Sample records for iii secretion substrate
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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...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Samudrala, Ram; Heffron, Fred; McDermott, Jason E.
2009-04-24
The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates, effector proteins, are not. We have used a machine learning approach to identify new secreted effectors. The method integrates evolutionary measures, such as the pattern of homologs in a range of other organisms, and sequence-based features, such as G+C content, amino acid composition and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from Salmonella typhimurium and validated on a corresponding set of effectors from Pseudomonas syringae, aftermore » eliminating effectors with detectable sequence similarity. The method was able to identify all of the known effectors in P. syringae with a specificity of 84% and sensitivity of 82%. The reciprocal validation, training on P. syringae and validating on S. typhimurium, gave similar results with a specificity of 86% when the sensitivity level was 87%. These results show that type III effectors in disparate organisms share common features. We found that maximal performance is attained by including an N-terminal sequence of only 30 residues, which agrees with previous studies indicating that this region contains the secretion signal. We then used the method to define the most important residues in this putative secretion signal. Finally, we present novel predictions of secreted effectors in S. typhimurium, some of which have been experimentally validated, and apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis. This approach is a novel and effective way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal.« less
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Akeda, Yukihiro; Okayama, Kanna; Kimura, Tomomi; Dryselius, Rikard; Kodama, Toshio; Oishi, Kazunori; Iida, Tetsuya; Honda, Takeshi
2009-07-01
Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus. In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30-100 amino acids and an amino terminal secretion signal encompassing the first 5-20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.
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Lara-Tejero, María; Bewersdorf, Jörg; Galán, Jorge E.
2017-01-01
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. PMID:28533372
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Frost, Stefan; Ho, Oanh; Login, Frédéric H.; Weise, Christoph F.; Wolf-Watz, Hans; Wolf-Watz, Magnus
2012-01-01
Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms. PMID:23185318
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Frost, Stefan; Ho, Oanh; Login, Frédéric H; Weise, Christoph F; Wolf-Watz, Hans; Wolf-Watz, Magnus
2012-01-01
Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC). Here we show that depletion of calcium induces intramolecular dissociation of YscU(CC) from YscU followed by secretion of the YscU(CC) polypeptide. Thus, YscU(CC) behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU(CC)in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU(CC) dissociation for Yop secretion. We propose that YscU(CC) orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.
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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.
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YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal*
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
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YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal.
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.
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Akeda, Yukihiro; Kodama, Toshio; Saito, Kazunobu; Iida, Tetsuya; Oishi, Kazunori; Honda, Takeshi
2011-11-01
The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H.; Wolf-Watz, Magnus
2017-01-01
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria. PMID:28039361
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Identification of secreted bacterial proteins by noncanonical amino acid tagging
Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.
2014-01-01
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637
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Ho, Oanh; Rogne, Per; Edgren, Tomas; Wolf-Watz, Hans; Login, Frédéric H; Wolf-Watz, Magnus
2017-02-24
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia , the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscU C binds to the inner rod protein YscI with a dissociation constant ( K d ) of 3.8 μm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T-mediated virulence.
Balachandran, Priya; Dragone, Leonard; Garrity-Ryan, Lynne; Lemus, Armando; Weiss, Arthur; Engel, Joanne
2007-02-01
Pseudomonas aeruginosa, an important cause of opportunistic infections in humans, delivers bacterial cytotoxins by type III secretion directly into the host cell cytoplasm, resulting in disruption of host cell signaling and host innate immunity. However, little is known about the fate of the toxins themselves following injection into the host cytosol. Here, we show by both in vitro and in vivo studies that the host ubiquitin ligase Cbl-b interacts with the type III-secreted effector exotoxin T (ExoT) and plays a key role in vivo in limiting bacterial dissemination mediated by ExoT. We demonstrate that, following polyubiquitination, ExoT undergoes regulated proteasomal degradation in the host cell cytosol. ExoT interacts with the E3 ubiquitin ligase Cbl-b and Crk, the substrate for the ExoT ADP ribosyltransferase (ADPRT) domain. The efficiency of degradation is dependent upon the activity of the ADPRT domain. In mouse models of acute pneumonia and systemic infection, Cbl-b is specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl plays no detectable role. To the best of our knowledge, this represents the first identification of a mammalian gene product that is specifically required for in vivo resistance to disease mediated by a type III-secreted effector.
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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
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2015-01-01
Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221
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LcrV Mutants That Abolish Yersinia Type III Injectisome Function
Ligtenberg, Katherine Given; Miller, Nathan C.; Mitchell, Anthony; Plano, Gregory V.
2013-01-01
LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR+] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV*327). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR− phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV*327 mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR+, LCR−, or dominant negative LCR− phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor. PMID:23222719
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Physiological and pathophysiological functions of SIRT1.
Wojcik, M; Mac-Marcjanek, K; Wozniak, L A
2009-03-01
The human SIRT1 is a nuclear enzyme from the class III histone deacetylases (HDACs) which is widely distributed in mammalian tissues. A variety of SIRT1 substrates hints that this protein is involved in the regulation of diverse biological processes, including cell survival, apoptosis, gluconeogenesis, adipogenesis, lipolysis, stress resistance, muscle differentiation, and insulin secretion. This review emphasizes catalytic properties of SIRT1 and its role in apoptosis, insulin pathway, and neuron survival.
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The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba.
Siddiqui, Ruqaiyyah; Malik, Huma; Sagheer, Mehwish; Jung, Suk-Yul; Khan, Naveed Ahmed
2011-08-01
The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P<0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts. Copyright © 2011 Elsevier Inc. All rights reserved.
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Regulation of the Yersinia type III secretion system: traffic control
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
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A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.
Ittig, Simon J; Schmutz, Christoph; Kasper, Christoph A; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R; Nigg, Erich A; Arrieumerlou, Cécile
2015-11-23
Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. © 2015 Ittig et al.
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Global impact of Salmonella type III secretion effector SteA on host cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cardenal-Muñoz, Elena, E-mail: e_cardenal@us.es; Gutiérrez, Gabriel, E-mail: ggpozo@us.es; Ramos-Morales, Francisco, E-mail: framos@us.es
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. Thesemore » 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.« less
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Li, Jiabao; Cai, Shichun; Luo, Yuanming; Dong, Xiuzhu
2011-09-01
Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.
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Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; ...
2015-04-09
We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increasemore » in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.« less
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Code of Federal Regulations, 2014 CFR
2014-07-01
... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the Superfund...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the Superfund...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the Superfund...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... INFORMATION: AND TRADE SECRET DISCLOSURES TO HEALTH PROFESSIONALS Trade Secrecy Claims § 350.1 Definitions... secret. Sanitized means a version of a document from which information claimed as trade secret or... support of a claim that chemical identity is a trade secret. Title III means Title III of the Superfund...
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NSAID-derived gamma-secretase modulators. Part III: Membrane anchoring.
Baumann, Stefanie; Höttecke, Nicole; Schubenel, Robert; Baumann, Karlheinz; Schmidt, Boris
2009-12-15
Selective lowering of Abeta(42) levels with small-molecule substrate targeting gamma-secretase modulators (sGSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. Here we present N-substituted carbazole- and O-substituted fenofibrate-derived sGSMs and their activity data. Seven out of 19 screened compounds exhibited promising activity against Abeta(42) secretion at a low micromolar level. We presume that the sGSMs interact with lys624 at the membrane interface and that the lipophilic substituents anchor the compound orientation in the membrane.
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Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.
2011-01-01
Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588
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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, ...
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Discovery of the type VII ESX-1 secretion needle?
Ates, Louis S; Brosch, Roland
2017-01-01
Mycobacterium tuberculosis, the etiological agent of human tuberculosis, harbours five ESAT-6/type VII secretion (ESX/T7S) systems. The first esx gene clusters were identified during the genome-sequencing project of M. tuberculosis H37Rv. Follow-up studies revealed additional genes playing important roles in ESX/T7S systems. Among the latter genes, one can find those that encode Pro-Glu (PE) and Pro-Pro-Glu (PPE) proteins as well as a gene cluster that is encoded >260 kb upstream of the esx-1 locus and encodes ESX-1 secretion-associated proteins EspA (Rv3616c), EspC (Rv3615c) and EspD (Rv3614c). The espACD cluster has been suggested to have an important function in ESX-1 secretion since EspA-EspC and EsxA-EsxB are mutually co-dependent on each other for secretion. However, the molecular mechanism of this co-dependence and interaction between the substrates remained unknown. In this issue of Molecular Microbiology, Lou and colleagues show that EspC forms high-molecular weight polymerization complexes that resemble selected components of type II, III and/or IV secretion systems of Gram-negative bacteria. Indeed, EspC-multimeric complexes form filamentous structures that could well represent a secretion needle of ESX-1 type VII secretion systems. This exciting observation opens new avenues for research to discover and characterize ESX/T7S components and elucidates the co-dependence of EsxA/B secretion with EspA/C. © 2016 John Wiley & Sons Ltd.
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Crystal structure of the Yersinia type III secretion protein YscE
DOE Office of Scientific and Technical Information (OSTI.GOV)
Phan, Jason; Austin, Brian P.; Waugh, David S.
2010-12-06
The plague-causing bacterium Yersinia pestis utilizes a contact-dependent (type III) secretion system (T3SS) to transport virulence factors from the bacterial cytosol directly into the interior of mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. The type III secretion apparatus is composed of 20-25 different Yersinia secretion (Ysc) proteins. We report here the structure of YscE, the smallest Ysc protein, which is a dimer in solution. The probable mode of oligomerization is discussed.
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Applications of β-gal-III isozyme from Bacillus coagulans RCS3, in lactose hydrolysis.
Batra, Navneet; Singh, Jagtar; Joshi, Amit; Bhatia, Sonu
2011-12-01
Bacillus coagulans RCS3 isolated from hot water springs secreted five isozymes i.e. β-gal I-V of β-galactosidase. β-gal III isozyme was purified using DEAE cellulose and Sephadex G 100 column chromatography. Its molecular weight characterization showed a single band at 315kD in Native PAGE, while two subunits of 50.1 and 53.7 kD in SDS PAGE. β-Gal III had pH optima in the range of 6-7 and temperature optima at 65°C. It preferred nitro-aryl-β-d-galactoside as substrate having K(m) of 4.16 mM with ONPG. More than 85% and 80% hydrolysis of lactose (1-5%, w/v) was recorded within 48 h of incubation at 55°C and 50°C respectively and pH range of 6-7. About 78-86% hydrolysis of lactose in various brands of standardized milk was recorded at incubation temperature of 50°C. These results marked the applications of β-gal III in processing of milk/whey industry. Copyright © 2011 Elsevier B.V. All rights reserved.
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Escherichia coli type III secretion system 2: a new kind of T3SS?
Zhou, Mingxu; Guo, Zhiyan; Duan, Qiangde; Hardwidge, Philip R; Zhu, Guoqiang
2014-03-19
Type III secretion systems (T3SSs) are employed by Gram-negative bacteria to deliver effector proteins into the cytoplasm of infected host cells. Enteropathogenic Escherichia coli use a T3SS to deliver effector proteins that result in the creation of the attaching and effacing lesions. The genome sequence of the Escherichia coli pathotype O157:H7 revealed the existence of a gene cluster encoding components of a second type III secretion system, the E. coli type III secretion system 2 (ETT2). Researchers have revealed that, although ETT2 may not be a functional secretion system in most (or all) strains, it still plays an important role in bacterial virulence. This article summarizes current knowledge regarding the E. coli ETT2, including its genetic characteristics, prevalence, function, association with virulence, and prospects for future work.
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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...
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Benz-de Bretagne, Isabelle; Respaud, Renaud; Vourc'h, Patrick; Halimi, Jean-Michel; Caille, Agnès; Hulot, Jean-Sébastien; Andres, Christian R; Le Guellec, Chantal
2011-01-01
MRP2 encoded by ABCC2 gene is involved in the secretion of numerous drugs and endogenous substrates. Patients with Dubin-Johnson syndrome due to mutation in ABCC2 gene have elevated urinary coproporphyrin ratio (UCP I/(I + III)). Here we investigated whether this ratio could serve as a biomarker of MRP2 function. Phenotype-genotype relationships were studied in 74 healthy subjects by measuring individual UCP I/(I + III) ratio obtained on 24-hour urine and by analyzing five common SNPs in ABCC2 gene. The UCP I/(I + III) ratio varied from 14.7% to 46.0% in our population. Subjects with 3972TT genotype had a higher ratio (P = .04) than those carrying the C allele. This higher UCP I/(I + III) ratio was correlated with a higher level of isomer I excretion. This study provides a proof of concept that UCP I/(I + III) ratio can be used as a biomarker of MRP2 function in clinical studies as it provides quantitative information about the in vivo activity of MRP2 in a given patient.
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Benz-de Bretagne, Isabelle; Respaud, Renaud; Vourc'h, Patrick; Halimi, Jean-Michel; Caille, Agnès; Hulot, Jean-Sébastien; Andres, Christian R.; Le Guellec, Chantal
2011-01-01
MRP2 encoded by ABCC2 gene is involved in the secretion of numerous drugs and endogenous substrates. Patients with Dubin-Johnson syndrome due to mutation in ABCC2 gene have elevated urinary coproporphyrin ratio (UCP I/(I + III)). Here we investigated whether this ratio could serve as a biomarker of MRP2 function. Phenotype-genotype relationships were studied in 74 healthy subjects by measuring individual UCP I/(I + III) ratio obtained on 24-hour urine and by analyzing five common SNPs in ABCC2 gene. The UCP I/(I + III) ratio varied from 14.7% to 46.0% in our population. Subjects with 3972TT genotype had a higher ratio (P = .04) than those carrying the C allele. This higher UCP I/(I + III) ratio was correlated with a higher level of isomer I excretion. This study provides a proof of concept that UCP I/(I + III) ratio can be used as a biomarker of MRP2 function in clinical studies as it provides quantitative information about the in vivo activity of MRP2 in a given patient. PMID:21541183
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Kassegne, Kokouvi; Hu, Weilin; Ojcius, David M; Sun, Dexter; Ge, Yumei; Zhao, Jinfang; Yang, X Frank; Li, Lanjuan; Yan, Jie
2014-04-01
Leptospirosis is a global zoonotic disease. Transmission of Leptospira from animals to humans occurs through contact with water contaminated with leptospire-containing urine of infected animals. However, the molecular basis for the invasiveness of Leptospira and transmission of leptospirosis remains unknown. Activity of Leptospira interrogans strain Lai colA gene product (ColA) to hydrolyze different collagenic substrates was determined by spectrophotometry. Expression and secretion of ColA during infection were detected by reverse-transcription quantitative polymerase chain reaction and Western blot assay. The colA gene-deleted (ΔcolA) and colA gene-complemented (CΔcolA) mutants were generated to determine the roles of ColA in transcytosis in vitro and virulence in hamsters. Recombinant or native ColA hydrolyzed all the tested substrates in which type III collagen was the favorite substrate with 2.16 mg/mL Km and 35.6 h(-)(1) Kcat values. Coincubation of the spirochete with HUVEC or HEK293 cells directly caused the significant elevation of ColA expression and secretion. Compared with wild-type strain, ΔcolA mutant displayed much-attenuated transcytosis through HEK293 and HUVEC monolayers, and less leptospires in blood, lung, liver, kidney and urine and 25-fold-decreased 50% lethal dose and milder histopathological injury in hamsters. The product of colA gene is a collagenase as a crucial virulence factor in the invasiveness and transmission of L. interrogans.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Douillet, Christelle; Currier, Jenna; Saunders, Jesse
Epidemiologic evidence has linked chronic exposure to inorganic arsenic (iAs) with an increased prevalence of diabetes mellitus. Laboratory studies have identified several mechanisms by which iAs can impair glucose homeostasis. We have previously shown that micromolar concentrations of arsenite (iAs{sup III}) or its methylated trivalent metabolites, methylarsonite (MAs{sup III}) and dimethylarsinite (DMAs{sup III}), inhibit the insulin-activated signal transduction pathway, resulting in insulin resistance in adipocytes. Our present study examined effects of the trivalent arsenicals on insulin secretion by intact pancreatic islets isolated from C57BL/6 mice. We found that 48-hour exposures to low subtoxic concentrations of iAs{sup III}, MAs{sup III} ormore » DMAs{sup III} inhibited glucose-stimulated insulin secretion (GSIS), but not basal insulin secretion. MAs{sup III} and DMAs{sup III} were more potent than iAs{sup III} as GSIS inhibitors with estimated IC{sub 50} ≤ 0.1 μM. The exposures had little or no effects on insulin content of the islets or on insulin expression, suggesting that trivalent arsenicals interfere with mechanisms regulating packaging of the insulin transport vesicles or with translocation of these vesicles to the plasma membrane. Notably, the inhibition of GSIS by iAs{sup III}, MAs{sup III} or DMAs{sup III} could be reversed by a 24-hour incubation of the islets in arsenic-free medium. These results suggest that the insulin producing pancreatic β-cells are among the targets for iAs exposure and that the inhibition of GSIS by low concentrations of the methylated metabolites of iAs may be the key mechanism of iAs-induced diabetes. - Highlights: ► Trivalent arsenicals inhibit glucose stimulated insulin secretion by pancreatic islets. ► MAs{sup III} and DMAs{sup III} are more potent inhibitors than arsenite with IC{sub 50} ∼ 0.1 μM. ► The arsenicals have little or no effects on insulin expression in pancreatic islets. ► The inhibition of insulin secretion by arsenite, MAs{sup III} or DMAs{sup III} is reversible. ► Thus, pancreatic β-cells may be primary targets for chronic exposure to arsenic.« less
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Protein export through the bacterial flagellar type III export pathway.
Minamino, Tohru
2014-08-01
For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V. All rights reserved.
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Electricity from methane by reversing methanogenesis
McAnulty, Michael J.; G. Poosarla, Venkata; Kim, Kyoung-Yeol; Jasso-Chávez, Ricardo; Logan, Bruce E.; Wood, Thomas K.
2017-01-01
Given our vast methane reserves and the difficulty in transporting methane without substantial leaks, the conversion of methane directly into electricity would be beneficial. Microbial fuel cells harness electrical power from a wide variety of substrates through biological means; however, the greenhouse gas methane has not been used with much success previously as a substrate in microbial fuel cells to generate electrical current. Here we construct a synthetic consortium consisting of: (i) an engineered archaeal strain to produce methyl-coenzyme M reductase from unculturable anaerobic methanotrophs for capturing methane and secreting acetate; (ii) micro-organisms from methane-acclimated sludge (including Paracoccus denitrificans) to facilitate electron transfer by providing electron shuttles (confirmed by replacing the sludge with humic acids), and (iii) Geobacter sulfurreducens to produce electrons from acetate, to create a microbial fuel cell that converts methane directly into significant electrical current. Notably, this methane microbial fuel cell operates at high Coulombic efficiency. PMID:28513579
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Electricity from methane by reversing methanogenesis
NASA Astrophysics Data System (ADS)
McAnulty, Michael J.; G. Poosarla, Venkata; Kim, Kyoung-Yeol; Jasso-Chávez, Ricardo; Logan, Bruce E.; Wood, Thomas K.
2017-05-01
Given our vast methane reserves and the difficulty in transporting methane without substantial leaks, the conversion of methane directly into electricity would be beneficial. Microbial fuel cells harness electrical power from a wide variety of substrates through biological means; however, the greenhouse gas methane has not been used with much success previously as a substrate in microbial fuel cells to generate electrical current. Here we construct a synthetic consortium consisting of: (i) an engineered archaeal strain to produce methyl-coenzyme M reductase from unculturable anaerobic methanotrophs for capturing methane and secreting acetate; (ii) micro-organisms from methane-acclimated sludge (including Paracoccus denitrificans) to facilitate electron transfer by providing electron shuttles (confirmed by replacing the sludge with humic acids), and (iii) Geobacter sulfurreducens to produce electrons from acetate, to create a microbial fuel cell that converts methane directly into significant electrical current. Notably, this methane microbial fuel cell operates at high Coulombic efficiency.
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Lountos, George T; Austin, Brian P; Nallamsetty, Sreedevi; Waugh, David S
2009-01-01
Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a β-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU. PMID:19165725
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Methods for fabricating thin film III-V compound solar cell
Pan, Noren; Hillier, Glen; Vu, Duy Phach; Tatavarti, Rao; Youtsey, Christopher; McCallum, David; Martin, Genevieve
2011-08-09
The present invention utilizes epitaxial lift-off in which a sacrificial layer is included in the epitaxial growth between the substrate and a thin film III-V compound solar cell. To provide support for the thin film III-V compound solar cell in absence of the substrate, a backing layer is applied to a surface of the thin film III-V compound solar cell before it is separated from the substrate. To separate the thin film III-V compound solar cell from the substrate, the sacrificial layer is removed as part of the epitaxial lift-off. Once the substrate is separated from the thin film III-V compound solar cell, the substrate may then be reused in the formation of another thin film III-V compound solar cell.
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Evaluating models of cellulose degradation by Fibrobacter succinogenes S85
Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; ...
2015-12-02
Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins,more » and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.« less
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Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85
Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.
2015-01-01
Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases. PMID:26629814
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Lee, Justin H.; Park, Soojin; Mun, Kevin; Boo, Yong Chool; Kim, Deok-Ho
2016-01-01
Endothelial physiology is regulated not only by humoral factors but also by mechanical factors such as fluid shear stress and the underlying cellular matrix microenvironment. The purpose of the present study was to examine the effects of matrix topographical cues on the endothelial secretion of cytokines/chemokines in vitro. Human endothelial cells were cultured on nanopatterned polymeric substrates with different ratios of ridge to groove widths (1:1, 1:2, and 1:5) and with different stiffnesses (6.7 MPa and 2.5 GPa) in the presence and absence of 1.0 ng/mL TNF-α. The levels of cytokines/chemokines secreted into the conditioned media were analyzed with a multiplexed bead-based sandwich immunoassay. Of the nano-patterns tested, the 1:1 and 1:2 type-patterns were found to induce the greatest degree of endothelial cell elongation and directional alignment. The 1:2 type nanopatterns lowered the secretion of inflammatory cytokines such as IL-1β, IL-3 and MCP-1, compared to unpatterned substrates. Additionally, of the two polymers tested, it was found that the stiffer substrate resulted in significant decreases in the secretion of IL-3 and MCP-1. These results suggest that substrates with specific extracellular nanotopographical cues or stiffnesses may provide anti-atherogenic effects like those seen with laminar shear stresses by suppressing the endothelial secretion of cytokines and chemokines involved in vascular inflammation and remodeling. PMID:25658848
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Maeda, Yoshinori; Kudo, Saki; Tsushima, Ken; Sato, Eri; Kubota, Chisato; Kayamori, Aika; Bochimoto, Hiroki; Koga, Daisuke; Torii, Seiji; Gomi, Hiroshi; Watanabe, Tsuyoshi; Hosaka, Masahiro
2018-02-01
Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors. Copyright © 2018 Endocrine Society.
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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...
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Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.
Castiblanco, Luisa F; Triplett, Lindsay R; Sundin, George W
2018-01-01
Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora .
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Watson, Douglas S.; Feng, Xizhi; Askew, David S.; Jambunathan, Kalyani; Kodukula, Krishna; Galande, Amit K.
2011-01-01
Background The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician's toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers. Methodology and Principal Findings As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures. Conclusions This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis. PMID:21695046
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Sutherland, Molly C.; Nguyen, Thuy Linh; Tseng, Victor; Vogel, Joseph P.
2012-01-01
Legionella pneumophila is a Gram-negative bacterium that replicates within human alveolar macrophages by evasion of the host endocytic pathway through the formation of a replicative vacuole. Generation of this vacuole is dependent upon the secretion of over 275 effector proteins into the host cell via the Dot/Icm type IVB secretion system (T4SS). The type IV coupling protein (T4CP) subcomplex, consisting of DotL, DotM, DotN, IcmS and IcmW, was recently defined. DotL is proposed to be the T4CP of the L. pneumophila T4SS based on its homology to known T4CPs, which function as inner-membrane receptors for substrates. As a result, DotL is hypothesized to play an integral role(s) in the L. pneumophila T4SS for the engagement and translocation of substrates. To elucidate this role, a genetic approach was taken to screen for dotL mutants that were unable to survive inside host cells. One mutant, dotLY725Stop, did not interact with the type IV adaptor proteins IcmS/IcmW (IcmSW) leading to the identification of an IcmSW-binding domain on DotL. Interestingly, the dotLY725Stop mutant was competent for export of one class of secreted effectors, the IcmSW-independent substrates, but exhibited a specific defect in secretion of IcmSW-dependent substrates. This differential secretion illustrates that DotL requires a direct interaction with the type IV adaptor proteins for the secretion of a major class of substrates. Thus, by identifying a new target for IcmSW, we have discovered that the type IV adaptors perform an additional role in the export of substrates by the L. pneumophila Dot/Icm T4SS. PMID:23028312
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Vincent, Carr D; Vogel, Joseph P
2006-08-01
Many bacterial pathogens require a functional type IV secretion system (T4SS) for virulence. Legionella pneumophila, the causative agent of Legionnaires' disease, employs the Dot/Icm T4SS to inject a large number of protein substrates into its host, thereby altering phagosome trafficking. The L. pneumophila T4SS substrate SdeA has been shown to require the accessory factor IcmS for its export. IcmS, defined as a type IV adaptor, exists as a heterodimer with IcmW and this complex functions in a manner similar to a type III secretion chaperone. Here we report an interaction between IcmS and the previously identified virulence factor LvgA. Similar to the icmS mutant, the lvgA mutant appears to assemble a fully functional Dot/Icm complex. Both LvgA and IcmS are small, acidic proteins localized to the cytoplasm and are not exported by the Dot/Icm system, suggesting they form a novel type IV adaptor complex. Inactivation of lvgA causes a minimal defect in growth in the human monocytic cell line U937 and the environmental host Acanthamoeba castellanii. However, the lvgA mutant was severely attenuated for intracellular growth of L. pneumophila in mouse macrophages, suggesting LvgA may be a critical factor that confers host specificity.
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Computational prediction of type III and IV secreted effectors in Gram-negative bacteria
DOE Office of Scientific and Technical Information (OSTI.GOV)
McDermott, Jason E.; Corrigan, Abigail L.; Peterson, Elena S.
2011-01-01
In this review, we provide an overview of the methods employed by four recent papers that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. The results of the studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVEserver (http://www.biopilot.org). Finally, we assess the accuracy of the three type III effector prediction methods onmore » a small set of proteins not known prior to the development of these tools that we have recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif, and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.« less
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Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon
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
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Structure of Salmonella FlhE, conserved member of a flagellar Type III secretion operon
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, Jaemin; Monzingo, Arthur F.; Keatinge-Clay, Adrian T.
In this paper, the bacterial flagellum is assembled by a multicomponent transport apparatus categorized as a type III secretion system. The secretion of proteins that assemble into the flagellum is driven by the proton motive force. The periplasmic protein FlhE is a member of the flhBAE operon in the majority of bacteria where FlhE is found. FlhA and FlhB are established components of the flagellar type III secretion system. The absence of FlhE results in a proton leak through the flagellar system, inappropriate secretion patterns, and cell death, indicating that FlhE regulates an important aspect of proper flagellar biosynthesis. Wemore » isolated FlhE from the periplasm of Salmonella and solved its structure to 1.5 Å resolution. The structure reveals a β-sandwich fold, with no close structural homologs. Finally, possible roles of FlhE, including that of a chaperone, are discussed.« less
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Investment in secreted enzymes during nutrient-limited growth is utility dependent.
Cezairliyan, Brent; Ausubel, Frederick M
2017-09-12
Pathogenic bacteria secrete toxins and degradative enzymes that facilitate their growth by liberating nutrients from the environment. To understand bacterial growth under nutrient-limited conditions, we studied resource allocation between cellular and secreted components by the pathogenic bacterium Pseudomonas aeruginosa during growth on a protein substrate that requires extracellular digestion by secreted proteases. We identified a quantitative relationship between the rate of increase of cellular biomass under nutrient-limiting growth conditions and the rate of increase in investment in secreted proteases. Production of secreted proteases is stimulated by secreted signals that convey information about the utility of secreted proteins during nutrient-limited growth. Growth modeling using this relationship recapitulated the observed kinetics of bacterial growth on a protein substrate. The proposed regulatory strategy suggests a rationale for quorum-sensing-dependent stimulation of the production of secreted enzymes whereby investment in secreted enzymes occurs in proportion to the utility they confer. Our model provides a framework that can be applied toward understanding bacterial growth in many environments where growth rate is limited by the availability of nutrients.
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Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis.
Ates, Louis S; Dippenaar, Anzaan; Ummels, Roy; Piersma, Sander R; van der Woude, Aniek D; van der Kuij, Kim; Le Chevalier, Fabien; Mata-Espinosa, Dulce; Barrios-Payán, Jorge; Marquina-Castillo, Brenda; Guapillo, Carolina; Jiménez, Connie R; Pain, Arnab; Houben, Edith N G; Warren, Robin M; Brosch, Roland; Hernández-Pando, Rogelio; Bitter, Wilbert
2018-02-01
Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems 1,2 . The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus 3,4 . However, the role of these proteins remains largely elusive 1 . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion 5 . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the 'modern' Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution 6,7 .
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Substrate adaptation of Trichophyton rubrum secreted endoproteases.
Chen, Jian; Yi, Jinling; Liu, Li; Yin, Songchao; Chen, Rongzhang; Li, Meirong; Ye, Congxiu; Zhang, Yu-qing; Lai, Wei
2010-02-01
Trichophyton rubrum is the most common pathogen caused the dermatophytosis of nail and skin in human. The secreted proteases were considered to be the most important virulence factors. However, the substrates adaptation of T. rubrum secreted proteases is largely unknown. For the first time, we use the keratins from human nail and skin stratum corneum as the growth medium to investigate the different expression patterns of T. rubrum secreted endoproteases genes. During grow in both keratin-containing media SUB7 and MEP2 were the highest expressed gene in each family. These results indicated that SUB7 and MEP2 may be the dominant endoproteases secreted by T. rubrum during host infection and the other proteases may play a supplementary role. The direct comparison of T. rubrum grown on skin and nail medium showed different substrate favorite of secreted endoproteases. The genes MEP2, SUB5, SUB2 and SUB3 were more active during growth in skin medium, possibly these proteases have a higher affinity for skin original keratins. While the structures of SUB1, SUB4, and MEP4 maybe more suitable for the degradation of nail original keratins. This work presents useful molecular details for further understanding the pathogenesis of secreted proteases and the wide adaptation of T. rubrum.
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The YopJ superfamily of type III efforts in plant-associated bacteria
USDA-ARS?s Scientific Manuscript database
Bacterial pathogens employ the type III secretion system to secrete and translocate effector proteins into their hosts. The primary function of these effector proteins is believed to be the suppression of host defense responses or innate immunity. However, some effector proteins may be recognized by...
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Type 1 Does The Two-Step: Type 1 Secretion Substrates With A Functional Periplasmic Intermediate.
