS. Several potential new interactions among T3SS components could help to understand the assembly and regulation of Yersinia T3SS. PMID:23349800

  11. Molecular mechanisms of two-component system RhpRS regulating type III secretion system in Pseudomonas syringae

    PubMed Central

    Deng, Xin; Liang, Haihua; Chen, Kai; He, Chuan; Lan, Lefu; Tang, Xiaoyan

    2014-01-01

    Pseudomonas syringae uses the two-component system RhpRS to regulate the expression of type III secretion system (T3SS) genes and bacterial virulence. However, the molecular mechanisms and the regulons of RhpRS have yet to be fully elucidated. Here, we show that RhpS functions as a kinase and a phosphatase on RhpR and as an autokinase upon itself. RhpR is phosphorylated by the small phosphodonor acetyl phosphate. A specific RhpR-binding site containing the inverted repeat (IR) motif GTATC-N6-GATAC, was mapped to its own promoter by a DNase I footprint analysis. Electrophoretic mobility shift assay indicated that P-RhpR has a higher binding affinity to the IR motif than RhpR. To identify additional RhpR targets in P. syringae, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) and detected 167 enriched loci including the hrpR promoter, suggesting the direct regulation of T3SS cascade genes by RhpR. A genome-wide microarray analysis showed that, in addition to the T3SS cascade genes, RhpR differentially regulates a large set of genes with various functions in response to different growth conditions. Together, these results suggested that RhpRS is a global regulator that allows P. syringae to sense and respond to environmental changes by coordinating T3SS expression and many other biological processes. PMID:25249629

  12. Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles.

    PubMed

    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

    2015-02-01

    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 (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III 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.

  13. The Bordetella bronchiseptica Type III Secretion System Is Required for Persistence and Disease Severity but Not Transmission in Swine

    PubMed Central

    Brockmeier, Susan L.; Loving, Crystal L.; Register, Karen B.; Kehrli, Marcus E.; Shore, Sarah M.

    2014-01-01

    Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica utilize isolates derived from hosts other than pigs in conjunction with rodent infection models. Based on previous in vivo mouse studies, we hypothesized that the B. bronchiseptica type III secretion system (T3SS) would be required for maximal disease severity and persistence in the swine lower respiratory tract. To examine the contribution of the T3SS to the pathogenesis of B. bronchiseptica in swine, we compared the abilities of a virulent swine isolate and an isogenic T3SS mutant to colonize, cause disease, and be transmitted from host to host. We found that the T3SS is required for maximal persistence throughout the lower swine respiratory tract and contributed significantly to the development of nasal lesions and pneumonia. However, the T3SS mutant and the wild-type parent are equally capable of transmission among swine by both direct and indirect routes, demonstrating that transmission can occur even with attenuated disease. Our data further suggest that the T3SS skews the adaptive immune response in swine by hindering the development of serum anti-Bordetella antibody levels and inducing an interleukin-10 (IL-10) cell-mediated response, likely contributing to the persistence of B. bronchiseptica in the respiratory tract. Overall, our results demonstrate that the Bordetella T3SS is required for maximal persistence and disease severity in pigs, but not for transmission. PMID:24366249

  14. Pseudomonas aeruginosa utilises its type III secretion system to kill the free-living amoeba Acanthamoeba castellanii.

    PubMed

    Abd, Hadi; Wretlind, Bengt; Saeed, Amir; Idsund, Eva; Hultenby, Kjell; Sandström, Gunnar

    2008-01-01

    Pseudomonas aeruginosa is a free-living and common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious human health problems. To overcome its predators, such as macrophages and environmental phagocytes, it utilises different survival strategies, such as the formation of microcolonies and the production of toxins mediated by a type III secretion system (TTSS). The aim of this study was to examine interaction of TTSS effector proteins of P. aeruginosa PA103 with Acanthamoeba castellanii by co-cultivation, viable count, eosin staining, electron microscopy, apoptosis assay, and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS effector proteins ExoU, ExoS, ExoT, and ExoY. In comparison, Acanthamoeba cultured alone and co-cultured with P. aeruginosa PA103 lacking the known four TTSS effector proteins were not killed. The results are consistent with P. aeruginosa being a strict extracellular bacterium that needs TTSS to survive in the environment, because the TTSS effector proteins are able to kill its eukaryotic predators, such as Acanthamoeba.

  15. Pre-clinical pharmacokinetics and anti-chlamydial activity of salicylidene acylhydrazide inhibitors of bacterial type III secretion.

    PubMed

    Ur-Rehman, Tofeeq; Slepenkin, Anatoly; Chu, Hencelyn; Blomgren, Anders; Dahlgren, Markus K; Zetterström, Caroline E; Peterson, Ellena M; Elofsson, Mikael; Gylfe, Asa

    2012-08-01

    Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (C(max)) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.

  16. Unique clustering genes in the bacterial chromosome affecting the type-III secretion of enterohaemorrhagic Escherichia coli.

    PubMed

    Lin, I-Ting; Chiou, Yi-Ming; Liang, Yen-Chia; Lin, Ching-Nan; Sun, Wei-Sheng W; Li, Shiaowen; Chang, Chuan-Hsiung; Syu, Wan-Jr; Chen, Jenn-Wei

    2016-10-01

    Bioinformatics analysis was used to search for unknown genes that might influence the phenotypic presentations of enterohaemorrhagic Escherichia coli (EHEC). By so doing and using the known genomic data from EHEC O157  : H7 and K-12, it has been deduced that genes Z4863 to Z4866 of EHEC do not exist in K-12 strains. These four gene sequences have low degrees of homology (18-40 % amino acid identities) to a set of genes in K-12, which have been known to encode fatty acid biosynthesis enzymes. We referred these four consecutive genes as a fasyn cluster and found that deletion of fasyn from EHEC resulted in a defective type-III secretion (T3S). This deletion apparently did not decrease the amounts of the T3S proteins ectopically expressed from plasmids. Examination of the corresponding mRNAs by real-time PCR revealed that the mRNAs readily decreased in the fasyn-deleted mutant and this suppressive effect on the mRNA levels appeared to spread across all lee operons. Complementation with fasyn reverted the T3S-deficient phenotype. Furthermore, this reversion was also seen when the mutant was supplemented with locus of enterocyte effacement activators (Ler or GrlA). Thus, these unique clustering genes located apart from locus of enterocyte effacement on the bacterial chromosome also play a role in affecting T3S of EHEC.

  17. The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri.

    PubMed

    Wang, Xia; Jiang, Feng; Zheng, Jianhua; Chen, Lihong; Dong, Jie; Sun, Lilian; Zhu, Yafang; Liu, Bo; Yang, Jian; Yang, Guowei; Jin, Qi

    2016-09-01

    Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens.

  18. Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum.

    PubMed

    Nahar, Kamrun; Matsumoto, Iyo; Taguchi, Fumiko; Inagaki, Yoshishige; Yamamoto, Mikihiro; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki; Mukaihara, Takafumi

    2014-04-01

    Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid-resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp-dependent manner. An Agrobacterium-mediated transient expression system revealed that Rip36, a putative Zn-dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn-binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002-derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn-dependent protease motif is essential for this activity.

  19. Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type III Secretion System Protein IpaD.

    PubMed

    Dey, Supratim; Anbanandam, Asokan; Mumford, Ben E; De Guzman, Roberto N

    2017-09-21

    Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. The Bordetella bronchiseptica type III secretion system is required for persistence and disease severity but not transmission in swine.

    PubMed

    Nicholson, Tracy L; Brockmeier, Susan L; Loving, Crystal L; Register, Karen B; Kehrli, Marcus E; Shore, Sarah M

    2014-03-01

    Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica utilize isolates derived from hosts other than pigs in conjunction with rodent infection models. Based on previous in vivo mouse studies, we hypothesized that the B. bronchiseptica type III secretion system (T3SS) would be required for maximal disease severity and persistence in the swine lower respiratory tract. To examine the contribution of the T3SS to the pathogenesis of B. bronchiseptica in swine, we compared the abilities of a virulent swine isolate and an isogenic T3SS mutant to colonize, cause disease, and be transmitted from host to host. We found that the T3SS is required for maximal persistence throughout the lower swine respiratory tract and contributed significantly to the development of nasal lesions and pneumonia. However, the T3SS mutant and the wild-type parent are equally capable of transmission among swine by both direct and indirect routes, demonstrating that transmission can occur even with attenuated disease. Our data further suggest that the T3SS skews the adaptive immune response in swine by hindering the development of serum anti-Bordetella antibody levels and inducing an interleukin-10 (IL-10) cell-mediated response, likely contributing to the persistence of B. bronchiseptica in the respiratory tract. Overall, our results demonstrate that the Bordetella T3SS is required for maximal persistence and disease severity in pigs, but not for transmission.

  1. Mathematical Model for Length Control by the Timing of Substrate Switching in the Type III Secretion System

    PubMed Central

    Nariya, Maulik K.; Israeli, Johnny; Shi, Jack J.; Deeds, Eric J.

    2016-01-01

    Type III Secretion Systems (T3SS) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism whereby they grow a needle-like structure in order to inject bacterial effector proteins into the cytoplasm of a host cell. Numerous experiments have been performed to understand the structural details of this nanomachine during the past decade. Despite the concerted efforts of molecular and structural biologists, several crucial aspects of the assembly of this structure, such as the regulation of the length of the needle itself, remain unclear. In this work, we used a combination of mathematical and computational techniques to better understand length control based on the timing of substrate switching, which is a possible mechanism for how bacteria ensure that the T3SS needles are neither too short nor too long. In particular, we predicted the form of the needle length distribution based on this mechanism, and found excellent agreement with available experimental data from Salmonella typhimurium with only a single free parameter. Although our findings provide preliminary evidence in support of the substrate switching model, they also make a set of quantitative predictions that, if tested experimentally, would assist in efforts to unambiguously characterize the regulatory mechanisms that control the growth of this crucial virulence factor. PMID:27078235

  2. Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

    PubMed

    Yang, Fan; Korban, Schuyler S; Pusey, P Lawrence; Elofsson, Michael; Sundin, George W; Zhao, Youfu

    2014-01-01

    The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease.

  3. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion.

    PubMed

    Nguyen, Tuyet Thi; Quan, Xianglan; Xu, Shanhua; Das, Ranjan; Cha, Seung-Kuy; Kong, In Deok; Shong, Minho; Wollheim, Claes B; Park, Kyu-Sang

    2016-12-01

    Elevated plasma levels of inorganic phosphate (Pi) are harmful, causing, among other complications, vascular calcification and defective insulin secretion. The underlying molecular mechanisms of these complications remain poorly understood. We demonstrated the role of Pi transport across the plasmalemma on Pi toxicity in INS-1E rat clonal β cells and rat pancreatic islet cells. Type III sodium-phosphate cotransporters (NaPis) are the predominant Pi transporters expressed in insulin-secreting cells. Transcript and protein levels of sodium-dependent phosphate transporter 1 and 2 (PiT-1 and -2), isotypes of type III NaPi, were up-regulated by high-Pi incubation. In patch-clamp experiments, extracellular Pi elicited a Na(+)-dependent, inwardly rectifying current, which was markedly reduced under acidic extracellular conditions. Cellular uptake of Pi elicited cytosolic alkalinization; intriguingly, this pH change facilitated Pi transport into the mitochondrial matrix. Increased mitochondrial Pi uptake accelerated superoxide generation, mitochondrial permeability transition (mPT), and endoplasmic reticulum stress-mediated translational attenuation, leading to reduced insulin content and impaired glucose-stimulated insulin secretion. Silencing of PiT-1/2 prevented Pi-induced superoxide generation and mPT, and restored insulin secretion. We propose that Pi transport across the plasma membrane and consequent cytosolic alkalinization could be a therapeutic target for protection from Pi toxicity in insulin-secreting cells, as well as in other cell types.-Nguyen, T. T., Quan, X., Xu, S., Das, R., Cha, S.-K., Kong, I. D., Shong, M., Wollheim, C. B., Park, K.-S. Intracellular alkalinization by phosphate uptake via type III sodium-phosphate cotransporter participates in high-phosphate-induced mitochondrial oxidative stress and defective insulin secretion. © FASEB.

  4. Analysis of the Crystal Structure of the ExsC.ExsE Complex Reveals Distinctive Binding Interactions of the Pseudomonas aeruginosa Type III Secretion Chaperone ExsC with ExsE and ExsD

    SciTech Connect

    Vogelaar, N.J.; Robinson, H.; Jing, X.; Schubot, F. D.

    2010-07-20

    Pseudomonas aeruginosa, like many Gram-negative bacterial pathogens, requires its type III secretion system (T3SS) to facilitate acute infections. In P. aeruginosa, the expression of all T3SS-related genes is regulated by the transcriptional activator ExsA. A signaling cascade involving ExsA and three additional proteins, ExsC, ExsD, and ExsE, directly ties the upregulation of ExsA-mediated transcription to the activation of the type III secretion apparatus. In order to characterize the events underlying the signaling process, the crystal structure of the T3SS chaperone ExsC in complex with its cognate effector ExsE has been determined. The structure reveals critical contacts that mediate the interactions between these two proteins. Particularly striking is the presence of two Arg-X-Val-X-Arg motifs in ExsE that form identical interactions along opposite sides of an ExsC dimer. The structure also provides insights into the interactions of ExsC with the antiactivator protein ExsD. It was shown that the amino-terminal 46 residues of ExsD are sufficient for ExsC binding. On the basis of these findings, a new model for the ExsC {center_dot} ExsD complex is proposed to explain its distinctive 2:2 stoichiometry and why ExsC displays a weaker affinity for ExsD than for ExsE.

  5. Long-Chain Fatty Acid Sensor, PsrA, Modulates the Expression of rpoS and the Type III Secretion exsCEBA Operon in Pseudomonas aeruginosa

    SciTech Connect

    Kang, Y.; Lunin, V. V.; Skarina, T.; Savchenko, A.; Schurr, M. J.; Hoang, T. T.

    2009-01-01

    The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5{beta}-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC. DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N-(butyryl)-l-homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa.

  6. Characterization of the Direct Interaction between Hybrid Sensor Kinases PA1611 and RetS That Controls Biofilm Formation and the Type III Secretion System in Pseudomonas aeruginosa.

    PubMed

    Bhagirath, Anjali Y; Pydi, Sai P; Li, Yanqi; Lin, Chen; Kong, Weina; Chelikani, Prashen; Duan, Kangmin

    2017-02-10

    One of the leading causes of morbidity and mortality in cystic fibrosis (CF) patients is pulmonary infection with Pseudomonas aeruginosa, and the pathophysiology of pulmonary infection in CF is affected by the lifestyle of this micro-organism. RetS-GacS/A-RsmA is a key regulatory pathway in P. aeruginosa that determines the bacterium's lifestyle choice. Previously, we identified PA1611, a hybrid sensor kinase, as a new player in this pathway that interacts with RetS and influences biofilm formation and type III secretion system. In this study, we explored the structural and mechanistic basis of the interaction between PA1611 and RetS. We identified the amino acid residues critical for PA1611-RetS interactions by molecular modeling. These residues were then targeted for site-directed mutagenesis. Amino acid substitutions were carried out at seven key positions in PA1611 and at six corresponding key positions in RetS. The influence of such substitutions in PA1611 on the interaction was analyzed by bacterial two-hybrid assays. We carried out functional analysis of these mutants in P. aeruginosa for their effect on specific phenotypes. Two residues, F269 and E276, located within the histidine kinase A and histidine kinase-like ATPase domains of PA1611 were found to play crucial roles in the PA1611-RetS interaction and had profound effects on phenotypes. Corresponding mutations in RetS demonstrated similar results. We further confirmed that these mutations in PA1611 function through the GacS/GacA-RsmY/Z signaling pathway. Collectively, our findings provide a noncognate sensor kinase direct interaction model for a signaling pathway, key for lifestyle selection in P. aeruginosa, and targeting such interaction may serve as a novel way of controlling infections with P. aeruginosa.

  7. Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD.

    PubMed

    Zwack, Erin E; Snyder, Annelise G; Wynosky-Dolfi, Meghan A; Ruthel, Gordon; Philip, Naomi H; Marketon, Melanie M; Francis, Matthew S; Bliska, James B; Brodsky, Igor E

    2015-02-17

    Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS. The innate immune response is critical to effective clearance of pathogens. Recognition of conserved virulence structures and activities by innate immune receptors such as NLRs

  8. Ultrasensitive fluorescence polarization DNA detection by target assisted exonuclease III-catalyzed signal amplification.

    PubMed

    Zhang, Min; Guan, Yi-Meng; Ye, Bang-Ce

    2011-03-28

    Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.

  9. Free fatty acid receptor 1 (FFAR1/GPR40) signaling affects insulin secretion by enhancing mitochondrial respiration during palmitate exposure.

    PubMed

    Kristinsson, Hjalti; Bergsten, Peter; Sargsyan, Ernest

    2015-12-01

    Fatty acids affect insulin secretion via metabolism and FFAR1-mediated signaling. Recent reports indicate that these two pathways act synergistically. Still it remains unclear how they interrelate. Taking into account the key role of mitochondria in insulin secretion, we attempted to dissect the metabolic and FFAR1-mediated effects of fatty acids on mitochondrial function. One-hour culture of MIN6 cells with palmitate significantly enhanced mitochondrial respiration. Antagonism or silencing of FFAR1 prevented the palmitate-induced rise in respiration. On the other hand, in the absence of extracellular palmitate FFAR1 agonists caused a modest increase in respiration. Using an agonist of the M3 muscarinic acetylcholine receptor and PKC inhibitor we found that in the presence of the fatty acid mitochondrial respiration is regulated via Gαq protein-coupled receptor signaling. The increase in respiration in palmitate-treated cells was largely due to increased glucose utilization and oxidation. However, glucose utilization was not dependent on FFAR1 signaling. Collectively, these results indicate that mitochondrial respiration in palmitate-treated cells is enhanced via combined action of intracellular metabolism of the fatty acid and the Gαq-coupled FFAR1 signaling. Long-term palmitate exposure reduced ATP-coupling efficiency of mitochondria and deteriorated insulin secretion. The presence of the FFAR1 antagonist during culture did not improve ATP-coupling efficiency, however, it resulted in enhanced mitochondrial respiration and improved insulin secretion after culture. Taken together, our study demonstrates that during palmitate exposure, integrated actions of fatty acid metabolism and fatty acid-induced FFAR1 signaling on mitochondrial respiration underlie the synergistic action of the two pathways on insulin secretion.

  10. LcrV delivered via type III secretion system of live attenuated Yersinia pseudotuberculosis enhances immunogenicity against pneumonic plague.

    PubMed

    Sun, Wei; Sanapala, Shilpa; Henderson, Jeremy C; Sam, Shandiin; Olinzock, Joseph; Trent, M Stephen; Curtiss, Roy

    2014-10-01

    Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression (araC P(BAD) crp) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δasd-206 ΔmsbB868::P(msbB) msbB(EC) ΔP(crp21)::TT araC P(BAD) crp] for use with a balanced-lethal Asd-positive (Asd(+)) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE(Nt138)-LcrV) was synthesized in χ10057 harboring an Asd(+) plasmid (pYA5199, yopE(Nt138)-lcrV) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ~240 median lethal doses (LD50) (2.4 × 10(4) CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Deconvolution of non-stationary physical signals: a smooth variance model for insulin secretion rate

    NASA Astrophysics Data System (ADS)

    Pillonetto, Gianluigi; Bell, Bradley M.

    2004-04-01

    Deconvolution is the process of estimating a system's input using measurements of a causally related output where the relationship between the input and output is known and linear. Regularization parameters are used to balance smoothness of the estimated input with accuracy of the measurement values. In this paper we present a maximum marginal likelihood method for estimating unknown regularization (and other) parameters used during deconvolution of dynamical systems. Our computational approach uses techniques that were developed for Kalman filters and smoothers. As an example application we consider estimating insulin secretion rate (ISR) following an intravenous glucose stimulus. This procedure is referred to in the medical literature as an intravenous glucose tolerance test (IVGTT). This estimation problem is difficult because ISR is a strongly non-stationary signal; it presents a fast peak in the first minutes of the experiment, followed by a smoother release. We use three regularization parameters to define a smooth model for ISR variance. This model takes into account the rapid variation of ISR during the beginning of an IVGTT and its slower variation as time progresses. Simulations are used to assess marginal likelihood estimation of these regularization parameters as well as of other parameters in the system. Simulations are also used to compare our model for ISR variance with other stochastic ISR models. In addition, we apply maximum marginal likelihood and our ISR variance model to real data that have previous ISR estimation results reported in the literature.

  12. Fucoidan Stimulates Monocyte Migration via ERK/p38 Signaling Pathways and MMP9 Secretion.

    PubMed

    Sapharikas, Elene; Lokajczyk, Anna; Fischer, Anne-Marie; Boisson-Vidal, Catherine

    2015-06-30

    Critical limb ischemia (CLI) induces the secretion of paracrine signals, leading to monocyte recruitment and thereby contributing to the initiation of angiogenesis and tissue healing. We have previously demonstrated that fucoidan, an antithrombotic polysaccharide, promotes the formation of new blood vessels in a mouse model of hindlimb ischemia. We examined the effect of fucoidan on the capacity of peripheral blood monocytes to adhere and migrate. Monocytes negatively isolated with magnetic beads from peripheral blood of healthy donors were treated with fucoidan. Fucoidan induced a 1.5-fold increase in monocyte adhesion to gelatin (p < 0.05) and a five-fold increase in chemotaxis in Boyden chambers (p < 0.05). Fucoidan also enhanced migration 2.5-fold in a transmigration assay (p < 0.05). MMP9 activity in monocyte supernatants was significantly enhanced by fucoidan (p < 0.05). Finally, Western blot analysis of fucoidan-treated monocytes showed upregulation of ERK/p38 phosphorylation. Inhibition of ERK/p38 phosphorylation abrogated fucoidan enhancement of migration (p < 0.01). Fucoidan displays striking biological effects, notably promoting monocyte adhesion and migration. These effects involve the ERK and p38 pathways, and increased MMP9 activity. Fucoidan could improve critical limb ischemia by promoting monocyte recruitment.

  13. The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola.

    PubMed

    Krijger, Jorrit-Jan; Horbach, Ralf; Behr, Michael; Schweizer, Patrick; Deising, Holger B; Wirsel, Stefan G R

    2008-10-01

    The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

  14. Embryonic Dorsal-Ventral Signaling: Secreted Frizzled-Related Proteins as Inhibitors of Tolloid Proteinases

    PubMed Central

    Lee, Hojoon X.; Ambrosio, Andrea L.; Reversade, Bruno; De Robertis, E.M.

    2008-01-01

    SUMMARY Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl ┤ Xlr ┤ Chd ┤ BMP → P-Smad1 → Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10−8 M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism. PMID:16413488

  15. Role of Epac2A/Rap1 signaling in interplay between incretin and sulfonylurea in insulin secretion.

    PubMed

    Takahashi, Harumi; Shibasaki, Tadao; Park, Jae-Hyung; Hidaka, Shihomi; Takahashi, Toshimasa; Ono, Aika; Song, Dae-Kyu; Seino, Susumu

    2015-04-01

    Incretin-related drugs and sulfonylureas are currently used worldwide for the treatment of type 2 diabetes. We recently found that Epac2A, a cAMP binding protein having guanine nucleotide exchange activity toward Rap, is a target of both incretin and sulfonylurea. This suggests the possibility of interplay between incretin and sulfonylurea through Epac2A/Rap1 signaling in insulin secretion. In this study, we examined the combinatorial effects of incretin and various sulfonylureas on insulin secretion and activation of Epac2A/Rap1 signaling. A strong augmentation of insulin secretion by combination of GLP-1 and glibenclamide or glimepiride, which was found in Epac2A(+/+) mice, was markedly reduced in Epac2A(-/-) mice. In contrast, the combinatorial effect of GLP-1 and gliclazide was rather mild, and the effect was not altered by Epac2A ablation. Activation of Rap1 was enhanced by the combination of an Epac-selective cAMP analog with glibenclamide or glimepiride but not gliclazide. In diet-induced obese mice, ablation of Epac2A reduced the insulin secretory response to coadministration of the GLP-1 receptor agonist liraglutide and glimepiride. These findings clarify the critical role of Epac2A/Rap1 signaling in the augmenting effect of incretin and sulfonylurea on insulin secretion and provide the basis for the effects of combination therapies of incretin-related drugs and sulfonylureas.

  16. 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.

  17. Fitness Cost of Fluoroquinolone Resistance in Clinical Isolates of Pseudomonas aeruginosa Differs by Type III Secretion Genotype.

    PubMed

    Agnello, Melissa; Finkel, Steven E; Wong-Beringer, Annie

    2016-01-01

    Fluoroquinolone (FQ) resistance is highly prevalent among clinical strains of Pseudomonas aeruginosa, limiting treatment options. We have reported previously that highly virulent strains containing the exoU gene of the type III secretion system are more likely to be FQ-resistant than strains containing the exoS gene, as well as more likely to acquire resistance-conferring mutations in gyrA/B and parC/E. We hypothesize that FQ-resistance imposes a lower fitness cost on exoU compared to exoS strains, thus allowing for better adaptation to the FQ-rich clinical environment. We created isogenic mutants containing a common FQ-resistance conferring point mutation in parC from three exoU to three exoS clinical isolates and tested fitness in vitro using head-to-head competition assays. The mutation differentially affected fitness in the exoU and exoS strains tested. While the addition of the parC mutation dramatically increased fitness in one of the exoU strains leaving the other two unaffected, all three exoS strains displayed a general decrease in fitness. In addition, we found that exoU strains may be able to compensate for the fitness costs associated with the mutation through better regulation of supercoiling compared to the exoS strains. These results may provide a biological explanation for the observed predominance of the virulent exoU genotype in FQ-resistant clinical subpopulations and represent the first investigation into potential differences in fitness costs of FQ-resistance that are linked to the virulence genotype of P. aeruginosa. Understanding the fitness costs of antibiotic resistance and possibilities of compensation for these costs is essential for the rational development of strategies to combat the problem of antibiotic resistance.

  18. The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

    PubMed

    Sullivan, Aaron B; Tam, K P Connie; Metruccio, Matteo M E; Evans, David J; Fleiszig, Suzanne M J

    2015-04-01

    Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ∼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88-/- epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88-/- mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain.

  19. Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces.

    PubMed

    Lee, Jiyoung; Teitzel, Gail M; Munkvold, Kathy; del Pozo, Olga; Martin, Gregory B; Michelmore, Richard W; Greenberg, Jean T

    2012-04-01

    The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GREEN FLUORESCENT PROTEIN-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and nonhost (tomato [Solanum lycopersicum]) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth, and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3, and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.