Smith, Timothy J; Sondermann, Holger; O'Toole, George A
2018-06-04
Bacteria have evolved several secretion strategies for polling and responding to environmental flux and insult. Of these, the type 1 secretion system (T1SS) is known to secrete an array of biologically diverse proteins - from small < 10 kDa bacteriocins to gigantic adhesins with a mass over 1 MDa. For the last several decades T1SS have been characterized as a one-step translocation strategy whereby the secreted substrate is transported directly into the extracellular environment from the cytoplasm with no periplasmic intermediate. Recent phylogenetic, biochemical, and genetic evidence point to a distinct sub-group of T1SS machinery linked with a bacterial transglutaminase-like cysteine proteinase (BTLCP), which uses a two-step secretion mechanism. BTLCP-linked T1SS transport a class of repeats-in-toxin (RTX) adhesins that are critical for biofilm formation. The prototype of this RTX adhesin group, LapA of Pseudomonas fluorescens Pf0-1, uses a novel N-terminal retention module to anchor the adhesin at the cell surface as a secretion intermediate threaded through the outer membrane-localized, TolC-like protein LapE. This secretion intermediate is post-translationally cleaved by the BTLCP family LapG protein to release LapA from its cognate T1SS pore. Thus, secretion of LapA and related RTX adhesins into the extracellular environment appears to be a T1SS-mediated, two-step process that involves a periplasmic intermediate. In this review, we contrast the T1SS machinery and substrates of the BLTCP-linked two-step secretion process with those of the classical one-step T1SS to better understand the newly recognized and expanded role of this secretion machinery. Copyright © 2018 American Society for Microbiology.
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The ubiquitin ligase Cbl-b limits Pseudomonas aeruginosa exotoxin T–mediated virulence
Balachandran, Priya; Dragone, Leonard; Garrity-Ryan, Lynne; Lemus, Armando; Weiss, Arthur; Engel, Joanne
2007-01-01
Pseudomonas aeruginosa, an important cause of opportunistic infections in humans, delivers bacterial cytotoxins by type III secretion directly into the host cell cytoplasm, resulting in disruption of host cell signaling and host innate immunity. However, little is known about the fate of the toxins themselves following injection into the host cytosol. Here, we show by both in vitro and in vivo studies that the host ubiquitin ligase Cbl-b interacts with the type III–secreted effector exotoxin T (ExoT) and plays a key role in vivo in limiting bacterial dissemination mediated by ExoT. We demonstrate that, following polyubiquitination, ExoT undergoes regulated proteasomal degradation in the host cell cytosol. ExoT interacts with the E3 ubiquitin ligase Cbl-b and Crk, the substrate for the ExoT ADP ribosyltransferase (ADPRT) domain. The efficiency of degradation is dependent upon the activity of the ADPRT domain. In mouse models of acute pneumonia and systemic infection, Cbl-b is specifically required to limit the dissemination of ExoT-producing bacteria whereas c-Cbl plays no detectable role. To the best of our knowledge, this represents the first identification of a mammalian gene product that is specifically required for in vivo resistance to disease mediated by a type III–secreted effector. PMID:17235393
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Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana
2016-01-01
Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms. © 2015 CSIC New Phytologist © 2015 New Phytologist Trust.
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Sangar, Vineet; Funk, Cory C; Kusebauch, Ulrike; Campbell, David S; Moritz, Robert L; Price, Nathan D
2014-10-01
Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Pinaud, Laurie; Ferrari, Mariana L.; Friedman, Robin; Jehmlich, Nico; von Bergen, Martin; Phalipon, Armelle; Sansonetti, Philippe J.
2017-01-01
Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed. PMID:29073283
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Analysis of the ZAR1 immune complex reveals determinants for immunity and molecular interactions
USDA-ARS?s Scientific Manuscript database
Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary function of most T3SEs is to supp...
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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 ...
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Diepold, Andreas; Kudryashev, Mikhail; Delalez, Nicolas J; Berry, Richard M; Armitage, Judith P
2015-01-01
Many gram-negative pathogens employ a type III secretion injectisome to translocate effector proteins into eukaryotic host cells. While the structure of the distal "needle complex" is well documented, the composition and role of the functionally important cytosolic complex remain less well understood. Using functional fluorescent fusions, we found that the C-ring, an essential and conserved cytosolic component of the system, is composed of ~22 copies of SctQ (YscQ in Yersinia enterocolitica), which require the presence of YscQC, the product of an internal translation initiation site in yscQ, for their cooperative assembly. Photoactivated localization microscopy (PALM) reveals that in vivo, YscQ is present in both a free-moving cytosolic and a stable injectisome-bound state. Notably, fluorescence recovery after photobleaching (FRAP) shows that YscQ exchanges between the injectisome and the cytosol, with a t½ of 68 ± 8 seconds when injectisomes are secreting. In contrast, the secretin SctC (YscC) and the major export apparatus component SctV (YscV) display minimal exchange. Under non-secreting conditions, the exchange rate of YscQ is reduced to t½ = 134 ± 16 seconds, revealing a correlation between C-ring exchange and injectisome activity, which indicates a possible role for C-ring stability in regulation of type III secretion. The stabilization of the C-ring depends on the presence of the functional ATPase SctN (YscN). These data provide new insights into the formation and composition of the injectisome and present a novel aspect of type III secretion, the exchange of C-ring subunits, which is regulated with respect to secretion.
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Symbiotic implications of type III protein secretion machinery in Rhizobium.
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.
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NASA Technical Reports Server (NTRS)
Ganong, William F.
1987-01-01
The present goal is to determine by the production of discrete lesions the parts of the hypothalamus and brainstem that are involved in serotonin-mediated increases in renin secretion. A variety of stimuli which act in different ways to increase renin stimuli were developed and standardized. The experiments with p-chloroamphetamine (PCA) demonstrated that there is a serotonergic pathway which projects from the dorsal raphe nuclei to the paraventricular nuclei and the vetromedial nuclei of the hypothalamus; that projection from paraventricular nuclei to the brainstem and spinal cord may be oxytocinergic; and that the pathway from the spinal cord to the renin secreting cells is sympathetic. The demonstration that paraventicular lesions lower circulating renin substrate is important because it raises the possibility that substrate secretion is under neural control, either via the pituitary or by direct neural pathways. The discovery that lesions of the ventromedial nuclei appear to abolish the increase in renin secretion produced by many different stimuli without affecting the concentration of renin substrate in the plasma makes the position of the hypothalamus in the regulation of fluid and electrolyte balance more prominent than previously suspected.
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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...
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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...
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Singh, G; Wu, B; Baek, M S; Camargo, A; Nguyen, A; Slusher, N A; Srinivasan, R; Wiener-Kronish, J P; Lynch, S V
2010-10-01
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 post-translational control, specifically governing type III cytotoxin secretion, exists in this species. Copyright 2010 Elsevier Ltd. All rights reserved.
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Kamke, Janine; Soni, Priya; Li, Yang; Ganesh, Siva; Kelly, William J; Leahy, Sinead C; Shi, Weibing; Froula, Jeff; Rubin, Edward M; Attwood, Graeme T
2017-08-08
Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH 4 /kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. This is the first report of bacterial type III secretion system genes being associated with high methane emissions in ruminants, and identifies these secretions systems as potential new targets for methane mitigation research. The effects of S. dextrinosolvens on the growth of rumen methanogens in co-cultures indicate that bacteria-methanogen interactions are important modulators of methane production in ruminant animals.
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Production and Characterization of Trametes versicolor Mutants Unable To Bleach Hardwood Kraft Pulp
Addleman, K.; Dumonceaux, T.; Paice, M. G.; Bourbonnais, R.; Archibald, F. S.
1995-01-01
Protoplasts of the monokaryotic strain 52J of Trametes versicolor were treated with UV light and screened for the inability to produce a colored precipitate on guaiacol-containing agar plates. Mutants unable to oxidize guaiacol had absent or very low secretion of laccase and manganese peroxidase (MnP) proteins. All isolates unable to secrete MnP were also unable to bleach or delignify kraft pulp. One mutant strain, M49, which grew normally but did not oxidize guaiacol, was tested further with a number of other substrates whose degradation has been associated with delignification by white rot fungi. Compared with the parent, 52J, mutant M49, secreting no MnP and low laccase, could not brighten or delignify kraft pulp, produced less ethylene from 2-keto methiolbutyric acid, released much less (sup14)CO(inf2) from [(sup14)C]DHP (a synthetic lignin-like polymerizate), and produced much less methanol from pulp. This mutant also displayed decreased abilities to oxidize the dyes poly B-411, poly R-478, and phenol red compared with the wild-type strain and was also unable to decolorize kraft bleachery effluent or mineralize its organochlorine. Addition of purified MnP in conjunction with H(inf2)O(inf2), MnSO(inf4), and an Mn(III) chelator to M49 cultures partially restored methanol production, pulp delignification, and biobleaching in some cases. PMID:16535150
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Prediction of type III secretion signals in genomes of gram-negative bacteria.
Löwer, Martin; Schneider, Gisbert
2009-06-15
Pathogenic bacteria infecting both animals as well as plants use various mechanisms to transport virulence factors across their cell membranes and channel these proteins into the infected host cell. The type III secretion system represents such a mechanism. Proteins transported via this pathway ("effector proteins") have to be distinguished from all other proteins that are not exported from the bacterial cell. Although a special targeting signal at the N-terminal end of effector proteins has been proposed in literature its exact characteristics remain unknown. In this study, we demonstrate that the signals encoded in the sequences of type III secretion system effectors can be consistently recognized and predicted by machine learning techniques. Known protein effectors were compiled from the literature and sequence databases, and served as training data for artificial neural networks and support vector machine classifiers. Common sequence features were most pronounced in the first 30 amino acids of the effector sequences. Classification accuracy yielded a cross-validated Matthews correlation of 0.63 and allowed for genome-wide prediction of potential type III secretion system effectors in 705 proteobacterial genomes (12% predicted candidates protein), their chromosomes (11%) and plasmids (13%), as well as 213 Firmicute genomes (7%). We present a signal prediction method together with comprehensive survey of potential type III secretion system effectors extracted from 918 published bacterial genomes. Our study demonstrates that the analyzed signal features are common across a wide range of species, and provides a substantial basis for the identification of exported pathogenic proteins as targets for future therapeutic intervention. The prediction software is publicly accessible from our web server (www.modlab.org).
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Berube, Bryan J.; Murphy, Katherine R.; Torhan, Matthew C.; Bowlin, Nicholas O.; Williams, John D.; Bowlin, Terry L.; Moir, Donald T.
2017-01-01
ABSTRACT Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or microabscess formation. The rising incidence of multidrug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro. In this study, we used a mouse model of P. aeruginosa abscess formation to test the in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of the abscesses, as neutrophil depletion resulted in an increased abscess size, the formation of dermonecrotic lesions on the skin, and the dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS-neutrophil interaction, P. aeruginosa bacteria lacking a functional T3SS were fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in the immune clearance of the bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together, our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection. PMID:28807906
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Qin, Wen; Sundaram, Meenakshi; Wang, Yuwei; Zhou, Hu; Zhong, Shumei; Chang, Chia-Ching; Manhas, Sanjay; Yao, Erik F; Parks, Robin J; McFie, Pamela J; Stone, Scot J; Jiang, Zhenghui G; Wang, Congrong; Figeys, Daniel; Jia, Weiping; Yao, Zemin
2011-08-05
Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.
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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
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lountos, George T.; Tropea, Joseph E.; Waugh, David S.
2012-09-17
The Yersinia pestis YscD protein is an essential component of the type III secretion system. YscD consists of an N-terminal cytoplasmic domain (residues 1-121), a transmembrane linker (122-142) and a large periplasmic domain (143-419). Both the cytoplasmic and the periplasmic domains are required for the assembly of the type III secretion system. Here, the structure of the YscD cytoplasmic domain solved by SAD phasing is presented. Although the three-dimensional structure is similar to those of forkhead-associated (FHA) domains, comparison with the structures of canonical FHA domains revealed that the cytoplasmic domain of YscD lacks the conserved residues that are requiredmore » for binding phosphothreonine and is therefore unlikely to function as a true FHA domain.« less
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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.
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Group I-III-VI.sub.2 semiconductor films for solar cell application
Basol, Bulent M.; Kapur, Vijay K.
1991-01-01
This invention relates to an improved thin film solar cell with excellent electrical and mechanical integrity. The device comprises a substrate, a Group I-III-VI.sub.2 semiconductor absorber layer and a transparent window layer. The mechanical bond between the substrate and the Group I-III-VI.sub.2 semiconductor layer is enhanced by an intermediate layer between the substrate and the Group I-III-VI.sub.2 semiconductor film being grown. The intermediate layer contains tellurium or substitutes therefor, such as Se, Sn, or Pb. The intermediate layer improves the morphology and electrical characteristics of the Group I-III-VI.sub.2 semiconductor layer.
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Yarchoan, R; Redfield, R R; Broder, S
1986-01-01
Patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) have hyperimmunoglobulinemia and increased numbers of circulating immunoglobulin-secreting cells. In this paper, we studied the basis for this B cell hyperactivity. Limiting dilution studies of B cells from seven patients with ARC and four with AIDS revealed that some B cells spontaneously produced antibodies to human T cell lymphotropic virus, type III/lymphadenopathy-associated virus (HTLV-III/LAV) (39:10(6) and 7:10(6) B cells, respectively), suggesting that chronic antigenic stimulation by HTLV-III/LAV was one contributing factor. The patients also had an increased number of spontaneously outgrowing B cells than did normals (6:10(6) vs. less than 2:10(6) B cells), suggesting that they had an increased number of Epstein-Barr virus (EBV)-infected B cells. However, fewer B cells from patients were immortalized by exogenously added EBV than were B cells from normals. In additional studies, HTLV-III/LAV induced immunoglobulin secretion (mean 2,860 ng/ml) by peripheral blood mononuclear cells from normals; this HTLV-III/LAV-induced immunoglobulin secretion required the presence of both B and T cells. Thus, antigenic stimulation by HTLV-III/LAV, increased numbers of EBV-infected B cells, and HTLV-III/LAV-induced T cell-dependent B cell activation all contribute to the B cell hyperactivity in patients with HTLV-III/LAV disease. PMID:3016028
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Phan, Jason; Tropea, Joseph E.; Waugh, David S.
2010-11-16
Pathogenic Yersinia species use a type III secretion system to inject cytotoxic effector proteins directly into the cytosol of mammalian cells, where they neutralize the innate immune response by interfering with the signal-transduction pathways that control phagocytosis and inflammation. To be exported efficiently, some effectors must transiently associate with cognate cytoplasmic secretion chaperones. SycH is the chaperone for YopH, a potent eukaryotic-like protein tyrosine phosphatase that is essential for virulence. SycH also binds two negative regulators of type III secretion, YscM1 and YscM2, both of which share significant sequence homology with the chaperone-binding domain of YopH. Here, the structure ofmore » a complex between SycH and a stable fragment of YscM2 that was designed on the basis of limited proteolysis experiments is presented. The overall fold of SycH is very similar to the structures of other homodimeric secretion chaperones that have been determined to date. YscM2 wraps around SycH in an extended fashion, with some secondary but no tertiary structure, assuming a conformation distinct from the globular fold that it is predicted to adopt in the absence of SycH.« less
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What's the point of the type III secretion system needle?
Blocker, Ariel J.; Deane, Janet E.; Veenendaal, Andreas K. J.; Roversi, Pietro; Hodgkinson, Julie L.; Johnson, Steven; Lea, Susan M.
2008-01-01
Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion system (T3SS) of Gram-negative bacteria. This perspective presents a structure-informed analysis of functional data and discusses three nonmutually exclusive models of how a key aspect of T3SS biology, the sensing of host cells, may be performed. PMID:18458349
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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.
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Nanoparticle-Seeding Approach to Buried (Semi) Metal Film Growth
2014-05-20
semimetals that can be grown epitaxially on zinc-blende III-V substrates, with thermodynamically stable interfaces. However, the rotational symmetry...epitaxially on zinc-blende III-V substrates, with thermodynamically stable interfaces. However, the rotational symmetry mismatch between the III-V and ErAs
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Dahan, Arik; Amidon, Gordon L
2009-01-01
The purpose of this study was to investigate the role of P-gp efflux in the in vivo intestinal absorption process of BCS class III P-gp substrates, i.e. high-solubility low-permeability drugs. The in vivo permeability of two H (2)-antagonists, cimetidine and famotidine, was determined by the single-pass intestinal perfusion model in different regions of the rat small intestine, in the presence or absence of the P-gp inhibitor verapamil. The apical to basolateral (AP-BL) and the BL-AP transport of the compounds in the presence or absence of various efflux transporters inhibitors (verapamil, erythromycin, quinidine, MK-571 and fumitremorgin C) was investigated across Caco-2 cell monolayers. P-gp expression levels in the different intestinal segments were confirmed by immunoblotting. Cimetidine and famotidine exhibited segmental dependent permeability through the gut wall, with decreased P(eff) in the distal ileum in comparison to the proximal regions of the intestine. Coperfusion of verapamil with the drugs significantly increased the permeability in the ileum, while no significant change in the jejunal permeability was observed. Both drugs exhibited significantly greater BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. Concentration dependent decrease of this secretion was obtained by the P-gp inhibitors verapamil, erythromycin and quinidine, while no effect was evident by the MRP2 inhibitor MK-571 and the BCRP inhibitor FTC, indicating that P-gp is the transporter mediates the intestinal efflux of cimetidine and famotidine. P-gp levels throughout the intestine were inversely related to the in vivo permeability of the drugs from the different segments. The data demonstrate that for these high-solubility low-permeability P-gp substrates, P-gp limits in vivo intestinal absorption in the distal segments of the small intestine; however P-gp plays a minimal role in the proximal intestinal segments due to significant lower P-gp expression levels in this region.
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Vertical group III-V nanowires on si, heterostructures, flexible arrays and fabrication
Wang, Deli; Soci, Cesare; Bao, Xinyu; Wei, Wei; Jing, Yi; Sun, Ke
2015-01-13
Embodiments of the invention provide a method for direct heteroepitaxial growth of vertical III-V semiconductor nanowires on a silicon substrate. The silicon substrate is etched to substantially completely remove native oxide. It is promptly placed in a reaction chamber. The substrate is heated and maintained at a growth temperature. Group III-V precursors are flowed for a growth time. Preferred embodiment vertical Group III-V nanowires on silicon have a core-shell structure, which provides a radial homojunction or heterojunction. A doped nanowire core is surrounded by a shell with complementary doping. Such can provide high optical absorption due to the long optical path in the axial direction of the vertical nanowires, while reducing considerably the distance over which carriers must diffuse before being collected in the radial direction. Alloy composition can also be varied. Radial and axial homojunctions and heterojunctions can be realized. Embodiments provide for flexible Group III-V nanowire structures. An array of Group III-V nanowire structures is embedded in polymer. A fabrication method forms the vertical nanowires on a substrate, e.g., a silicon substrate. Preferably, the nanowires are formed by the preferred methods for fabrication of Group III-V nanowires on silicon. Devices can be formed with core/shell and core/multi-shell nanowires and the devices are released from the substrate upon which the nanowires were formed to create a flexible structure that includes an array of vertical nanowires embedded in polymer.
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Composition and substrate-dependent strength of the silken attachment discs in spiders
Grawe, Ingo; Wolff, Jonas O.; Gorb, Stanislav N.
2014-01-01
Araneomorph spiders have evolved different silks with dissimilar material properties, serving different purposes. The two-compound pyriform secretion is used to glue silk threads to substrates or to other threads. It is applied in distinct patterns, called attachment discs. Although ubiquitously found in spider silk applications and hypothesized to be strong and versatile at low material consumption, the performance of attachment discs on different substrates remains unknown. Here, we analyse the detachment forces and fracture mechanics of the attachment discs spun by five different species on three different substrates, by pulling on the upstream part of the attached thread. Results show that although the adhesion of the pyriform glue is heavily affected by the substrate, even on Teflon it is frequently strong enough to hold the spider's weight. As plant surfaces are often difficult to wet, they are hypothesized to be the major driving force for evolution of the pyriform secretion. PMID:25030386
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Nasser, William; Reverchon, Sylvie; Vedel, Regine; Boccara, Martine
2005-11-01
Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.
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Santos, Anderson F.; Valle, Roberta S.; Pacheco, Clarissa A.; Alvarez, Vanessa M.; Seldin, Lucy; Santos, André L.S.
2013-01-01
Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties. PMID:24688526
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Santos, Anderson F; Valle, Roberta S; Pacheco, Clarissa A; Alvarez, Vanessa M; Seldin, Lucy; Santos, André L S
2013-12-01
Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.
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Lattice matched crystalline substrates for cubic nitride semiconductor growth
Norman, Andrew G; Ptak, Aaron J; McMahon, William E
2015-02-24
Disclosed embodiments include methods of fabricating a semiconductor layer or device and devices fabricated thereby. The methods include, but are not limited to, providing a substrate having a cubic crystalline surface with a known lattice parameter and growing a cubic crystalline group III-nitride alloy layer on the cubic crystalline substrate by coincident site lattice matched epitaxy. The cubic crystalline group III-nitride alloy may be prepared to have a lattice parameter (a') that is related to the lattice parameter of the substrate (a). The group III-nitride alloy may be a cubic crystalline In.sub.xGa.sub.yAl.sub.1-x-yN alloy. The lattice parameter of the In.sub.xGa.sub.yAl.sub.1-x-yN or other group III-nitride alloy may be related to the substrate lattice parameter by (a')= 2(a) or (a')=(a)/ 2. The semiconductor alloy may be prepared to have a selected band gap.
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NASA Astrophysics Data System (ADS)
Gryzia, Aaron; Predatsch, Hans; Brechling, Armin; Hoeke, Veronika; Krickemeyer, Erich; Derks, Christine; Neumann, Manfred; Glaser, Thorsten; Heinzmann, Ulrich
2011-08-01
We report on the characterization of various salts of [ Mn III 6 Cr III ] 3+ complexes prepared on substrates such as highly oriented pyrolytic graphite (HOPG), mica, SiO2, and Si3N4. [ Mn III 6 Cr III ] 3+ is a single-molecule magnet, i.e., a superparamagnetic molecule, with a blocking temperature around 2 K. The three positive charges of [ Mn III 6 Cr III ] 3+ were electrically neutralized by use of various anions such as tetraphenylborate (BPh4 -), lactate (C3H5O3 -), or perchlorate (ClO4 -). The molecule was prepared on the substrates out of solution using the droplet technique. The main subject of investigation was how the anions and substrates influence the emerging surface topology during and after the preparation. Regarding HOPG and SiO2, flat island-like and hemispheric-shaped structures were created. We observed a strong correlation between the electronic properties of the substrate and the analyzed structures, especially in the case of mica where we observed a gradient in the analyzed structures across the surface.
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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
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Notch, Emily G; Goodale, Britton C; Barnaby, Roxanna; Coutermarsh, Bonita; Berwin, Brent; Taylor, Vivien F; Jackson, Brian P; Stanton, Bruce A
2015-01-01
Arsenic is the number one contaminant of concern with regard to human health according to the World Health Organization. Epidemiological studies on Asian and South American populations have linked arsenic exposure with an increased incidence of lung disease, including pneumonia, and chronic obstructive pulmonary disease, both of which are associated with bacterial infection. However, little is known about the effects of low dose arsenic exposure, or the contributions of organic arsenic to the innate immune response to bacterial infection. This study examined the effects on Pseudomonas aeruginosa (P. aeruginosa) induced cytokine secretion by human bronchial epithelial cells (HBEC) by inorganic sodium arsenite (iAsIII) and two major metabolites, monomethylarsonous acid (MMAIII) and dimethylarsenic acid (DMAV), at concentrations relevant to the U.S. Neither iAsIII nor DMAV altered P. aeruginosa induced cytokine secretion. By contrast, MMAIII increased P. aeruginosa induced secretion of IL-8, IL-6 and CXCL2. A combination of iAsIII, MMAIII and DMAV (10 pbb total) reduced IL-8 and CXCL1 secretion. These data demonstrate for the first time that exposure to MMAIII alone, and a combination of iAsIII, MMAIII and DMAV at levels relevant to the U.S. may have negative effects on the innate immune response of human bronchial epithelial cells to P. aeruginosa.
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Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells
Cavet, M E; West, M; Simmons, N L
1997-01-01
Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 μM) or bromosulphophthalein (100 μM). Similarly tetraethylammonium and N-′methylnicotinamide (10 mM) were without effect. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS, 400 μM). Net secretion of ciprofloxacin was partially inhibited by 100 μM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretion in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl− or K+ in the bathing media. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia. PMID:9283689
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Sullivan, Eva; Bensman, Joyce; Lou, Mimi; Agnello, Melissa; Shriner, Kimberly; Wong-Beringer, Annie
2014-01-01
To determine the differential association of host characteristics, antimicrobial resistance, and type III secretion system virulence of Pseudomonas aeruginosa isolates with respiratory syndromes in hospitalized adult patients. Retrospective, cohort study. Community teaching hospital. Two hundred eighteen consecutive adult patients with respiratory culture positive for P. aeruginosa between January 2005 to January 2010. Medical charts were reviewed to obtain demographic, laboratory, radiographic, and clinical information. Isolates were assayed by polymerase chain reaction for genes encoding the type III secretion system effectors (ExoU, ExoS, and PcrV) and for strain relatedness using randomly amplified polymorphic DNA analysis. Levofloxacin susceptibility was determined by broth microdilution. Patients were grouped by colonization, bronchitis, or pneumonia and were compared for differential risk of developing the clinical syndrome with respect to host and microbial characteristics. Half of the study cohort (54%, 117 of 218) had pneumonia, 32% (70 of 218) had bronchitis, and 14% (31 of 218) had colonization; in-hospital mortality was 35%, 11%, and 0%, respectively. Host factors strongly associated with pneumonia development were residence in long-term care facility, healthcare-associated acquisition of P. aeruginosa, higher Acute Physiology and Chronic Health Evaluation II score, presence of enteral feeding tube, mechanical ventilation, and recent history of pneumonia. Fluoroquinolone-resistant (57% vs 34%, 16%; p < 0.0001) and multidrug-resistant (36% vs 26%, 7%; p = 0.0045) strains were more likely to cause pneumonia than bronchitis or colonization, respectively. Analysis of host and microbial factors in a multivariate regression model yielded the combined traits of fluoroquinolone resistance and gene encoding the type III secretion system ExoU effector in P. aeruginosa as the single most significant predictor of pneumonia development. These results suggest that fluoroquinolone-resistant phenotype in a type III secretion system exoU strain background contributes toward the pathogenesis of P. aeruginosa in pneumonia.
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Ramsubramaniam, Nikhil; Harris, Steven D; Marten, Mark R
2014-11-01
We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra-long reverse phase gradient, and identified using a "high-high" strategy (high mass accuracy on the parent and fragment ions) with higher-energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: "sites of polar growth," "vesicle mediated transport," and "cytoskeleton organization." In these same GO categories, kinase-substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase-substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An-Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 (http://proteomecentral.proteomexchange.org/dataset/PXD000715). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Meshcheryakov, Vladimir A.; Kitao, Akio; Core Research for Evolutionary Science and Technology, Tokyo 113-0032
2013-05-01
Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, themore » crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.« less
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Warfare between Host Immunity and Bacterial Weapons.
Yu, Manda; Lai, Erh-Min
2017-01-11
Bacterial pathogens deploy protein secretion systems to facilitate infection and colonization of their hosts. In this issue of Cell Host & Microbe, Chen et al. (2017) report a new role for a type VI secretion effector in promoting bacterial colonization by preventing inflammasome activation induced by a type III secretion system. Copyright © 2017 Elsevier Inc. All rights reserved.
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Bardill, J Patrick; Miller, Jennifer L; Vogel, Joseph P
2005-04-01
Legionella pneumophila replicates inside alveolar macrophages and causes an acute, potentially fatal pneumonia called Legionnaires' disease. The ability of this bacterium to grow inside of macrophages is dependent on the presence of a functional dot/icm type IV secretion system (T4SS). Proteins secreted by the Dot/Icm T4SS are presumed to alter the host endocytic pathway, allowing L. pneumophila to establish a replicative niche within the host cell. Here we show that a member of the SidE family of proteins interacts with IcmS and is required for full virulence in the protozoan host Acanthamoeba castellanii. Using immunofluorescence microscopy and adenylate cyclase fusions, we show that SdeA is secreted into host cells by L. pneumophila in an IcmS-dependent manner. The SidE-like proteins are secreted very early during macrophage infection, suggesting that they are important in the initial formation of the replicative phagosome. Secreted SidE family members show a similar localization to other Dot/Icm substrates, specifically, to the poles of the replicative phagosome. This common localization of secreted substrates of the Dot/Icm system may indicate the formation of a multiprotein complex on the cytoplasmic face of the replicative phagosome.
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Process for preparing group Ib-IIIa-VIa semiconducting films
Birkmire, Robert W.; Schultz, Jerold M.; Marudachalam, Matheswaran; Hichri, Habib
1997-01-01
Methods are provided for the production of supported monophasic group I-III-VI semiconductor films. In the subject methods, a substrate is coated with group I and III elements and then contacted with a reactive group VI element containing atmosphere under conditions sufficient to produce a substrate coated with a composite of at least two different group I-III-IV alloys. The resultant composite coated substrate is then annealed in an inert atmosphere under conditions sufficient to convert the composite coating to a monophasic group I-III-VI semiconductor film. The resultant supported semiconductor films find use in photovoltaic applications, particularly as absorber layers in solar cells.
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Process for preparing group Ib-IIIa-VIa semiconducting films
Birkmire, R.W.; Schultz, J.M.; Marudachalam, M.; Hichri, H.
1997-10-07
Methods are provided for the production of supported monophasic group I-III-VI semiconductor films. In the subject methods, a substrate is coated with group I and III elements and then contacted with a reactive group VI element containing atmosphere under conditions sufficient to produce a substrate coated with a composite of at least two different group I-III-IV alloys. The resultant composite coated substrate is then annealed in an inert atmosphere under conditions sufficient to convert the composite coating to a monophasic group I-III-VI semiconductor film. The resultant supported semiconductor films find use in photovoltaic applications, particularly as absorber layers in solar cells. 4 figs.
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Holtappels, Michelle; Noben, Jean-Paul; Valcke, Roland
2016-09-01
Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram-negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D-DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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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
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Microscale optical cryptography using a subdiffraction-limit optical key
NASA Astrophysics Data System (ADS)
Ogura, Yusuke; Aino, Masahiko; Tanida, Jun
2018-04-01
We present microscale optical cryptography using a subdiffraction-limit optical pattern, which is finer than the diffraction-limit size of the decrypting optical system, as a key and a substrate with a reflectance distribution as an encrypted image. Because of the subdiffraction-limit spatial coding, this method enables us to construct a secret image with the diffraction-limit resolution. Simulation and experimental results demonstrate, both qualitatively and quantitatively, that the secret image becomes recognizable when and only when the substrate is illuminated with the designed key pattern.
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Seredyński, Rafał; Wolna, Dorota; Kędzior, Mateusz; Gutowicz, Jan
2017-01-01
Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolytic activity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Shanks, John; Burtnick, Mary N; Brett, Paul J; Waag, David M; Spurgers, Kevin B; Ribot, Wilson J; Schell, Mark A; Panchal, Rekha G; Gherardini, Frank C; Wilkinson, Keith D; Deshazer, David
2009-04-01
Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of approximately 60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). The B. mallei tssM gene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in a virAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. The tssM gene was expressed within 1 h after uptake of B. mallei into RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provide B. mallei a selective advantage in the intracellular environment during infection.