  20. Type III secretion system-dependent translocation of ectopically expressed Yop effectors into macrophages by intracellular Yersinia pseudotuberculosis.

    PubMed

    Zhang, Yue; Romanov, Galina; Bliska, James B

    2011-11-01

    Yersinia pseudotuberculosis is a Gram-negative bacterial pathogen. Virulence in Y. pseudotuberculosis requires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected with Y. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete and Y. pseudotuberculosis is internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently when Yersinia is extracellular. However, it is not clear whether the T3SS can be utilized by intracellular Y. pseudotuberculosis to translocate Yops. This possibility was investigated here using Y. pseudotuberculosis strains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viable Y. pseudotuberculosis in macrophages. Collectively, our results suggest that the Ysc T3SS of Y. pseudotuberculosis can function within macrophage phagosomes to translocate Yops into the host cytosol.

  1. Fitness Cost of Fluoroquinolone Resistance in Clinical Isolates of Pseudomonas aeruginosa Differs by Type III Secretion Genotype

    PubMed Central

    Agnello, Melissa; Finkel, Steven E.; Wong-Beringer, Annie

    2016-01-01

    Fluoroquinolone (FQ) resistance is highly prevalent among clinical strains of Pseudomonas aeruginosa, limiting treatment options. We have reported previously that highly virulent strains containing the exoU gene of the type III secretion system are more likely to be FQ-resistant than strains containing the exoS gene, as well as more likely to acquire resistance-conferring mutations in gyrA/B and parC/E. We hypothesize that FQ-resistance imposes a lower fitness cost on exoU compared to exoS strains, thus allowing for better adaptation to the FQ-rich clinical environment. We created isogenic mutants containing a common FQ-resistance conferring point mutation in parC from three exoU to three exoS clinical isolates and tested fitness in vitro using head-to-head competition assays. The mutation differentially affected fitness in the exoU and exoS strains tested. While the addition of the parC mutation dramatically increased fitness in one of the exoU strains leaving the other two unaffected, all three exoS strains displayed a general decrease in fitness. In addition, we found that exoU strains may be able to compensate for the fitness costs associated with the mutation through better regulation of supercoiling compared to the exoS strains. These results may provide a biological explanation for the observed predominance of the virulent exoU genotype in FQ-resistant clinical subpopulations and represent the first investigation into potential differences in fitness costs of FQ-resistance that are linked to the virulence genotype of P. aeruginosa. Understanding the fitness costs of antibiotic resistance and possibilities of compensation for these costs is essential for the rational development of strategies to combat the problem of antibiotic resistance. PMID:27757111

  2. Solving the supply of resveratrol tetramers from Papua New Guinean rainforest anisoptera species that inhibit bacterial type III secretion systems.

    PubMed

    Davis, Rohan A; Beattie, Karren D; Xu, Min; Yang, Xinzhou; Yin, Sheng; Holla, Harish; Healy, Peter C; Sykes, Melissa; Shelper, Todd; Avery, Vicky M; Elofsson, Mikael; Sundin, Charlotta; Quinn, Ronald J

    2014-12-26

    The supply of (-)-hopeaphenol (1) was achieved via enzymatic biotransformation in order to provide material for preclinical investigation. High-throughput screening of a prefractionated natural product library aimed to identify compounds that inhibit the bacterial virulence type III secretion system (T3SS) identified several fractions derived from two Papua New Guinean Anisoptera species, showing activity against Yersinia pseudotuberculosis outer proteins E and H (YopE and YopH). Bioassay-directed isolation from the leaves of A. thurifera, and similarly A. polyandra, resulted in three known resveratrol tetramers, (-)-hopeaphenol (1), vatalbinoside A (2), and vaticanol B (3). Compounds 1-3 displayed IC50 values of 8.8, 12.5, and 9.9 μM in a luminescent reporter-gene assay (YopE) and IC50 values of 2.9, 4.5, and 3.3 μM in an enzyme-based YopH assay, respectively, which suggested that they could potentially act against the T3SS in Yersinia. The structures of 1-3 were confirmed through a combination of spectrometric, chemical methods, and single-crystal X-ray structure determinations of the natural product 1 and the permethyl ether analogue of 3. The enzymatic hydrolysis of the β-glycoside 2 to the aglycone 1 was achieved through biotransformation using the endogenous leaf enzymes. This significantly enhanced the yield of the target bioactive natural product from 0.08% to 1.3% and facilitates ADMET studies of (-)-hopeaphenol (1).

  3. Interactions between CdsD, CdsQ, and CdsL, Three Putative Chlamydophila pneumoniae Type III Secretion Proteins▿

    PubMed Central

    Johnson, Dustin L.; Stone, Chris B.; Mahony, James B.

    2008-01-01

    Chlamydophila pneumoniae is a gram-negative obligate intracellular bacterial pathogen that causes pneumonia and bronchitis and may contribute to atherosclerosis. The developmental cycle of C. pneumoniae includes a morphological transition from an infectious extracellular elementary body (EB) to a noninfectious intracellular reticulate body (RB) that divides by binary fission. The C. pneumoniae genome encodes a type III secretion (T3S) apparatus that may be used to infect eukaryotic cells and to evade the host immune response. In the present study, Cpn0712 (CdsD), Cpn0704 (CdsQ), and Cpn0826 (CdsL), three C. pneumoniae genes encoding yersiniae T3S YscD, YscQ, and YscL homologs, respectively, were cloned and expressed as histidine- and glutathione S-transferase (GST)-tagged proteins in Escherichia coli. Purified recombinant proteins were used to raise hyper-immune polyclonal antiserum and were used in GST pull-down and copurification assays to identify protein-protein interactions. CdsD was detected in both EB and RB lysates by Western blot analyses, and immunofluorescent staining demonstrated the presence of CdsD within inclusions. Triton X-114 solubilization and phase separation of chlamydial EB proteins indicated that CdsD partitions with cytoplasmic proteins, suggesting it is not an integral membrane protein. GST pull-down assays indicated that recombinant CdsD interacts with CdsQ and CdsL, and copurification assays with chlamydial lysates confirmed that native CdsD interacts with CdsQ and CdsL. To the best of our knowledge, this is the first report demonstrating interactions between YscD, YscQ, and YscL homologs of bacterial T3S systems. These novel protein interactions may play important roles in the assembly or function of the chlamydial T3S apparatus. PMID:18281400

  4. Transcriptome Wide Annotation of Eukaryotic RNase III Reactivity and Degradation Signals

    PubMed Central

    Gagnon, Jules; Lavoie, Mathieu; Catala, Mathieu; Malenfant, Francis; Elela, Sherif Abou

    2015-01-01

    Detection and validation of the RNA degradation signals controlling transcriptome stability are essential steps for understanding how cells regulate gene expression. Here we present complete genomic and biochemical annotations of the signals required for RNA degradation by the dsRNA specific ribonuclease III (Rnt1p) and examine its impact on transcriptome expression. Rnt1p cleavage signals are randomly distributed in the yeast genome, and encompass a wide variety of sequences, indicating that transcriptome stability is not determined by the recurrence of a fixed cleavage motif. Instead, RNA reactivity is defined by the sequence and structural context in which the cleavage sites are located. Reactive signals are often associated with transiently expressed genes, and their impact on RNA expression is linked to growth conditions. Together, the data suggest that Rnt1p reactivity is triggered by malleable RNA degradation signals that permit dynamic response to changes in growth conditions. PMID:25680180

  5. Vaccination with a single CD4 T cell peptide epitope from a Salmonella type III-secreted effector protein provides protection against lethal infection.

    PubMed

    Kurtz, Jonathan R; Petersen, Hailey E; Frederick, Daniel R; Morici, Lisa A; McLachlan, James B

    2014-06-01

    Salmonella infections affect millions worldwide and remain a significant cause of morbidity and mortality. It is known from mouse studies that CD4 T cells are essential mediators of immunity against Salmonella infection, yet it is not clear whether targeting CD4 T cell responses directly with peptide vaccines against Salmonella can be effective in combating infection. Additionally, it is not known whether T cell responses elicited against Salmonella secreted effector proteins can provide protective immunity against infection. In this study, we investigated both of these possibilities using prime-boost immunization of susceptible mice with a single CD4 T cell peptide epitope from Salmonella secreted effector protein I (SseI), a component of the Salmonella type III secretion system. This immunization conferred significant protection against lethal oral infection, equivalent to that conferred by whole heat-killed Salmonella bacteria. Surprisingly, a well-characterized T cell epitope from the flagellar protein FliC afforded no protection compared to immunization with an irrelevant control peptide. The protective response appeared to be most associated with polyfunctional CD4 T cells raised against the SseI peptide, since no antibodies were produced against any of the peptides and very little CD8 T cell response was observed. Overall, this study demonstrates that eliciting CD4 T cell responses against components of the Salmonella type III secretion system can contribute to protection against infection and should be considered in the design of future Salmonella subunit vaccines.

  6. Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris.

    PubMed

    Aw, Rochelle; McKay, Paul F; Shattock, Robin J; Polizzi, Karen M

    2017-12-01

    The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 μg mL(-1) in small scale expression. VRC01 was expressed using both the α-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly, using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the α-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.

  7. 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.

  8. A Ralstonia solanacearum type III effector directs the production of the plant signal metabolite trehalose-6-phosphate.

    PubMed

    Poueymiro, M; Cazalé, A C; François, J M; Parrou, J L; Peeters, N; Genin, S

    2014-12-23

    The plant pathogen Ralstonia solanacearum possesses two genes encoding a trehalose-6-phosphate synthase (TPS), an enzyme of the trehalose biosynthetic pathway. One of these genes, named ripTPS, was found to encode a protein with an additional N-terminal domain which directs its translocation into host plant cells through the type 3 secretion system. RipTPS is a conserved effector in the R. solanacearum species complex, and homologues were also detected in other bacterial plant pathogens. Functional analysis of RipTPS demonstrated that this type 3 effector synthesizes trehalose-6-phosphate and identified residues essential for this enzymatic activity. Although trehalose-6-phosphate is a key signal molecule in plants that regulates sugar status and carbon assimilation, the disruption of ripTPS did not alter the virulence of R. solanacearum on plants. However, heterologous expression assays showed that this effector specifically elicits a hypersensitive-like response on tobacco that is independent of its enzymatic activity and is triggered by the C-terminal half of the protein. Recognition of this effector by the plant immune system is suggestive of a role during the infectious process. Ralstonia solanacearum, the causal agent of bacterial wilt disease, infects more than two hundred plant species, including economically important crops. The type III secretion system plays a major role in the pathogenicity of this bacterium, and approximately 70 effector proteins have been shown to be translocated into host plant cells. This study provides the first description of a type III effector endowed with a trehalose-6-phosphate synthase enzymatic activity and illustrates a new mechanism by which the bacteria may manipulate the plant metabolism upon infection. In recent years, trehalose-6-phosphate has emerged as an essential signal molecule in plants, connecting plant metabolism and development. The finding that a bacterial pathogen could induce the production of trehalose-6

  9. Effects of Lutein and Zeaxanthin on LPS-Induced Secretion of IL-8 by Uveal Melanocytes and Relevant Signal Pathways.

    PubMed

    Chao, Shih-Chun; Vagaggini, Tommaso; Nien, Chan-Wei; Huang, Sheng-Chieh; Lin, Hung-Yu

    2015-01-01

    The effects of lutein and zeaxanthin on lipopolysaccharide- (LPS-) induced secretion of IL-8 by uveal melanocytes (UM) were tested in cultured human UM. MTT assay revealed that LPS (0.01-1 μg/mL) and lutein and zeaxanthin (1-10 μM) did not influence the cell viability of cultured UM. LPS caused a dose-dependent increase of secretion of IL-8 by cultured UM. Lutein and zeaxanthin did not affect the constitutive secretion of IL-8. However, lutein and zeaxanthin decreased LPS-induced secretion of IL-8 in cultured UM in a dose-dependent manner. LPS significantly increased NF-κB levels in cell nuclear extracts and p-JNK levels in the cell lysates from UM, but not p-p38 MAPK and p-ERG. Lutein or zeaxanthin significantly reduced LPS-induced increase of NF-κB and p-JNK levels, but not p38 MAPK and ERG levels. The present study demonstrated that lutein and zeaxanthin inhibited LPS-induced secretion of IL-8 in cultured UM via JNK and NF-κB signal pathways. The anti-inflammatory effects of lutein and zeaxanthin might be explored as a therapeutic approach in the management of uveitis and other inflammatory diseases of the eye.

  10. Effects of Lutein and Zeaxanthin on LPS-Induced Secretion of IL-8 by Uveal Melanocytes and Relevant Signal Pathways

    PubMed Central

    Chao, Shih-Chun; Vagaggini, Tommaso; Nien, Chan-Wei; Huang, Sheng-Chieh; Lin, Hung-Yu

    2015-01-01

    The effects of lutein and zeaxanthin on lipopolysaccharide- (LPS-) induced secretion of IL-8 by uveal melanocytes (UM) were tested in cultured human UM. MTT assay revealed that LPS (0.01–1 μg/mL) and lutein and zeaxanthin (1–10 μM) did not influence the cell viability of cultured UM. LPS caused a dose-dependent increase of secretion of IL-8 by cultured UM. Lutein and zeaxanthin did not affect the constitutive secretion of IL-8. However, lutein and zeaxanthin decreased LPS-induced secretion of IL-8 in cultured UM in a dose-dependent manner. LPS significantly increased NF-κB levels in cell nuclear extracts and p-JNK levels in the cell lysates from UM, but not p-p38 MAPK and p-ERG. Lutein or zeaxanthin significantly reduced LPS-induced increase of NF-κB and p-JNK levels, but not p38 MAPK and ERG levels. The present study demonstrated that lutein and zeaxanthin inhibited LPS-induced secretion of IL-8 in cultured UM via JNK and NF-κB signal pathways. The anti-inflammatory effects of lutein and zeaxanthin might be explored as a therapeutic approach in the management of uveitis and other inflammatory diseases of the eye. PMID:26609426

  11. The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis.

    PubMed

    Pollard, Dominic J; Young, Joanna C; Covarelli, Valentina; Herrera-León, Silvia; Connor, Thomas R; Fookes, Maria; Walker, Danielle; Echeita, Aurora; Thomson, Nicholas R; Berger, Cedric N; Frankel, Gad

    2016-12-01

    Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae.

  12. Cinnamaldehyde inhibits pro-inflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling.

    PubMed

    Chao, Louis Kuoping; Hua, Kuo-Feng; Hsu, Hsien-Yeh; Cheng, Sen-Sung; Lin, I-Fan; Chen, Chia-Jung; Chen, Shui-Tein; Chang, Shang-Tzen

    2008-01-01

    We investigated the in vitro anti-inflammatory effects of Cinnamaldehyde, a cytokine production inhibitor isolated from an essential oil produced from the leaves of Cinnamomum osmophloeum Kaneh, and its mechanism of action. Although Cinnamaldehyde has been reported to have contact sensitizing properties at high concentration (mM), we found that low concentration of Cinnamaldehyde (muM) inhibited the secretion of interleukin-1beta and tumor necrosis factor alpha within lipopolysaccharide (LPS) or lipoteichoic acid (LTA) stimulated murine J774A.1 macrophages. Cinnamaldehyde also suppressed the production of these cytokines from LPS stimulated human blood monocytes derived primary macrophages and human THP-1 monocytes. Furthermore, Cinnamaldehyde also inhibited the production of prointerleukin-1beta within LPS or LTA stimulated human THP-1 monocytes. Reactive oxygen species release from LPS stimulated J774A.1 macrophages was reduced by Cinnamaldehyde. The phosphorylation of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase 1/2 induced by LPS was also inhibited by Cinnamaldehyde; however, Cinnamaldehyde neither antagonize the binding of LPS to the cells nor alter the cell surface expression of toll-like receptor 4 and CD14. In addition, we also noted that Cinnamaldehyde appeared to elicit no cytotoxic effect upon J774A.1 macrophages under our experimental conditions, although Cinnamaldehyde reduced J774A.1 macrophages proliferation as analysed by MTT assay. Our current results have demonstrated the anti-oxidation and anti-inflammatory properties of Cinnamaldehyde that could provide the possibility for Cinnamaldehyde's future pharmaceutical application in the realm of immuno-modulation.

  13. Mutations in the Yersinia pseudotuberculosis Type III Secretion System Needle Protein, YscF, That Specifically Abrogate Effector Translocation into Host Cells▿ †

    PubMed Central

    Davis, Alison J.; Mecsas, Joan

    2007-01-01

    The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface. PMID:17071752

  14. Mutations in the Yersinia pseudotuberculosis type III secretion system needle protein, YscF, that specifically abrogate effector translocation into host cells.

    PubMed

    Davis, Alison J; Mecsas, Joan

    2007-01-01

    The trafficking of effectors, termed Yops, from Yersinia spp. into host cells is a multistep process that requires the type III secretion system (TTSS). The TTSS has three main structural parts: a base, a needle, and a translocon, which work together to ensure the polarized movement of Yops directly from the bacterial cytosol into the host cell cytosol. To understand the interactions that take place at the interface between the tip of the TTSS needle and the translocon, we developed a screen to identify mutations in the needle protein YscF that separated its function in secretion from its role in translocation. We identified 25 translocation-defective (TD) yscF mutants, which fall into five phenotypic classes. Some classes exhibit aberrant needle structure and/or reduced levels of Yop secretion, consistent with known functions for YscF. Strikingly, two yscF TD classes formed needles and secreted Yops normally but displayed distinct translocation defects. Class I yscF TD mutants showed diminished pore formation, suggesting incomplete pore insertion and/or assembly. Class II yscF TD mutants formed pores but showed nonpolar translocation, suggesting unstable needle-translocon interactions. These results indicate that YscF functions in Yop secretion and translocation can be genetically separated. Furthermore, the identification of YscF residues that are required for the assembly of the translocon and/or productive interactions with the translocon has allowed us to initiate the mapping of the needle-translocon interface.

  15. Evaluation of Recombinant Human Growth Hormone Secretion in E. coli using the L-asparaginase II Signal Peptide

    PubMed Central

    Zamani, Mozhdeh; Nezafat, Navid; Ghasemi, Younes

    2016-01-01

    Background: In the recent years, there has been an increasing interest in secretory production of recombinant proteins, due to its various advantages compared with intracellular expression. Signal peptides play a critical role in prosperous secretion of recombinant proteins. Accordingly, different signal peptides have been assessed for their ability to produce secretory proteins by trial-and-error experiments. The aim of this study was to evaluate the effect of L-asparaginase II signal peptide on the recombinant human Growth Hormone (hGH) protein secretion in the Escherichia coli (E. coli) host. Methods: Cloning and expression of a synthetic hGH gene, containing L-asparaginase II signal sequence was performed in E. coli BL21 (DE3) using 0.1mM IPTG as an inducer at 23°C overnight. Periplasmic protein extraction was performed using three methods, including osmotic shock, osmotic shock in the presence of glycine and combined Lysozyme/EDTA osmotic shock. Afterwards, the hGH expression was determined by SDS-PAGE. Results: Based on experimentally obtained results, hGH protein is expressed as inclusion body even in the presence of L-asparaginase II signal peptide. Conclusion: Therefore, this signal peptide is not effective for secretory production of the recombinant hGH. PMID:27920886

  16. Feedback regulation of PRL secretion is mediated by the transcription factor, signal transducer, and activator of transcription 5b.

    PubMed

    Grattan, D R; Xu, J; McLachlan, M J; Kokay, I C; Bunn, S J; Hovey, R C; Davey, H W

    2001-09-01

    PRL secretion from the anterior pituitary gland is inhibited by dopamine produced in the tuberoinfundibular dopamine neurons of the hypothalamus. The activity of tuberoinfundibular dopamine neurons is stimulated by PRL; thus, PRL regulates its own secretion by a negative feedback mechanism. PRL receptors are expressed on tuberoinfundibular dopamine neurons, but the intracellular signaling pathway is not known. We have observed that mice with a disrupted signal transducer and activator of transcription 5b gene have grossly elevated serum PRL concentrations. Despite this hyperprolactinemia, mRNA levels and immunoreactivity of tyrosine hydroxylase, the key enzyme in dopamine synthesis, were significantly lower in the tuberoinfundibular dopamine neurons of these signal transducer and activator of transcription 5b-deficient mice. Concentrations of the dopamine metabolite dihydroxyphenylacetic acid in the median eminence were also significantly lower in signal transducer and activator of transcription 5b-deficient mice than in wild-type mice. No changes were observed in nonhypothalamic dopaminergic neuronal populations, indicating that the effects were selective to tuberoinfundibular dopamine neurons. These data indicate that in the absence of signal transducer and activator of transcription 5b, PRL signal transduction in tuberoinfundibular dopamine neurons is impaired, and they demonstrate that this transcription factor plays an obligatory and nonredundant role in mediating the negative feedback action of PRL on tuberoinfundibular dopamine neurons.

  17. HMGB1 is secreted by 3T3-L1 adipocytes through JNK signaling and the secretion is partially inhibited by adiponectin.

    PubMed

    Shimizu, Toshiaki; Yamakuchi, Munekazu; Biswas, Kamal Krishna; Aryal, Bibek; Yamada, Shingo; Hashiguchi, Teruto; Maruyama, Ikuro

    2016-09-01

    Obesity is a chronic inflammatory disease, and adipocytes contribute to obesity-associated inflammation by releasing inflammatory mediators. High mobility group box 1 (HMGB1), a highly conserved DNA-binding protein, mainly localized to cell nuclei, has been recently recognized as an innate pro-inflammatory mediator when released extracellularly. It was hypothesized that HMGB1 is an adipocytokine that acts as an innate pro-inflammatory mediator in white adipose tissue (WAT) of patients with obesity and is associated with insulin resistance. Additionally, it was hypothesized that HMGB1 secretion is regulated by adiponectin. 3T3-L1 cells were differentiated into mature adipocytes. After tumor necrosis factor-α (TNF-α) stimulation, HMGB1 in culture media was measured. Localizations of HMGB1 in 3T3-L1 adipocytes and human WAT were examined by immunostaining. HMGB1 was secreted from TNF-α-induced 3T3-L1 adipocytes through JNK signaling. HMGB1-activated MAP kinases (ERK1/2, JNK) and suppressed insulin-stimulated Akt phosphorylation in 3T3-L1 adipocytes. The cytoplasm in 3T3-L1 adipocytes and adipocytes of WAT from a patient with obesity was intensely stained with HMGB1. Adiponectin partially inhibited TNF-α-induced HMGB1 secretion from 3T3-L1 adipocytes. These findings suggest that HMGB1 is a pro-inflammatory adipocytokine involved in WAT inflammation and insulin resistance in patients with obesity, which may contribute to the progression of metabolic syndrome, and that adiponectin protects against HMGB1-induced adipose tissue inflammation. © 2016 The Obesity Society.

  18. The Ruler Protein EscP of the Enteropathogenic Escherichia coli Type III Secretion System Is Involved in Calcium Sensing and Secretion Hierarchy Regulation by Interacting with the Gatekeeper Protein SepL

    PubMed Central

    Shaulov, Lihi; Gershberg, Jenia; Deng, Wanyin; Finlay, B. Brett

    2017-01-01

    ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. PMID:28049143

  19. Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

    PubMed Central

    Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-01-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  20. Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri

    PubMed Central

    Epler, Chelsea R.; Dickenson, Nicholas E.; Bullitt, Esther; Picking, Wendy L.

    2012-01-01

    Shigella flexneri is a Gram-negative enteric pathogen that is the predominant cause of bacillary dysentery. Shigella uses a type III secretion system (T3SS) to deliver effector proteins that alter normal target cell functions to promote pathogen invasion. The type III secretion apparatus (T3SA) consists of a basal body, an extracellular needle, and a tip complex that is responsible for delivering effectors into the host cell cytoplasm. Invasion plasmid antigen D (IpaD) is the first protein to localize to the T3SA needle tip, where it prevents premature effector secretion and serves as an environmental sensor for triggering recruitment of the translocator protein IpaB to the needle tip. Thus, IpaD would be expected to form a stable structure whose overall architecture supports its functions. It is not immediately obvious from the published IpaD crystal structure (pdb 2j0o) how a multimer of IpaD would be incorporated at the tip of the first static T3SA intermediate, nor what its functional role would be in building a mature T3SA. Here we produce three-dimensional reconstructions from transmission electron microscopy images of IpaD localized at the Shigella T3SA needle tip for comparison to needle tips from a Shigella ipaD-null mutant. The results demonstrate that IpaD resides as a homopentamer at the needle tip of the T3SA. Furthermore, comparison to tips assembled from the distal domain IpaDΔ192-267 mutation shows that IpaD adopts an elongated conformation that facilitates its ability to control type III secretion and stepwise assembly of the T3SA needle tip complex. PMID:22480614

  1. Vectorial secretion of interleukin-8 mediates autocrine signalling in intestinal epithelial cells via apically located CXCR1

    PubMed Central

    2013-01-01

    Background In the intestinal mucosa, several adaptations of TLR signalling have evolved to avoid chronic inflammatory responses to the presence of commensal microbes. Here we investigated whether polarized monolayers of intestinal epithelial cells might regulate inflammatory responses by secreting IL-8 in a vectorial fashion (i.e. apical versus basolateral) depending on the location of the TLR stimulus. Results In the Caco-2 BBE model of polarized villus-like epithelium, apical stimulation with TLR2 and TLR5 ligands resulted in the apical secretion of IL-8. The CXCR1 receptor for IL-8 was expressed only on the apical membrane of Caco-2 BBE cells and differentiated epithelial cells in the human small intestine and colon. Transcriptome analyses revealed that Caco-2 BBE cells respond to stimulation with IL-8 supporting the hypothesis that IL-8 induces G protein-coupled receptor signalling. Conclusions These results show that IL-8 induces autocrine signalling via an apical CXCR1 in Caco-2 BBE intestinal epithelial cells and that this receptor is also expressed on the apical surface of differentiated human intestinal epithelial cells in vivo, suggesting an autocrine function for IL-8 secreted in the lumen. PMID:24164922

  2. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    PubMed Central

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-01-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. PMID:27541856

  3. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

    PubMed

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-11-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

  4. Mycobacterium tuberculosis Cyclophilin A Uses Novel Signal Sequence for Secretion and Mimics Eukaryotic Cyclophilins for Interaction with Host Protein Repertoire

    PubMed Central

    Bhaduri, Asani; Misra, Richa; Maji, Abhijit; Bhetaria, Preetida J.; Mishra, Sonakshi; Arora, Gunjan; Singh, Lalit Kumar; Dhasmana, Neha; Dubey, Neha; Virdi, Jugsharan Singh; Singh, Yogendra

    2014-01-01

    Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins. PMID:24505389

  5. Mycobacterium tuberculosis cyclophilin A uses novel signal sequence for secretion and mimics eukaryotic cyclophilins for interaction with host protein repertoire.