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Li, Jing; O'Connor, Kathleen L; Greeley, George H; Blackshear, Perry J; Townsend, Courtney M; Evers, B Mark
2005-03-04
Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-alpha and -delta, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-delta siRNA, ROKalpha siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-delta and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-delta was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-delta downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-delta proteins, in stimulated gut peptide secretion.
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The SdiA-Regulated Gene srgE Encodes a Type III Secreted Effector
Habyarimana, Fabien; Sabag-Daigle, Anice
2014-01-01
Salmonella enterica serovar Typhimurium is a food-borne pathogen that causes severe gastroenteritis. The ability of Salmonella to cause disease depends on two type III secretion systems (T3SSs) encoded in two distinct Salmonella pathogenicity islands, 1 and 2 (SPI1 and SPI2, respectively). S. Typhimurium encodes a solo LuxR homolog, SdiA, which can detect the acyl-homoserine lactones (AHLs) produced by other bacteria and upregulate the rck operon and the srgE gene. SrgE is predicted to encode a protein of 488 residues with a coiled-coil domain between residues 345 and 382. In silico studies have provided conflicting predictions as to whether SrgE is a T3SS substrate. Therefore, in this work, we tested the hypothesis that SrgE is a T3SS effector by two methods, a β-lactamase activity assay and a split green fluorescent protein (GFP) complementation assay. SrgE with β-lactamase fused to residue 40, 100, 150, or 300 was indeed expressed and translocated into host cells, but SrgE with β-lactamase fused to residue 400 or 488 was not expressed, suggesting interference by the coiled-coil domain. Similarly, SrgE with GFP S11 fused to residue 300, but not to residue 488, was expressed and translocated into host cells. With both systems, translocation into host cells was dependent upon SPI2. A phylogenetic analysis indicated that srgE is found only within Salmonella enterica subspecies. It is found sporadically within both typhoidal and nontyphoidal serovars, although the SrgE protein sequences found within typhoidal serovars tend to cluster separately from those found in nontyphoidal serovars, suggesting functional diversification. PMID:24727228
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Structure of a Type-1 Secretion System ABC Transporter.
Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen
2017-03-07
Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Engineering Escherichia coli into a protein delivery system for mammalian cells.
Reeves, Analise Z; Spears, William E; Du, Juan; Tan, Kah Yong; Wagers, Amy J; Lesser, Cammie F
2015-05-15
Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.
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Veith, Paul D; Glew, Michelle D; Gorasia, Dhana G; Reynolds, Eric C
2017-10-01
The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Proteins secreted by the T9SS have an N-terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C-terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C-terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components. © 2017 John Wiley & Sons Ltd.
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A Repulsive Electrostatic Mechanism for Protein Export through the Type III Secretion Apparatus
Rathinavelan, Thenmalarchelvi; Zhang, Lingling; Picking, Wendy L.; Weis, David D.; De Guzman, Roberto N.; Im, Wonpil
2010-01-01
Abstract Many Gram-negative bacteria initiate infections by injecting effector proteins into host cells through the type III secretion apparatus, which is comprised of a basal body, a needle, and a tip. The needle channel is formed by the assembly of a single needle protein. To explore the export mechanisms of MxiH needle protein through the needle of Shigella flexneri, an essential step during needle assembly, we have performed steered molecular dynamics simulations in implicit solvent. The trajectories reveal a screwlike rotation motion during the export of nativelike helix-turn-helix conformations. Interestingly, the channel interior with excessive electronegative potential creates an energy barrier for MxiH to enter the channel, whereas the same may facilitate the ejection of the effectors into host cells. Structurally known basal regions and ATPase underneath the basal region also have electronegative interiors. Effector proteins also have considerable electronegative potential patches on their surfaces. From these observations, we propose a repulsive electrostatic mechanism for protein translocation through the type III secretion apparatus. Based on this mechanism, the ATPase activity and/or proton motive force could be used to energize the protein translocation through these nanomachines. A similar mechanism may be applicable to macromolecular channels in other secretion systems or viruses through which proteins or nucleic acids are transported. PMID:20141759
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Sitia, Roberto; Rubartelli, Anna
2018-04-04
Interleukin 1β (IL-1β) is a major mediator of inflammation, with a causative role in many diseases. Unlike most other cytokines, however, it lacks a secretory signal sequence, raising intriguing mechanistic, functional and evolutionary questions. Despite decades of strenuous efforts in many laboratories, how IL-1β is secreted is still a matter of intense debate. Here, we summarize the different mechanisms and pathways that have been proposed for IL-1β secretion. At least two of them, namely the endolysosomal vesicle-based and gasdermin D-dependent pathways (types III and I in the recent Rabouille's classification of unconventional protein secretion), can be triggered in monocytes, the main source of IL-1β in humans, according to the type and strength of the pro-inflammatory stimuli. As during the escalation of human conflicts, monocytes deploy secretory mechanisms of increasing efficiency and dangerousness, shifting from the specific and controlled type III pathway to the much faster release of type I. Thus, the different mechanisms are activated depending on the severity of the conditions, from the self-limiting type III pathways in response of low pathogen load or small trauma, to the uncontrolled responses that underlie autoinflammatory disorders and sepsis. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Hu, Xuewei; Yang, Lei; Lai, Xinke; Yao, Qi; Chen, Kai
2017-10-03
This paper presented the influence of Al(III) on biodegradability, micromorphology, composition and functional groups characteristics of the biofilm extracellular polymeric substances (EPS) during different growth phases. The sequencing batch biofilm reactors were developed to cultivate biofilms under different Al(III) dosages. The results elucidated that Al(III) affected biofilm development adversely at the beginning of biofilm growth, but promoted the biofilm mass and improved the biofilm activity with the growth of the biofilm. The micromorphological observation indicated that Al(III) led to a reduction of the filaments and promotion of the EPS secretion in growth phases of the biofilm, also Al(III) could promote microorganisms to form larger colonies for mature biofilm. Then, the analysis of EPS contents and components suggested that Al(III) could increase the protein (PN) of tightly bound EPS (TB-EPS) which alleviated the metal toxicity inhibition on the biofilm during the initial phases of biofilm growth. The biofilm could gradually adapt to the inhibition caused by Al(III) at the biofilm maturation moment. Finally, through the Fourier transform infrared spectroscopy, it was found that Al(III) was beneficial for the proliferation and secretion of TB-EPS functional groups, especially the functional groups of protein and polysaccharides.
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Photodetectors using III-V nitrides
Moustakas, T.D.; Misra, M.
1997-10-14
A photodetector using a III-V nitride and having predetermined electrical properties is disclosed. The photodetector includes a substrate with interdigitated electrodes formed on its surface. The substrate has a sapphire base layer, a buffer layer formed from a III-V nitride and a single crystal III-V nitride film. The three layers are formed by electron cyclotron resonance microwave plasma-assisted molecular beam epitaxy (ECR-assisted MBE). Use of the ECR-assisted MBE process allows control and predetermination of the electrical properties of the photodetector. 24 figs.
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Photodetectors using III-V nitrides
Moustakas, Theodore D.; Misra, Mira
1997-01-01
A photodetector using a III-V nitride and having predetermined electrical properties is disclosed. The photodetector includes a substrate with interdigitated electrodes formed on its surface. The substrate has a sapphire base layer, a buffer layer formed from a III-V nitride and a single crystal III-V nitride film. The three layers are formed by electron cyclotron resonance microwave plasma-assisted molecular beam epitaxy (ECR-assisted MBE). Use of the ECR-assisted MBE process allows control and predetermination of the electrical properties of the photodetector.
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2010-01-01
Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei. PMID:20540813
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Takasuka, Taichi E; Acheson, Justin F; Bianchetti, Christopher M; Prom, Ben M; Bergeman, Lai F; Book, Adam J; Currie, Cameron R; Fox, Brian G
2014-01-01
β-Mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.
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Jacobs, Jonathan M.; Pesce, Céline; Lefeuvre, Pierre; Koebnik, Ralf
2015-01-01
Pathogenic bacteria in the genus Xanthomonas cause diseases on over 350 plant species, including cannabis (Cannabis sativa L.). Because of regulatory limitations, the biology of the Xanthomonas-cannabis pathosystem remains largely unexplored. To gain insight into the evolution of Xanthomonas strains pathogenic to cannabis, we sequenced the genomes of two geographically distinct Xanthomonas strains, NCPPB 3753 and NCPPB 2877, which were previously isolated from symptomatic plant tissue in Japan and Romania. Comparative multilocus sequence analysis of housekeeping genes revealed that they belong to Group 2, which comprises most of the described species of Xanthomonas. Interestingly, both strains lack the Hrp Type III secretion system and do not contain any of the known Type III effectors. Yet their genomes notably encode two key Hrp pathogenicity regulators HrpG and HrpX, and hrpG and hrpX are in the same genetic organization as in the other Group 2 xanthomonads. Promoter prediction of HrpX-regulated genes suggests the induction of an aminopeptidase, a lipase and two polygalacturonases upon plant colonization, similar to other plant-pathogenic xanthomonads. Genome analysis of the distantly related Xanthomonas maliensis strain 97M, which was isolated from a rice leaf in Mali, similarly demonstrated the presence of HrpG, HrpX, and a HrpX-regulated polygalacturonase, and the absence of the Hrp Type III secretion system and known Type III effectors. Given the observation that some Xanthomonas strains across distinct taxa do not contain hrpG and hrpX, we speculate a stepwise evolution of pathogenicity, which involves (i) acquisition of key regulatory genes and cell wall-degrading enzymes, followed by (ii) acquisition of the Hrp Type III secretion system, which is ultimately accompanied by (iii) successive acquisition of Type III effectors. PMID:26136759
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Inhibition of polygylcine hydrolases by substrate analog peptides
USDA-ARS?s Scientific Manuscript database
Polyglycine hydrolases are proteases secreted by fungal pathogens that target corn defense chitinases. They cleave interdomain glycine-glycine bonds within a polyglycine linker, separating substrate chitinases into two single domain proteins. Polyglycine hydrolases consist of 640 amino acids with a ...
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Lu, Jin Fang; Wang, Wei Na; Wang, Gai Ling; Zhang, He; Zhou, Ying; Gao, Zhi Peng; Nie, Pin; Xie, Hai Xia
2016-01-01
The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD50) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Chakraborty, Smarajit; Mizusaki, Hideaki; Kenney, Linda J.
2015-01-01
In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed. PMID:25875623
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Shanks, John; Burtnick, Mary N.; Brett, Paul J.; Waag, David M.; Spurgers, Kevin B.; Ribot, Wilson J.; Schell, Mark A.; Panchal, Rekha G.; Gherardini, Frank C.; Wilkinson, Keith D.; DeShazer, David
2009-01-01
Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of ∼60 genes, including a large virulence gene cluster encoding a type VI secretion system (T6SS). The B. mallei tssM gene encodes a putative ubiquitin-specific protease that is physically linked to, and transcriptionally coregulated with, the T6SS gene cluster. Mass spectrometry and immunoblot analysis demonstrated that TssM was secreted in a virAG-dependent manner in vitro. Surprisingly, the T6SS was found to be dispensable for the secretion of TssM. The C-terminal half of TssM, which contains Cys and His box motifs conserved in eukaryotic deubiquitinases, was purified and biochemically characterized. Recombinant TssM hydrolyzed multiple ubiquitinated substrates and the cysteine at position 102 was critical for enzymatic activity. The tssM gene was expressed within 1 h after uptake of B. mallei into RAW 264.7 murine macrophages, suggesting that the TssM deubiquitinase is produced in this intracellular niche. Although the physiological substrate(s) is currently unknown, the TssM deubiquitinase may provide B. mallei a selective advantage in the intracellular environment during infection. PMID:19168747
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The role of Shewanella oneidensis MR-1 outer surface structures in extracellular electron transfer
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bouhenni, Rachida; Vora, Gary J.; Biffinger, Justin C.
2010-04-20
Shewanella oneidensis is a facultative anaerobe that uses more than 14 different terminal electron acceptors for respiration. These include metal oxides and hydroxyoxides, and toxic metals such as uranium and chromium. Mutants deficient in metal reduction were isolated using the mariner transposon derivative, minihimar RB1. These included mutants with transposon insertions in the prepilin peptidase and type II secretion system genes. All mutants were deficient in Fe(III) and Mn(IV) reduction, and exhibited slow growth when DMSO was used as the electron acceptor. The genome sequence of S. oneidensis contains one prepilin peptidase gene, pilD. A similar prepilin peptidase that maymore » function in the processing of type II secretion prepilins was not found. Single and multiple chromosomal deletions of four putative type IV pilin genes did not affect Fe(III) and Mn(IV) reduction. These results indicate that PilD in S. oneidensis is responsible for processing both type IV and type II secretion prepilin proteins. Type IV pili do not appear to be required for Fe(III) and Mn(IV) reduction.« less
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Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B.
2012-01-01
Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity. PMID:22563435
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Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B
2012-01-01
Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.
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The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation.
Ninio, Shira; Zuckman-Cholon, Deborah M; Cambronne, Eric D; Roy, Craig R
2005-02-01
The intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two-hybrid screen. It was determined that the IcmS-IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS-IcmW complex being involved in the recognition and Dot/Icm-dependent translocation of substrate proteins during Legionella infection of host cells.
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Bengtsson, Oskar; Arntzen, Magnus Ø; Mathiesen, Geir; Skaugen, Morten; Eijsink, Vincent G H
2016-01-10
Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates. Copyright © 2015 Elsevier B.V. All rights reserved.
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Molecular architectures of benzoic acid-specific type III polyketide synthases
Stewart, Charles; Woods, Kate; Macias, Greg; Allan, Andrew C.; Noel, Joseph P.
2017-01-01
Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs. In order to gain a better understanding of structure–function relationships within the type III PKS family, the crystal structures of biphenyl synthase from Malus × domestica and benzophenone synthase from Hypericum androsaemum were compared with the structure of an archetypal type III PKS: chalcone synthase from Malus × domestica. Both biphenyl synthase and benzophenone synthase contain mutations that reshape their active-site cavities to prevent the binding of 4-coumaroyl-CoA and to favor the binding of small hydrophobic substrates. The active-site cavities of biphenyl synthase and benzophenone synthase also contain a novel pocket associated with their chain-elongation and cyclization reactions. Collectively, these results illuminate structural determinants of benzoic acid-specific type III PKSs and expand the understanding of the evolution of specialized metabolic pathways in plants. PMID:29199980
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Chracterization of class III peroxidases from switchgrass
USDA-ARS?s Scientific Manuscript database
Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specifici...
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III-nitrides on oxygen- and zinc-face ZnO substrates
NASA Astrophysics Data System (ADS)
Namkoong, Gon; Burnham, Shawn; Lee, Kyoung-Keun; Trybus, Elaissa; Doolittle, W. Alan; Losurdo, Maria; Capezzuto, Pio; Bruno, Giovanni; Nemeth, Bill; Nause, Jeff
2005-10-01
The characteristics of III-nitrides grown on zinc- and oxygen-face ZnO by plasma-assisted molecular beam epitaxy were investigated. The reflection high-energy electron diffraction pattern indicates formation of a cubic phase at the interface between III-nitride and both Zn- and O-face ZnO. The polarity indicates that Zn-face ZnO leads to a single polarity, while O-face ZnO forms mixed polarity of III-nitrides. Furthermore, by using a vicinal ZnO substrate, the terrace-step growth of GaN was realized with a reduction by two orders of magnitude in the dislocation-related etch pit density to ˜108cm-2, while a dislocation density of ˜1010cm-2 was obtained on the on-axis ZnO substrates.
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Vincent, Carr D; Friedman, Jonathan R; Jeong, Kwang Cheol; Sutherland, Molly C; Vogel, Joseph P
2012-07-01
Legionella pneumophila, the causative agent of Legionnaires' disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella type IV secretion system substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. © 2012 Blackwell Publishing Ltd.
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Tyson, Jessica Y; Vargas, Paloma; Cianciotto, Nicholas P
2014-12-01
The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts. © 2014 The Authors.
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Veenendaal, Andreas K J; Hodgkinson, Julie L; Schwarzer, Lynn; Stabat, David; Zenk, Sebastian F; Blocker, Ariel J
2007-03-01
Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.
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Heinz, Dirk W
2013-12-03
Secretins are major constituents of bacterial type III secretion systems (T3SS). In this issue of Structure, Kowal and colleagues report on the cryo-EM structure of the native YscC secretin from Yersinia, revealing its internal symmetry and mode of length adaptation. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Storbeck, Sonja; Saha, Sayantan; Krausze, Joern; Klink, Björn U.; Heinz, Dirk W.; Layer, Gunhild
2011-01-01
During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate. PMID:21632530
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Creze, Christophe; Castang, Sandra; Derivery, Emmanuel; Haser, Richard; Hugouvieux-Cotte-Pattat, Nicole; Shevchik, Vladimir E; Gouet, Patrice
2008-06-27
The crystallographic structure of the family 3 polysaccharide lyase (PL-3) PelI from Erwinia chrysanthemi has been solved to 1.45 A resolution. It consists of an N-terminal domain harboring a fibronectin type III fold linked to a catalytic domain displaying a parallel beta-helix topology. The N-terminal domain is located away from the active site and is not involved in the catalytic process. After secretion in planta, the two domains are separated by E. chrysanthemi proteases. This event turns on the hypersensitive response of the host. The structure of the single catalytic domain determined to 2.1 A resolution shows that the domain separation unveils a "Velcro"-like motif of asparagines, which might be recognized by a plant receptor. The structure of PelI in complex with its substrate, a tetragalacturonate, has been solved to 2.3 A resolution. The sugar binds from subsites -2 to +2 in one monomer of the asymmetric unit, although it lies on subsites -1 to +3 in the other. These two "Michaelis complexes" have never been observed simultaneously before and are consistent with the dual mode of bond cleavage in this substrate. The bound sugar adopts a mixed 2(1) and 3(1) helical conformation similar to that reported in inactive mutants from families PL-1 and PL-10. However, our study suggests that the catalytic base in PelI is not a conventional arginine but a lysine as proposed in family PL-9.
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Vincent, Carr D.; Friedman, Jonathan R.; Jeong, Kwang Cheol; Sutherland, Molly C.; Vogel, Joseph P.
2012-01-01
Summary Legionella pneumophila, the causative agent of Legionnaires’ disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella T4SS substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. PMID:22694730
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A study of over production and enhanced secretion of enzymes. Quarterly report 1
DOE Office of Scientific and Technical Information (OSTI.GOV)
Dashek, W.V.
1992-12-28
The current project is concerned with the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. The project is divided into two segments: over-production of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electron microscopical techniques. The former approach employs recombinant DNA procedures, ligation of appropriate nuclease generated DNA fragments into a vector and the subsequent transformation of Escherichia coli to yield E. coli harboring a C. versicolor DNA insert. The biochemistry/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO inhibitors to elevate C.versicolor`s ability to synthesizemore » and secrete lignocellulosic enzymes. In this connection, cell fractionation/kinetic analysis, TEM immunoelectron microscopic localization and TEM substrate localization of PPO are being employed to assess the route of secretion. Both approaches will culminate in the batch culture of either E. coli or C. versicolor, in a fermentor with the subsequent development of rapid isolation and purification procedures to yield elevated quantities of pure lignocellulosic enzymes. During the past year, research effort were directed toward determining the route of polyphenol oxidase (PPO) secretion by the wood-decay fungus, Coriolus versicolor. In addition, research activities were continued to over-produce and to purify PPO as well as define the time-dependent intra- and extra-cellular appearances of C. versicolor ligninases and cellulases.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Niemann, George; Brown, Roslyn N.; Gustin, Jean K.
The intracellular pathogen Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis in the world. This pathogen has two type-III secretion systems (TTSS) necessary for virulence that are encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) and are expressed during extracellular or intracellular infectious states, respectively, to deliver virulence factors (effectors) to the host cell cytoplasm. While many have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this mass spectrometry-based proteomics study, we identified effector proteins secreted under minimal acidic medium growth conditions that induced themore » SPI-2 TTSS and its effectors, and compared the secretome from the parent strain to the secretome from strains missing either essential (SsaK) or regulatory components (SsaL) of the SPI-2 secretion apparatus. We identified 75% of the known TTSS effector repertoire. Excluding translocon components, 95% of the known effectors were biased for identification in the ssaL mutant background, which demonstrated that SsaL regulates SPI-2 type III secretion. To confirm secretion to animal cells, we made translational fusions of several of the best candidates to the calmodulin-dependent adenylate cyclase of Bordetella pertussis and assayed cAMP levels of infected J774 macrophage-like cells. From these infected cells we identified six new TTSS effectors and two others that are secreted independent of TTSS. Our results substantiate reports of additional secretion systems encoded by Salmonella other than TTSS.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ward, J. Scott; Remo, Timothy; Horowitz, Kelsey
The high cost of wafers suitable for epitaxial deposition of III-V solar cells has been a primary barrier to widespread use of these cells in low-concentration and one-sun terrestrial solar applications. A possible solution is to reuse the substrate many times, thus spreading its cost across many cells. We performed a bottom-up techno-economic analysis of three different strategies for substrate reuse in high-volume manufacturing: epitaxial lift-off, spalling, and the use of a porous germanium release layer. The analysis shows that the potential cost reduction resulting from substrate reuse is limited in all three strategies--not by the number of reuse cyclesmore » achievable, but by the costs that are incurred in each cycle to prepare the substrate for another epitaxial deposition. The dominant substrate-preparation cost component is different for each of the three strategies, and the cost-ranking of these strategies is subject to change if future developments substantially reduce the cost of epitaxial deposition.« less
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Hormonal substrate of estrous odor preference in beagles.
Anisko, J J
1977-01-01
The effect of testosterone propionate (TP), injected during pregnancy and/or implanted on Days 1-3 of postpartum, on a behavioral preference for estrous vaginal secretions was assessed in beagles. Males castrated in adulthood and intact, untreated males were used to test the attractability of vaginal secretions. Intact and castrated males showed a marked preference for estrous vaginal secretions. Castrated males showed a greater preference for estrous secretions following treatment with TP. Further, females treated with TP before and/or after birth also showed a marked preference for estrous vaginal secretions. The results suggest that the preference for estrous odor is a sexually dimorphic pattern which depends upon developmental and concurrent androgenic stimulation for its manifestation.
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Lu, Jian-ping; Zhang, Xiao-hui; Yu, Xiao-yun
2006-01-01
The structural change of the oviduct of freshwater shrimp (Macrobrachium nipponense) during spawning was examined by electron microscopy. The oviduct wall structural characteristics seem to be influenced significantly by the spawning process. Before the parturition and ovulation, two types of epithelial cells (types I and II) are found in the epithelium. The free surfaces of type I and type II cells have very dense long microvilli. Under the type I and type II cells, are a relatively thick layer of secreting material and a layer of mostly dead cells. After ovulation, two other types of epithelial cells (types III and IV) are found in the oviduct wall epithelium. The free surface of type III cells only has short microvilli scattered on the surface. The thick layer with secreting material and the dead cell layer disappeared at this stage. In some type III cells, the leaking out of cytoplasm from broken cell membrane led to the death of these type III cells. The transformation of all four types of epithelial cells was in the order: IV→I→II→III. PMID:16365928
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Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Morzadec, Claudie; Macoch, Mélinda; Robineau, Marc
2012-08-01
Trivalent inorganic arsenic [As(III)] is an efficient anticancer agent used to treat patients suffering from acute promyelocytic leukemia. Recently, experimental studies have clearly demonstrated that this metalloid can also cure lymphoproliferative and/or pro-inflammatory syndromes in different murine models of chronic immune-mediated diseases. T helper (Th) 1 and Th17 lymphocytes play a central role in development of these diseases, in mice and humans, especially by secreting the potent pro-inflammatory cytokine interferon-γ and IL-17A, respectively. As(III) impairs basic functions of human T cells but its ability to modulate secretion of pro-inflammatory cytokines by differentiated Th lymphocytes is unknown. In the present study,more » we demonstrate that As(III), used at concentrations clinically achievable in plasma of patients, has no effect on the secretion of interferon-γ from Th1 cells but almost totally blocks the expression and the release of IL-17A from human Th17 lymphocytes co-stimulated for five days with anti-CD3 and anti-CD28 antibodies, in the presence of differentiating cytokines. In addition, As(III) specifically reduces mRNA levels of the retinoic-related orphan receptor (ROR)C gene which encodes RORγt, a key transcription factor controlling optimal IL-17 expression in fully differentiated Th17 cells. The metalloid also blocks initial expression of IL-17 gene induced by the co-stimulation, probably in part by impairing activation of the JNK/c-Jun pathway. In conclusion, our results demonstrate that As(III) represses expression of the major pro-inflammatory cytokine IL-17A produced by human Th17 lymphocytes, thus strengthening the idea that As(III) may be useful to treat inflammatory immune-mediated diseases in humans. -- Highlights: ► Arsenic inhibits secretion of IL-17A from human naïve and memory Th17 lymphocytes. ► Arsenic represses early expression of IL-17A gene in human activated T lymphocytes. ► Arsenic interferes with activation of the JNK/c-Jun pathway in human T lymphocytes.« less
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Oliveira, G S; Ulhoa, C J; Silveira, M H L; Andreaus, J; Silva-Pereira, I; Poças-Fonseca, M J; Faria, F P
2013-01-01
Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.
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Huang, Ming-Der; Chen, Tung-Ling L.; Huang, Anthony H.C.
2013-01-01
Lipid transfer proteins (LTPs) are small secretory proteins in plants with defined lipid-binding structures for possible lipid exocytosis. Special groups of LTPs unique to the anther tapetum are abundant, but their functions are unclear. We studied a special group of LTPs, type III LTPs, in Arabidopsis (Arabidopsis thaliana). Their transcripts were restricted to the anther tapetum, with levels peaking at the developmental stage of maximal pollen-wall exine synthesis. We constructed an LTP-Green Fluorescent Protein (LTP-GFP) plasmid, transformed it into wild-type plants, and monitored LTP-GFP in developing anthers with confocal laser scanning microscopy. LTP-GFP appeared in the tapetum and was secreted via the endoplasmic reticulum-trans-Golgi network machinery into the locule. It then moved to the microspore surface and remained as a component of exine. Immuno-transmission electron microscopy of native LTP in anthers confirmed the LTP-GFP observations. The in vivo association of LTP-GFP and exine in anthers was not observed with non-type III or structurally modified type III LTPs or in transformed exine-defective mutant plants. RNA interference knockdown of individual type III LTPs produced no observable mutant phenotypes. RNA interference knockdown of two type III LTPs produced microscopy-observable morphologic changes in the intine underneath the exine (presumably as a consequence of changes in the exine not observed by transmission electron microscopy) and pollen susceptible to dehydration damage. Overall, we reveal a novel transfer pathway of LTPs in which LTPs bound or nonbound to exine precursors are secreted from the tapetum to become microspore exine constituents; this pathway explains the need for plentiful LTPs to incorporate into the abundant exine. PMID:24096413
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Espina, Marianela; Ausar, S. Fernando; Middaugh, C. Russell; Baxter, M. Aaron; Picking, William D.; Picking, Wendy L.
2007-01-01
Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are α-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins. PMID:17327391
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Investigation of Low Cost Substrate Approaches for III-V Solar Cells
NASA Astrophysics Data System (ADS)
Lichty, Marlene Lydia
With the need for cleaner energy sources, which can displace fossil fuel, the solar cell industry is of particular interest due to the abundancy of the Sun. Silicon currently dominates terrestrial applications, but efficiency improvements have saturated. III-V based solar cells have reported the highest efficiencies, however, high costs due to substrates and fabrication processes have limited these devices to specialty applications, such as space. In order to reduce the cost associated with fabricating III-V semiconductor substrate material, two different approaches were taken in this work with a particular focus on making III-Vs more applicable outside of specialty applications, including InP, InAsnd Ge. Typical material characterization techniques were used to analyze the samples and processes studied in this thesis. The first process examined was the direct epitaxial growth of III-V materials by MOCVD on cheaper substrates. More specifically, the direct growth of InP and InAs on metal foils. A growth time study and surface coverage analysis was performed for the growth of InP. A characterization study was then conducted on the second process, the aluminum- induced crystallization of germanium to determine the effects this process had on the surface. Crystalline InP, InAs and Ge were successfully characterized in this work, and show promise for use in cheaper III-V alternatives to terrestrial energy solutions.
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Controlled Spalling in (100)-Oriented Germanium by Electroplating
NASA Astrophysics Data System (ADS)
Crouse, Dustin Ray
This work investigates controlled spalling as a method to exfoliate thin films of various thickness from rigid, crystalline germanium (Ge) substrates and to enable substrate reuse for III-V single junction photovoltaic devices. Technological limitations impeding wide-spread adoption of flexible electronics and high-material-cost photovoltaic devices have motivated significant interest in a method to remove devices from their substrates. DC magnetron sputtering has been previously utilized to remove semiconductor devices of various thicknesses from Ge substrates, but this method is expensive and time-consuming. Controlled spalling via high-speed electrodeposition is a fast, inexpensive exfoliation method that utilizes a tensile-stressed metal layer deposited on a (100)-oriented Ge substrate and an external force to mechanically propagate a crack parallel to the surface at a desired depth in the substrate material. Suo and Hutchinson's quantitative models describe critical combinations of film thickness and strain mismatch between a film and substrate at which a stressed bilayer system spontaneously spalls; however, fine control over a wide steady-state spall depth range has been limited by the ability to experimentally tailor strain mismatch caused by residual stress within deposited stressor layers. This work investigates the effect of tuning electroplating current density and electrolyte chemistry on the residual stress in a nickel stressor film and their impact on the achievable spall depth range. Steady-state spall depth is found to increase with increasing stressor layer thickness and decrease with increasing residual stress. By tailoring residual stress through adjusting plating conditions and the electrolyte's phosphorous concentration, wide control over spall depth within Ge substrates from sub-micron to 76microm-thicknesses were achieved. To assess the viability of utilizing controlled spalling for substrate reuse, this dissertation demonstrates the first III-V solar cells (GaInAsP, Eg 1.7 eV) grown directly on a spalled-Ge substrate without any additional surface preparation. Widespread adoption of high-efficiency III-V solar cells has been limited by expensive deposition processes and high material cost of substrates. Substrate reuse offers a promising route towards enabling III-V devices to become cost-competitive for one-sun terrestrial applications. In this study, the quality of spalled Ge surfaces is characterized to assess lattice matching capability between the device layer materials and the substrate. GaAs films grown on spalled Ge substrates by hydride vapor phase epitaxy were single-crystal in nature. III-V solar cells grown on spalled and pristine Ge substrates show nearly equivalent efficiency of 8%, despite the roughness of the spalled-Ge substrate. Principles of fractography were used to deduce that surface roughness originated from non-uniform crack propagation and mixed-mode loading during the spalling process.
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Develoment and Evaluation of Immunomodulators of Hemopoietic and Immunologic Mechanisms.