    PubMed

    Bhaduri, Asani; Misra, Richa; Maji, Abhijit; Bhetaria, Preetida J; Mishra, Sonakshi; Arora, Gunjan; Singh, Lalit Kumar; Dhasmana, Neha; Dubey, Neha; Virdi, Jugsharan Singh; Singh, Yogendra

    2014-01-01

    Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

  6. A conserved domain in type III secretion links the cytoplasmic domain of InvA to elements of the basal body

    SciTech Connect

    Lilic, Mirjana; Quezada, Cindy M.; Stebbins, C. Erec

    2010-06-01

    The cytoplasmic domain of Salmonella InvA shares homology to a recurring scaffold in the membrane-spanning components of the type II and type III secretion systems. Protein type III secretion systems (T3SSs) are organic nanosyringes that achieve an energy-dependent translocation of bacterial proteins through the two membranes of Gram-negative organisms. Examples include the pathogenic systems of animals, plants and symbiotic bacteria that inject factors into eukaryotic cells, and the flagellar export system that secretes flagellin. T3SSs possess a core of several membrane-associated proteins that are conserved across all known bacterial species that use this system. The Salmonella protein InvA is one of the most highly conserved proteins of this core of critical T3SS components. The crystal structure of a C-terminal domain of InvA reveals an unexpected homology to domains that have been repeatedly found as building blocks of other elements of the T3SS apparatus. This suggests the surprising hypothesis that evolution has produced a significant component of the apparatus structure through a series of gene-duplication and gene-rearrangement events.

  7. The mechanisms of insulin secretion and calcium signaling in pancreatic β-cells exposed to fluoroquinolones.

    PubMed

    Bito, Motoki; Tomita, Takashi; Komori, Mika; Taogoshi, Takanori; Kimura, Yasuhiro; Kihira, Kenji

    2013-01-01

    Fluoroquinolones reportedly induce hypoglycemia through stimulation of insulin secretion from pancreatic β-cells via inhibition of K(ATP) channels and activation of L-type voltage-dependent Ca(2+) channels. In physiological condition, the cytosolic Ca(2+) concentration ([Ca(2+)](c)) is also regulated by release of Ca(2+) from intracellular Ca(2+) stores. In this study, we investigated the mechanism of insulin secretion induced by fluoroquinolones, with respect to intracellular Ca(2+) stores. Even where the absence of supplemental extracellular Ca(2+), insulin secretion and [Ca(2+)](c) were increased by gatifloxacin, levofloxacin or tolbutamide. Insulin secretion and the rise of [Ca(2+)](c) induced by fluoroquinolones were reduced by depleting of Ca(2+) in endoplasmic reticumum (ER) by thapsigargin, and inhibiting ryanodine receptor of ER by dantrolene. Inhibition of inositol 1,4,5-triphosphate receptor of ER by xestospongin C suppressed insulin secretion induced by fluoroquinolones, whereas it did not affect [Ca(2+)](c). Destruction of acidic Ca(2+) stores such as lysosome and lysosome-related organelles by glycyl-L-phenylalanine-2-nephthylamide (GPN) did not affect insulin secretion and the rise of [Ca(2+)](c) induced by fluoroquinolones. The increase in insulin and [Ca(2+)](c) induced by tolbutamide were reduced by thapsigargin, dantrolene, and GPN but not by xestospongin C. In conclusion, fluoroquinolones induces Ca(2+) release from ER mediated by the ryanodine receptor, and the reaction might involve in insulin secretion. Sulfonylureas induce Ca(2+) release from GPN-sensitive acidic Ca(2+) stores, but fluoroquinolones did not.

  8. Chronic effects of palmitate overload on nutrient-induced insulin secretion and autocrine signalling in pancreatic MIN6 beta cells.

    PubMed

    Watson, Maria L; Macrae, Katherine; Marley, Anna E; Hundal, Harinder S

    2011-01-01

    Sustained exposure of pancreatic β cells to an increase in saturated fatty acids induces pleiotropic effects on β-cell function, including a reduction in stimulus-induced insulin secretion. The objective of this study was to investigate the effects of chronic over supply of palmitate upon glucose- and amino acid-stimulated insulin secretion (GSIS and AASIS, respectively) and autocrine-dependent insulin signalling with particular focus on the importance of ceramide, ERK and CaMKII signalling. GSIS and AASIS were both stimulated by >7-fold resulting in autocrine-dependent activation of protein kinase B (PKB, also known as Akt). Insulin release was dependent upon nutrient-induced activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) as their pharmacological inhibition suppressed GSIS/AASIS significantly. Chronic (48 h, 0.4 mM) palmitate treatment blunted glucose/AA-induced activation of CaMKII and ERK and caused a concomitant reduction (~75%) in GSIS/AASIS and autocrine-dependent activation of PKB. This inhibition could not be attributed to enhanced mitochondrial fatty acid uptake/oxidation or ceramide synthesis, which were unaffected by palmitate. In contrast, diacylglycerol synthesis was elevated suggesting increased palmitate esterification rather than oxidation may contribute to impaired stimulus-secretion coupling. Consistent with this, 2-bromopalmitate, a non-oxidisable palmitate analogue, inhibited GSIS as effectively as palmitate. Our results exclude changes in ceramide content or mitochondrial fatty acid handling as factors initiating palmitate-induced defects in insulin release from MIN6 β cells, but suggest that reduced CaMKII and ERK activation associated with palmitate overload may contribute to impaired stimulus-induced insulin secretion.

  9. Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

    PubMed Central

    Watson, Maria L.; Macrae, Katherine; Marley, Anna E.; Hundal, Harinder S.

    2011-01-01

    Background Sustained exposure of pancreatic β cells to an increase in saturated fatty acids induces pleiotropic effects on β-cell function, including a reduction in stimulus-induced insulin secretion. The objective of this study was to investigate the effects of chronic over supply of palmitate upon glucose- and amino acid-stimulated insulin secretion (GSIS and AASIS, respectively) and autocrine-dependent insulin signalling with particular focus on the importance of ceramide, ERK and CaMKII signalling. Principal Findings GSIS and AASIS were both stimulated by >7-fold resulting in autocrine-dependent activation of protein kinase B (PKB, also known as Akt). Insulin release was dependent upon nutrient-induced activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) as their pharmacological inhibition suppressed GSIS/AASIS significantly. Chronic (48 h, 0.4 mM) palmitate treatment blunted glucose/AA-induced activation of CaMKII and ERK and caused a concomitant reduction (∼75%) in GSIS/AASIS and autocrine-dependent activation of PKB. This inhibition could not be attributed to enhanced mitochondrial fatty acid uptake/oxidation or ceramide synthesis, which were unaffected by palmitate. In contrast, diacylglycerol synthesis was elevated suggesting increased palmitate esterification rather than oxidation may contribute to impaired stimulus-secretion coupling. Consistent with this, 2-bromopalmitate, a non-oxidisable palmitate analogue, inhibited GSIS as effectively as palmitate. Conclusions Our results exclude changes in ceramide content or mitochondrial fatty acid handling as factors initiating palmitate-induced defects in insulin release from MIN6 β cells, but suggest that reduced CaMKII and ERK activation associated with palmitate overload may contribute to impaired stimulus-induced insulin secretion. PMID:21998735

  10. EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD.

    PubMed

    Zheng, Jun; Li, Nan; Tan, Yuen Peng; Sivaraman, J; Mok, Yu-Keung; Mo, Zhao Lan; Leung, Ka Yin

    2007-06-01

    Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by coils. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of DeltaescC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

  11. Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli

    PubMed Central

    Monjarás Feria, Julia; García-Gómez, Elizabeth; Espinosa, Norma; Minamino, Tohru; Namba, Keiichi

    2012-01-01

    Enteropathogenic Escherichia coli employs a type III secretion system (T3SS) to translocate virulence effector proteins directly into enterocyte host cells, leading to diarrheal disease. The T3SS is encoded within the chromosomal locus of enterocyte effacement (LEE). The function of some of the LEE-encoded proteins remains unknown. Here we investigated the role of the Orf16 protein in T3SS biogenesis and function. An orf16 deletion mutant showed translocator and effector protein secretion profiles different from those of wild-type cells. The orf16 null strain produced T3S structures with abnormally long needles and filaments that caused weak hemolysis of red blood cells. Furthermore, the number of fully assembled T3SSs was also reduced in the orf16 mutant, indicating that Orf16, though not essential, is required for efficient T3SS assembly. Analysis of protein secretion revealed that Orf16 is a T3SS-secreted substrate and regulates the secretion of the inner rod component EscI. Both pulldown and yeast two-hybrid assays showed that Orf16 interacts with the C-terminal domain of an inner membrane component of the secretion apparatus, EscU; the inner rod protein EscI; the needle protein EscF; and the multieffector chaperone CesT. These results suggest that Orf16 regulates needle length and, along with EscU, participates in a substrate specificity switch from early substrates to translocators. Taken together, our results suggest that Orf16 acts as a molecular measuring device in a way similar to that of members of the Yersinia YscP and flagellar FliK protein family. Therefore, we propose that this protein be renamed EscP. PMID:22923600

  12. The type III secreted effector DspE is required early in Solanum tuberosum leaf infection by Pectobacterium carotovorum to elicit cell death, and requires Wx(3-6)D/E motifs

    USDA-ARS?s Scientific Manuscript database

    Pectobacterium species are enterobacterial plant-pathogens that cause soft rot disease in diverse plant species. Unlike hemi-biotrophic plant pathogenic bacteria, the type III secretion system (T3SS) of Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) appears to secrete only one effect...

  13. Increased adrenergic signaling is responsible for decreased glucose-stimulated insulin secretion in the chronically hyperinsulinemic ovine fetus.

    PubMed

    Andrews, Sasha E; Brown, Laura D; Thorn, Stephanie R; Limesand, Sean W; Davis, Melissa; Hay, William W; Rozance, Paul J

    2015-01-01

    Insulin may stimulate its own insulin secretion and is a potent growth factor for the pancreatic β-cell. Complications of pregnancy, such as diabetes and intrauterine growth restriction, are associated with changes in fetal insulin concentrations, secretion, and β-cell mass. However, glucose concentrations are also abnormal in these conditions. The direct effect of chronic fetal hyperinsulinemia with euglycemia on fetal insulin secretion and β-cell mass has not been tested. We hypothesized that chronic fetal hyperinsulinemia with euglycemia would increase glucose-stimulated insulin secretion (GSIS) and β-cell mass in the ovine fetus. Singleton ovine fetuses were infused with iv insulin to produce high physiological insulin concentrations, or saline for 7-10 days. The hyperinsulinemic animals also received a direct glucose infusion to maintain euglycemia. GSIS, measured at 133 ± 1 days of gestation, was significantly attenuated in the hyperinsulinemic fetuses (P < .05). There was no change in β-cell mass. The hyperinsulinemic fetuses also had decreased oxygen (P < .05) and higher norepinephrine (1160 ± 438 vs 522 ± 106 pg/mL; P < .005). Acute pharmacologic adrenergic blockade restored GSIS in the hyperinsulinemic-euglycemic fetuses, demonstrating that increased adrenergic signaling mediates decreased GSIS in these fetuses.

  14. Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis.

    PubMed

    Wu, Bo; He, Ming-Xiong; Feng, Hong; Shui, Zong-Xia; Tang, Xiao-Yu; Hu, Qi-Chun; Zhang, Yi-Zheng

    2014-01-01

    In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD's SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD's SP were also higher than the other two strains containing PhoC or ZMO0331's SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.

  15. Uropygial gland-secreted alkanols contribute to olfactory sex signals in budgerigars.

    PubMed

    Zhang, Jian-Xu; Wei, Wei; Zhang, Jin-Hua; Yang, Wei-He

    2010-06-01

    The possible role of uropygial gland-secreted compounds in olfactory discrimination of sex or sex attractants in the budgerigar, Melopsittacus undulatus, was investigated using behavioral 2-choice tests and gas chromatography-mass spectrometry analysis. Our data showed that female budgerigars were capable of distinguishing males from females in a Y maze via body odor, indicating its sexual dimorphism. When we conducted a chemical assay of the uropygial preen gland secretions, we found 4 times more volatile octadecanol, nonadecanol, and eicosanol in ratios in males than in females, making them putative male pheromone candidates. Female birds also showed overt preferences for the odor of male preen gland secretions or the 3-alkanol blend equivalent preened onto the plumage of a male over that of female counterparts. Removal of any one alkanol was associated with a loss of attractiveness to the female. In another test device (a test cage) with visible male bird stimulus, females chose the male with the 3-alkanol blend of males over the other male with female preen gland secretion, whereas did not differentiate their responses between the males with either this blend or male preen gland secretions. The behavioral data robustly suggested that the 3 alkanols synergistically created a female attractant odor or male pheromone in the budgerigar and that bird uropygial glands have broader implications in sexual behavior than previously known. This is the first investigation with bioassay of components of the gland in a bird species.

  16. Simultaneous spectrophotometric determination of Fe(III) and Al(III) using orthogonal signal correction-partial least squares calibration method after solidified floating organic drop microextraction

    NASA Astrophysics Data System (ADS)

    Rohani Moghadam, Masoud; Haji Shabani, Ali Mohammad; Dadfarnia, Shayessteh

    2015-01-01

    A solidified floating organic drop microextraction (SFODME) procedure was developed for the simultaneous extraction and preconcentration of Fe(III) and Al(III) from water samples. The method was based on the formation of cationic complexes between Fe(III) and Al(III) and 3,5,7,2‧,4‧-pentahydroxyflavone (morin) which were extracted into 1-undecanol as ion pairs with perchlorate ions. The absorbance of the extracted complexes was then measured in the wavelength range of 300-450 nm. Finally, the concentration of each metal ion was determined by the use of the orthogonal signal correction-partial least squares (OSC-PLS) calibration method. Several experimental parameters that may be affected on the extraction process such as the type and volume of extraction solvent, pH of the aqueous solution, morin and perchlorate concentration and extraction time were optimized. Under the optimum conditions, Fe(III) and Al(III) were determined in the ranges of 0.83-27.00 μg L-1 (R2 = 0.9985) and 1.00-32.00 μg L-1 (R2 = 0.9979) of Fe(III) and Al(III), respectively. The relative standard deviations (n = 6) at 12.80 μg L-1 of Fe(III) and 17.00 μg L-1 of Al(III) were 3.2% and 3.5%, respectively. An enhancement factors of 102 and 96 were obtained for Fe(III) and Al(III) ions, respectively. The procedure was successfully applied to determination of iron and aluminum in steam and water samples of thermal power plant; and the accuracy was assessed through the recovery experiments and independent analysis by electrothermal atomic absorption spectroscopy (ETAAS).

  17. Simultaneous spectrophotometric determination of Fe(III) and Al(III) using orthogonal signal correction-partial least squares calibration method after solidified floating organic drop microextraction.

    PubMed

    Rohani Moghadam, Masoud; Haji Shabani, Ali Mohammad; Dadfarnia, Shayessteh

    2015-01-25

    A solidified floating organic drop microextraction (SFODME) procedure was developed for the simultaneous extraction and preconcentration of Fe(III) and Al(III) from water samples. The method was based on the formation of cationic complexes between Fe(III) and Al(III) and 3,5,7,2',4'-pentahydroxyflavone (morin) which were extracted into 1-undecanol as ion pairs with perchlorate ions. The absorbance of the extracted complexes was then measured in the wavelength range of 300-450 nm. Finally, the concentration of each metal ion was determined by the use of the orthogonal signal correction-partial least squares (OSC-PLS) calibration method. Several experimental parameters that may be affected on the extraction process such as the type and volume of extraction solvent, pH of the aqueous solution, morin and perchlorate concentration and extraction time were optimized. Under the optimum conditions, Fe(III) and Al(III) were determined in the ranges of 0.83-27.00 μg L(-1) (R(2)=0.9985) and 1.00-32.00 μg L(-1) (R(2)=0.9979) of Fe(III) and Al(III), respectively. The relative standard deviations (n=6) at 12.80 μg L(-1) of Fe(III) and 17.00 μg L(-)(1) of Al(III) were 3.2% and 3.5%, respectively. An enhancement factors of 102 and 96 were obtained for Fe(III) and Al(III) ions, respectively. The procedure was successfully applied to determination of iron and aluminum in steam and water samples of thermal power plant; and the accuracy was assessed through the recovery experiments and independent analysis by electrothermal atomic absorption spectroscopy (ETAAS).

  18. 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

  19. Structural and Functional Analysis of the Type III Secretion System from Pseudomonas fluorescens Q8r1-96▿ §

    PubMed Central

    Mavrodi, Dmitri V.; Joe, Anna; Mavrodi, Olga V.; Hassan, Karl A.; Weller, David M.; Paulsen, Ian T.; Loper, Joyce E.; Alfano, James R.; Thomashow, Linda S.

    2011-01-01

    Pseudomonas fluorescens Q8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogen Pseudomonas syringae. We also screened a collection of 30 closely related P. fluorescens strains and detected the T3SS genes in all but one of them. The Q8r1-96 genome contained ropAA and ropM type III effector genes, which are orthologs of the P. syringae effector genes hopAA1-1 and hopM1, as well as a novel type III effector gene designated ropB. These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by both P. syringae and Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses. PMID:20971913

  20. Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants.

    PubMed Central

    Li, H; Nicholson, A W

    1996-01-01

    Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop. Images PMID:8635475

  1. Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

    PubMed

    Sandoval, Alejandro; Duran, Paz; Gandini, María A; Andrade, Arturo; Almanza, Angélica; Kaja, Simon; Felix, Ricardo

    2017-09-01

    cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca(2+) currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca(2+) channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca(2+) macroscopic currents and impair insulin release stimulated with high K(+). In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine.

    PubMed

    Schreiber, Rainer; Faria, Diana; Skryabin, Boris V; Wanitchakool, Podchanart; Rock, Jason R; Kunzelmann, Karl

    2015-06-01

    Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca(2+) and opening of apical Cl(-) channels. In infants and young animals, but not in adults, Ca(2+)-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca(2+)-dependent Cl(-) channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca(2+)-dependent Cl(-) secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca(2+)-activated Cl(-) secretion. In contrast, Ano10 is essential for Ca(2+)-dependent Cl(-) secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl(-) secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca(2+) increase, while the essential and possibly only apical Cl(-) channel is CFTR. These results define a new role of Ano1 for intestinal Ca(2+)-dependent Cl(-) secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

  3. DsbA directs efficient expression of outer membrane secretin EscC of the enteropathogenic Escherichia coli type III secretion apparatus.

    PubMed

    Miki, Tsuyoshi; Okada, Nobuhiko; Kim, Yeongsuk; Abe, Akio; Danbara, Hirofumi

    2008-02-01

    The formation of disulfide bond is essential for the folding, activity, and stability of many secreted proteins of Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into exported proteins from the cytoplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we investigated the role of DsbA in enteropathogenic Escherichia coli. Here, we show that the DsbA is required for stable expression of outer membrane secretin EscC. DsbA has no effect on LEE transcription as measured with LEE-lacZ fusions. Replacement of either cysteine residue 136 or 155 of EscC with a serine resulted in reduced level of EscC, similar to the effect of the dsbA mutation. These results demonstrate the role of DsbA in assembly of the type III secretion apparatus.

  4. The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry.

    PubMed

    Zhou, Z; Xu, L; Sennepin, A; Federici, C; Ganor, Y; Tudor, D; Damotte, D; Barry Delongchamps, N; Zerbib, M; Bomsel, M

    2017-04-26

    The complexity of signal transduction resulting from the contact of human immunodeficiency virus type 1 (HIV-1)-infected cells and mucosal cells has hampered our comprehension of HIV-1 mucosal entry. Such process is driven efficiently only by viral synapse contacts, whereas cell-free HIV-1 remains poorly infectious. Using CD4(+) T-cells expressing only HIV-1 envelope inoculated on human adult foreskin tissues, we designed methodologies to identify the signals transduced in foreskin keratinocytes following HIV-1-envelope-dependent viral synapse formation. We find that the viral synapse activates the MyD88-independent TLR-4-nuclear factor (NfκB) signaling pathway in keratinocytes and the subsequent secretion of cytokines including thymic stromal lymphopoietin (TSLP), a cytokine linking innate and T-helper type 2-adaptive immune responses. Moreover, the viral synapse upregulates the non-coding microRNA miR-375, known to control TSLP, and transfection of keratinocytes with anti-miR-375 blocks significantly TSLP secretion. Thus, the secretion of TSLP by keratinocytes is induced by the viral synapse in a miR-375 controlled manner. At the tissue level, these signals translate into the epidermal redistribution of Langerhans cells and formation of conjugates with T-cells, recapitulating the initial events observed in human foreskin infection by HIV-1. These results open new possibilities for designing strategies to block mucosal HIV-1 transmission, the major pathway by which HIV-1 spreads worldwide.Mucosal Immunology advance online publication 26 April 2017; doi:10.1038/mi.2017.23.

  5. Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

    PubMed Central

    Cappelletti, Paola A.; dos Santos, Rafael Freitas; do Amaral, Alexandre M.; Homem, Rafael Augusto; dos Santos Souza, Thaís; Machado, Marcos A.; Farah, Chuck S.

    2011-01-01

    Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcUAAAH) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the ΔhrcU mutant with HrcUAAAH produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the ΔhrcU mutant complemented with HrcUAAAH, suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the ΔhrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta. PMID:21408079

  6. The Type III Secretion System (T3SS) is a Determinant for Rice-Endophyte Colonization by Non-Photosynthetic Bradyrhizobium.

    PubMed

    Piromyou, Pongdet; Songwattana, Pongpan; Greetatorn, Teerana; Okubo, Takashi; Kakizaki, Kaori Chiba; Prakamhang, Janpen; Tittabutr, Panlada; Boonkerd, Nantakorn; Teaumroong, Neung; Minamisawa, Kiwamu

    2015-01-01

    Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6-24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection.

  7. The Type III Secretion System (T3SS) is a Determinant for Rice-Endophyte Colonization by Non-Photosynthetic Bradyrhizobium

    PubMed Central

    Piromyou, Pongdet; Songwattana, Pongpan; Greetatorn, Teerana; Okubo, Takashi; Kakizaki, Kaori Chiba; Prakamhang, Janpen; Tittabutr, Panlada; Boonkerd, Nantakorn; Teaumroong, Neung; Minamisawa, Kiwamu

    2015-01-01

    Plant associations by bradyrhizobia have been detected not only in leguminous plants, but also in non-leguminous species including rice. Bradyrhizobium sp. SUTN9-2 was isolated from Aeschynomene americana L., which is a leguminous weed found in the rice fields of Thailand. This strain promoted the highest total rice (Oryza sativa L. cultivar Pathum Thani 1) dry weight among the endophytic bradyrhizobial strains tested, and was, thus, employed for the further characterization of rice-Bradyrhizobium interactions. Some known bacterial genes involved in bacteria-plant interactions were selected. The expression of the type III secretion component (rhcJ), type IV secretion component (virD4), and pectinesterase (peces) genes of the bacterium were up-regulated when the rice root exudate was added to the culture. When SUTN9-2 was inoculated into rice seedlings, the peces, rhcJ, virD4, and exopolysaccharide production (fliP) genes were strongly expressed in the bacterium 6–24 h after the inoculation. The gene for glutathione-S-transferase (gst) was slightly expressed 12 h after the inoculation. In order to determine whether type III secretion system (T3SS) is involved in bradyrhizobial infections in rice plants, wild-type SUTN9-2 and T3SS mutant strains were inoculated into the original host plant (A. americana) and a rice plant (cultivar Pathum Thani 1). The ability of T3SS mutants to invade rice tissues was weaker than that of the wild-type strain; however, their phenotypes in A. americana were not changed by T3SS mutations. These results suggest that T3SS is one of the important determinants modulating rice infection; however, type IV secretion system and peces may also be responsible for the early steps of rice infection. PMID:26582551

  8. Plant food anthocyanins inhibit platelet granule secretion in hypercholesterolaemia: Involving the signalling pathway of PI3K-Akt.

    PubMed

    Song, Fenglin; Zhu, Yanna; Shi, Zhenyin; Tian, Jinju; Deng, Xiujuan; Ren, Jing; Andrews, Marc C; Ni, Heyu; Ling, Wenhua; Yang, Yan

    2014-11-01

    Controlling platelet granule secretion has been considered an effective strategy to dampen thrombosis and prevent atherosclerosis. Anthocyanins are natural plant pigments and possess a wide range of biological activities, including cardiovascular protective activity. In the present study we explored the effects and the potential mechanisms of anthocyanins on platelet granule secretion in hypercholesterolemia. In a randomised, double-blind clinical trial, 150 hypercholesterolaemic individuals were treated with purified anthocyanins (320 mg/day) or placebo for 24 weeks. Anthocyanins consumption significantly reduced plasma levels of β-thromboglobulin (β-TG), soluble P-selectin, and of Regulated on Activation Normal T cell Expressed and Secreted (RANTES) as compared with the placebo. A minor reduction in platelet factor 4 (PF4) and transforming growth factor β1 (TGF-β1) levels were also observed. In in vitro experiments, we observed that puriӿed anthocyanin mixture, as well as its two main anthocyanin components, delphinidin-3-glucoside (Dp-3-g) and cyanidin-3-glucoside (Cy-3g) directly inhibited platelet á-granule, dense granule, and lysosome secretion evaluated by P-selectin, RANTES, β-TG, PF4, TGF-β1, serotonin, ATP, and CD63 release. Further, anthocyanins inhibited platelet PI3K/Akt activation and consequently attenuated eNOS phosphorylation and cGMP production, thus interrupting MAPK activation. LY294002, a PI3K inhibitor, did not cause additional inhibitory efficacy, indicating that anthocyanin-induced effects may be involved in inhibition of the PI3K/Akt signalling pathway. These results provide evidence that by inhibiting platelet granule secretion, anthocyanins may be a potent cardioprotective agent.

  9. YscP and YscU switch the substrate specificity of the Yersinia type III secretion system by regulating export of the inner rod protein YscI.

    PubMed

    Wood, Sarah E; Jin, Jin; Lloyd, Scott A

    2008-06-01

    Pathogenic yersiniae utilize a type III secretion system to inject antihost factors, called Yops, directly into the cytosol of eukaryotic cells. The Yops are injected via a needle-like structure, comprising the YscF protein, on the bacterial surface. While the needle is being assembled, Yops cannot be secreted. YscP and YscU switch the substrate specificity of the secretion system to enable Yop export once the needle attains its proper length. Here, we demonstrate that the inner rod protein YscI plays a critical role in substrate specificity switching. We show that YscI is secreted by the type III secretion system and that YscI secretion by a yscP mutant is abnormally elevated. Furthermore, we show that mutations in the cytoplasmic domain of YscU reduce YscI secretion by the yscP null strain. We also demonstrate that mutants expressing one of three forms of YscI (those with mutations Q84A, L87A, and L96A) secrete substantial amounts of Yops yet exhibit severe defects in needle formation. In the absence of YscP, mutants with the same changes in YscI assemble needles but are unable to secrete Yops. Together, these results suggest that the formation of the inner rod, not the needle, is critical for substrate specificity switching and that YscP and YscU exert their effects on substrate export by controlling the secretion of YscI.