1991-07-02
secretion was much more marked : in the presence of IL-4 cultuie supernatants. these effects were dissociated to some culture supernatants had average IgE...required to induce IgE secretion. and 25. Cobbold . S.. Hale. G. & Waldman. H. 11987) in Leukocvte Tvpine 4 iii) these effects are not inhibited by a
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Whitlock, Gregory C; Estes, D Mark; Young, Glenn M; Young, Briana; Torres, Alfredo G
2008-12-01
Burkholderia mallei, the aetiological agent of glanders disease, is a Gram-negative facultative intracellular bacterium. Despite numerous studies, the detailed mechanism of its pathogenesis is almost unknown. The presence of a type III secretion system (TTSS) is one of the known mechanisms associated with virulence. An intact TTSS indicates that B. mallei is able to secrete proteins in response to different environmental conditions, which could play an important role in pathogenesis. Therefore, characterization of the TTSS and identification of the secreted proteins associated with bacterial pathogenesis could provide crucial information for the development of a candidate vaccine. In the current study, we used an enzymatic reporter system to establish some of the conditions enabling TTS. Construction of the TTSS bopA mutant revealed that BopA is important for B. mallei invasion and intracellular survival. Overall, our study elucidates how BopA can aid in the optimization of TTS and defines the function of TTS effectors in bacterial intracellular survival and invasion.
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Salmonella-secreted Virulence Factors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Heffron, Fred; Niemann, George; Yoon, Hyunjin
In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellentmore » reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.« less
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Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger
2017-01-01
There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.
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Self-Chaperoning of the Type III Secretion System needle tip proteins IpaD and BipD
Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E.; Birket, Susan; Field, Terry; Picking, William D.; Blocker, Ariel; Galyov, Edouard E.; Picking, Wendy L.; Lea, Susan M.
2007-01-01
Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence; IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intra-molecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure. PMID:17077085
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Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD.
Johnson, Steven; Roversi, Pietro; Espina, Marianela; Olive, Andrew; Deane, Janet E; Birket, Susan; Field, Terry; Picking, William D; Blocker, Ariel J; Galyov, Edouard E; Picking, Wendy L; Lea, Susan M
2007-02-09
Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.
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Method to grow group III-nitrides on copper using passivation layers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Li, Qiming; Wang, George T; Figiel, Jeffrey T
Group III-nitride epilayers can be grown directly on copper substrates using intermediate passivation layers. For example, single crystalline c-plane GaN can be grown on Cu (110) substrates with MOCVD. The growth relies on a low temperature AlN passivation layer to isolate any alloying reaction between Ga and Cu.
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Quantum cascade lasers grown on silicon.
Nguyen-Van, Hoang; Baranov, Alexei N; Loghmari, Zeineb; Cerutti, Laurent; Rodriguez, Jean-Baptiste; Tournet, Julie; Narcy, Gregoire; Boissier, Guilhem; Patriarche, Gilles; Bahriz, Michael; Tournié, Eric; Teissier, Roland
2018-05-08
Technological platforms offering efficient integration of III-V semiconductor lasers with silicon electronics are eagerly awaited by industry. The availability of optoelectronic circuits combining III-V light sources with Si-based photonic and electronic components in a single chip will enable, in particular, the development of ultra-compact spectroscopic systems for mass scale applications. The first circuits of such type were fabricated using heterogeneous integration of semiconductor lasers by bonding the III-V chips onto silicon substrates. Direct epitaxial growth of interband III-V laser diodes on silicon substrates has also been reported, whereas intersubband emitters grown on Si have not yet been demonstrated. We report the first quantum cascade lasers (QCLs) directly grown on a silicon substrate. These InAs/AlSb QCLs grown on Si exhibit high performances, comparable with those of the devices fabricated on their native InAs substrate. The lasers emit near 11 µm, the longest emission wavelength of any laser integrated on Si. Given the wavelength range reachable with InAs/AlSb QCLs, these results open the way to the development of a wide variety of integrated sensors.
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Meyer, Damien; Cunnac, Sébastien; Guéneron, Mareva; Declercq, Céline; Van Gijsegem, Frédérique; Lauber, Emmanuelle; Boucher, Christian; Arlat, Matthieu
2006-01-01
Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum. PMID:16788199
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Zeng, Quan; McNally, R Ryan; Sundin, George W
2013-04-01
Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora.
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Zeng, Quan; McNally, R. Ryan
2013-01-01
Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora. PMID:23378513
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Yang, Yi; Ling, Yichuan; Wang, Gongming; Lu, Xihong; Tong, Yexiang; Li, Yat
2013-03-07
We report a general strategy for synthesis of gallium nitride (GaN) and indium nitride (InN) nanowires on conductive and flexible carbon cloth substrates. GaN and InN nanowires were prepared via a nanocluster-mediated growth method using a home built chemical vapor deposition (CVD) system with Ga and In metals as group III precursors and ammonia as a group V precursor. Electron microscopy studies reveal that the group III-nitride nanowires are single crystalline wurtzite structures. The morphology, density and growth mechanism of these nanowires are determined by the growth temperature. Importantly, a photoelectrode fabricated by contacting the GaN nanowires through a carbon cloth substrate shows pronounced photoactivity for photoelectrochemical water oxidation. The ability to synthesize group III-nitride nanowires on conductive and flexible substrates should open up new opportunities for nanoscale photonic, electronic and electrochemical devices.
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NASA Astrophysics Data System (ADS)
Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.
2011-09-01
Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.
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Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.
2011-01-01
Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603
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Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R
2011-09-01
Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kirimura, Kohtaro, E-mail: kkohtaro@waseda.jp; Watanabe, Shotaro; Kobayashi, Keiichi
Type III polyketide synthases (PKSs) catalyze the formation of pyrone- and resorcinol-types aromatic polyketides. The genomic analysis of the filamentous fungus Aspergillus niger NRRL 328 revealed that this strain has a putative gene (chr-8-2: 2978617–2979847) encoding a type III PKS, although its functions are unknown. In this study, for functional analysis of this putative type III PKS designated as An-CsyA, cloning and heterologous expression of the An-CsyA gene (An-csyA) in Escherichia coli were performed. Recombinant His-tagged An-CsyA was successfully expressed in E. coli BL21 (DE3), purified by Ni{sup 2+}-affinity chromatography, and used for in vitro assay. Tests on the substrate specificity ofmore » the His-tagged An-CsyA with myriad acyl-CoAs as starter substrates and malonyl-CoA as extender substrate showed that His-tagged An-CsyA accepted fatty acyl-CoAs (C2-C14) and produced triketide pyrones (C2-C14), tetraketide pyrones (C2-C10), and pentaketide resorcinols (C10-C14). Furthermore, acetoacetyl-CoA, malonyl-CoA, isobutyryl-CoA, and benzoyl-CoA were also accepted as starter substrates, and both of triketide pyrones and tetraketide pyrones were produced. It is noteworthy that the His-tagged An-CsyA produced polyketides from malonyl-CoA as starter and extender substrates and produced tetraketide pyrones from short-chain fatty acyl-CoAs as starter substrates. Therefore, this is the first report showing the functional properties of An-CsyA different from those of other fungal type III PKSs. -- Highlights: •Type III PKS from Aspergillus niger NRRL 328, An-CsyA, was cloned and characterized. •An-CsyA produced triketide pyrones, tetraketide pyrones and pentaketide resorcinols. •Functional properties of An-CsyA differs from those of other fungal type III PKSs.« less
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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
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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.
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Takasuka, Taichi E.; Acheson, Justin F.; Bianchetti, Christopher M.; Prom, Ben M.; Bergeman, Lai F.; Book, Adam J.; Currie, Cameron R.; Fox, Brian G.
2014-01-01
β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity. PMID:24710170
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Veenendaal, Andreas K J; Sundin, Charlotta; Blocker, Ariel J
2009-01-01
Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.
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Sarre, Aili; Ökvist, Mats; Klar, Tobias; Hall, David R; Smalås, Arne O; McSweeney, Sean; Timmins, Joanna; Moe, Elin
2015-08-01
While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity. Copyright © 2015 Elsevier Inc. All rights reserved.
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Sajó, Ráchel; Liliom, Károly; Muskotál, Adél; Klein, Agnes; Závodszky, Péter; Vonderviszt, Ferenc; Dobó, József
2014-11-01
Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate. Copyright © 2014 Elsevier B.V. All rights reserved.
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Parasitoid-host endocrine relations: self-reliance or co-optation?
Cole, T J; Beckage, N E; Tan, F F; Srinivasan, A; Ramaswamy, S B
2002-12-01
High titers of juvenile hormone (JH) maintain developmental arrest in Manduca sexta larvae parasitized by Cotesia congregata. Parasitized hosts exhibit up to 9.5 times greater amounts of total hemolymph JH (from 0.6+/-0.09 to 2.51+/-0.43ng/ml) compared to non-parasitized controls. Elevated titers are observed throughout the fifth instar, even beyond egression of the parasitoids on day 5. GC-MS analysis revealed that in hemolymph of unparasitized control larvae, JH I is the major homolog and levels of JH III are negligible; in parasitized individuals the amounts of JH I, II, and III rise, and JH III predominates. Neck ligation ensured separation of M. sexta's corpora allata from the posterior section, which contained most of the parasitoids in the infected insects. When the posterior region was sampled, JHs were not detected in the non-parasitzed larvae, but in those parasitized, JH III was found (1.98+/-0.29ng/ml, 24 h post-ligation). JH III was the only homolog produced and secreted by the parasitoid in in vitro culture. This is the first report stating that a parasitoid secretes JH III and may contribute, at least in part, to the circulating titer in the host hemocoel, concurrently promoting host production of JH I and II.
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Type VI Secretion System in Pseudomonas aeruginosa
Hachani, Abderrahman; Lossi, Nadine S.; Hamilton, Alexander; Jones, Cerith; Bleves, Sophie; Albesa-Jové, David; Filloux, Alain
2011-01-01
Pseudomonas aeruginosa is a Gram-negative bacterium causing chronic infections in cystic fibrosis patients. Such infections are associated with an active type VI secretion system (T6SS), which consists of about 15 conserved components, including the AAA+ ATPase, ClpV. The T6SS secretes two categories of proteins, VgrG and Hcp. Hcp is structurally similar to a phage tail tube component, whereas VgrG proteins show similarity to the puncturing device at the tip of the phage tube. In P. aeruginosa, three T6SSs are known. The expression of H1-T6SS genes is controlled by the RetS sensor. Here, 10 vgrG genes were identified in the PAO1 genome, among which three are co-regulated with H1-T6SS, namely vgrG1a/b/c. Whereas VgrG1a and VgrG1c were secreted in a ClpV1-dependent manner, secretion of VgrG1b was ClpV1-independent. We show that VgrG1a and VgrG1c form multimers, which confirmed the VgrG model predicting trimers similar to the tail spike. We demonstrate that Hcp1 secretion requires either VgrG1a or VgrG1c, which may act independently to puncture the bacterial envelope and give Hcp1 access to the surface. VgrG1b is not required for Hcp1 secretion. Thus, VgrG1b does not require H1-T6SS for secretion nor does H1-T6SS require VgrG1b for its function. Finally, we show that VgrG proteins are required for secretion of a genuine H1-T6SS substrate, Tse3. Our results demonstrate that VgrG proteins are not only secreted components but are essential for secretion of other T6SS substrates. Overall, we emphasize variability in behavior of three P. aeruginosa VgrGs, suggesting that, although very similar, distinct VgrGs achieve specific functions. PMID:21325275
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Chan, Dick C; Wong, Annette T Y; Pang, Jing; Barrett, P Hugh R; Watts, Gerald F
2015-03-01
Postprandial lipaemia, due to elevated plasma apolipoprotein (apo) B-48 concentrations, contributes to increased cardiovascular (CV) risk in obesity. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and apoC-III may play a role in regulating triacylglycerol-rich lipoprotein (TRL)-apoB-48 metabolism. We investigated the associations between plasma PCSK9 and apoC-III concentrations and the kinetics of apoB-48 in obese subjects. Seventeen obese subjects were given an oral fat load. ApoB-48 tracer/tracee ratios were measured after an intravenous 2H3-leucine administration using GC-MS. Kinetic parameters, including secretion and fractional catabolic rates (FCRs), were derived using a multi-compartmental model. Plasma PCSK9 and apoC-III concentrations were significantly and positively (P<0.05 in all) associated with the total area-under-curve (AUC) and incremental AUC for apoB-48 and inversely with TRL-apoB-48 FCR. Plasma PCSK9 and apoC-III concentrations were not correlated (P>0.05 in all) with basal secretion or the number of TRL-apoB-48 secreted over the postprandial period. In the stepwise regression analysis, plasma PCSK9 was the best predictor of the total and incremental AUCs for plasma apoB-48 and the FCR of TRL-apoB-48. The association between plasma PCSK9 and apoC-III and TRL-apoB-48 FCR remained significant (P<0.05 in all) after adjusting for age, homoeostasis model assessment (HOMA) score, hepatic lipase or lipoprotein lipase (LPL). In a multiple regression model, 31% of variance in TRL-apoB-48 FCR was accounted for by plasma PCSK9 and apoC-III concentrations (adjusted R2=0.306, P<0.05). However, their associations with TRL-apoB-48 FCR were not independent of each other. Our results suggest that the catabolism of TRL-apoB-48 in the postprandial state may be co-ordinated by PCSK9 and apoC-III in obese individuals.
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Chaves, Daniela Fojo Seixas; de Souza, Emanuel Maltempi; Monteiro, Rose Adele; de Oliveira Pedrosa, Fábio
2009-11-02
Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant-bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.
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PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System
Veith, Paul D.; Butler, Catherine A.; Nor Muhammad, Nor A.; Chen, Yu-Yen; Slakeski, Nada; Peng, Benjamin; Zhang, Lianyi; Dashper, Stuart G.; Cross, Keith J.; Cleal, Steven M.; Moore, Caroline; Reynolds, Eric C.
2016-01-01
Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS. PMID:27711252
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Dahms, Sven O.; Arciniega, Marcelino; Steinmetzer, Torsten; Huber, Robert; Than, Manuel E.
2016-01-01
Proprotein convertases (PCs) are highly specific proteases required for the proteolytic modification of many secreted proteins. An unbalanced activity of these enzymes is connected to pathologies like cancer, atherosclerosis, hypercholesterolaemia, and infectious diseases. Novel protein crystallographic structures of the prototypical PC family member furin in different functional states were determined to 1.8–2.0 Å. These, together with biochemical data and modeling by molecular dynamics calculations, suggest essential elements underlying its unusually high substrate specificity. Furin shows a complex activation mechanism and exists in at least four defined states: (i) the “off state,” incompatible with substrate binding as seen in the unliganded enzyme; (ii) the active “on state” seen in inhibitor-bound furin; and the respective (iii) calcium-free and (iv) calcium-bound forms. The transition from the off to the on state is triggered by ligand binding at subsites S1 to S4 and appears to underlie the preferential recognition of the four-residue sequence motif of furin. The molecular dynamics simulations of the four structural states reflect the experimental observations in general and provide approximations of the respective stabilities. Ligation by calcium at the PC-specific binding site II influences the active-site geometry and determines the rotamer state of the oxyanion hole-forming Asn295, and thus adds a second level of the activity modulation of furin. The described crystal forms and the observations of different defined functional states may foster the development of new tools and strategies for pharmacological intervention targeting furin. PMID:27647913
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III-V semiconductor solid solution single crystal growth
NASA Technical Reports Server (NTRS)
Gertner, E. R.
1982-01-01
The feasibility and desirability of space growth of bulk IR semiconductor crystals for use as substrates for epitaxial IR detector material were researched. A III-V ternary compound (GaInSb) and a II-VI binary compound were considered. Vapor epitaxy and quaternary epitaxy techniques were found to be sufficient to permit the use of ground based binary III-V crystals for all major device applications. Float zoning of CdTe was found to be a potentially successful approach to obtaining high quality substrate material, but further experiments were required.
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NASA Astrophysics Data System (ADS)
Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam
2017-10-01
In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.
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Fukuda, N; Ontko, J A
1984-08-01
In a series of experiments with male rat livers perfused with or without 5-tetradecyloxy-2-furoic acid (TOFA) in the presence and absence of oleate, the relationships between fatty acid synthesis, oxidation, and esterification from newly synthesized and exogenous fatty acid substrates have been examined. When livers from fed rats were perfused without exogenous fatty acid substrate, 20% of the triglyceride secreted was derived from de novo fatty acid synthesis. Addition of TOFA caused immediate and nearly complete inhibition of fatty acid synthesis, measured by incorporation of 3H2O into fatty acids. Concurrently, ketone body production increased 140% and triglyceride secretion decreased 84%. These marked reciprocal alterations in fatty acid synthesis and oxidation in the liver almost completely abolished the production of very low density lipoproteins (VLDL). Cholesterol synthesis was also depressed by TOFA, suggesting that this drug also inhibited lipid synthesis at a site other than acetyl-CoA carboxylase. When livers from fed rats were supplied with a continuous infusion of [1-14C]oleate as exogenous substrate, similar proportions, about 45-47%, of both ketone bodies and triglyceride in the perfusate were derived from the infused [1-14C]oleate. The production of ketone bodies was markedly increased by TOFA; the secretion of triglyceride and cholesterol were decreased. Altered conversion of [1-14C]oleate into these products occurred in parallel. While TOFA decreased esterification of oleate into triglyceride, incorporation of [1-14C]oleate into liver phospholipid was increased, indicating that TOFA also affected glycerolipid synthesis at the stage of diglyceride processing. The decreased secretion of triglyceride and cholesterol following TOFA treatment was localized almost exclusively in VLDL. The specific activities of 3H and of 14C fatty acids in triglyceride of the perfusate were greater than those of liver triglyceride, indicating preferential secretion of triglyceride produced from both de novo fatty acid synthesis and from infused free fatty acid substrate. These observations suggest the following chain of events in the liver following TOFA treatment: inhibition of fatty acid and cholesterol synthesis; increased fatty acid oxidation and ketogenesis; decreased triglyceride synthesis as a result of inhibition of fatty acid synthesis, stimulation of fatty acid oxidation, and altered partition of diglyceride between triglyceride and phospholipid synthesis; and decreased production of VLDL. These comparative rat liver perfusion experiments indicate that free fatty acids provide the major source of substrate for the hepatic production of triglyceri
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Gross, R L
1999-01-01
PURPOSE: This study was performed to better understand the differences between patients in specific components of wound healing as it may pertain to glaucoma filtration surgery, including the use of antimetabolites. METHODS: Human Tenon's capsule fibroblasts were obtained at the time of glaucoma filtering surgery and established in individual cell cultures from 35 glaucoma patients. The dose-response to 5-fluorouracil (5FU) and mitomycin C (MMC) was determined. The individual cell lines were exposed to the antimetabolites and ascorbic acid with measurement of collagen type I and III production by an ELISA-type dot blot assay. These results were then statistically compared to the individual patient characteristics including age, race, previous surgery and medications, and type of glaucoma. RESULTS: 5-FU had little effect on collagen type I and III production or protein synthesis. MMC had an inhibitory effect on collagen secretion and total protein synthesis with increasing concentration. Photomicrographs of the cells after each treatment condition revealed characteristic morphologic changes when compared to controls. There was a large range of collagen type I and III production with correlation between the amounts of each collagen type secreted in response to the antimetabolites. However, there was no correlation with accepted risk factors for filtration failure. CONCLUSION: These antimetabolites act similarly on different cell lines in a nonspecific manner. The results suggest that the increased risk of filtration failure due to age, race, diagnosis, and previous conjunctival surgery is not due to differences in secretion of collagen types I and III by Tenon's capsule fibroblasts. Images FIGURE 3 PMID:10703140
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Liu, Guanghua; Wang, Fen; Jiang, Jun; Yan, Zhaoli; Zhang, Dianxi; Zhang, Yinsheng
2016-01-01
Context: To date, all the familial hyperaldosteronism type III (FH-III) patients reported presenting with typical primary aldosteronism (PA), without showing other adrenal hormone abnormalities. Objective: This study characterized a novel phenotype of FH-III and explored the possible pathogenesis. Patients and Methods: A male patient presented with severe hypertension and hypokalemia at the age of 2 years and developed Cushing's syndrome at 20 years. He was diagnosed with PA and Cushing's syndrome on the basis of typical biochemical findings. He had massive bilateral adrenal hyperplasia and underwent left adrenalectomy. KCNJ5 was sequenced, and secretion of aldosterone and cortisol were observed both in vivo and in vitro. Results: A heterozygous germline p.Glu145Gln mutation of KCNJ5 was identified. ARMC5, PRKAR1A, PDE8B, PDE11A, and PRKACA genes and β-catenin, P53 immunoactivity were normal in the adrenal. CYP11B2 was highly expressed, whereas mRNA expression of CYP11B1, CYP17A1, and STAR was relatively low in the hyperplastic adrenal, compared with normal adrenal cortex and other adrenal diseases. In the primary cell culture of the resected hyperplastic adrenal, verapamil and nifedipine, two calcium channel blockers, markedly inhibited the secretion of both aldosterone and cortisol and the mRNA expression of CYP11B1, CYP11B2, CYP17A1, and STAR. Conclusions: We presented the first FH-III patient who had both severe PA and Cushing's syndrome. Hypersecretion of cortisol might be ascribed to overly large size of the hyperplastic adrenal because CYP11B1 expression was relatively low in his adrenal. Like aldosterone, synthesis and secretion of cortisol in the mutant adrenal may be mediated by voltage-gated Ca2+ channels. PMID:27403928
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Sundaram, Meenakshi; Zhong, Shumei; Bou Khalil, Maroun; Zhou, Hu; Jiang, Zhenghui G; Zhao, Yang; Iqbal, Jahangir; Hussain, M Mahmood; Figeys, Daniel; Wang, Yuwei; Yao, Zemin
2010-06-01
We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [(3)H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [(3)H]TAG from C3AT cells was markedly decreased. The lowered [(3)H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL(1). Moreover, [(3)H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [(3)H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [(3)H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL(1) maturation. Furthermore, lumenal [(35)S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL(1) maturation.
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Vascular trauma and prostacyclin release.
Jeremy, J Y; Mikhailidis, D P; Dandona, P
1984-10-01
The effect of trauma on prostacyclin (PGI2) secretion by rat aorta was examined. Whereas cutting and puncturing markedly increased PGI2 secretion, sonication and stretch had no effect. Cutting and puncturing were also effective in stimulating further endogenous secretion of PGI2 from 'exhausted' aortic segments in whom PGI2 production had dwindled to a negligible rate. These experiments show that trauma is an important stimulator of PGI2 secretion, the exhaustion of PGI2 secretion by vascular segments is not due to the depletion of substrate (arachidonic acid) and that an unusually traumatic venepuncture may invalidate the subsequent assays of PGI2 and its stable metabolite, 6-oxo-PGF1 alpha, in plasma. The close relationship of vascular trauma to PGI2 release also suggests a possible cytoprotective effect of this prostanoid on vascular endothelium.
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Reduction of structural Fe(III) in nontronite by methanogen Methanosarcina barkeri
Liu, D.; Dong, Hailiang H.; Bishop, M.E.; Wang, Hongfang; Agrawal, A.; Tritschler, S.; Eberl, D.D.; Xie, S.
2011-01-01
Clay minerals and methanogens are ubiquitous and co-exist in anoxic environments, yet it is unclear whether methanogens are able to reduce structural Fe(III) in clay minerals. In this study, the ability of methanogen Methanosarcina barkeri to reduce structural Fe(III) in iron-rich smectite (nontronite NAu-2) and the relationship between iron reduction and methanogenesis were investigated. Bioreduction experiments were conducted in growth medium using three types of substrate: H2/CO2, methanol, and acetate. Time course methane production and hydrogen consumption were measured by gas chromatography. M. barkeri was able to reduce structural Fe(III) in NAu-2 with H2/CO2 and methanol as substrate, but not with acetate. The extent of bioreduction, as measured by the 1,10-phenanthroline method, was 7-13% with H2/CO2 as substrate, depending on nontronite concentration (5-10g/L). The extent was higher when methanol was used as a substrate, reaching 25-33%. Methanogenesis was inhibited by Fe(III) reduction in the H2/CO2 culture, but enhanced when methanol was used. High charge smectite and biogenic silica formed as a result of bioreduction. Our results suggest that methanogens may play an important role in biogeochemical cycling of iron in clay minerals and may have important implications for the global methane budget. ?? 2010 Elsevier Ltd.
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GaN-based light-emitting diodes on various substrates: a critical review.
Li, Guoqiang; Wang, Wenliang; Yang, Weijia; Lin, Yunhao; Wang, Haiyan; Lin, Zhiting; Zhou, Shizhong
2016-05-01
GaN and related III-nitrides have attracted considerable attention as promising materials for application in optoelectronic devices, in particular, light-emitting diodes (LEDs). At present, sapphire is still the most popular commercial substrate for epitaxial growth of GaN-based LEDs. However, due to its relatively large lattice mismatch with GaN and low thermal conductivity, sapphire is not the most ideal substrate for GaN-based LEDs. Therefore, in order to obtain high-performance and high-power LEDs with relatively low cost, unconventional substrates, which are of low lattice mismatch with GaN, high thermal conductivity and low cost, have been tried as substitutes for sapphire. As a matter of fact, it is not easy to obtain high-quality III-nitride films on those substrates for various reasons. However, by developing a variety of techniques, distincts progress has been made during the past decade, with high-performance LEDs being successfully achieved on these unconventional substrates. This review focuses on state-of-the-art high-performance GaN-based LED materials and devices on unconventional substrates. The issues involved in the growth of GaN-based LED structures on each type of unconventional substrate are outlined, and the fundamental physics behind these issues is detailed. The corresponding solutions for III-nitride growth, defect control, and chip processing for each type of unconventional substrate are discussed in depth, together with a brief introduction to some newly developed techniques in order to realize LED structures on unconventional substrates. This is very useful for understanding the progress in this field of physics. In this review, we also speculate on the prospects for LEDs on unconventional substrates.
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Better Back Contacts for Solar Cells on Flexible Substrates
NASA Technical Reports Server (NTRS)
Woods, Lawrence M.; Ribelin, Rosine M.
2006-01-01
Improved low-resistance, semitransparent back contacts, and a method of fabricating them, have been developed for solar photovoltaic cells that are made from thin films of I-III-VI2 semiconductor materials on flexible, high-temperatureresistant polyimide substrates or superstrates. The innovative aspect of the present development lies in the extension, to polyimide substrates or superstrates, of a similar prior development of improved low-resistance, semitransparent back contacts for I-III-VI2 solar cells on glass substrates or superstrates. A cell incorporating this innovation can be used either as a stand-alone photovoltaic device or as part of a monolithic stack containing another photovoltaic device that utilizes light of longer wavelengths.
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Fiber optic detector and method for using same for detecting chemical species
Baylor, Lewis C.; Buchanan, Bruce R.
1995-01-01
An optical sensing device for uranyl and other substances, a method for making an optical sensing device and a method for chemically binding uranyl and other indicators to glass, quartz, cellulose and similar substrates. The indicator, such as arsenazo III, is immobilized on the substrate using a chemical binding process. The immobilized arsenazo III causes uranyl from a fluid sample to bind irreversibly to the substrate at its active sites, thus causing absorption of a portion of light transmitted through the substrate. Determination of the amount of light absorbed, using conventional means, yields the concentration of uranyl present in the sample fluid. The binding of uranyl on the substrate can be reversed by subsequent exposure of the substrate to a solution of 2,6-pyridinedicarboxylic acid. The chemical binding process is suitable for similarly binding other indicators, such as bromocresol green.
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Yao, Yufeng; Xie, Yi; Perace, Donna; Zhong, Yi; Lu, Jie; Tao, Jing; Guo, Xiaokui; Kim, Kwang Sik
2009-11-01
Type III secretion systems (T3SSs) have been documented in many Gram-negative bacteria, including enterohemorrhagic Escherichia coli. We have previously shown the existence of a putative T3SS in meningitis-causing E. coli K1 strains, referred to as E. coli type III secretion 2 (ETT2). The sequence of ETT2 in meningitis-causing E. coli K1 strain EC10 (O7:K1) revealed that ETT2 comprises the epr, epa and eiv genes, but bears mutations, deletions and insertions. We constructed the EC10 mutants deleted of ETT2 or eivA gene, and their contributions to bacterial pathogenesis were evaluated in human brain microvascular endothelial cells (HBMECs). The deletion mutant of ETT2 exhibited defects in invasion and intracellular survival compared with the parental E. coli K1 strain EC10. The mutant deleted of eivA within ETT2 was also significantly defective in invasion and intracellular survival in HBMECs, and the defects of the eiv mutant were restored to the levels of the parent strain EC10 by transcomplementation. These findings suggest that ETT2 plays a role in the pathogenesis of E. coli K1 infection, including meningitis.
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Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture
Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.
2009-01-01
Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357
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PPARδ inhibits UVB-induced secretion of MMP-1 through MKP-7-mediated suppression of JNK signaling.
Ham, Sun A; Kang, Eun S; Lee, Hanna; Hwang, Jung S; Yoo, Taesik; Paek, Kyung S; Park, Chankyu; Kim, Jin-Hoi; Lim, Dae-Seog; Seo, Han G
2013-11-01
In the present study, we investigated the role of peroxisome proliferator-activated receptor (PPAR) δ in modulating matrix-degrading metalloproteinases and other mechanisms underlying photoaging processes in the skin. In human dermal fibroblasts (HDFs), activation of PPARδ by its specific ligand GW501516 markedly attenuated UVB-induced secretion of matrix metalloproteinase (MMP)-1, concomitant with decreased generation of reactive oxygen species. These effects were significantly reduced in the presence of PPARδ small interfering RNA and GSK0660. Furthermore, c-Jun N-terminal kinase (JNK), but not p38 or extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-1 secretion in HDFs exposed to UVB. PPARδ-mediated messenger RNA stabilization of mitogen-activated protein kinase phosphatase (MKP)-7 was responsible for the GW501516-mediated inhibition of JNK signaling. Inhibition of UVB-induced secretion of MMP-1 by PPARδ was associated with the restoration of types I and III collagen to levels approaching those in cells not exposed to UVB. Finally, in HR-1 hairless mice exposed to UVB, administration of GW501516 significantly reduced wrinkle formation and skin thickness, downregulated MMP-1 and JNK phosphorylation, and restored the levels of MKP-7, types I and III collagen. These results suggest that PPARδ-mediated inhibition of MMP-1 secretion prevents some effects of photoaging and maintains the integrity of skin by inhibiting the degradation of the collagenous extracellular matrix.
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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.
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Lambeir, A M; Durinx, C; Proost, P; Van Damme, J; Scharpé, S; De Meester, I
2001-11-02
Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.
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The effect of V/III ratio on the morphology and structure of GaAs nanowires by MOCVD
NASA Astrophysics Data System (ADS)
Liu, Yan; Peng, Yan; Guo, Jingwei; La, Dongsheng; Xu, Zhaopeng
2018-05-01
In this paper, GaAs nanowires with different V/III ratios (70, 140, 280 and 560) were vertically grown from bottom to top on GaAs substrates by using metal organic chemical vapor deposition based on gold assisted vapor-liquid-solid mechanism. It is found that the growth rate of nanowires is inversely proportional to their V/III ratio. And the V/III ratio can also change nanowire growth type. For the nanowire with small V/III ratios (≤280), the reactants are most from those atoms merged in the catalyst. But, for the nanowire with V/III ratio 560, the contribution mainly comes from the diffusions of atoms pyrolyzed on the surface of the nanowire and the substrate. A shrunken neck under the catalyst is observed in TEM characterizations. These results will provide a theoretical basis for potential practical applications of nanowire-based devices.