  10. Serum Amyloid A3 Secreted by Preosteoclasts Inhibits Parathyroid Hormone-stimulated cAMP Signaling in Murine Osteoblasts*

    PubMed Central

    Choudhary, Shilpa; Goetjen, Alexandra; Estus, Thomas; Jacome-Galarza, Christian E.; Aguila, Hector L.; Lorenzo, Joseph; Pilbeam, Carol

    2016-01-01

    Continuous parathyroid hormone (PTH) blocks its own osteogenic actions in marrow stromal cell cultures by inducing Cox2 and receptor activator of nuclear factor κB ligand (RANKL) in the osteoblastic lineage cells, which then cause the hematopoietic lineage cells to secrete an inhibitor of PTH-stimulated osteoblast differentiation. To identify this inhibitor, we used bone marrow macrophages (BMMs) and primary osteoblasts (POBs) from WT and Cox2 knock-out (KO) mice. Conditioned medium (CM) from RANKL-treated WT, but not KO, BMMs blocked PTH-stimulated cAMP production in POBs. Inhibition was reversed by pertussis toxin (PTX), which blocks Gαi/o activation. Saa3 was the most highly differentially expressed gene in a microarray comparison of RANKL-treated WT versus Cox2 KO BMMs, and RANKL induced Saa3 protein secretion only from WT BMMs. CM from RANKL-stimulated BMMs with Saa3 knockdown did not inhibit PTH-stimulated responses in POBs. SAA added to POBs inhibited PTH-stimulated cAMP responses, which was reversed by PTX. Selective agonists and antagonists of formyl peptide receptor 2 (Fpr2) suggested that Fpr2 mediated the inhibitory actions of Saa3 on osteoblasts. In BMMs committed to become osteoclasts by RANKL treatment, Saa3 expression peaked prior to appearance of multinucleated cells. Flow sorting of WT marrow revealed that Saa3 was secreted only from the RANKL-stimulated B220− CD3−CD11b−/low CD115+ preosteoclast population. We conclude that Saa3 secretion from preosteoclasts, induced by RANKL in a Cox2-dependent manner, inhibits PTH-stimulated cAMP signaling and osteoblast differentiation via Gαi/o signaling. The induction of Saa3 by PTH may explain the suppression of bone formation when PTH is applied continuously and may be a new therapeutic target for osteoporosis. PMID:26703472

  11. Signal pathways regulating hyaluronan secretion into static and cycled synovial joints of rabbits.

    PubMed

    Ingram, K R; Wann, A K T; Wingate, R M; Coleman, P J; McHale, N; Levick, J R

    2009-09-01

    Joint lubrication, synovial fluid conservation and many pathophysiological processes depend on hyaluronan (HA). Intra-articular HA injection and exercise, which stimulates articular HA production, ameliorate osteoarthritis. We therefore investigated the pathways regulating movement-stimulated articular HA secretion rate ( ) in vivo. Endogenous HA was removed from the knee joint cavity of anaesthetised rabbits by washout. Joints were then cycled passively or remained static for 5 h, with/without intra-articular agonist/inhibitor, after which newly secreted HA was harvested for analysis. Movement almost doubled . Similar or larger increases were elicited in static joints by the intra-articular Ca(2+) ionophore ionomycin, prostaglandin E(2), cAMP-raising agents, serine/threonine phosphatase inhibitor and activation of protein kinase C (PKC). PKC-stimulated secretion was inhibited by the PKC inhibitor bisindolylmaleimide I and inhibitors of the downstream kinases MEK-ERK (U0126, PD98059). These agents inhibited movement-stimulated secretion of HA (MSHA) only when the parallel p38 kinase path was simultaneously inhibited by SB203580 (ineffective alone). The phospholipase C inhibitor U73122 almost fully blocked MSHA (P = 0.001, n = 10), without affecting static . The ENaC channel blocker amiloride inhibited MSHA, whereas other inhibitors of stretch-activated channels (Gd(3+), ruthenium red, SKF96365) did not. It is proposed that MSHA may be mediated by PLC activation, leading to activation of parallel PKC-MEK-ERK and p38 kinase pathways.

  12. The Animal Pathogen-Like Type III Secretion System is Required for the Intracellular Survival of Burkholderia mallei within J774.2 Macrophages

    DTIC Science & Technology

    2006-03-30

    E -mail: Ricky.Ulrich @AMEDD.ARMY.MIL. 4349 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of...counterbioterrorism. The Humana Press, Inc., Totowa, N.J. 4. DeShazer, D., D. M. Waag, D. L. Fritz, and D. E . Woods. 2001. Identification of a Burkholderia...Microb. Pathog. 30:253–269. 5. Galan, J. E . 2001. Salmonella interactions with host cells: type III secretion at work. Annu. Rev. Cell Dev. Biol. 17:53

  13. TNF phase III signalling in tolerant cells is tightly controlled by A20 and CYLD.

    PubMed

    Bikker, Rolf; Christmann, Martin; Preuß, Katharina; Welz, Bastian; Friesenhagen, Judith; Dittrich-Breiholz, Oliver; Huber, René; Brand, Korbinian

    2017-09-01

    Following the acute phase of an inflammatory reaction, a strictly controlled resolution of inflammation is necessary. A dysregulation of this process leads to hyperinflammation, chronic inflammatory disease, or immune paralysis. Different mechanisms participate in the coordinated termination of the inflammatory process, e.g. the expression of antiinflammatory molecules and different forms of tolerance. To better understand the processes which mediate resolution of TNF-dependent inflammation and induce tolerance, it is necessary to characterize the signal transduction quality during TNF long-term (pre)incubation. Within a time frame from 12 to 48h, designated as phase III of the TNF response, we measured an ongoing, constitutive activation of TNFR1/NF-κB-dependent pathways in monocytic cells. Phase III signalling which was also named "constitutive signaling in TNF tolerant cells" induces the expression of low- and high-sensitive target genes including A20 which is differentially regulated by transcriptional and proteolytic events. A20 strictly controls TNF long-term constitutive signalling in an IκB kinase complex- and partially RIP-dependent manner supported by adjuvant ABIN1. In addition, CYLD proteins participate in the regulation of this late-phase signal transduction, whereas downstream molecules such as Bcl3 and p50 are not involved. A20 and CYLD are expressed with different mRNA kinetics resulting in a strong or only a modest increase in protein levels, respectively. The identification of mechanisms which contribute to the termination of inflammation will provide additional diagnostic and therapeutic aspects to specifically diagnose certain aspects of inflammation and specifically modulate them. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Signal Perception by the Secretion Stress-Responsive CssRS Two-Component System in Bacillus subtilis

    PubMed Central

    Botella, Eric; Butler, Clodagh; Hansen, Annette; Jende, Inga; Devine, Kevin M.

    2012-01-01

    The CssRS two-component system responds to heat and secretion stresses in Bacillus subtilis by controlling expression of HtrA and HtrB chaperone-type proteases and positively autoregulating its own expression. Here we report on the features of the CssS extracellular loop domain that are involved in signal perception and on CssS subcellular localization. Individual regions of the CssS extracellular loop domain contribute differently to signal perception and activation. The conserved hydrophilic 26-amino-acid segment juxtaposed to transmembrane helix 1 is involved in the switch between the deactivated and activated states, while the conserved 19-amino-acid hydrophobic segment juxtaposed to transmembrane 2 is required for signal perception and/or transduction. Perturbing the size of the extracellular loop domain increases CssS kinase activity and makes it unresponsive to secretion stress. CssS is localized primarily at the septum but is also found in a punctate pattern with lower intensity throughout the cell cylinder. Moreover, the CssRS-controlled HtrA and HtrB proteases are randomly distributed in foci throughout the cell surface, with more HtrB than HtrA foci in unstressed cells. PMID:22307758

  15. The Type III Secretion System Effector SeoC of Salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and Inhibits Opsonophagocytosis

    PubMed Central

    Pollard, Dominic J.; Young, Joanna C.; Covarelli, Valentina; Herrera-León, Silvia; Connor, Thomas R.; Fookes, Maria; Walker, Danielle; Echeita, Aurora; Thomson, Nicholas R.; Berger, Cedric N.

    2016-01-01

    Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S. Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC. SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae. The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro. Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae. PMID:27736780

  16. ER stress in adipocytes inhibits insulin signaling, represses lipolysis, and alters the secretion of adipokines without inhibiting glucose transport.

    PubMed

    Xu, L; Spinas, G A; Niessen, M

    2010-08-01

    The endoplasmic reticulum (ER) is the intra-cellular site, where secreted and membrane proteins are synthesized. ER stress and activation of the unfolded protein response (UPR) contribute to insulin resistance and the development of diabetes in obesity. It was shown previously in hepatocytes that the UPR activates c-jun N-terminal kinase (JNK), which phosphorylates insulin receptor substrate (IRS) proteins on serine residues thereby inhibiting insulin signal transduction. Here we describe how ER stress affects insulin signaling and the biological function of adipocytes. In addition to inhibition of IRS we found that ER stress downregulates the expression of the insulin receptor. Concomitantly, insulin-induced activation of Akt/PKB and of ERK1/2 was strongly inhibited. Ectopic expression of IRS1 or IRS2 strongly counteracted the inhibitory effect of ER stress on insulin signaling while pharmacological inhibition of JNK with SP600125 resulted only in a mild improvement. ER stress decreased the secretion of the adipokines adiponectin and leptin, but strongly increased secretion of IL-6. ER stress inhibited expression and insulin-induced phosphorylation of AS160, reduced lipolysis but did not inhibit glucose transport. Finally, supernatants collected from 3T3-L1 adipocytes undergoing ER stress improved or impaired proliferation when used to condition the culture medium of INS-1E beta-cells dependent on the degree of ER stress. It appears that ER stress in adipocytes might initially lead to changes resembling early prediabetic stages, which at least in part support the regulation of systemic energy homeostasis. Copyright Georg Thieme Verlag KG Stuttgart New York.

  17. Harnessing Novel Secreted Inhibitors of EGF Receptor Signaling for Breast Cancer Treatment

    DTIC Science & Technology

    2006-04-01

    of EGF Receptor Signaling for Breast Cancer Treatment PRINCIPAL INVESTIGATOR: Mark A. Lemmon, Ph.D. CONTRACTING...Receptor Signaling for Breast Cancer Treatment INTRODUCTION The aim of this research project is to develop novel inhibitors of signaling through receptors...related proteins), thus providing the essential groundwork for developing an innovative approach for breast cancer treatment that will attack the

  18. Intracellular Signaling Mechanisms Pharmacological Action of Jasminum amplexicaule Buch.-Ham. (Oleaceae) on Gastrointestinal Secretion.

    PubMed

    Gao, Zhenhua; Yin, Junqiang; Xie, Xiaolin; Long, Hanwu; Qi, Xiang; Lin, Changhu; Wu, Liangcai

    2014-01-01

    Jasminum amplexicaule Buch-Ham. (Oleaceae) has been commonly used in the traditional medicine in dysentery, diarrhoea and bellyache in China. In the present work, the methanol extract of Jasminum amplexicaule (JME) was examined for pharmacology on human colonic epithelial cell line T84 by the short-circuit current technique. The results showed that pretreatment of T84 cells with JME produced a concentration-dependent (0-1000 μg/mL. EC50 = 0.055 mg/ mL) inhibition effect on adrenalin (Adr.)-induced Cl- secretion. The maximal response was observed at 200 μg/mL. It has been demonstrated that JME has a direct effect on the enterocyte. Our results also demonstrated that the JME exerted inhibitory effect on gastrointestinal Cl(-)secretion that effected by acting on basolateral β-adrenoreceptors. These results suggested that the Chinese traditional medicine of JME can be used for the treatment of acute diarrhea and bellyache.

  19. PACAP stimulation of maturational gonadotropin secretion in goldfish involves extracellular signal-regulated kinase, but not nitric oxide or guanylate cyclase, signaling.

    PubMed

    Chang, John P; Sawisky, Grant R; Mitchell, Gabriel; Uretsky, Aubrey D; Kwong, Patrick; Grey, Caleb L; Meints, Amanda N; Booth, Morgan

    2010-01-01

    In goldfish, nitric oxide synthase (NOS) immunoreactivity is present in gonadotropes and extracellular signal-regulated protein kinase (ERK) mediates GnRH stimulation of gonadotropin release and synthesis. In this study, we tested the possible involvement of nitric oxide (NO) and ERK in mediating PACAP-stimulated maturational gonadotropin (GTH-II) release from primary cultures of dispersed goldfish pituitary cells. In static incubation experiments, PACAP-induced GTH-II release was unaffected by two inhibitors of NOS synthase, AGH and 1400W; whereas addition of a NO donor, SNAP, elevated GTH-II secretion. In perifusion experiments, neither NOS inhibitors (AGH, 1400W and 7-Ni) nor NO scavengers (PTIO and rutin hydrate) attenuated the GTH-II response to pulse applications of PACAP. In addition, the GTH-II responses to PACAP and the NO donor SNP were additive while PTIO blocked SNP action. Although dibutyryl cGMP increased GTH-II secretion in static incubation, inhibition of guanylate cyclase (GC), a known down-stream target for NO signaling, did not reduce the GTH-II response to pulse application of PACAP. On the other hand, GTH-II responses to PACAP in perifusion were attenuated in the presence of two inhibitors of ERK kinase (MEK), U 0126 and PD 98059. These results suggest that although increased availability of NO and cGMP can lead to increased GTH-II secretion, MEK/ERK signaling, rather than NOS/NO/GC activation, mediates PACAP action on GTH-II release in goldfish.