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Stephenson, Jason R.; Stacey, Julie A.; Morgenthaler, Justin B.; Friesen, Jon A.; Lash, Timothy D.; Jones, Marjorie A.
2007-01-01
Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the Km value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher Km for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was ∼3% compared to wild-type CPO, with a threefold increase in the Km value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation. PMID:17242372
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Stephenson, Jason R; Stacey, Julie A; Morgenthaler, Justin B; Friesen, Jon A; Lash, Timothy D; Jones, Marjorie A
2007-03-01
Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.
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Brickman, Timothy J.; Cummings, Craig A.; Liew, Sin-Yee; Relman, David A.; Armstrong, Sandra K.
2011-01-01
Serological studies of patients with pertussis and the identification of antigenic Bordetella pertussis proteins support the hypothesis that B. pertussis perceives an iron starvation cue and expresses multiple iron source utilization systems in its natural human host environment. Furthermore, previous studies using a murine respiratory tract infection model showed that several of these B. pertussis iron systems are required for colonization and persistence and are differentially expressed over the course of infection. The present study examined genome-wide changes in B. pertussis gene transcript abundance in response to iron starvation in vitro. In addition to known iron source utilization genes, we identified a previously uncharacterized iron-repressed cytoplasmic membrane transporter system, fbpABC, that is required for the utilization of multiple structurally distinct siderophores including alcaligin, enterobactin, ferrichrome, and desferrioxamine B. Expression of type III secretion system genes was also found to be upregulated during iron starvation in both B. pertussis strain Tohama I and Bordetella bronchiseptica strain RB50. In a survey of type III secretion system protein production by an assortment of B. pertussis laboratory-adapted and low-passage clinical isolate strains, iron limitation increased the production and secretion of the type III secretion system-specific translocation apparatus tip protein Bsp22 in all Bvg-proficient strains. These results indicate that iron starvation in the infected host is an important environmental cue influencing not only Bordetella iron transport gene expression but also the expression of other important virulence-associated genes. PMID:21742863
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Veenendaal, Andreas K. J.; Sundin, Charlotta; Blocker, Ariel J.
2009-01-01
Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds. PMID:18996990
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Agrobacterium VirB10, an ATP energy sensor required for type IV secretion.
Cascales, Eric; Christie, Peter J
2004-12-07
Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa. Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate [T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] of the Agrobacterium tumefaciens VirB/D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel. Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility. VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation. Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9. We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 "bridge" between inner and outer membrane subassemblies of the VirB/D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A. tumefaciens cell envelope.
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Tomić, A; Berynskyy, M; Wade, R C; Tomić, S
2015-11-01
The experimentally determined structures of human dipeptidyl peptidase III (DPP III) for the wild-type protein and for the complex of its E451A mutant with the peptide substrate, tynorphin, differ significantly in their overall shape. The two domains of the enzyme are separated by a wide cleft in the structure of the ligand-free enzyme, while in the ligand-bound mutant they are very close to each other, and the protein structure is extremely compact. Here, we applied a range of molecular dynamics simulation techniques to investigate the DPP III conformational landscape and the influence of ligand binding on the protein structure and dynamics. We used conventional, accelerated and steered methods to simulate DPP III and its complexes with tynorphin and with the preferred, synthetic, substrate Arg-Arg-2-naphthylamide. We found that DPP III can adopt a number of different forms in solution. The compact forms are more stable, but the open and partially closed states, spanning a wide range of conformations, can more effectively recognize the substrate which preferentially binds to the five-stranded β-core of the lower DPP III domain. The simulations indicated the existence of a dynamic equilibrium between open and semi-closed states and revealed two ways that the protein can close, leading to two distinct compact structures. The way in which the protein closes depends on the presence of the ligand.
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Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition
Kumar, Prashant; Reithofer, Viktoria; Reisinger, Manuel; Wallner, Silvia; Pavkov-Keller, Tea; Macheroux, Peter; Gruber, Karl
2016-01-01
Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors. PMID:27025154
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Ford, David D.; Nielsen, Lars P. C.; Zuend, Stephan J.; Jacobsen, Eric N.
2013-01-01
In the (salen)Co(III)-catalyzed hydrolytic kinetic resolution (HKR) of terminal epoxides, the rate- and stereoselectivity-determining epoxide ring-opening step occurs by a cooperative bimetallic mechanism with one Co(III) complex acting as a Lewis acid and another serving to deliver the hydroxide nucleophile. In this paper, we analyze the basis for the extraordinarily high stereoselectivity and broad substrate scope observed in the HKR. We demonstrate that the stereochemistry of each of the two (salen)Co(III) complexes in the rate-determining transition structure is important for productive catalysis: a measurable rate of hydrolysis occurs only if the absolute stereochemistry of each of these (salen)Co(III) complexes is the same. Experimental and computational studies provide strong evidence that stereochemical communication in the HKR is mediated by the stepped conformation of the salen ligand, and not the shape of the chiral diamine backbone of the ligand. A detailed computational analysis reveals that the epoxide binds the Lewis acidic Co(III) complex in a well-defined geometry imposed by stereoelectronic, rather than steric effects. This insight serves as the basis of a complete stereochemical and transition structure model that sheds light on the reasons for the broad substrate generality of the HKR. PMID:24041239
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Ford, David D; Nielsen, Lars P C; Zuend, Stephan J; Musgrave, Charles B; Jacobsen, Eric N
2013-10-16
In the (salen)Co(III)-catalyzed hydrolytic kinetic resolution (HKR) of terminal epoxides, the rate- and stereoselectivity-determining epoxide ring-opening step occurs by a cooperative bimetallic mechanism with one Co(III) complex acting as a Lewis acid and another serving to deliver the hydroxide nucleophile. In this paper, we analyze the basis for the extraordinarily high stereoselectivity and broad substrate scope observed in the HKR. We demonstrate that the stereochemistry of each of the two (salen)Co(III) complexes in the rate-determining transition structure is important for productive catalysis: a measurable rate of hydrolysis occurs only if the absolute stereochemistry of each of these (salen)Co(III) complexes is the same. Experimental and computational studies provide strong evidence that stereochemical communication in the HKR is mediated by the stepped conformation of the salen ligand, and not the shape of the chiral diamine backbone of the ligand. A detailed computational analysis reveals that the epoxide binds the Lewis acidic Co(III) complex in a well-defined geometry imposed by stereoelectronic rather than steric effects. This insight serves as the basis of a complete stereochemical and transition structure model that sheds light on the reasons for the broad substrate generality of the HKR.
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Chandrashekharaiah, K S; Swamy, N Ramachandra; Murthy, K R Siddalinga
2011-12-01
Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB). Copyright © 2011 Elsevier Ltd. All rights reserved.
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Angiotensin II-producing enzyme III from acidified serum of nephrectomized dogs.
Haas, E; Lewis, L; Koshy, T J; Varde, A U; Renerts, L; Bagai, R C
1989-09-01
A highly active angiotensin-producing enzyme (enzyme III) was obtained from the serum of bilaterally nephrectomized dogs by acid treatment and ammonium sulfate fractionation. An inactive precursor (proenzyme III) was converted to enzyme III during prolonged storage (or by treatment with acid or with cathepsin G or by incubation at 38 degrees C as described in the following paper). Enzyme III reacted maximally at pH 7.7 and it produced up to 400 ng of angiotensin II/mL serum/h (i.e., amounts 4000 times higher than that generated by the endogenous renin present in serum after bilateral nephrectomy). Enzyme III produced angiotensin II at identical rates when either dog angiotensinogen or angiotensin I was used as substrate, but the rate was 710 times higher with synthetic tetradecapeptide renin substrate. Enzyme III is not identical to renin, cathepsin G, tonin, enzyme I, enzyme II, the calcium-dependent angiotensin I-converting enzyme, or the calcium-independent carboxy peptidase, which acts by sequential cleavage of angiotensin I. Enzyme III was inhibited by alpha-1-antitrypsin, diisopropyl fluorophosphate, and lima bean trypsin inhibitor (hence it is a serine proteinase). It was not inhibited by Captopril, Teprotide, or Enalapril. It had been reported previously that cathepsin G released from neutrophil granulocytes, by producing high local concentrations of angiotensin II, may provide a mobile means for modulating blood flow in tissue microvasculature during the inflammatory response. The present study offers a new, additional pathway, by enzyme III, for a similar rapid formation of angiotensin II from serum protein substrate or angiotensin I.
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USDA-ARS?s Scientific Manuscript database
In a recent study we demonstrated that in comparison to the wild-type enterohemorrhagic Escherichia coli (EHEC) O157:H7, a motility-compromised hha deletion mutant with an up-regulated type III secretion system and increased secretion of adherence proteins showed reduced fecal shedding in cattle. In...
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Oh, Chang-Sik; Carpenter, Sara C D; Hayes, Marshall L; Beer, Steven V
2010-04-01
DspA/E is a type III effector of Erwinia amylovora, the bacterial pathogen that causes fire blight disease in roseaceous plants. This effector is indispensable for disease development, and it is translocated into plant cells. A DspA/E-specific chaperone, DspB/F, is necessary for DspA/E secretion and possibly for its translocation. In this work, DspB/F-binding sites and secretion and translocation signals in the DspA/E protein were determined. Based on yeast two-hybrid assays, DspB/F was found to bind DspA/E within the first 210 amino acids of the protein. Surprisingly, both DspB/F and OrfA, the putative chaperone of Eop1, also interacted with the C-terminal 1059 amino acids of DspA/E; this suggests another chaperone-binding site. Secretion and translocation assays using serial N-terminal lengths of DspA/E fused with the active form of AvrRpt2 revealed that at least the first 109 amino acids, including the first N-terminal chaperone-binding motif and DspB/F, were required for efficient translocation of DspA/E, although the first 35 amino acids were sufficient for its secretion and the presence of DspB/F was not required. These results indicate that secretion and translocation signals are present in the N terminus of DspA/E, and that at least one DspB/F-binding motif is required for efficient translocation into plant cells.
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Norepinephrine is coreleased with serotonin in mouse taste buds.
Huang, Yijen A; Maruyama, Yutaka; Roper, Stephen D
2008-12-03
ATP and serotonin (5-HT) are neurotransmitters secreted from taste bud receptor (type II) and presynaptic (type III) cells, respectively. Norepinephrine (NE) has also been proposed to be a neurotransmitter or paracrine hormone in taste buds. Yet, to date, the specific stimulus for NE release in taste buds is not well understood, and the identity of the taste cells that secrete NE is not known. Chinese hamster ovary cells were transfected with alpha(1A) adrenoceptors and loaded with fura-2 ("biosensors") to detect NE secreted from isolated mouse taste buds and taste cells. Biosensors responded to low concentrations of NE (>or=10 nm) with a reliable fura-2 signal. NE biosensors did not respond to stimulation with KCl or taste compounds. However, we recorded robust responses from NE biosensors when they were positioned against mouse circumvallate taste buds and the taste buds were stimulated with KCl (50 mm) or a mixture of taste compounds (cycloheximide, 10 microm; saccharin, 2 mm; denatonium, 1 mm; SC45647, 100 microm). NE biosensor responses evoked by stimulating taste buds were reversibly blocked by prazosin, an alpha(1A) receptor antagonist. Together, these findings indicate that taste bud cells secrete NE when they are stimulated. We isolated individual taste bud cells to identify the origin of NE release. NE was secreted only from presynaptic (type III) taste cells and not receptor (type II) cells. Stimulus-evoked NE release depended on Ca(2+) in the bathing medium. Using dual biosensors (sensitive to 5-HT and NE), we found all presynaptic cells secrete 5-HT and 33% corelease NE with 5-HT.
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Oinen, Mark Emil; Yu, Robert T.; Rovis, Tomislav
2009-01-01
Excess substrate has been identified as an unintended spectator ligand affecting enantioselectivity in the [2+2+2] cycloaddition of alkenyl isocyanates with tolanes. Replacement of excess substrate with an exogenous additive affords products with consistent and higher ee’s. The increase in enantioselectivity is the result of a change in composition of a proposed rhodium(III) intermediate on the catalytic cycle. The net result is a rational probe of a short-lived rhodium(III) intermediate, and gives insight that may have applications in many rhodium catalyzed reactions. PMID:19803471
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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.
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Brennan, T.M.; Hammons, B.E.; Tsao, J.Y.
1992-12-15
A method for on-line accurate monitoring and precise control of molecular beam epitaxial growth of Groups III-III-V or Groups III-V-V layers in an advanced semiconductor device incorporates reflection mass spectrometry. The reflection mass spectrometry is responsive to intentional perturbations in molecular fluxes incident on a substrate by accurately measuring the molecular fluxes reflected from the substrate. The reflected flux is extremely sensitive to the state of the growing surface and the measurements obtained enable control of newly forming surfaces that are dynamically changing as a result of growth. 3 figs.
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Brennan, Thomas M.; Hammons, B. Eugene; Tsao, Jeffrey Y.
1992-01-01
A method for on-line accurate monitoring and precise control of molecular beam epitaxial growth of Groups III-III-V or Groups III-V-V layers in an advanced semiconductor device incorporates reflection mass spectrometry. The reflection mass spectrometry is responsive to intentional perturbations in molecular fluxes incident on a substrate by accurately measuring the molecular fluxes reflected from the substrate. The reflected flux is extremely sensitive to the state of the growing surface and the measurements obtained enable control of newly forming surfaces that are dynamically changing as a result of growth.
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Assessing the ability of Salmonella enterica to translocate Type III effectors into plant cells
USDA-ARS?s Scientific Manuscript database
Salmonella enterica, a human enteric pathogen, has the ability to multiply and survive endophytically in plants, and mutations in genes encoding the type III secretion system (T3SS) or its effectors (T3Es) may contribute to this colonization. Two reporter plasmids for T3E translocation into plant ce...
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Structural Characterization of the N Terminus of IpaC from Shigella flexneri
Harrington, Amanda T.; Hearn, Patricia D.; Picking, Wendy L.; Barker, Jeffrey R.; Wessel, Andrew; Picking, William D.
2003-01-01
The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere. PMID:12595440
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Chen, Chen; Gao, George F.
2012-01-01
A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans. PMID:23056296
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Deformable L-shaped microwell array for trapping pairs of heterogeneous cells
NASA Astrophysics Data System (ADS)
Lee, Gi-Hun; Kim, Sung-Hwan; Kang, AhRan; Takayama, Shuichi; Lee, Sang-Hoon; Park, Joong Yull
2015-03-01
To study cell-to-cell interactions, there has been a continuous demand on developing microsystems for trapping pairs of two different cells in microwell arrays. Here, we propose an L-shaped microwell (L-microwell) array that relies on the elasticity of a polydimethylsiloxane (PDMS) substrate for trapping and pairing heterogeneous cells. We designed an L-microwell suitable for trapping single cell in each branch via stretching/releasing the PDMS substrate, and also performed 3D time-dependent diffusion simulations to visualize how cell-secreted molecules diffuse in the L-microwell and communicate with the partner cell. The computational results showed that the secreted molecule first contacted the partner cell after 35 min, and the secreted molecule fully covered the partner cell in 4 h (when referenced to 10% of the secreted molecular concentration). The molecules that diffused to the outside of the L-microwell were significantly diluted by the bulk solution, which prevented unwanted cellular communication between neighboring L-microwells. We produced over 5000 cell pairs in one 2.25 cm2 array with about 30 000 L-microwells. The proposed L-microwell array offers a versatile and convenient cell pairing method to investigate cell-to-cell interactions in, for example, cell fusion, immune reactions, and cancer metastasis.
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Yersinia Type III Secretion System Master Regulator LcrF
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
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Libério, Michelle da Silva; Bastos, Izabela M. D.; Pires Júnior, Osmindo R.; Fontes, Wagner; Santana, Jaime M.; Castro, Mariana S.
2014-01-01
Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology. PMID:24906116
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Ates, Louis S.; van der Woude, Aniek D.; Bestebroer, Jovanka; van Stempvoort, Gunny; Musters, René J. P.; Garcia-Vallejo, Juan J.; Picavet, Daisy I.; van de Weerd, Robert; Maletta, Massimiliano; Kuijl, Coenraad P.; van der Wel, Nicole N.; Bitter, Wilbert
2016-01-01
Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation. PMID:27280885
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Gu, Shuang; Shevchik, Vladimir E; Shaw, Rosie; Pickersgill, Richard W; Garnett, James A
2017-10-01
Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS. The central role of intrinsic disorder-order transitions in these processes may be a particular feature of type II secretion. Copyright © 2017 Elsevier B.V. All rights reserved.
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Truchan, Hilary K; Christman, Harry D; White, Richard C; Rutledge, Nakisha S; Cianciotto, Nicholas P
2017-06-20
Legionella pneumophila replicates in macrophages in a host-derived phagosome, termed the Legionella- containing vacuole (LCV). While the translocation of type IV secretion (T4S) effectors into the macrophage cytosol is well established, the location of type II secretion (T2S) substrates in the infected host cell is unknown. Here, we show that the T2S substrate ProA, a metalloprotease, translocates into the cytosol of human macrophages, where it associates with the LCV membrane (LCVM). Translocation is detected as early as 10 h postinoculation (p.i.), which is approximately the midpoint of the intracellular life cycle. However, it is detected as early as 6 h p.i. if ProA is hyperexpressed, indicating that translocation depends on the timing of ProA expression and that any other factors necessary for translocation are in place by that time point. Translocation occurs with all L. pneumophila strains tested and in amoebae, natural hosts for L. pneumophila It was absent in murine bone marrow-derived macrophages and murine macrophage cell lines. The ChiA chitinase also associated with the cytoplasmic face of the LCVM at 6 h p.i. and in a T2S-dependent manner. Galectin-3 and galectin-8, eukaryotic proteins whose localization is influenced by damage to host membranes, appeared within the LCV of infected human but not murine macrophages beginning at 6 h p.i. Thus, we hypothesize that ProA and ChiA are first secreted into the vacuolar lumen by the activity of the T2S and subsequently traffic into the macrophage cytosol via a novel mechanism that involves a semipermeable LCVM. IMPORTANCE Infection of macrophages and amoebae plays a central role in the pathogenesis of L. pneumophila , the agent of Legionnaires' disease. We have previously demonstrated that the T2S system of L. pneumophila greatly contributes to intracellular infection. However, the location of T2S substrates within the infected host cell is unknown. This report presents the first evidence of a L. pneumophila T2S substrate in the host cell cytosol and, therefore, the first evidence of a non-T4S effector trafficking out of the LCV. We also provide the first indication that the LCV is not completely intact but is instead semipermeable and that this occurs in human but not murine macrophages. Given this permeability, we hypothesize that other T2S substrates and LCV lumenal contents can escape into the host cell cytosol. Thus, these substrates may represent a battery of previously unidentified effectors that can interact with host factors and contribute to intracellular infection by L. pneumophila . Copyright © 2017 Truchan et al.
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Na Ayutthaya, Pratchaya Pramoj; Chanchao, Chanpen; Chunsrivirot, Surasak
2018-01-01
Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.
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Amyot, Whitney M.; deJesus, Dennise
2013-01-01
Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system. PMID:23798536
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Cline, Gary W., E-mail: gary.cline@yale.edu; Department of Surgery, University of Minnesota-Twin Cities, Minneapolis, MN 55455; Pongratz, Rebecca L.
Highlights: Black-Right-Pointing-Pointer We studied media effects on mechanisms of insulin secretion of INS-1 cells. Black-Right-Pointing-Pointer Insulin secretion was higher in DMEM than KRB despite identical ATP synthesis rates. Black-Right-Pointing-Pointer Insulin secretion rates correlated with rates of anaplerosis and TCA cycle. Black-Right-Pointing-Pointer Mitochondria metabolism and substrate cycles augment secretion signal of ATP. -- Abstract: Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as amore » surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with {sup 31}P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by {sup 13}C NMR isotopomer analysis of the fate of [U-{sup 13}C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media.« less
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Zhang, Chao; Shan, Liwei; Su, Shuaikun; Nan, Yanni; Guo, Zhongyu; Fan, Sanhong
2012-07-01
Wheat grain peroxidase 1 (WP1) belonged to class III plant peroxidase with cofactor heme, which not only has antifungal activity, but also influences the processing quality of flour. In order to enhance functional expression of WP1 in prokaryotic system by increasing endogenous heme synthesis, we constructed a recombinant plasmid pACYC-A-L containing hemA and hemL of Esherichia coli. Then, we co-transformed it into host strain T7 Express with secretive expression vector (pMAL-p4x-WP1) or non-secretive expression vector (pET21a-MBP-WP1), respectively. The MBP-WP1 fusion protein was further purified by amylose affinity chromatography and its peroxidase activity was assayed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate. At 12 h after induction at 28 degree, the extracellular 5-aminolevulinic acid (5-ALA) production of T7 Express/pACYC-A-L was up to 146.73 mg/L, simultaneously the extracellular porphrins also increased dramatically. The peroxidase activity of functional MBP-WP1 obtained from T7 Express/ (pACYC-A-L + pMAL-p4x-WP1) was 14.6-folds of that purified from T7 Express/ pET21a-MBP-WP1. This study not only successfully enhanced functional expression of wheat peroxidase 1 in Esherichia coli, but also provided beneficial references for other important proteins with cofactor heme.
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Computational and Experimental Analysis of the Secretome of Methylococcus capsulatus (Bath)
Indrelid, Stine; Mathiesen, Geir; Jacobsen, Morten; Lea, Tor; Kleiveland, Charlotte R.
2014-01-01
The Gram-negative methanotroph Methylococcus capsulatus (Bath) was recently demonstrated to abrogate inflammation in a murine model of inflammatory bowel disease, suggesting interactions with cells involved in maintaining mucosal homeostasis and emphasizing the importance of understanding the many properties of M. capsulatus. Secreted proteins determine how bacteria may interact with their environment, and a comprehensive knowledge of such proteins is therefore vital to understand bacterial physiology and behavior. The aim of this study was to systematically analyze protein secretion in M. capsulatus (Bath) by identifying the secretion systems present and the respective secreted substrates. Computational analysis revealed that in addition to previously recognized type II secretion systems and a type VII secretion system, a type Vb (two-partner) secretion system and putative type I secretion systems are present in M. capsulatus (Bath). In silico analysis suggests that the diverse secretion systems in M.capsulatus transport proteins likely to be involved in adhesion, colonization, nutrient acquisition and homeostasis maintenance. Results of the computational analysis was verified and extended by an experimental approach showing that in addition an uncharacterized protein and putative moonlighting proteins are released to the medium during exponential growth of M. capsulatus (Bath). PMID:25479164
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Substrate effects on endothelial cell adherence rates.
Scott, W J; Mann, P
1990-01-01
Endothelial cell attachment to a synthetic substrate was studied using an in vitro model system. Attachment rate was defined as the number of tritium-labeled endothelial cells attached to a synthetic substrate after 30 minutes. The surface of the synthetic substrate was chemically modified with either laminin or fibronectin. Labeled endothelial cells attached more rapidly to synthetic substrate, chemically modified with biomolecules, as compared with the untreated substrate controls. Unlabeled endothelial cells were grown to confluency on a second set of modified and untreated substrates. The cells were removed with 1% Triton, and the rate of re-endothelialization with tritium-labeled endothelial cells was determined. The rate was 11-13 times that of the same cells on untreated substrate. These data confirm that biomolecules increase the attachment rate of endothelial cells to synthetic substrate, and also suggest that endothelial cells may secrete a Triton-insoluble product (Sigma, St. Louis, MO) into subendothelial matrix that increases re-endothelialization.
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Subcellular localization of pituitary enzymes
NASA Technical Reports Server (NTRS)
Smith, R. E.
1970-01-01
A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.
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Jia, Bin; Song, Yanzi; Wu, Min; Lin, Baicheng; Xiao, Kang; Hu, Zhangli; Huang, Ying
2016-01-01
Microalgae biofuel has become the most promising renewable energy over the past few years. But limitations still exist because of its high cost. Although, efforts have been made in enhancement of lipid productivity, the major cost problem in harvesting and oil extraction is still intractable. Thus, the idea of fatty acids (FAs) secretion which can massively facilitate algae harvesting and oil extraction was investigated here. The cDNAs of two long-chain acyl-CoA synthetases (LACSs) genes were cloned from Chlamydomonas reinhardtii and named as cracs1 and cracs2. They showed different substrate adaptation in the yeast complementation experiments. Cracs2 could utilize FAs C12:0, C14:0, C16:0, C18:0, C16:1 and C18:1, while crac1 could only utilize substrate C14:0, C16:1 and C18:1. Knockdown of cracs1 and cracs2 in C. reinhardtii resulted in accumulation of intracellular lipids. The total intracellular lipids contents of transgenic algae q-15 (knockdown of cracs1) and p-13 (knockdown of cracs2) were 45 and 55 %, respectively higher than that of cc849. Furthermore, FAs secretion was discovered in both transgenic algae. Secreted FAs can reach 8.19 and 9.66 mg/10(9) cells in q-15 and p-13, respectively. These results demonstrated the possibility of FAs secretion by microalgae and may give a new strategy of low-cost oil extraction. According to our findings, we proposed that FAs secretion may also be achieved in other species besides Chlamydomonas reinhardtii by knocking-down cracs genes, which may promote the future industrial application of microalgae biofuels.
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Progressive quality control of secretory proteins in the early secretory compartment by ERp44
Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto
2014-01-01
ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval via interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here we show that also conserved histidines in the C-terminal tail regulate ERp44 in vivo. Mutants lacking these histidines are hyperactive in retaining substrates. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon over-expression of different partners. The ensuing gradients may help optimising folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. PMID:25097228
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Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph
2017-01-03
The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio
2012-01-01
Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182
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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
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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
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini
2012-03-26
Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SSs) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) that is responsible for over one million deaths per year. The Shigella type III secretion apparatus is composed of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC.more » While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 {angstrom} and 2.8 {angstrom} limiting resolution, respectively. These newly identified domains are composed of extended-length (114 {angstrom} in IpaB and 71 {angstrom} in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably, colicin Ia. This suggests that these mechanistically separate and functionally distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events.« less
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Hawken, P A R; Luckins, N; Tilbrook, A; Fiol, C; Martin, G B; Blache, D
2013-01-01
We investigated the effect of genetic selection for temperament on the way that stressors affect the behaviour and the adrenal and reproductive axes of sheep. We tested three hypotheses: (i) isolation would increase cortisol secretion and decrease luteinising hormone (LH) secretion more in nervous sheep than in calm sheep; (ii) isolation combined with simulated human presence would increase cortisol secretion and decrease LH secretion more in nervous sheep than in calm sheep and (iii) isolation combined with stressors that were not specific to the selection process (i.e. non-selection stressors) would increase cortisol secretion and decrease LH secretion equally in calm and nervous sheep. Isolation alone increased cortisol secretion and decreased LH secretion in nervous sheep but not in calm sheep. Compared to calm sheep, nervous sheep were more agitated during the first 2 h of isolation but not during the second 2 h of isolation. Exposure to non-selection stressors increased cortisol secretion, decreased LH pulse amplitude and the mean plasma concentrations of LH in both calm and nervous sheep. We conclude that genetic selection for temperament affects the behavioural expression of the stress response and the secretion of adrenal and reproductive hormones during isolation, but has less impact on their reactivity to non-selection stressors.
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Effect of Parainfluenza-3 Neuraminidase on Bovine Nasal Secretion
Morein, Bror; Bergman, Rune
1972-01-01
Three samples of bovine nasal secretion were each separated into a sol phase and a surface gel phase. In all samples, the gel phase contained an approximately four times greater amount of bound N-acetylneuraminic acid (NANA) than the sol phase. From the gel phase, bound NANA could be released by exposure to parainfluenza-3 virus neuraminidase. The surface gel appears to be a natural substrate for this enzyme. PMID:4347547
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DBSecSys: A Database of Burkholderia mallei Secretion Systems
2014-07-16
toxins and the lipases, as well as non-proteinaceous substrates, e.g., cyclic β- glucans and polysaccharides. 2* - Represents a Sec/Tat-dependent system...divided into three types: 1) the archetypal bacterial proteins exported into the periplasm via the Sec system; 2) trimeric proteins with a single beta ...barrel domain; and 3) pairs of proteins in which one partner carries the beta barrel domain and the other partner is the secreted protein. 6
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Adsorption of biometals to monosodium titanate in biological environments
DOE Office of Scientific and Technical Information (OSTI.GOV)
HOBBS, D.T.; MESSER, R. L. W.; LEWIS, J. B.
2005-06-06
Monosodium titanate (MST) is an inorganic sorbent/ion exchanger developed for the removal of radionuclides from nuclear wastes. We investigated the ability of MST to bind Cd(II), Hg(II), or Au(III) to establish the utility of MST for applications in environmental decontamination or medical therapy (drug delivery). Adsorption isotherms for MST were determined at pH 7-7.5 in water or phosphate-buffered saline. The extent of metal binding was determined spectroscopically by measuring the concentrations of the metals in solution before and after contact with the MST. Cytotoxic responses to MST were assessed using THP1 monocytes and succinate dehydrogenase activity. Monocytic activation by MSTmore » was assessed by TNF{alpha} secretion (ELISA) with or without lipopolysaccharide (LPS) activation. MST sorbed Cd(II), Hg(II), and Au(III) under conditions similar to that in physiological systems. MST exhibited the highest affinity for Cd(II) followed by Hg(II) and Au (III). MST (up to 100 mg/L) exhibited only minor (< 25% suppression of succinate dehydrogenase) cytotoxicity and did not trigger TNF{alpha} secretion nor modulate LPS-induced TNF{alpha} secretion from monocytes. MST exhibits high affinity for biometals with no significant biological liabilities in these introductory studies. MST deserves further scrutiny as a substance with the capacity to decontaminate biological environments or deliver metals in a controlled fashion.« less
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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
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Cormet-Boyaka, Estelle; Huneau, Jean-François; Mordrelle, Agnès; Boyaka, Prosper N.; Carbon, Claude; Rubinstein, Ethan; Tomé, Daniel
1998-01-01
The mechanism of intestinal secretion of the difluorinated quinolone sparfloxacin was investigated with the epithelial cell line Caco-2 and was compared to that of the P-glycoprotein (P-gp) substrate vinblastine. The P-gp inhibitors verapamil and progesterone significantly increased the epithelial cell accumulation of both vinblastine and sparfloxacin. This increase is likely to result from an inhibition of drug secretion since both vinblastine uptake and sparfloxacin uptake are known to proceed through a passive transmembrane diffusion. The unidirectional fluxes across cell monlayers grown on permeable filters indicated that a net secretion of sparfloxacin and vinblastine occurred across Caco-2 cells. These secretions were significantly inhibited by the MDR-reversing agent verapamil. We conclude that the P-gp is likely to be involved in the intestinal elimination of the difluorinated quinolone sparfloxacin. PMID:9756763
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Zhou, Xiaohui; Addwebi, Tarek; Davis, Margaret A.; Orfe, Lisa; Call, Douglas R.; Guard, Jean; Besser, Thomas E.
2011-01-01
Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins. PMID:21292746
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Bergagnini-Kolev, Mackenzie C; Hebert, Mary F; Easterling, Thomas R; Lin, Yvonne S
2017-03-01
N 1 -methylnicotinamide (1-NMN) has been investigated as an endogenous probe for the renal transporter activity of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2-K (MATE1 and MATE2-K). As pregnancy increased the renal secretion of metformin, a substrate for OCT2, MATE1, and MATE2-K, we hypothesized that the renal secretion of 1-NMN would be similarly affected. Blood and urine samples collected from women prescribed metformin for type 2 diabetes, gestational diabetes, and polycystic ovarian syndrome during early, mid, and late pregnancy ( n = 34 visits) and postpartum ( n = 14 visits) were analyzed for 1-NMN using liquid chromatography-mass spectrometry. The renal clearance and secretion clearance, using creatinine clearance to correct for glomerular filtration, were estimated for 1-NMN and correlated with metformin renal clearance. 1-NMN renal clearance was higher in both mid (504 ± 293 ml/min, P < 0.01) and late pregnancy (557 ± 305 ml/min, P < 0.01) compared with postpartum (240 ± 106 ml/min). The renal secretion of 1-NMN was 3.5-fold higher in mid pregnancy (269± 267, P < 0.05) and 4.5-fold higher in late pregnancy compared with postpartum (342 ± 283 versus 76 ± 92 ml/min, P < 0.01). Because creatinine is also a substrate of OCT2, MATE1, and MATE2-K, creatinine clearance likely overestimates filtration clearance, whereas the calculated 1-NMN secretion clearance is likely underestimated. Metformin renal clearance and 1-NMN renal clearance were positively correlated (r s = 0.68, P < 0.0001). 1-NMN renal clearance increases during pregnancy due to increased glomerular filtration and net secretion by renal transporters. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.
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Fritsch, Maximilian J.; Trunk, Katharina; Diniz, Juliana Alcoforado; Guo, Manman; Trost, Matthias; Coulthurst, Sarah J.
2013-01-01
It has recently become apparent that the Type VI secretion system (T6SS) is a complex macromolecular machine used by many bacterial species to inject effector proteins into eukaryotic or bacterial cells, with significant implications for virulence and interbacterial competition. “Antibacterial” T6SSs, such as the one elaborated by the opportunistic human pathogen, Serratia marcescens, confer on the secreting bacterium the ability to rapidly and efficiently kill rival bacteria. Identification of secreted substrates of the T6SS is critical to understanding its role and ability to kill other cells, but only a limited number of effectors have been reported so far. Here we report the successful use of label-free quantitative mass spectrometry to identify at least eleven substrates of the S. marcescens T6SS, including four novel effector proteins which are distinct from other T6SS-secreted proteins reported to date. These new effectors were confirmed as antibacterial toxins and self-protecting immunity proteins able to neutralize their cognate toxins were identified. The global secretomic study also unexpectedly revealed that protein phosphorylation-based post-translational regulation of the S. marcescens T6SS differs from that of the paradigm, H1-T6SS of Pseudomonas aeruginosa. Combined phosphoproteomic and genetic analyses demonstrated that conserved PpkA-dependent threonine phosphorylation of the T6SS structural component Fha is required for T6SS activation in S. marcescens and that the phosphatase PppA can reverse this modification. However, the signal and mechanism of PpkA activation is distinct from that observed previously and does not appear to require cell–cell contact. Hence this study has not only demonstrated that new and species-specific portfolios of antibacterial effectors are secreted by the T6SS, but also shown for the first time that PpkA-dependent post-translational regulation of the T6SS is tailored to fit the needs of different bacterial species. PMID:23842002
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Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I; Hill, Christopher P
2015-05-22
The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I.; Hill, Christopher P.
2015-01-01
The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. PMID:25833946
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.
Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.
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Prevalence of type III secretion system in effective biocontrol pseudomonads.
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.
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Tsai, Chi -Lin; Burkinshaw, Brianne J.; Strynadka, Natalie C. J.; ...
2014-12-08
Bacteria hijack eukaryotic cells by injecting virulence effectors into host cytosol with a type III secretion system (T3SS). Effectors are targeted with their cognate chaperones to hexameric T3SS ATPase at the bacterial membrane's cytosolic face. In this issue of the Journal of Bacteriology, Roblin et al. (P. Roblin, F. Dewitte, V. Villeret, E. G. Biondi, and C. Bompard, J Bacteriol 197:688–698, 2015, http://dx.doi.org/10.1128/JB.02294-14) show that the T3SS chaperone SigE of Salmonella can form hexameric rings rather than dimers when bound to its cognate effector, SopB, implying a novel multimeric association for chaperone/effector complexes with their ATPase.
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Goodrum, K J
1987-01-01
Complement levels and complement activation are key determinants in streptococcus-induced inflammatory responses. Activation of macrophage functions, such as complement synthesis, by group B streptococci (GBS) was examined as a possible component of GBS-induced chronic inflammation. Using an enzyme-linked immunosorbent assay, secreted C3 from mouse macrophagelike cell lines (PU5-1.8 and J774A.1) was monitored after cultivation with GBS. Whole, heat-killed GBS (1 to 10 CFU per macrophage) of both type Ia and III strains induced 25 to 300% increases in secreted C3 in both cell lines after a 24-h cultivation. GBS-treated cell lines exhibited increases in secreted lysozyme (10%) and in cellular protein (25 to 50%). Inhibition of macrophage phagocytosis by cytochalasin B inhibited GBS stimulation of C3. Purified cell walls of GBS type III strain 603-79 (1 to 10 micrograms/ml) also enhanced C3 synthesis. Local enhancement of macrophage C3 production by ingested streptococci or by persistent cell wall antigens may serve to promote chronic inflammatory responses. PMID:3552987
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[Cephalic phase of insulin secretion and the variety of the food in rat feed].
Louis-Sylvestre, J; Le Magnen, J
1983-01-01
Immunologically reactive insulin levels were determined in freely-moving normal rats offered three different test-meals. In test I, they were offered their normal diet for 4.5 min. In test II, they first ate the same diet for the same amount of time, then a cookie for 6 min, and finally they had free access to lard. In test III, the normal diet was followed by a synthetic sweetener and then by vaseline. It was shown that in a varied meal (tests II and III), the ingestion of each new food was immediately followed by a peak of insulin secretion superimposed on normal postprandial hyperinsulinemia. This resulted in an overall increase in insulinemia over the 15-min period measured. It is well known that in a varied meal more food is consumed. Thus, it may be that when a varied meal is offered, final satiety is postponed. This could be due to sensorially-triggered peaks of insulin secretion which would explain why the "cafeteria diet" induces hyperphagia and obesity.
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Type III secretion system effector proteins: double agents in bacterial disease and plant defense.
Alfano, James R; Collmer, Alan
2004-01-01
Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.
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Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation.
Bahrani, F K; Sansonetti, P J; Parsot, C
1997-01-01
The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins. PMID:9316999
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Molecular modeling of fibronectin adsorption on topographically nanostructured rutile (110) surfaces
NASA Astrophysics Data System (ADS)
Guo, Chuangqiang; Wu, Chunya; Chen, Mingjun; Zheng, Ting; Chen, Ni; Cummings, Peter T.
2016-10-01
To investigate the topographical dependency of protein adsorption, molecular dynamics simulations were employed to describe the adsorption behavior of the tenth type-III module of fibronectin (FN-III10) on nanostructured rutile (110) surfaces. The results indicated that the residence time of adsorbed FN-III10 largely relied on its binding mode (direct or indirect) with the substrate and the region for protein migration on the periphery (protrusion) or in the interior (cavity or groove) of nanostructures. In the direct binding mode, FN-III10 molecules were found to be 'trapped' at the anchoring sites of rutile surface, or even penetrate deep into the interior of nanostructures, regardless of the presented geometrical features. In the indirect binding mode, FN-III10 molecules were indirectly connected to the substrate via a hydrogen-bond network (linking FN-III10 and interfacial hydrations). The facets created by nanostructures, which exerted restraints on protein migration, were suggested to play an important role in the stability of indirect FN-III10-rutile binding. However, a doubly unfavorable situation - indirect FN-III10-rutile connections bridged by a handful of mediating waters and few constraints on movement of protein provided by nanostructures - would result in an early desorption of protein.
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Vertical III-nitride thin-film power diode
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wierer, Jr., Jonathan; Fischer, Arthur J.; Allerman, Andrew A.
2017-03-14
A vertical III-nitride thin-film power diode can hold off high voltages (kV's) when operated under reverse bias. The III-nitride device layers can be grown on a wider bandgap template layer and growth substrate, which can be removed by laser lift-off of the epitaxial device layers grown thereon.
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Faponle, Abayomi S; Quesne, Matthew G; Sastri, Chivukula V; Banse, Frédéric; de Visser, Sam P
2015-01-01
Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high-valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O–O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L52). Direct C–O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L52)FeIII(OOH)]2+ should be able to initiate an aromatic hydroxylation process, although a low-energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O–O bond, whereas a heterolytic O–O bond breaking in heme iron(III)–hydroperoxo is found. PMID:25399782
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Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis.
Mitchell, Anthony; Tam, Christina; Elli, Derek; Charlton, Thomas; Osei-Owusu, Patrick; Fazlollahi, Farbod; Faull, Kym F; Schneewind, Olaf
2017-05-16
Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys 273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys 273 Ala in lcrV Moreover, the lcrV C273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys 273 Ala substitution. Furthermore, macrophages infected by the lcrV C273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB , which encodes glutathione synthetase of Y. pestis , resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis Δ gshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione. IMPORTANCE Yersinia pestis , the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis. Copyright © 2017 Mitchell et al.
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Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon
2017-12-01
Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Kitamura, Akira; Ishida, Yoshihito; Kubota, Hiroshi; Pack, Chan-Gi; Homma, Takayuki; Ito, Shinya; Araki, Kazutaka; Kinjo, Masataka; Nagata, Kazuhiro
2018-02-26
Heat shock protein 47 kDa (HSP47), an ER-resident and collagen-specific molecular chaperone, recognizes collagenous hydrophobic amino acid sequences (Gly-Pro-Hyp) and assists in secretion of correctly folded collagen. Elevated collagen production is correlated with HSP47 expression in various diseases, including fibrosis and keloid. HSP47 knockdown ameliorates liver fibrosis by inhibiting collagen secretion, and inhibition of the interaction of HSP47 with procollagen also prevents collagen secretion. Therefore, a high-throughput system for screening of drugs capable of inhibiting the interaction between HSP47 and collagen would aid the development of novel therapies for fibrotic diseases. In this study, we established a straightforward method for rapidly and quantitatively measuring the interaction between HSP47 and collagen in solution using fluorescence correlation spectroscopy (FCS). The diffusion rate of HSP47 labeled with Alexa Fluor 488 (HSP47-AF), a green fluorescent dye, decreased upon addition of type I or III collagen, whereas that of dye-labeled protein disulfide isomerase (PDI) or bovine serum albumin (BSA) did not, indicating that specific binding of HSP47 to collagen could be detected using FCS. Using this method, we calculated the dissociation constant of the interaction between HSP47 and collagen. The binding ratio between HSP47-AF and collagen did not change in the presence of sodium chloride, confirming that the interaction was hydrophobic in nature. In addition, we observed dissociation of collagen from HSP47 at low pH and re-association after recovery to neutral pH. These observations indicate that this system is appropriate for detecting the interaction between HSP47 and collagen, and could be applied to high-throughput screening for drugs capable of suppressing and/or curing fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.
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Expression of and secretion through the Aeromonas salmonicida type III secretion system.
Ebanks, Roger O; Knickle, Leah C; Goguen, Michel; Boyd, Jessica M; Pinto, Devanand M; Reith, Michael; Ross, Neil W
2006-05-01
Aeromonas salmonicida subsp. salmonicida is the aetiological agent of furunculosis, a disease of farmed and wild salmonids. The type III secretion system (TTSS) is one of the primary virulence factors in A. salmonicida. Using a combination of differential proteomic analysis and reverse transcriptase (RT)-PCR, it is shown that A. salmonicida A449 induces the expression of TTSS proteins at 28 degrees C, but not at its more natural growth temperature of 17 degrees C. More modest increases in expression occur at 24 degrees C. This temperature-induced up-regulation of the TTSS in A. salmonicida A449 occurs within 30 min of a growth temperature increase from 16 to 28 degrees C. Growth conditions such as low-iron, low pH, low calcium, growth within the peritoneal cavity of salmon and growth to high cell densities do not induce the expression of the TTSS in A. salmonicida A449. The only other known growth condition that induces expression of the TTSS is growth of the bacterium at 16 degrees C in salt concentrations ranging from 0.19 to 0.38 M NaCl. It is also shown that growth at 28 degrees C followed by exposure to low calcium results in the secretion of one of the TTSS effector proteins. This study presents a simple in vitro model for the expression of TTSS proteins in A. salmonicida.
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GaAs Solar Cells Grown on Unpolished, Spalled Ge Substrates: Preprint
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cavalli, Alessandro; Johnston, Steven; Sulas, Dana
Decreasing the cost of single-crystal substrates by wafer reuse techniques has long been sought for III-V solar cells. Controlled spalling of III-V devices is a possible pathway for epitaxial liftoff, which would help reduce costs, but chemo- mechanical polishing after liftoff tends to limit the potential cost savings. Growth on an unpolished spalled surface would be an additional step toward lower costs, but it is crucial to show high efficiency solar cell devices on these unprocessed substrates. In this study, we spalled 2-inch Ge wafers using a Ni stressor layer, and then grew GaAs solar cells by HVPE on themore » spalled Ge surface without any other surface treatment. We show a 12.8% efficient single-junction device, without anti-reflection coating, with quantum efficiency very close to identical devices grown by HVPE on non-spalled GaAs substrates. Demonstrating a high carrier collection on unpolished spalled wafers is a step toward reducing substrate-related liftoff and reuse costs.« less
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Transfer of InP epilayers by wafer bonding
NASA Astrophysics Data System (ADS)
Hjort, Klas
2004-08-01
Wafer bonding increases the freedom of design in the integration of dissimilar materials. For example, it is interesting to combine III-V compounds that have direct band gap and high mobility with silicon (Si) that is extensively used in microelectronic applications. The interest to integrate III-V-based materials with Si arises primarily from two types of applications: smart pixels for optical intra- and inter-chip interconnects in the so-called optoelectronic integrated circuits, and optoelectronic devices using some material advantages of combining III-V with Si. Also, in the III-V industry larger substrates are crucial for higher efficiency in high-volume production, and especially so for monolithic microwave integrated circuits (MMIC). For indium phosphide (InP) the development of large-area substrates has not been able to keep up with market demands. One way to circumvent this problem is to use silicon substrates that are large-area, low-cost, and mechanically strong with high thermal conductivity. In addition, silicon is transparent at the emission wavelengths most often used in InP-based optoelectronics. Unfortunately, the large lattice-mismatch, 8.1%, between silicon and InP, has limited the success of heteroepitaxial growth. Hence, one alternative to be reviewed is InP-to-Si wafer bonding. When a direct semiconductor interface is not needed there are several other means of wafer bonding, e.g. adhesive, eutectic, and solid-state. These processes can be used for direct integration of small islets of epitaxially thin InP microelectronics onto other substrates, e.g. by transferring of InP-based epilayers to a Si-based microwave circuit by pick-and-place, BCB resist adhesive bonding and sacrificing of the InP substrate.
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Paumann-Page, Martina; Katz, Romy-Sophie; Bellei, Marzia; Schwartz, Irene; Edenhofer, Eva; Sevcnikar, Benjamin; Soudi, Monika
2017-01-01
Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV. PMID:28154175
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Paumann-Page, Martina; Katz, Romy-Sophie; Bellei, Marzia; Schwartz, Irene; Edenhofer, Eva; Sevcnikar, Benjamin; Soudi, Monika; Hofbauer, Stefan; Battistuzzi, Gianantonio; Furtmüller, Paul G; Obinger, Christian
2017-03-17
Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H 2 O 2 -mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Ruan, Huaqin; Hu, Meijuan; Chen, Jingyu; Li, Xue; Li, Ting; Lai, Yongxiu; Wang, En Tao; Gu, Jun
2018-04-08
The distribution of rhcRST and rhcJ-C1 fragments located in different loci of the type III secretion system (T3SS) gene cluster in the peanut-nodulating bradyrhizobia isolated from Guangdong Province, China was investigated by PCR-based sequencing. T3SS was detected in approximately one-third of the peanut bradyrhizobial strains and the T3SS-harboring strains belonging to different Bradyrhizobium genomic species. Diverse T3SS groups corresponding to different symbiotic gene types were defined among the 23 T3SS-harboring strains. The same or similar T3SS genes were detected in different genospecies, indicating that interspecies horizontal transfer of symbiotic genes had occurred in the Bradyrhizobium genus. Copyright © 2018. Published by Elsevier GmbH.
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Cooper, Colin A; Zhang, Kun; Andres, Sara N; Fang, Yuan; Kaniuk, Natalia A; Hannemann, Mandy; Brumell, John H; Foster, Leonard J; Junop, Murray S; Coombes, Brian K
2010-02-05
Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.
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Bernard, Abram R; Duarte, Shari M; Kumar, Prashant; Dickenson, Nicholas E
2016-07-01
Shigella rely on a type III secretion system as the primary virulence factor for invasion and colonization of human hosts. Although there are an estimated 90 million Shigella infections, annually responsible for more than 100,000 deaths worldwide, challenges isolating and stabilizing many type III secretion system proteins have prevented a full understanding of the Shigella invasion mechanism and additionally slowed progress toward a much needed Shigella vaccine. Here, we show that the non-denaturing zwitterionic detergent N, N-dimethyldodecylamine N-oxide (LDAO) and non-ionic detergent n-octyl-oligo-oxyethylene efficiently isolated the hydrophobic Shigella translocator protein IpaC from the co-purified IpaC/IpgC chaperone-bound complex. Both detergents resulted in monomeric IpaC that exhibits strong membrane binding and lysis characteristics while the chaperone-bound complex does not, suggesting that the stabilizing detergents provide a means of following IpaC "activation" in vitro. Additionally, biophysical characterization found that LDAO provides significant thermal and temporal stability to IpaC, protecting it for several days at room temperature and brief exposure to temperatures reaching 90°C. In summary, this work identified and characterized conditions that provide stable, membrane active IpaC, providing insight into key interactions with membranes and laying a strong foundation for future vaccine formulation studies taking advantage of the native immunogenicity of IpaC and the stability provided by LDAO. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis
Mitchell, Anthony; Tam, Christina; Elli, Derek; Charlton, Thomas; Osei-Owusu, Patrick; Fazlollahi, Farbod; Faull, Kym F.
2017-01-01
ABSTRACT Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV. Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione. PMID:28512097
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Methods for improved growth of group III nitride buffer layers
DOE Office of Scientific and Technical Information (OSTI.GOV)
Melnik, Yurity; Chen, Lu; Kojiri, Hidehiro
Methods are disclosed for growing high crystal quality group III-nitride epitaxial layers with advanced multiple buffer layer techniques. In an embodiment, a method includes forming group III-nitride buffer layers that contain aluminum on suitable substrate in a processing chamber of a hydride vapor phase epitaxy processing system. A hydrogen halide or halogen gas is flowing into the growth zone during deposition of buffer layers to suppress homogeneous particle formation. Some combinations of low temperature buffers that contain aluminum (e.g., AlN, AlGaN) and high temperature buffers that contain aluminum (e.g., AlN, AlGaN) may be used to improve crystal quality and morphologymore » of subsequently grown group III-nitride epitaxial layers. The buffer may be deposited on the substrate, or on the surface of another buffer. The additional buffer layers may be added as interlayers in group III-nitride layers (e.g., GaN, AlGaN, AlN).« less
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Progressive quality control of secretory proteins in the early secretory compartment by ERp44.
Sannino, Sara; Anelli, Tiziana; Cortini, Margherita; Masui, Shoji; Degano, Massimo; Fagioli, Claudio; Inaba, Kenji; Sitia, Roberto
2014-10-01
ERp44 is a pH-regulated chaperone of the secretory pathway. In the acidic milieu of the Golgi, its C-terminal tail changes conformation, simultaneously exposing the substrate-binding site for cargo capture and the RDEL motif for ER retrieval through interactions with cognate receptors. Protonation of cysteine 29 in the active site allows tail movements in vitro and in vivo. Here, we show that conserved histidine residues in the C-terminal tail also regulate ERp44 in vivo. Mutants lacking these histidine residues retain substrates more efficiently. Surprisingly, they are also O-glycosylated and partially secreted. Co-expression of client proteins prevents secretion of the histidine mutants, forcing tail opening and RDEL accessibility. Client-induced RDEL exposure allows retrieval of proteins from distinct stations along the secretory pathway, as indicated by the changes in O-glycosylation patterns upon overexpression of different partners. The ensuing gradients might help to optimize folding and assembly of different cargoes. Endogenous ERp44 is O-glycosylated and secreted by human primary endometrial cells, suggesting possible pathophysiological roles of these processes. © 2014. Published by The Company of Biologists Ltd.
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Induction of KIAA1199/CEMIP is associated with colon cancer phenotype and poor patient survival
Fink, Stephen P.; Myeroff, Lois L.; Kariv, Revital; Platzer, Petra; Xin, Baozhong; Mikkola, Debra; Lawrence, Earl; Morris, Nathan; Nosrati, Arman; Willson, James K. V.; Willis, Joseph; Veigl, Martina; Barnholtz-Sloan, Jill S.; Wang, Zhenghe; Markowitz, Sanford D.
2015-01-01
Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. We originally identified KIAA1199 (now officially called CEMIP) as a transcript highly induced in colon cancer: initially designating the transcript as Colon Cancer Secreted Protein 1. We molecularly characterized CEMIP expression both at the mRNA and protein level and found it is a secreted protein induced an average of 54-fold in colon cancer. Knockout of CEMIPreduced the ability of human colon cancer cells to form xenograft tumors in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. We find CEMIP mRNA overexpression correlates with poorer patient survival. In stage III only (n = 31) or in combined stage II plus stage III colon cancer cases (n = 73), 5-year overall survival was significantly better (p = 0.004 and p = 0.0003, respectively) among patients with low CEMIP expressing tumors than those with high CEMIP expressing tumors. These results demonstrate that CEMIP directly facilitates colon tumor growth, and high CEMIP expression correlates with poor outcome in stage III and in stages II+III combined cohorts. We present CEMIP as a candidate prognostic marker for colon cancer and a potential therapeutic target. PMID:26437221
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Kitahara, Tadashi; Okamoto, Hidehiko; Fukushima, Munehisa; Sakagami, Masaharu; Ito, Taeko; Yamashita, Akinori; Ota, Ichiro; Yamanaka, Toshiaki
2016-01-01
Meniere's disease, a common inner ear condition, has an incidence of 15-50 per 100,000. Because mental/physical stress and subsequent increase in the stress hormone vasopressin supposedly trigger Meniere's disease, we set a pilot study to seek new therapeutic interventions, namely management of vasopressin secretion, to treat this disease. We enrolled 297 definite Meniere's patients from 2010 to 2012 in a randomized-controlled and open-label trial, assigning Group-I (control) traditional oral medication, Group-II abundant water intake, Group-III tympanic ventilation tubes and Group-IV sleeping in darkness. Two hundred sixty-three patients completed the planned 2-year-follow-up, which included assessment of vertigo, hearing, plasma vasopressin concentrations and changes in stress/psychological factors. At 2 years, vertigo was completely controlled in 54.3% of patients in Group-I, 81.4% in Group-II, 84.1% in Group-III, and 80.0% in Group-IV (statistically I < II = III = IV). Hearing was improved in 7.1% of patients in Group-I, 35.7% in Group-II, 34.9% in Group-III, and 31.7% in Group-IV (statistically I < II = III = IV). Plasma vasopressin concentrations decreased more in Groups-II, -III, and -IV than in Groups-I (statistically I < II = III = IV), although patients' stress/psychological factors had not changed. Physicians have focused on stress management for Meniere's disease. However, avoidance of stress is unrealistic for patients who live in demanding social environments. Our findings in this pilot study suggest that interventions to decrease vasopressin secretion by abundant water intake, tympanic ventilation tubes and sleeping in darkness is feasible in treating Meniere's disease, even though these therapies did not alter reported mental/physical stress levels. ClinicalTrials.gov NCT01099046.
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Miller, Nathan C; Quenee, Lauriane E; Elli, Derek; Ciletti, Nancy A; Schneewind, Olaf
2012-04-01
Current efforts to develop plague vaccines focus on LcrV, a polypeptide that resides at the tip of type III secretion needles. LcrV-specific antibodies block Yersinia pestis type III injection of Yop effectors into host immune cells, thereby enabling phagocytes to kill the invading pathogen. Earlier work reported that antibodies against Y. pestis LcrV cannot block type III injection by Yersinia enterocolitica strains and suggested that lcrV polymorphisms may provide for escape from LcrV-mediated plague immunity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIM D27 LcrV (LcrV(D27)) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrV(W22703)) or O:8 strain WA-314 (LcrV(WA-314)) but are otherwise unable to block type III injection by Y. enterocolitica strains. Replacing the lcrV gene on the pCD1 virulence plasmid of Y. pestis KIM D27 with either lcrV(W22703) or lcrV(WA-314) does not affect the ability of plague bacteria to secrete proteins via the type III pathway, to inject Yops into macrophages, or to cause lethal plague infections in mice. LcrV(D27)-specific antibodies blocked type III injection by Y. pestis expressing lcrV(W22703) or lcrV(WA-314) and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrV(D27) are unable to block the type III injection of Y. enterocolitica strains, expression of lcrV(W22703) or lcrV(WA-314) in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.
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Nørgaard, Hanne; Warui, Douglas M.; Rajakovich, Lauren J.; Chang, Wei-chen; Booker, Squire J.; Krebs, Carsten; Bollinger, J. Martin
2013-01-01
Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or mono-unsaturated Cn fatty aldehydes to formate and the corresponding Cn-1 alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 co-substrate to the oxidation state of water and incorporates one O-atom from O2 into the formate co-product. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2III/III complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2III/III center with resolved sub-sites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t1/2 ~ 400 s at 5 °C) to produce a very modest yield of formate (< 0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazine (MeOPMS) to yield product, albeit at only ~ 50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or hetero-dinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2III/III-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1–C2 radical fragmentation and formation of the alk(a/e)ne and formate products. PMID:23987523
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Brand, Martin D
2016-11-01
This review examines the generation of reactive oxygen species by mammalian mitochondria, and the status of different sites of production in redox signaling and pathology. Eleven distinct mitochondrial sites associated with substrate oxidation and oxidative phosphorylation leak electrons to oxygen to produce superoxide or hydrogen peroxide: oxoacid dehydrogenase complexes that feed electrons to NAD + ; respiratory complexes I and III, and dehydrogenases, including complex II, that use ubiquinone as acceptor. The topologies, capacities, and substrate dependences of each site have recently clarified. Complex III and mitochondrial glycerol 3-phosphate dehydrogenase generate superoxide to the external side of the mitochondrial inner membrane as well as the matrix, the other sites generate superoxide and/or hydrogen peroxide exclusively in the matrix. These different site-specific topologies are important for redox signaling. The net rate of superoxide or hydrogen peroxide generation depends on the substrates present and the antioxidant systems active in the matrix and cytosol. The rate at each site can now be measured in complex substrate mixtures. In skeletal muscle mitochondria in media mimicking muscle cytosol at rest, four sites dominate, two in complex I and one each in complexes II and III. Specific suppressors of two sites have been identified, the outer ubiquinone-binding site in complex III (site III Qo ) and the site in complex I active during reverse electron transport (site I Q ). These suppressors prevent superoxide/hydrogen peroxide production from a specific site without affecting oxidative phosphorylation, making them excellent tools to investigate the status of the sites in redox signaling, and to suppress the sites to prevent pathologies. They allow the cellular roles of mitochondrial superoxide/hydrogen peroxide production to be investigated without catastrophic confounding bioenergetic effects. They show that sites III Qo and I Q are active in cells and have important roles in redox signaling (e.g. hypoxic signaling and ER-stress) and in causing oxidative damage in a variety of biological contexts. Copyright © 2016 Elsevier Inc. All rights reserved.
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Protein secretion and surface display in Gram-positive bacteria
Schneewind, Olaf; Missiakas, Dominique M.
2012-01-01
The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983
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Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III*
Kuznetsov, Nikita A.; Kladova, Olga A.; Kuznetsova, Alexandra A.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Zharkov, Dmitry O.; Fedorova, Olga S.
2015-01-01
Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. PMID:25869130
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Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.
Lavoie, Mathieu; Abou Elela, Sherif
2008-08-19
Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.
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Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ.
Jeong, Kwang Cheol; Sexton, Jessica A; Vogel, Joseph P
2015-03-01
Modulation of host cell function is vital for intracellular pathogens to survive and replicate within host cells. Most commonly, these pathogens utilize specialized secretion systems to inject substrates (also called effector proteins) that function as toxins within host cells. Since it would be detrimental for an intracellular pathogen to immediately kill its host cell, it is essential that secreted toxins be inactivated or degraded after they have served their purpose. The pathogen Legionella pneumophila represents an ideal system to study interactions between toxins as it survives within host cells for approximately a day and its Dot/Icm type IVB secretion system (T4SS) injects a vast number of toxins. Previously we reported that the Dot/Icm substrates SidE, SdeA, SdeB, and SdeC (known as the SidE family of effectors) are secreted into host cells, where they localize to the cytoplasmic face of the Legionella containing vacuole (LCV) in the early stages of infection. SidJ, another effector that is unrelated to the SidE family, is also encoded in the sdeC-sdeA locus. Interestingly, while over-expression of SidE family proteins in a wild type Legionella strain has no effect, we found that their over-expression in a ∆sidJ mutant completely inhibits intracellular growth of the strain. In addition, we found expression of SidE proteins is toxic in both yeast and mammalian HEK293 cells, but this toxicity can be suppressed by co-expression of SidJ, suggesting that SidJ may modulate the function of SidE family proteins. Finally, we were able to demonstrate both in vivo and in vitro that SidJ acts on SidE proteins to mediate their disappearance from the LCV, thereby preventing lethal intoxication of host cells. Based on these findings, we propose that SidJ acts as a metaeffector to control the activity of other Legionella effectors.
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NASA Astrophysics Data System (ADS)
Thomas, Paul M.
Understanding of quantum tunneling phenomenon in semiconductor systems is increasingly important as CMOS replacement technologies are investigated. This work studies a variety of heterojunction materials and types to increase tunnel currents to CMOS competitive levels and to understand how integration onto Si substrates affects performance. Esaki tunnel diodes were grown by Molecular Beam Epitaxy (MBE) on Si substrates via a graded buffer and control Esaki tunnel diodes grown on lattice matched substrates for this work. Peak current density for each diode is extracted and benchmarked to build an empirical data set for predicting diode performance. Additionally, statistics are used as tool to show peak to valley ratio for the III-V on Si sample and the control perform similarly below a threshold area. This work has applications beyond logic, as multijunction solar cell, heterojunction bipolar transistor, and light emitting diode designs all benefit from better tunnel contact design.