  20. The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

    PubMed Central

    Hausner, Jens; Hartmann, Nadine; Jordan, Michael

    2016-01-01

    ABSTRACT The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. PMID:27895129

  1. The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation.

    PubMed

    Hausner, Jens; Hartmann, Nadine; Jordan, Michael; Büttner, Daniela

    2017-02-01

    The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system, which spans both bacterial membranes and translocates effector proteins into plant cells. The assembly of the T3S system presumably involves the predicted lytic transglycosylase (LT) HpaH, which is encoded adjacent to the T3S gene cluster. Bacterial LTs degrade peptidoglycan and often promote the formation of membrane-spanning macromolecular protein complexes. In the present study, we show that HpaH localizes to the bacterial periplasm and binds to peptidoglycan as well as to components of the T3S system, including the predicted periplasmic inner rod proteins HrpB1 and HrpB2 as well as the pilus protein HrpE. In vivo translocation assays revealed that HpaH promotes the translocation of various effector proteins and of early substrates of the T3S system, suggesting a general contribution of HpaH to type III-dependent protein export. Mutant studies and the analysis of reporter fusions showed that the N-terminal region of HpaH contributes to protein function and is proteolytically cleaved. The N-terminally truncated HpaH cleavage product is secreted into the extracellular milieu by a yet-unknown transport pathway, which is independent of the T3S system. Copyright © 2017 American Society for Microbiology.

  2. Structural and Biochemical Characterization of SrcA, a Multi-cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

    SciTech Connect

    Cooper, C.; Zhang, K; Andres, S; Fnag, Y; Kaniuk, N; Hannemann, M; Brumell, J; Foster, L; Junop, M; Coombes, B

    2010-01-01

    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 {angstrom} 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.

  3. Expression, purification, crystallization and preliminary crystallographic analysis of MxiH, a subunit of the Shigella flexneri type III secretion system needle

    SciTech Connect

    Deane, Janet E.; Cordes, Frank S.; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D.; Picking, Wendy L.; Lea, Susan M.; Blocker, Ariel

    2006-03-01

    A monodisperse truncation mutant of MxiH, the subunit of the S. flexneri type III secretion system needle, has been crystallized. SeMet derivatives and a uranyl derivative have undergone preliminary crystallographic analysis. A monodisperse truncation mutant of MxiH, the subunit of the needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH{sub CΔ5} and diffraction data were collected to 1.9 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 Å, β = 96.5°. An anomalous difference Patterson map calculated with the data from the SeMet-labelled crystals revealed a single peak on the Harker section v = 0. Inspection of a uranyl derivative also revealed one peak in the isomorphous difference Patterson map on the Harker section v = 0. Analysis of the self-rotation function indicates the presence of a twofold non-crystallographic symmetry axis approximately along a. The calculated Matthews coefficient is 1.9 Å{sup 3} Da{sup −1} for two molecules per asymmetric unit, corresponding to a solvent content of 33%.

  4. An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination

    PubMed Central

    Tobe, Toru; Beatson, Scott A.; Taniguchi, Hisaaki; Abe, Hiroyuki; Bailey, Christopher M.; Fivian, Amanda; Younis, Rasha; Matthews, Sophie; Marches, Olivier; Frankel, Gad; Hayashi, Tetsuya; Pallen, Mark J.

    2006-01-01

    Several pathogenic strains of Escherichia coli exploit type III secretion to inject “effector proteins” into human cells, which then subvert eukaryotic cell biology to the bacterium's advantage. We have exploited bioinformatics and experimental approaches to establish that the effector repertoire in the Sakai strain of enterohemorrhagic E. coli (EHEC) O157:H7 is much larger than previously thought. Homology searches led to the identification of >60 putative effector genes. Thirteen of these were judged to be likely pseudogenes, whereas 49 were judged to be potentially functional. In total, 39 proteins were confirmed experimentally as effectors: 31 through proteomics and 28 through translocation assays. At the protein level, the EHEC effector sequences fall into >20 families. The largest family, the NleG family, contains 14 members in the Sakai strain alone. EHEC also harbors functional homologs of effectors from plant pathogens (HopPtoH, HopW, AvrA) and from Shigella (OspD, OspE, OspG), and two additional members of the Map/IpgB family. Genes encoding proven or predicted effectors occur in >20 exchangeable effector loci scattered throughout the chromosome. Crucially, the majority of functional effector genes are encoded by nine exchangeable effector loci that lie within lambdoid prophages. Thus, type III secretion in E. coli is linked to a vast phage “metagenome,” acting as a crucible for the evolution of pathogenicity. PMID:16990433

  5. Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycD in complex with a peptide of the minor translocator YopD

    PubMed Central

    2012-01-01

    Background Type III secretion systems are used by Gram-negative bacteria as “macromolecular syringes” to inject effector proteins into eukaryotic cells. Two hydrophobic proteins called translocators form the necessary pore in the host cell membrane. Both translocators depend on binding to a single chaperone in the bacterial cytoplasm to ensure their stability and efficient transport through the secretion needle. It was suggested that the conserved chaperones bind the more divergent translocators via a hexapeptide motif that is found in both translocators and conserved between species. Results We crystallized a synthetic decapeptide from the Yersinia enterocolitica minor type III secretion translocator YopD bound to its cognate chaperone SycD and determined the complex structure at 2.5 Å resolution. The structure of peptide-bound SycD is almost identical to that of apo SycD with an all helical fold consisting of three tetratricopeptide repeats (TPRs) and an additional C-terminal helix. Peptide-bound SycD formed a kinked head-to-head dimer that had previously been observed for the apo form of SycD. The homodimer interface comprises both helices of the first tetratricopeptide repeat. The YopD peptide bound in extended conformation into a mainly hydrophobic groove on the concave side of SycD. TPRs 1 and 2 of SycD form three hydrophobic pockets that accommodated the conserved hydrophobic residues at position 1, 3 and 6 of the translocator hexapeptide sequence. Two tyrosines that are highly conserved among translocator chaperones contribute to the hydrophobic patches but also form hydrogen bonds to the peptide backbone. Conclusions The interaction between SycD and YopD is very similar to the binding of the Pseudomonas minor translocator PopD to its chaperone PcrH and the Shigella major translocator IpaB to its chaperone IpgC. This confirms the prediction made by Kolbe and co-workers that a hexapeptide with hydrophobic residues at three positions is a conserved

  6. Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycD in complex with a peptide of the minor translocator YopD.

    PubMed

    Schreiner, Madeleine; Niemann, Hartmut H

    2012-06-18

    Type III secretion systems are used by Gram-negative bacteria as "macromolecular syringes" to inject effector proteins into eukaryotic cells. Two hydrophobic proteins called translocators form the necessary pore in the host cell membrane. Both translocators depend on binding to a single chaperone in the bacterial cytoplasm to ensure their stability and efficient transport through the secretion needle. It was suggested that the conserved chaperones bind the more divergent translocators via a hexapeptide motif that is found in both translocators and conserved between species. We crystallized a synthetic decapeptide from the Yersinia enterocolitica minor type III secretion translocator YopD bound to its cognate chaperone SycD and determined the complex structure at 2.5 Å resolution. The structure of peptide-bound SycD is almost identical to that of apo SycD with an all helical fold consisting of three tetratricopeptide repeats (TPRs) and an additional C-terminal helix. Peptide-bound SycD formed a kinked head-to-head dimer that had previously been observed for the apo form of SycD. The homodimer interface comprises both helices of the first tetratricopeptide repeat. The YopD peptide bound in extended conformation into a mainly hydrophobic groove on the concave side of SycD. TPRs 1 and 2 of SycD form three hydrophobic pockets that accommodated the conserved hydrophobic residues at position 1, 3 and 6 of the translocator hexapeptide sequence. Two tyrosines that are highly conserved among translocator chaperones contribute to the hydrophobic patches but also form hydrogen bonds to the peptide backbone. The interaction between SycD and YopD is very similar to the binding of the Pseudomonas minor translocator PopD to its chaperone PcrH and the Shigella major translocator IpaB to its chaperone IpgC. This confirms the prediction made by Kolbe and co-workers that a hexapeptide with hydrophobic residues at three positions is a conserved chaperone binding motif. Because the

  7. 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.

  8. Host-Pathogen Interaction and Signaling Molecule Secretion Are Modified in the dpp3 Knockout Mutant of Candida lusitaniae

    PubMed Central

    Sabra, Ayman; Bessoule, Jean-Jacques; Atanasova-Penichon, Vessela; Noël, Thierry

    2014-01-01

    Candida lusitaniae is an emerging opportunistic yeast and an attractive model to discover new virulence factors in Candida species by reverse genetics. Our goal was to create a dpp3Δ knockout mutant and to characterize the effects of this gene inactivation on yeast in vitro and in vivo interaction with the host. The secretion of two signaling molecules in Candida species, phenethyl alcohol (PEA) and tyrosol, but not of farnesol was surprisingly altered in the dpp3Δ knockout mutant. NO and reactive oxygen species (ROS) production as well as tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) secretion were also modified in macrophages infected with this mutant. Interestingly, we found that the wild-type (WT) strain induced an increase in IL-10 secretion by zymosan-activated macrophages without the need for physical contact, whereas the dpp3Δ knockout mutant lost this ability. We further showed a striking role of PEA and tyrosol in this modulation. Last, the DPP3 gene was found to be an essential contributor to virulence in mice models, leading to an increase in TNF-α secretion and brain colonization. Although reinsertion of a WT DPP3 copy in the dpp3Δ knockout mutant was not sufficient to restore the WT phenotypes in vitro, it allowed a restoration of those observed in vivo. These data support the hypothesis that some of the phenotypes observed following DPP3 gene inactivation may be directly dependent on DPP3, while others may be the indirect consequence of another genetic modification that systematically arises when the DPP3 gene is inactivated. PMID:24191303

  9. Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

    PubMed Central

    Dickenson, Nicholas E; Choudhari, Shyamal P; Adam, Philip R; Kramer, Ryan M; Joshi, Sangeeta B; Middaugh, C Russell; Picking, Wendy L; Picking, William D

    2013-01-01

    The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligo-oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N-dimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore. PMID:23456854

  10. 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

  11. S-resistin, a non secretable resistin isoform, impairs the insulin signalling pathway in 3T3-L1 adipocytes.

    PubMed

    Rodríguez, María; Moltó, Eduardo; Aguado, Lidia; Gallardo, Nilda; Andrés, Antonio; Arribas, Carmen

    2015-09-01

    S-resistin is a non-secretable resistin spliced variant, which is expressed mainly in the white adipose tissue from Wistar rats. Previous results confirmed that 3T3-L1 cells expressing s-resistin (3T3-L1-s-res) showed an inflammatory response and exhibited a decrease in glucose transport, both basal and insulin-stimulated. Here we present evidences demonstrating for the first time that s-resistin, like resistin, blocks insulin signalling pathway by inhibiting insulin receptor, insulin receptor substrate 1, protein kinase B/Akt and the mammalian target of rapamycin phosphorylation, and increasing the suppressor of cytokine signalling 3 levels being the later probably due to augmented of leptin expression. Thus, our data suggest that s-resistin could act by a still unknown intracrine pathway as an intracellular sensor, regulating the adipocyte insulin sensitivity.

  12. Secretion of endothelin converting enzyme-1a: the hydrophobic signal anchor domain alone is not sufficient to promote membrane localization.

    PubMed

    Brooks, S C; Fernandez, L; Ergul, A

    2000-05-01

    Endothelin converting enzyme-1 (ECE-1) is a type II membrane protein that is important for the proteolytic activation of big endothelin-1 to endothelin-1. Although the highly conserved zinc-binding motif is known to be located in the extracellular domain, the role(s) of the N-terminal and membrane-spanning signal anchor domains in the biosynthesis and function of ECE-1 isoforms, ECE-1a, ECE-1b, and ECE-1c, remain undetermined. In this study, we provide evidence that the deletion of the cytoplasmic N-terminal tail (residues 1-55) of ECE-1a results in the cleavage of a potential signal peptide located in the signal anchor domain leading to the partial secretion of the recombinant enzyme into the media. However, the truncation of N-terminal and/or signal anchor domain does not affect the activity of ECE-1a. Therefore, our results demonstrate that the hydrophobic signal anchor domain alone is not sufficient for the membrane anchoring of ECE-1a and that the N-terminal domain of ECE-1a is important for membrane targeting as well as the intracellular localization of the enzyme.

  13. EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

    PubMed

    da Rosa, Marina Rolo Pinheiro; Falcão, Aline Semblano Carreira; Fuzii, Hellen Thais; da Silva Kataoka, Maria Sueli; Ribeiro, André L R; Boccardo, Enrique; de Siqueira, Adriane Sousa; Jaeger, Ruy G; de Jesus Viana Pinheiro, João; de Melo Alves Júnior, Sérgio

    2014-11-01

    Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion

  14. Mechano-signal transduction in mesenchymal stem cells induces prosaposin