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A gatekeeper chaperone complex directs translocator secretion during Type Three Secretion
Archuleta, Tara L.; Spiller, Benjamin W.; Kubori, Tomoko
2014-11-06
Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ~20 individual protein components thatmore » form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Thus, structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.« less
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Structural and thermochemical Aspects of (III-V)IV3 Material Assembly from First Principles
NASA Astrophysics Data System (ADS)
Chizmeshya, Andrew; Kouvetakis, John
2014-03-01
Alloys with (III-V)-(IV) compositions, including Si3(AlP), Si5-2y(AlP)y, Si3Al(As1-xNx), Si5-2yAl(P1-xNx)y and Ge5-2y(InP)y and have recently been synthesized as mono-crystalline films on Si substrates, using a synthesis route specifically designed to avoid phase separation between the III-V and IV constituents. Molecular ``building blocks'' containing group-V-centered III-V-IV3 cores, formed via interactions of group-III atoms and reactive silyly/germyl hydride precursors of desired composition (e.g, P(SiH3)3 , P(GeH3)3 , etc), assemble to form stable, covalent, diamond-like materials with the inherent tetrahedral symmetry and composition of the III-V-IV3 units. The resulting systems may provide access to a broad range of new semiconductor systems with extended optoelectronic properties, provided that the required molecular sources are available, the thermodynamic processes are viable, and the resulting alloy composition can be tuned to lattice-match the growth substrate. Molecular/solid-state simulations are used to identify promising synthetic pathways and guide the epitaxial creation of new (III-V)-(IV) materials. The thermodynamics of gas phase synthesis reactions, energetic stability of the alloys, and their epitaxial/chemical compatibility with the substrate are combined to form a global figure of merit. The latter corroborates the synthesis of known systems and predicts that formation of GaPSi3/Si(100), GaAsSi3/SiGe(100), AlPGe3/Ge(100) and InAsSi3/Ge(100) may also be favorable. Supported by NSF-DMR under SusChEM award #1309090.
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Schaal, René; Kupfahl, Claudio; Buchheidt, Dieter; Neumaier, Michael; Findeisen, Peter
2007-11-01
Reliable and early diagnosis of life-threatening invasive mycoses in neutropenic patients caused by fungi of the Aspergillus species remains challenging because current clinical diagnostic tools lack in sensitivity and/or specificity. During invasive growth a variety of fungal proteases are secreted into the bloodstream and protease profiling with reporter peptides might improve diagnosis of invasive aspergillosis in serum specimens. To characterise the specific protease activity of Aspergillus fumigatus and Aspergillus niger we analyzed Aspergillus culture supernatants, human serum and the mixture of both. A systematic screening for optimised protease substrates was performed using a random peptide library consisting of 360 synthetic peptides featuring fluorescence resonance energy transfer (FRET). We could identify numerous peptides that are selectively cleaved by fungus-specific proteases. These reporter peptides might be feasible for future protease profiling of serum specimens to improve diagnosis and monitoring of invasive aspergillosis.
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Lenis, Yasser Y; Wang, Xiaoqiu; Tang, Wanjin; Wu, Guoyao; Bazer, Fuller W
2016-10-01
Embryonic survival requires histotrophic nutrition, including molecules secreted or transported into the uterine lumen by uterine epithelia. L-Arginine (Arg) is a common substrate for synthesis of nitric oxide, ornithine, proline, glutamate, creatinine, urea, polyamines and agmatine. Agmatine (Agm) is a product of arginine decarboxylation and it is a substrate for agmatinase for synthesis of putrescine and other polyamines in the ovine conceptus. Polyamines are essential for conceptus development. Therefore, this study compared effects of Arg and Agm on the behavior of ovine trophectoderm (oTr1) cells cultured in vitro. Arg, but not Agm, increased proliferation and migration of oTr1 cells, but neither Arg nor Agm affected cell adhesion. The total amount of IFNT in culture medium of oTr1 cells was increased by Arg, but Agm increased the IFNT production per oTr1 cell. Arg and Agm plus Arg decreased secretion of dopamine and norepinephrine by oTr1 cells. Agm upregulates expression of mRNAs SLC7A1, agmatinase and OAZ2 while the combination of Arg and Agm decreased expression of mRNAs for ODC1, SLC7A1, OAZ1 and OAZ3 by oTr1 cells. Although Agm does not stimulate proliferation, migration or adhesion of oTr1 cells or their secretion of catecholamines, Agm did increase transcription of SLC7A1, agmatinase and OAZ2 genes which would increase the capacity of oTr1 cells to produce polyamines. Collectively, our findings suggest a role for Arg and Agm in the regulation of transport of basic amino acids (including Arg), polyamine synthesis, and secretion of catecholamines by oTr1 cells.
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Johnson, Michelle H; de Mejia, Elvira Gonzalez
2016-03-30
Berries are a rich source of bioactive phenolic compounds that are able to bind and inhibit the enzyme dipeptidyl peptidase-IV (DPP-IV), a current target for type-2 diabetes therapy. The objectives were to determine the role of berry phenolic compounds to modulate incretin-cleaving DPP-IV and its substrate glucagon-like peptide-1 (GLP-1), insulin secretion from pancreatic β-cells, and genes and proteins involved in the insulin secretion pathway using cell culture. Anthocyanins (ANC) from 50% blueberry-50% blackberry (Blu-Bla) and 100% blackberry (Bla) fermented beverages at 50 μM cyanidin-3-glucoside equivalents increased (p < 0.05) glucose-stimulated insulin secretion from pancreatic β-cells (iNS-1E) both when applied directly and following simulated absorption through Caco-2 cells (by 233 and 100 μIU insulin/mL, respectively). ANC 50%Blu-Bla and ANC 100%Bla upregulated the gene for incretin hormone GLP-1 (fold-change 3.0 ± 1.4 and 2.0 ± 0.3, respectively) and genes in the insulin secretory pathway including insulin-like growth factor 1 receptor (iGF1R, 2.3 ± 0.6 and 1.6 ± 0.3, respectively), and increased (p < 0.05) the protein expression of insulin-like growth factor 2 (IGF-II), insulin-like growth factor binding proteins (IGFBP-2 and 3), and vascular endothelial growth factor (VEGF) in iNS-1E cells. Taken together, anthocyanins, predominantly delphinidin-3-arabinoside, from fermented berry beverages have the potential to modulate DPP-IV and its substrate GLP-1, to increase insulin secretion, and to upregulate expression of mRNA of insulin-receptor associated genes and proteins in pancreatic β-cells.
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Genetically engineered T cells to target EGFRvIII expressing glioblastoma.
Bullain, Szofia S; Sahin, Ayguen; Szentirmai, Oszkar; Sanchez, Carlos; Lin, Ning; Baratta, Elizabeth; Waterman, Peter; Weissleder, Ralph; Mulligan, Richard C; Carter, Bob S
2009-09-01
Glioblastoma remains a significant therapeutic challenge, warranting further investigation of novel therapies. We describe an immunotherapeutic strategy to treat glioblastoma based on adoptive transfer of genetically modified T-lymphocytes (T cells) redirected to kill EGFRvIII expressing gliomas. We constructed a chimeric immune receptor (CIR) specific to EGFRvIII, (MR1-zeta). After in vitro selection and expansion, MR1-zeta genetically modified primary human T-cells specifically recognized EGFRvIII-positive tumor cells as demonstrated by IFN-gamma secretion and efficient tumor lysis compared to control CIRs defective in EGFRvIII binding (MRB-zeta) or signaling (MR1-delzeta). MR1-zeta expressing T cells also inhibited EGFRvIII-positive tumor growth in vivo in a xenografted mouse model. Successful targeting of EGFRvIII-positive tumors via adoptive transfer of genetically modified T cells may represent a new immunotherapy strategy with great potential for clinical applications.
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Akeda, Yukihiro; Kimura, Tomomi; Yamasaki, Aiko; Kodama, Toshio; Iida, Tetsuya; Honda, Takeshi; Oishi, Kazunori
2012-10-19
The type III secretion system (T3SS) of gram-negative bacteria involves dedicated protein translocation machinery that directly injects proteins into target cells. Pathogenic bacteria already benefit from this unique system. The successful functional cloning of this useful tool into non-pathogenic bacteria would help establish novel clinical and basic biotechnology strategies in areas such as vaccine administration, the development of screening systems for anti-T3SS drugs and the target-specific delivery of bioactive compounds. In this study, we successfully cloned the Vibrio parahaemolyticus T3SS1 genetic locus into a non-pathogenic Escherichia coli K-12 strain. Assays performed here revealed that the T3SS1 cloned into the E. coli K-12 strain has the ability to translocate V. parahaemolyticus T3SS1 secreted proteins. Importantly, we also observed this system to allow the E. coli K-12 strain to inject foreign protein, as well as the V. parahaemolyticus T3SS effector, into cultured cells. These results demonstrate a prospective useful tool with experimental and therapeutic applications. Copyright © 2012 Elsevier Inc. All rights reserved.
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[Effect of different nutritional support on pancreatic secretion in acute pancreatitis].
Achkasov, E E; Pugaev, A V; Nabiyeva, Zh G; Kalachev, S V
To develop and justify optimal nutritional support in early phase of acute pancreatitis (AP). 140 AP patients were enrolled. They were divided into groups depending on nutritional support: group I (n=70) - early enteral tube feeding (ETF) with balanced mixtures, group II (n=30) - early ETF with oligopeptide mixture, group III (n=40) - total parenteral nutrition (TPN). The subgroups were also isolated depending on medication: A - Octreotide, B - Quamatel, C - Octreotide + Quamatel. Pancreatic secretion was evaluated by using of course of disease, instrumental methods, APUD-system hormone levels (secretin, cholecystokinin, somatostatin, vasointestinal peptide). ETF was followed by pancreas enlargement despite ongoing therapy, while TPN led to gradual reduction of pancreatic size up to normal values. α-amylase level progressively decreased in all groups, however in patients who underwent ETF (I and II) mean values of the enzyme were significantly higher compared with TPN (group III). Secretin, cholecystokinin and vasointestinal peptide were increasing in most cases, while the level of somatostatin was below normal in all groups. Enteral tube feeding (balanced and oligopeptide mixtures) contributes to pancreatic secretion compared with TPN, but this negative impact is eliminated by antisecretory therapy. Dual medication (Octreotide + Quamatel) is more preferable than monotherapy (Octreotide or Quamatel).
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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.
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Structure of the heterotrimeric complex that regulates type III secretion needle formation
Quinaud, Manuelle; Plé, Sophie; Job, Viviana; Contreras-Martel, Carlos; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa
2007-01-01
Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-Å x-ray crystal structure of the PscE-PscF55–85-PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus- or flagellum-forming molecules. Disruption of specific PscG–PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials. PMID:17470796
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A new transcriptional role for matrix metalloproteinase-12 in antiviral immunity.
Marchant, David J; Bellac, Caroline L; Moraes, Theo J; Wadsworth, Samuel J; Dufour, Antoine; Butler, Georgina S; Bilawchuk, Leanne M; Hendry, Reid G; Robertson, A Gordon; Cheung, Caroline T; Ng, Julie; Ang, Lisa; Luo, Zongshu; Heilbron, Karl; Norris, Michael J; Duan, Wenming; Bucyk, Taylor; Karpov, Andrei; Devel, Laurent; Georgiadis, Dimitris; Hegele, Richard G; Luo, Honglin; Granville, David J; Dive, Vincent; McManus, Bruce M; Overall, Christopher M
2014-05-01
Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.
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Gomez-Valero, Laura; Rusniok, Christophe; Rolando, Monica; Neou, Mario; Dervins-Ravault, Delphine; Demirtas, Jasmin; Rouy, Zoe; Moore, Robert J; Chen, Honglei; Petty, Nicola K; Jarraud, Sophie; Etienne, Jerome; Steinert, Michael; Heuner, Klaus; Gribaldo, Simonetta; Médigue, Claudine; Glöckner, Gernot; Hartland, Elizabeth L; Buchrieser, Carmen
2014-01-01
The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.
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Pérez-Sotelo, D; Roca-Rivada, A; Baamonde, I; Baltar, J; Castro, A I; Domínguez, E; Collado, M; Casanueva, F F; Pardo, M
2017-11-24
Irisin is a browning-stimulating molecule secreted from the fibronectin type III domain containing 5 precursor (FNDC5) by muscle tissue upon exercise stimulation. Despite its beneficial role, there is an unmet and clamorous need to discern many essential aspects of this protein and its mechanism of action not only as a myokine but also as an adipokine. Here we contribute to address this topic by revealing the nature and role of FNDC5/irisin in adipose tissue. First, we show that FNDC5/irisin expression and secretion are induced by adipocyte differentiation and confirm its over-secretion by human obese visceral (VAT) and subcutaneous (SAT) adipose tissues. Second, we show how secreted factors from human obese VAT and SAT decrease PGC1α, FNDC5 and UCP1 gene expression on differentiating adipocytes; this effect over UCP1 is blunted by blocking irisin in obese secretomes. Finally, by stable gene silencing FNDC5 we reveal that FNDC5-KO adipocytes show reduced UCP1 expression and enhanced adipogenesis.
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Vawter, G. Allen
2013-11-12
An optical XOR gate is formed as a photonic integrated circuit (PIC) from two sets of optical waveguide devices on a substrate, with each set of the optical waveguide devices including an electroabsorption modulator electrically connected in series with a waveguide photodetector. The optical XOR gate utilizes two digital optical inputs to generate an XOR function digital optical output. The optical XOR gate can be formed from III-V compound semiconductor layers which are epitaxially deposited on a III-V compound semiconductor substrate, and operates at a wavelength in the range of 0.8-2.0 .mu.m.
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Skogen, Erik J [Albuquerque, NM; Tauke-Pedretti, Anna [Albuquerque, NM
2011-09-06
An optical NOR gate is formed from two pair of optical waveguide devices on a substrate, with each pair of the optical waveguide devices consisting of an electroabsorption modulator electrically connected in series with a waveguide photodetector. The optical NOR gate utilizes two digital optical inputs and a continuous light input to provide a NOR function digital optical output. The optical NOR gate can be formed from III-V compound semiconductor layers which are epitaxially deposited on a III-V compound semiconductor substrate, and operates at a wavelength in the range of 0.8-2.0 .mu.m.
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Liu, Tingqiang; Yu, Hongshan; Liu, Chunying; Bao, Yongming; Hu, Xiangchun; Wang, Yuanhao; Liu, Bing; Fu, Yaoyao; Tang, Sihui; Jin, Fengxie
2013-05-01
Progenin III, one of the most active spirostanol saponins, is a potential candidate for anti-cancer therapy due to its strong antitumor activity and low hemolytic activity. However, the concentration of progenin III is extremely low in natural Dioscorea plants. In this paper, the progenin III production from total steroidal saponins of Dioscorea nipponica Makino was studied using the crude enzyme from Aspergillus oryzae DLFCC-38. The crude enzyme converting total steroidal saponins into progenin III was obtained from the A. oryzae DLFCC-38 culture. For enzyme production, the strain was cultured for 72 h at 30 °C with shaking at 150 rpm in 5 % (w/v) malt extract medium containing 2 % (v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for 9 h at 50 °C and pH 5.0 with the 20 mg/ml of substrate. In the preparation of progenin III, 117 g of crude progenin III was obtained from 160 g of substrate, and the crude product was purified with silica gel column to obtain 60.3 g progenin III of 93.4 % purity.
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Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok
2011-02-01
RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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NASA Astrophysics Data System (ADS)
Hurley, Belinda Louise
Surface enhanced Raman scattering was used to observe interactions of dilute CrVI solutions with silver and copper surfaces in situ. Using silver as a model surface, CrIII was observed at the near monolayer level, and the spectra were compared to those from CrIII oxyhydroxide species and CrIII/Cr VI mixed oxide. Similar experiments were conducted with copper surfaces and 785 nm excitation. Upon exposure of a copper surface to CrVI solution, the characteristic copper oxide Raman bands disappeared, and a Cr III band increased in intensity over a period of ˜20 hours. The intensity of the CrIII band on copper became self limiting after the formation of several CrIII monolayers, as supported by chronoamperometry experiments. This CrIII spectrum was stable after CrVI was removed from the solution provided the potential remained negative of -200 mV vs. Ag/AgCl. The results support the conclusion that CrVI is reductively adsorbed to copper at the near neutral pH and open circuit potentials expected for Cu/Al alloys in field applications. The CrIII film is stable and strongly inhibits oxygen reduction at the treated copper surface. Copper surfaces and polished Aluminum Alloy 2024 T3 substrates were derivatized at open circuit potential with arenediazonium salts in both aprotic and aqueous media. Raman spectroscopy confirmed the presence of a derivatized film on the substrates before and after exposure to boiling water and sonication in acetone. Preliminary experiments to test these films for corrosion inhibition proved unsuccessful. Aluminum and copper substrates were prepared and used for x-ray photoelectron spectroscopy (XPS) analysis of the derivatization results. In the copper experiments, one surface was native oxide copper, predominantly in the form of Cu2O, and one surface was predominantly Cu 0. Results of the XPS analysis indicate the presence of a Cu-O-C linkage and possibly a Cu-C covalent bond between the aryl ring and the copper substrate. XPS results also indicate the formation of multilayers on both types of copper surfaces with different percentages of azo coupling within the multilayers on the two surfaces. These easily prepared, covalently bonded organic films could be used for applications currently fulfilled with self-absorbed monolayers and Langmuir Blodgett films.
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Aromatase inhibitor (anastrozole) affects growth of endometrioma cells in culture.
Badawy, Shawky Z A; Brown, Shereene; Kaufman, Lydia; Wojtowycz, Martha A
2015-05-01
To study the effects of aromatase inhibitor (anastrozole) on the growth and estradiol secretion of endometrioma cells in culture. Endometrioma cells are grown in vitro until maximum growth before used in this study. This was done in the research laboratory for tissue culture, in an academic hospital. Testosterone at a concentration of 10 μg/mL was added as a substrate for the intracellular aromatase. In addition, aromatase inhibitor was added at a concentration of 200 and 300 μg/mL. The effect on cell growth and estradiol secretion is evaluated using Student's t-test. The use of testosterone increased estradiol secretion by endometrioma cells in culture. The use of aromatase inhibitor significantly inhibited the growth of endometrioma cells, and estradiol secretion. Aromatase inhibitor (anastrozole) may be an effective treatment for endometriosis due to inhibition of cellular aromatase. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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[Alterations in tears aqueous layer during cytostatics treatment].
Wojciechowska, Katarzyna; Wieckowska-Szakiel, Marzena; Rózalska, Barbara; Jurowski, Piotr
2013-01-01
The aim of the study was to evaluate tears secretion, pH and lysozyme activity in tears aqueous layer during chemotherapy in lung, breast and bowel cancer. 36 patients were enrolled to the study. Depending on the type of cancer and type of chemotherapy patients were divided into three groups. Group I (12 patients) diagnosed with non-small-cell lung cancer treated with PE schema (cisplatin, etoposide), Group II (12 patients) with breast cancer treated with FAC schema (fluorouracil, doxorubicin, cyclophosphamide), Group III (12 patients) with bowel cancer treated with FU/LV schema (fluorouracil, leucovorin). In all the patients: Schirmer's I test, pH measurements and lysozyme test were performed. Patients were examined before chemotherapy, after 2nd, 4th, 6th cycle. In group I and II lowering of tears secretion (p < 0.001) was revealed. In group III there was higher tears secretion (p < 0.001). PH was lowered after 2nd chemotherapy course in group I and II. In further treatment pH value were in the same lower level as after the second course. In group III there was higher pH--more alkaline (p < 0.001) after 2nd cycle of treatment and it was on the same level to the end of the examination process. Lowering of lysozyme activity in the tears film in all groups (p < 0.001) was established. The higher alterations of the lysozyme activity were observed in group treated with FAC schema. Cytostatic treatment has major influence on tears aqueous layer causing alterations of tears secretions. PH alterations depending on type of chemotherapy was observed. Lowering of lysozyme activity in tears was observed. All the deteriorations aggravate with duration of chemotherapy. Alterations of tears film parameters during chemotherapy may influence upon eye surface homeostasis and infectious complication. tears aqueous layer, Schirmer's test, lysozyme activity, tears pH.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sun, Ping; Tropea, Joseph E.; Austin, Brian P.
2008-05-03
The plague-causing bacterium Yersinia pestis utilizes a type III secretion system to deliver effector proteins into mammalian cells where they interfere with signal transduction pathways that mediate phagocytosis and the inflammatory response. Effector proteins are injected through a hollow needle structure composed of the protein YscF. YscG and YscE act as 'chaperones' to prevent premature polymerization of YscF in the cytosol of the bacterium prior to assembly of the needle. Here, we report the crystal structure of the YscEFG protein complex at 1.8 {angstrom} resolution. Overall, the structure is similar to that of the analogous PscEFG complex from the Pseudomonasmore » aeruginosa type III secretion system, but there are noteworthy differences. The structure confirms that, like PscG, YscG is a member of the tetratricopeptide repeat family of proteins. YscG binds tightly to the C-terminal half of YscF, implying that it is this region of YscF that controls its polymerization into the needle structure. YscE interacts with the N-terminal tetratricopeptide repeat motif of YscG but makes very little direct contact with YscF. Its function may be to stabilize the structure of YscG and/or to participate in recruiting the complex to the secretion apparatus. No electron density could be observed for the 49 N-terminal residues of YscF. This and additional evidence suggest that the N-terminus of YscF is disordered in the complex with YscE and YscG. As expected, conserved residues in the C-terminal half of YscF mediate important intra- and intermolecular interactions in the complex. Moreover, the phenotypes of some previously characterized mutations in the C-terminal half of YscF can be rationalized in terms of the structure of the heterotrimeric YscEFG complex.« less
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Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.
Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT,more » NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of action.« less
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Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C B; Lakshmipriya, Thangavel; Tang, Thean-Hock
2015-01-01
Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.
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Pena-Bello, Lara; Pertega-Diaz, Sonia; Outeiriño-Blanco, Elena; Garcia-Buela, Jesus; Tovar, Sulay; Sangiao-Alvarellos, Susana; Dieguez, Carlos; Cordido, Fernando
2015-01-01
Metabolic substrates and nutritional status play a major role in growth hormone (GH) secretion. Uncovering the mechanisms involved in GH secretion following oral glucose (OG) administration in normal and obese patients is a pending issue. The aim of this study was to investigate GH after OG in relation with adiposity, insulin secretion and action, and ghrelin secretion in obese and healthy women, to further elucidate the mechanism of GH secretion after OG and the altered GH secretion in obesity. We included 64 healthy and obese women. After an overnight fast, 75 g of OG were administered; GH, glucose, insulin and ghrelin were obtained during 300 minutes. Insulin secretion and action indices and the area under the curve (AUC) were calculated for GH, glucose, insulin and ghrelin. Univariate and multivariate linear regression analyses were employed. The AUC of GH (μg/L•min) was lower in obese (249.8±41.8) than in healthy women (490.4±74.6), P=0.001. The AUC of total ghrelin (pg/mL•min) was lower in obese (240995.5±11094.2) than in healthy women (340797.5±37757.5), P=0.042. There were significant correlations between GH secretion and the different adiposity, insulin secretion and action, and ghrelin secretion indices. After multivariate analysis only ghrelin AUC remained a significant predictor for fasting and peak GH.
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Pena-Bello, Lara; Pertega-Diaz, Sonia; Outeiriño-Blanco, Elena; Garcia-Buela, Jesus; Tovar, Sulay; Sangiao-Alvarellos, Susana; Dieguez, Carlos; Cordido, Fernando
2015-01-01
Context Metabolic substrates and nutritional status play a major role in growth hormone (GH) secretion. Uncovering the mechanisms involved in GH secretion following oral glucose (OG) administration in normal and obese patients is a pending issue. Objective The aim of this study was to investigate GH after OG in relation with adiposity, insulin secretion and action, and ghrelin secretion in obese and healthy women, to further elucidate the mechanism of GH secretion after OG and the altered GH secretion in obesity. Participants and Methods We included 64 healthy and obese women. After an overnight fast, 75 g of OG were administered; GH, glucose, insulin and ghrelin were obtained during 300 minutes. Insulin secretion and action indices and the area under the curve (AUC) were calculated for GH, glucose, insulin and ghrelin. Univariate and multivariate linear regression analyses were employed. Results The AUC of GH (μg/L•min) was lower in obese (249.8±41.8) than in healthy women (490.4±74.6), P=0.001. The AUC of total ghrelin (pg/mL•min) was lower in obese (240995.5±11094.2) than in healthy women (340797.5±37757.5), P=0.042. There were significant correlations between GH secretion and the different adiposity, insulin secretion and action, and ghrelin secretion indices. After multivariate analysis only ghrelin AUC remained a significant predictor for fasting and peak GH. PMID:25782001
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Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël
2013-01-01
Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488
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The SPI-1-like Type III secretion system: more roles than you think
Egan, Frank; Barret, Matthieu; O’Gara, Fergal
2014-01-01
The type III secretion system (T3SS) is a protein delivery system which is involved in a wide spectrum of interactions, from mutualism to pathogenesis, between Gram negative bacteria and various eukaryotes, including plants, fungi, protozoa and mammals. Various phylogenetic families of the T3SS have been described, including the Salmonella Pathogenicity Island 1 family (SPI-1). The SPI-1 T3SS was initially associated with the virulence of enteric pathogens, but is actually found in a diverse array of bacterial species, where it can play roles in processes as different as symbiotic interactions with insects and colonization of plants. We review the multiple roles of the SPI-1 T3SS and discuss both how these discoveries are changing our perception of the SPI-1 family and what impacts this has on our understanding of the specialization of the T3SS in general. PMID:24575107
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Bacterial flagella and Type III secretion: case studies in the evolution of complexity.
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.
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The relationship between the structures of four beta-lactamases obtained from Bacillus cereus.
Cid, H; Carrillo, O; Bunster, M; Martínez, J; Vargas, V
1988-06-01
Bacillus cereus has proved to be one of the most interesting microorganisms in the study of beta-lactamases. It secrets these enzymes very efficiently and, frequently, in multiple forms. Three different forms are produced by strain 569/H; mutant 5/B of the same microorganism is constitutive for the secretion of beta-lactamases I and II. The present study, based on secondary structure prediction by two independent methods, states the relationship among the structures of beta-lactamases I, II and III produced by B. cereus 569/H and beta-lactamase I from the strain 5/B of this microorganism. A strong similarity is also established for the enzyme type III of B. cereus and the enzyme type I produced by B. licheniformis which could have an evolutionary explanation. A structural analysis of the leader peptide regions of these enzymes by the method of Mohana and Argos is also reported.
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The SPI-1-like Type III secretion system: more roles than you think.
Egan, Frank; Barret, Matthieu; O'Gara, Fergal
2014-01-01
The type III secretion system (T3SS) is a protein delivery system which is involved in a wide spectrum of interactions, from mutualism to pathogenesis, between Gram negative bacteria and various eukaryotes, including plants, fungi, protozoa and mammals. Various phylogenetic families of the T3SS have been described, including the Salmonella Pathogenicity Island 1 family (SPI-1). The SPI-1 T3SS was initially associated with the virulence of enteric pathogens, but is actually found in a diverse array of bacterial species, where it can play roles in processes as different as symbiotic interactions with insects and colonization of plants. We review the multiple roles of the SPI-1 T3SS and discuss both how these discoveries are changing our perception of the SPI-1 family and what impacts this has on our understanding of the specialization of the T3SS in general.
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III/V nano ridge structures for optical applications on patterned 300 mm silicon substrate
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kunert, B.; Guo, W.; Mols, Y.
We report on an integration approach of III/V nano ridges on patterned silicon (Si) wafers by metal organic vapor phase epitaxy (MOVPE). Trenches of different widths (≤500 nm) were processed in a silicon oxide (SiO{sub 2}) layer on top of a 300 mm (001) Si substrate. The MOVPE growth conditions were chosen in a way to guarantee an efficient defect trapping within narrow trenches and to form a box shaped ridge with increased III/V volume when growing out of the trench. Compressively strained InGaAs/GaAs multi-quantum wells with 19% indium were deposited on top of the fully relaxed GaAs ridges as an activemore » material for optical applications. Transmission electron microcopy investigation shows that very flat quantum well (QW) interfaces were realized. A clear defect trapping inside the trenches is observed whereas the ridge material is free of threading dislocations with only a very low density of planar defects. Pronounced QW photoluminescence (PL) is detected from different ridge sizes at room temperature. The potential of these III/V nano ridges for laser integration on Si substrates is emphasized by the achieved ridge volume which could enable wave guidance and by the high crystal quality in line with the distinct PL.« less
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Effect of long-term administration of dietary fiber on the exocrine pancreas in the rat.
Sommer, H; Kasper, H
1984-08-01
Male Sprague-Dawley rats (50--70g) were fed a standard laboratory diet containing 6% dietary fiber substances (diet I), the same diet supplemented with 5% guar (diet II), 10% wheat bran (diet III), or 5% pectin of high (76%) methylic esterification (diet IV), or a fiber-free diet (diet V). After a 6-week feeding period, the body weight of the animals had increased to 300--350g. The common bile duct was then canulated and the exocrine pancreatic function tested under urethane anesthesia (1.5 g/kg body weight). The tested fiber substances had no effect on the basal pancreatic secretion of volume, bicarbonate, lipase, amylase or protein, or on the wet weight and histological appearance of the organ. However, the fiber substances influenced the pancreatic response to maximal exogenous stimulation with secretin (3.0 CU/100 g X hour) and cholecystokinin (0.6 IDU/100 g X hour) and the enzyme content of the gland significantly. Compared with diet V, diet I increased the DNA content of the pancreas and its secretion of bicarbonate and protein, and decreased the protein concentration in the gland. Diet II reduced the pancreatic content of trypsinogen and protein. Diet III decreased the protein content, but increased the bicarbonate secretion, which was also increased by diet IV. -- We conclude that fiber substances influence stimulated secretion and the enzyme content of the pancrease to a varying degree.
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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.
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Raymond, Wilfred W; Su, Sharon; Makarova, Anastasia; Wilson, Todd M; Carter, Melody C; Metcalfe, Dean D; Caughey, George H
2009-05-01
Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by mast cells. When secreted from degranulating cells, it can interact with a variety of circulating antipeptidases, but is mostly captured by alpha(2)-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that alpha(2)-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used alpha(2)-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of approximately 1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. alpha(2)-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and it appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin-converting enzyme. These findings suggest that chymase bound to alpha(2)-macroglobulin is active, that the complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that alpha(2)-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases.
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Raymond, Wilfred W.; Su, Sharon; Makarova, Anastasia; Wilson, Todd M.; Carter, Melody C.; Metcalfe, Dean D.; Caughey, George H.
2009-01-01
Human chymase is a highly efficient angiotensin II-generating serine peptidase expressed by the MCTC subset of mast cells. When secreted from degranulating cells, it can interact with a variety of circulating anti-peptidases, but is mostly captured by α2-macroglobulin, which sequesters peptidases in a cage-like structure that precludes interactions with large protein substrates and inhibitors, like serpins. The present work shows that α2-macroglobulin-bound chymase remains accessible to small substrates, including angiotensin I, with activity in serum that is stable with prolonged incubation. We used α2-macroglobulin capture to develop a sensitive, microtiter plate-based assay for serum chymase, assisted by a novel substrate synthesized based on results of combinatorial screening of peptide substrates. The substrate has low background hydrolysis in serum and is chymase-selective, with minimal cleavage by the chymotryptic peptidases cathepsin G and chymotrypsin. The assay detects activity in chymase-spiked serum with a threshold of ~1 pM (30 pg/ml), and reveals native chymase activity in serum of most subjects with systemic mastocytosis. α2-Macroglobulin-bound chymase generates angiotensin II in chymase-spiked serum, and appears in native serum as chymostatin-inhibited activity, which can exceed activity of captopril-sensitive angiotensin converting enzyme. These findings suggest that chymase bound to α2-macroglobulin is active, that the circulating complex is an angiotensin-converting enzyme inhibitor-resistant reservoir of angiotensin II-generating activity, and that α2-macroglobulin capture may be exploited in assessing systemic release of secreted peptidases. PMID:19380825
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Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying
2015-02-28
Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal "off" state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an "open" configuration of the hairpin probe and a CRET signal "on" state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.
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Kang, Peng; Bobyr, Elena; Dustman, John; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I; Stack, T Daniel P
2010-12-06
N,N,N',N'-tetramethylethylenediamine (TMED), the simplest and most extensively used peralkylated diamine ligand, is conspicuously absent from those known to form a bis(μ-oxo)dicopper(III) (O) species, [(TMED)(2)Cu(III)(2)(μ(2)-O)(2)](2+), upon oxygenation of its Cu(I) complex. Presented here is the characterization of this O species and its reactivity toward exogenous substrates. Its formation is complicated both by the instability of the [(TMED)Cu(I)](1+) precursor and by competitive formation of a presumed mixed-valent trinuclear [(TMED)(3)Cu(III)Cu(II)(2)(μ(3)-O)(2)](3+) (T) species. Under most reaction conditions, the T species dominates, yet, the O species can be formed preferentially (>80%) upon oxygenation of acetone solutions, if the copper concentration is low (<2 mM) and [(TMED)Cu(I)](1+) is prepared immediately before use. The experimental data of this simplest O species provide a benchmark by which to evaluate density functional theory (DFT) computational methods for geometry optimization and spectroscopic predictions. The enhanced thermal stability of [(TMED)(2)Cu(III)(2)(μ(2)-O)(2)](2+) and its limited steric demands compared to other O species allows more efficient oxidation of exogenous substrates, including benzyl alcohol to benzaldehyde (80% yield), highlighting the importance of ligand structure to not only enhance the oxidant stability but also maintain accessibility to the nascent metal/O(2) oxidant.
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Costa, José M.; Loper, Joyce E.
1994-01-01
Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out- mutants of E. carotovora subsp. carotovora. Out- mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out- mutants of Ecb168 were complemented to the Out+ phenotype by introduction of the corresponding cloned HindIII fragment. Out- mutants of Ecb168 were less virulent than the Out+ parental strain on potato tubers. Strain Ecb168 and Out- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out- derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora. PMID:16349316
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Mehta-Kolte, Misha G.
2012-01-01
The current understanding of dissimilatory metal reduction is based primarily on isolates from the proteobacterial genera Geobacter and Shewanella. However, environments undergoing active Fe(III) reduction often harbor less-well-studied phyla that are equally abundant. In this work, electrochemical techniques were used to analyze respiratory electron transfer by the only known Fe(III)-reducing representative of the Acidobacteria, Geothrix fermentans. In contrast to previously characterized metal-reducing bacteria, which typically reach maximal rates of respiration at electron acceptor potentials of 0 V versus standard hydrogen electrode (SHE), G. fermentans required potentials as high as 0.55 V to respire at its maximum rate. In addition, G. fermentans secreted two different soluble redox-active electron shuttles with separate redox potentials (−0.2 V and 0.3 V). The compound with the lower midpoint potential, responsible for 20 to 30% of electron transfer activity, was riboflavin. The behavior of the higher-potential compound was consistent with hydrophilic UV-fluorescent molecules previously found in G. fermentans supernatants. Both electron shuttles were also produced when cultures were grown with Fe(III), but not when fumarate was the electron acceptor. This study reveals that Geothrix is able to take advantage of higher-redox-potential environments, demonstrates that secretion of flavin-based shuttles is not confined to Shewanella, and points to the existence of high-potential-redox-active compounds involved in extracellular electron transfer. Based on differences between the respiratory strategies of Geothrix and Geobacter, these two groups of bacteria could exist in distinctive environmental niches defined by redox potential. PMID:22843516
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Site of secretion of the trail marker of the eastern tent caterpillar.
Fitzgerald, T D; Edgerly, J S
1982-01-01
A new site of secretion of a chemical trail marker was found on the sternum at the tip of the last abdominal segment of the larva of the eastern tent caterpillarMalacosoma americanum. Larvae marked from this site by drawing their sterna along the substrate when they extended existing trails in search of food and again when they established recruitment trails to food-finds. Differences in the quantity or quality of the marker deposited by exploring and recruiting caterpillars may account for the greater activity of the recruitment trails.
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Sharma, V K; Sacco, R E; Kunkle, R A; Bearson, S M D; Palmquist, D E
2012-04-01
The locus of enterocyte effacement (LEE) of Escherichia coli O157:H7 (O157) encodes a type III secretion system (T3SS) for secreting LEE-encoded and non-LEE-encoded virulence proteins that promote the adherence of O157 to intestinal epithelial cells and the persistence of this food-borne human pathogen in bovine intestines. In this study, we compared hha sepB and hha mutants of O157 for LEE transcription, T3SS activity, adherence to HEp-2 cells, persistence in bovine intestines, and the ability to induce changes in the expression of proinflammatory cytokines. LEE transcription was upregulated in the hha sepB and hha mutant strains compared to that in the wild-type strain, but the secretion of virulence proteins in the hha sepB mutant was severely compromised. This reduced secretion resulted in reduced adherence of the hha sepB mutant to Hep-2 cells, correlating with a significantly shorter duration and lower magnitude of fecal shedding in feces of weaned (n = 4 per group) calves inoculated with this mutant strain. The levels of LEE transcription, T3SS activity, and adherence to HEp-2 cells were much lower in the wild-type strain than in the hha mutant, but no significant differences were observed in the duration or the magnitude of fecal shedding in calves inoculated with these strains. Examination of the rectoanal junction (RAJ) tissues from three groups of calves showed no adherent O157 bacteria and similar proinflammatory cytokine gene expression, irrespective of the inoculated strain, with the exception that interleukin-1β was upregulated in calves inoculated with the hha sepB mutant. These results indicate that the T3SS is essential for intestinal colonization and prolonged shedding, but increased secretion of virulence proteins did not enhance the duration and magnitude of fecal shedding of O157 in cattle or have any significant impact on the cytokine gene expression in RAJ tissue compared with that in small intestinal tissue from the same calves.
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Engineering of III-Nitride Semiconductors on Low Temperature Co-fired Ceramics.
Mánuel, J M; Jiménez, J J; Morales, F M; Lacroix, B; Santos, A J; García, R; Blanco, E; Domínguez, M; Ramírez, M; Beltrán, A M; Alexandrov, D; Tot, J; Dubreuil, R; Videkov, V; Andreev, S; Tzaneva, B; Bartsch, H; Breiling, J; Pezoldt, J; Fischer, M; Müller, J
2018-05-02
This work presents results in the field of advanced substrate solutions in order to achieve high crystalline quality group-III nitrides based heterostructures for high frequency and power devices or for sensor applications. With that objective, Low Temperature Co-fired Ceramics has been used, as a non-crystalline substrate. Structures like these have never been developed before, and for economic reasons will represent a groundbreaking material in these fields of Electronic. In this sense, the report presents the characterization through various techniques of three series of specimens where GaN was deposited on this ceramic composite, using different buffer layers, and a singular metal-organic chemical vapor deposition related technique for low temperature deposition. Other single crystalline ceramic-based templates were also utilized as substrate materials, for comparison purposes.
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Guo, Dingyi; Qi, Jing; Zhang, Wei; Cao, Rui
2017-01-20
The slow kinetics of water oxidation greatly jeopardizes the efficiency of water electrolysis for H 2 production. Developing highly active water oxidation electrodes with affordable fabrication costs is thus of great importance. Herein, a Ni II Fe III surface species on Ni metal substrate was generated by electrochemical modification of Ni in a ferrous solution by a fast, simple, and cost-effective procedure. In the prepared Ni II Fe III catalyst film, Fe III was incorporated uniformly through controlled oxidation of Fe II cations on the electrode surface. The catalytically active Ni II originated from the Ni foam substrate, which ensured the close contact between the catalyst and the support toward improved charge-transfer efficiency. The as-prepared electrode exhibited high activity and long-term stability for electrocatalytic water oxidation. The overpotentials required to reach water oxidation current densities of 50, 100, and 500 mA cm -2 are 276, 290, and 329 mV, respectively. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Molecular beam epitaxy of InN nanowires on Si
NASA Astrophysics Data System (ADS)
Golam Sarwar, A. T. M.; Carnevale, Santino D.; Kent, Thomas F.; Laskar, Masihhur R.; May, Brelon J.; Myers, Roberto C.
2015-10-01
We report on a systematic growth study of the nucleation process of InN nanowires on Si(1 1 1) substrates using plasma assisted molecular beam epitaxy (PAMBE). Samples are grown with various substrate temperatures and III/V ratios. Scanning electron microscopy, X-ray diffraction spectroscopy, energy dispersive X-ray spectroscopy, and photoluminescence are carried out to map out the variation in structural and optical properties versus growth conditions. Statistical averages of areal density, height, and radius are mapped as a function of substrate temperature and III/V ratio. Three different morphological phases are identified on the growth surface: InN, α-In and β-In. Based on SEM image analysis of samples grown at different conditions, the formation mechanism of these phases is proposed. Finally, the growth phase diagram of PAMBE grown InN on Si under N-rich condition is presented, and tapered versus non-tapered growth conditions are identified. It is found that high growth temperature and low III/V ratio plays a critical role in the growth of non-tapered InN nanowires.
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NASA Astrophysics Data System (ADS)
O'Steen, M. L.; Fedler, F.; Hauenstein, R. J.
1999-10-01
Reflection high-energy electron diffraction (RHEED) and laterally spatially resolved high resolution x-ray diffraction (HRXRD) have been used to identify and characterize rf plasma-assisted molecular-beam epitaxial growth factors which strongly affect the efficiency of In incorporation into InxGa1-xN epitaxial materials. HRXRD results for InxGa1-xN/GaN superlattices reveal a particularly strong dependence of average alloy composition x¯ upon both substrate growth temperature and incident V/III flux ratio. For fixed flux ratio, results reveal a strong thermally activated behavior, with over an order-of-magnitude decrease in x¯ with increasing growth temperature within the narrow range 590-670 °C. Within this same range, a further strong dependence upon V/III flux ratio is observed. The decreased In incorporation at elevated substrate temperatures is tentatively attributed to In surface-segregation and desorption processes. RHEED observations support this segregation/desorption interpretation to account for In loss.
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Behind the lines–actions of bacterial type III effector proteins in plant cells
Büttner, Daniela
2016-01-01
Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:28201715
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NASA Technical Reports Server (NTRS)
Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.;
1997-01-01
The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.
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1987-10-27
individuals and patients with acute EBV infectious mononucleosis . Of 124 mouthwash specimens from III patients, 33% were positive for EBV DNA with...controls and persons with acute EBV infectious mononucleosis , including those with oropharyngeal ERV detected by the bioassay. By the quantitative...oropharyngeal secretions compared to patients with acute infectious " mononucleosis (Table 5). EBV genotyping findings. Southern analysis using the Epstein-Barr
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Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications
Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, SangHyeon; Choi, Won Jun
2016-01-01
Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called “Si photonics”). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates. PMID:26864968
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Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis.
Jones, J T; Reavy, B; Smant, G; Prior, A E
2004-01-07
We report the cloning and characterisation of full-length DNAs complementary to RNA (cDNAs) encoding two glutathione peroxidases (GpXs) from a plant parasitic nematode, the potato cyst nematode (PCN) Globodera rostochiensis. One protein has a functional signal peptide that targets the protein for secretion from animal cells while the other is predicted to be intracellular. Both genes are expressed in all parasite stages tested. The mRNA encoding the intracellular GpX is present throughout the nematode second stage juvenile and is particularly abundant in metabolically active tissues including the genital primordia. The mRNA encoding the secreted GpX is restricted to the hypodermis, the outermost cellular layer of the nematode, a location from which it is likely to be secreted to the parasite surface. Biochemical studies confirmed the secreted protein as a functional GpX and showed that, like secreted GpXs of other parasitic nematodes, it does not metabolise hydrogen peroxide but has a preference for larger hydroperoxide substrates. The intracellular protein is likely to have a role in metabolism of active oxygen species derived from internal body metabolism while the secreted protein may protect the parasite from host defences. Other functional roles for this protein are discussed.
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Sat1 is dispensable for active oxalate secretion in mouse duodenum
Ko, Narae; Knauf, Felix; Jiang, Zhirong; Markovich, Daniel
2012-01-01
Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process. PMID:22517357
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Dudnik, Alexey; Dudler, Robert
2014-01-01
The Pseudomonas syringae species complex has recently been named the number one plant pathogen, due to its economic and environmental impacts, as well as for its role in scientific research. The bacterium has been repeatedly reported to cause outbreaks on bean, cucumber, stone fruit, kiwi and olive tree, as well as on other crop and non-crop plants. It also serves as a model organism for research on the Type III secretion system (T3SS) and plant-pathogen interactions. While most of the current work on this pathogen is either carried out on one of three model strains found on dicot plants with completely sequenced genomes or on isolates obtained from recent outbreaks, not much is known about strains isolated from grasses (Poaceae). Here, we use comparative genomics in order to identify putative virulence-associated genes and other Poaceae-specific adaptations in several newly available genome sequences of strains isolated from grass species. All strains possess only a small number of known Type III effectors, therefore pointing to the importance of non-Type III secreted virulence factors. The implications of this finding are discussed. PMID:25437611
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YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity
2016-01-01
SUMMARY Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted “effector” proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. PMID:27784797
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Dual luciferase assay for secreted luciferases based on Gaussia and NanoLuc.
Heise, Kerstin; Oppermann, Henry; Meixensberger, Jürgen; Gebhardt, Rolf; Gaunitz, Frank
2013-05-01
Just recently, NanoLuc, a new engineered luciferase based on the small subunit of the luciferase from Oplophorus gracilirostris was introduced. Like the luciferase from Gaussia princeps, this luciferase is secreted into the medium. Both luciferases are the smallest and brightest luciferases known and well-suited for reporter assays. In our experiments, we demonstrate that both luciferases can be used together in a dual-reporter assay by solving the problem that NanoLuc produces a significant signal with coelenterazine, which is the substrate for Gaussia luciferase. We found that the background signal from NanoLuc with coelenterazine can be calculated from the determination of NanoLuc activity in the presence of its substrate furimazine. This in turn allows the precise determination of the activity of Gaussia which does not produce light in the presence of furimazine. Based on this observation, we developed a high sensitive dual secreted luciferase assay which allows the determination of both activities in a single cotransfection experiment. We demonstrate the versatility and robustness of the assay for the normalization of reporter gene activities. Since Gaussia luciferase and NanoLuc are nonhomologous reporters, the method to determine both luciferase activities may also be useful for coincidence reporter gene systems for high-throughput screening.
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Liu, Ruihua; Zuo, Zhenqiang; Xu, Yingming; Song, Cunjiang; Jiang, Hong; Qiao, Chuanling; Xu, Ping; Zhou, Qixing; Yang, Chao
2014-04-02
The twin-arginine translocation (Tat) pathway exports folded proteins across the cytoplasmic membranes of bacteria and archaea. Two parallel Tat pathways (TatAdCd and TatAyCy systems) with distinct substrate specificities have previously been discovered in Bacillus subtilis. In this study, to secrete methyl parathion hydrolase (MPH) into the growth medium, the twin-arginine signal peptide of B. subtilis YwbN was used to target MPH to the Tat pathway of B. subtilis. Western blot analysis and MPH assays demonstrated that active MPH was secreted into the culture supernatant of wild-type cells. No MPH secretion occurred in a total-tat2 mutant, indicating that the observed export in wild-type cells was mediated exclusively by the Tat pathway. Export was fully blocked in a tatAyCy mutant. In contrast, the tatAdCd mutant was still capable of secreting MPH. These results indicated that the MPH secretion directed by the YwbN signal peptide was specifically mediated by the TatAyCy system. The N-terminal sequence of secreted MPH was determined as AAPQVR, demonstrating that the YwbN signal peptide had been processed correctly. This is the first report of functional secretion of a heterologous protein via the B. subtilis TatAyCy system. This study highlights the potential of the TatAyCy system to be used for secretion of other heterologous proteins in B. subtilis.
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Bacterial Exopolysaccharides For Corrosion Inhibition on Metal Substrates
USDA-ARS?s Scientific Manuscript database
Biofilms, composed of extra-cellular polymers secreted by bacteria, have been observed to both increase as well as decrease the rate of metal corrosion. Exopolysaccharides derived from Leuconostoc mesenteroides cultures have been shown to inhibit corrosion on corrosion-sensitive metals. The substa...
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Photoinduced Miyaura Borylation by a Rare Earth Photoreductant: the Hexachlorocerate(III) Anion.
Qiao, Yusen; Yang, Qiaomu; Schelter, Eric
2018-05-12
The first photoinduced sp2 carbon-heteroatom bond forming reaction by a rare earth photoreductant, a Miyaura borylation, has been achieved. This simple, scalable, and novel borylation method that makes use of the hexachlorocerate(III) anion, [CeIIICl6]3-, has a broad substrate scope and functional group tolerance and can be conducted at room temperature. Combined with Suzuki-Miyaura cross-coupling, the methodology is applicable to the synthesis of various biaryl products, including through the use of aryl chloride substrates. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Modulation of insulin secretion by fatty acyl analogs.
Las, Guy; Mayorek, Nina; Dickstein, Kobie; Bar-Tana, Jacob
2006-12-01
The secretagogue, the incretin-like, and the suppressive activities of long-chain fatty acids (LCFAs) in modulating insulin secretion in vivo and in cultured islets were simulated here by beta,beta'-tetramethyl-hexadecanedioic acid (M16) and alpha,alpha'-tetrachloro-tetradecanedioic acid (Cl-DICA). M16, but not Cl-DICA, serves as a substrate for ATP-dependent CoA thioesterification but is not further metabolized. M16, but not Cl-DICA, acted as a potent insulin secretagogue in islets cultured in basal but not high glucose. Short-term exposure to M16 or Cl-DICA resulted in activation of glucose- but not arginine-stimulated insulin secretion. Long-term exposure to M16, but not to Cl-DICA, resulted in suppression of glucose-, arginine-, and K(+)-stimulated insulin secretion; inhibition of glucose-induced proinsulin biosynthesis; and depletion of islets insulin. beta-Cell mass and islet ATP content remained unaffected. Hence, nonmetabolizable LCFA analogs may highlight discrete LCFA metabolites and pathways involved in modulating insulin secretion, which could be overlooked due to the rapid turnover of natural LCFA.
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An ultrahigh vacuum, low-energy ion-assisted deposition system for III-V semiconductor film growth
NASA Astrophysics Data System (ADS)
Rohde, S.; Barnett, S. A.; Choi, C.-H.
1989-06-01
A novel ion-assisted deposition system is described in which the substrate and growing film can be bombarded with high current densities (greater than 1 mA/sq cm) of very low energy (10-200 eV) ions. The system design philosophy is similar to that used in III-V semiconductor molecular-beam epitaxy systems: the chamber is an all-metal ultrahigh vacuum system with liquid-nitrogen-cooled shrouds, Knudsen-cell evaporation sources, a sample insertion load-lock, and a 30-kV reflection high-energy electron diffraction system. III-V semiconductor film growth is achieved using evaporated group-V fluxes and group-III elemental fluxes sputtered from high-purity targets using ions extracted from a triode glow discharge. Using an In target and an As effusion cell, InAs deposition rates R of 2 microns/h have been obtained. Epitaxial growth of InAs was observed on both GaSb(100) and Si(100) substrates.
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Fuel of the Bacterial Flagellar Type III Protein Export Apparatus.
Minamino, Tohru; Kinoshita, Miki; Namba, Keiichi
2017-01-01
The flagellar type III export apparatus utilizes ATP and proton motive force (PMF) across the cytoplasmic membrane as the energy sources and transports flagellar component proteins from the cytoplasm to the distal growing end of the growing structure to construct the bacterial flagellum beyond the cellular membranes. The flagellar type III export apparatus coordinates flagellar protein export with assembly by ordered export of substrates to parallel with their order of the assembly. The export apparatus is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. Since the ATPase complex is dispensable for flagellar protein export, PMF is the primary fuel for protein unfolding and translocation. Interestingly, the export gate complex can also use sodium motive force across the cytoplasmic membrane in addition to PMF when the ATPase complex does not work properly. Here, we describe experimental protocols, which have allowed us to identify the export substrate class and the primary fuel of the flagellar type III protein export apparatus in Salmonella enterica serovar Typhimurium.
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Understanding Self-Catalyzed Epitaxial Growth of III-V Nanowires toward Controlled Synthesis.
Zi, Yunlong; Suslov, Sergey; Yang, Chen
2017-02-08
The self-catalyzed growth of III-V nanowires has drawn plenty of attention due to the potential of integration in current Si-based technologies. The homoparticle-assisted vapor-liquid-solid growth mechanism has been demonstrated for self-catalyzed III-V nanowire growth. However, the understandings of the preferred growth sites of these nanowires are still limited, which obstructs the controlled synthesis and the applications of self-catalyzed nanowire arrays. Here, we experimentally demonstrated that thermally created pits could serve as the preferred sites for self-catalyzed InAs nanowire growth. On that basis, we performed a pregrowth annealing strategy to promote the nanowire density by enhancing the pits formation on the substrate surface and enable the nanowire growth on the substrate that was not capable to facilitate the growth. The discovery of the preferred self-catalyzed nanowire growth sites and the pregrowth annealing strategy have shown great potentials for controlled self-catalyzed III-V nanowire array growth with preferred locations and density.
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NASA Astrophysics Data System (ADS)
Suvarna, Puneet Harischandra
Solar-blind ultraviolet avalanche photodiodes are an enabling technology for applications in the fields of astronomy, communication, missile warning systems, biological agent detection and particle physics research. Avalanche photodiodes (APDs) are capable of detecting low-intensity light with high quantum efficiency and signal-to-noise ratio without the need for external amplification. The properties of III-N materials (GaN and AlGaN) are promising for UV photodetectors that are highly efficient, radiation-hard and capable of visible-blind or solar-blind operation without the need for external filters. However, the realization of reliable and high performance III-N APDs and imaging arrays has several technological challenges. The high price and lack of availability of bulk III-N substrates necessitates the growth of III-Ns on lattice mismatched substrates leading to a high density of dislocations in the material that can cause high leakage currents, noise and premature breakdown in APDs. The etched sidewalls of III-N APDs and high electric fields at contact edges are also detrimental to APD performance and reliability. In this work, novel technologies have been developed and implemented that address the issues of performance and reliability in III-Nitride based APDs. To address the issue of extended defects in the bulk of the material, a novel pulsed MOCVD process was developed for the growth of AlGaN. This process enables growth of high crystal quality AlxGa1-xN with excellent control over composition, doping and thickness. The process has also been adapted for the growth of high quality III-N materials on silicon substrate for devices such as high electron mobility transistors (HEMTs). A novel post-growth defect isolation technique is also discussed that can isolate the impact of conductive defects from devices. A new sidewall passivation technique using atomic layer deposition (ALD) of dielectric materials was developed for III-N APDs that is effective in reducing the dark-current and trap states at sidewalls by close to an order of magnitude, leading to improved APD performance. Development and implementation of an ion implantation based contact edge termination technique for III-N APDs that helps prevent premature breakdown from the contact edge of the devices, has further lead to improved reliability. Finally novel improved III-N APD device designs are proposed using preliminary experiments and numerical simulations for future implementations.
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Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun
2014-03-01
Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.
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Tuazon, Marc A; Campbell, Sara C; Klein, Dylan J; Shapses, Sue A; Anacker, Keith R; Anthony, Tracy G; Uzumcu, Mehmet; Henderson, Gregory C
2018-06-01
Menopause is associated with fatty liver, glucose dysregulation, increased body fat, and impaired bone quality. Previously, it was demonstrated that single sessions of high-intensity interval exercise (HIIE) are more effective than distance- and duration-matched continuous exercise (CE) on altering hepatic triglyceride (TG) metabolism and very-low density lipoprotein-TG (VLDL-TG) secretion. Six weeks training using these modalities was examined for effects on hepatic TG metabolism/secretion, glucose tolerance, body composition, and bone mineral density (BMD) in ovariectomized (OVX) and sham-operated (SHAM) mice. OVX and SHAM were assigned to distance- and duration-matched CE and HIIE, or sedentary control. Energy expenditure during exercise was confirmed to be identical between CE and HIIE and both similarly reduced post-exercise absolute carbohydrate oxidation and spontaneous physical activity (SPA). OVX vs. SHAM displayed impaired glucose tolerance and greater body fat despite lower hepatic TG, and these outcomes were not affected by training. Only HIIE increased hepatic AMPK in OVX and SHAM, but neither training type impacted VLDL-TG secretion. As expected, BMD was lower in OVX, and training did not affect long bones. The results reveal intensity-dependent effects on hepatic AMPK expression and general exercise effects on subsequent SPA and substrate oxidation that is independent of estrogen status. These findings support the notion that HIIE can impact aspects of liver physiology in females while the effects of exercise on whole body substrate selection appear to be independent of training intensity. However, neither exercise approach mitigated the impairment in glucose tolerance and elevated body fat occurring in OVX mice. Copyright © 2018 Elsevier Inc. All rights reserved.
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Pérez-Ramos, Adrian; Werning, Maria L.; Prieto, Alicia; Russo, Pasquale; Spano, Giuseppe; Mohedano, Mari L.; López, Paloma
2017-01-01
Pediococcus parvulus 2.6 secretes a 2-substituted (1,3)-β-D-glucan with prebiotic and immunomodulatory properties. It is synthesized by the GTF glycosyltransferase using UDP-glucose as substrate. Analysis of the P. parvulus 2.6 draft genome revealed the existence of a sorbitol utilization cluster of six genes (gutFRMCBA), whose products should be involved in sorbitol utilization and could generate substrates for UDP-glucose synthesis. Southern blot hybridization analysis showed that the cluster is located in a plasmid. Analysis of metabolic fluxes and production of the exopolysaccharide revealed that: (i) P. parvulus 2.6 is able to metabolize sorbitol, (ii) sorbitol utilization is repressed in the presence of glucose and (iii) sorbitol supports the synthesis of 2-substituted (1,3)-β-D-glucan. The sorbitol cluster encodes two putative regulators, GutR and GutM, in addition to a phosphoenolpyruvate-dependent phosphotransferase transport system and sorbitol-6-phosphate dehydrogenase. Therefore, we investigated the involvement of GutR and GutM in the expression of gutFRMCBA. The promoter-probe vector pRCR based on the mrfp gene, which encodes the fluorescence protein mCherry, was used to test the potential promoter of the cluster (Pgut) and the genes encoding the regulators. This was performed by transferring by electrotransformation the recombinant plasmids into two hosts, which metabolize sorbitol: Lactobacillus plantarum and Lactobacillus casei. Upon growth in the presence of sorbitol, but not of glucose, only the presence of Pgut was required to support expression of mrfp in L. plantarum. In L. casei the presence of sorbitol in the growth medium and the pediococcal gutR or gutR plus gutM in the genome was required for Pgut functionality. This demonstrates that: (i) Pgut is required for expression of the gut cluster, (ii) Pgut is subjected to catabolic repression in lactobacilli, (iii) GutR is an activator, and (iv) in the presence of sorbitol, trans-complementation for activation of Pgut exists in L. plantarum but not in L. casei. PMID:29259592
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Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway
DOE Office of Scientific and Technical Information (OSTI.GOV)
Pohlschroder Mechthild
2009-02-03
Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tatmore » pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.« less
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Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter
2013-01-01
The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400
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Bartho, Joseph D.; Bellini, Dom; Wuerges, Jochen; Demitri, Nicola; Toccafondi, Mirco; Schmitt, Armin O.; Zhao, Youfu; Walsh, Martin A.
2017-01-01
AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family. PMID:28426806
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Bartho, Joseph D; Bellini, Dom; Wuerges, Jochen; Demitri, Nicola; Toccafondi, Mirco; Schmitt, Armin O; Zhao, Youfu; Walsh, Martin A; Benini, Stefano
2017-01-01
AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.
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Hindle, Zoë; Chatfield, Steven N.; Phillimore, Jo; Bentley, Matthew; Johnson, Julie; Cosgrove, Catherine A.; Ghaem-Maghami, Marjan; Sexton, Amy; Khan, Mohammad; Brennan, Frank R.; Everest, Paul; Wu, Tao; Pickard, Derek; Holden, David W.; Dougan, Gordon; Griffin, George E.; House, Deborah; Santangelo, Joseph D.; Khan, Shahid A.; Shea, Jaqueline E.; Feldman, Robert G.; Lewis, David J. M.
2002-01-01
The attenuation and immunogenicity of two novel Salmonella vaccine strains, Salmonella enterica serovar Typhi (Ty2 ΔaroC ΔssaV, designated ZH9) and S. enterica serovar Typhimurium (TML ΔaroC ΔssaV, designated WT05), were evaluated after their oral administration to volunteers as single escalating doses of 107, 108, or 109 CFU. ZH9 was well tolerated, not detected in blood, nor persistently excreted in stool. Six of nine volunteers elicited anti-serovar Typhi lipopolysaccharide (LPS) immunoglobulin A (IgA) antibody-secreting cell (ASC) responses, with three of three vaccinees receiving 108 and two of three receiving 109 CFU which elicited high-titer LPS-specific serum IgG. WT05 was also well tolerated with no diarrhea, although the administration of 108 and 109 CFU resulted in shedding in stools for up to 23 days. Only volunteers immunized with 109 CFU of WT05 mounted detectable serovar Typhimurium LPS-specific ASC responses and serum antibody responses were variable. These data indicate that mutations in type III secretion systems may provide a route to the development of live vaccines in humans and highlight significant differences in the potential use of serovars Typhimurium and Typhi. PMID:12065485
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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.
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Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Vaca, Pilar; Berna, Genoveva; Araujo, Raquel
2008-03-10
The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice.more » Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.« less
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Harnessing Novel Secreted Inhibitors of EGF Receptor Signaling for Breast Cancer Treatment
2007-04-01
the original proposal, we described approaches for displaying the basic Argos and Dkk scaffolds as pIII fusions on M13 phage , so that we could...deal of effort into displaying Argos and other relevant proteins as pIII fusions on M13 phage (Task 2a). This has been technically very challenging... display function Argos on the surface of phage M13 , requiring a change in experimental strategy • To replace the phage display , we have established
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Vawter, G Allen [Corrales, NM
2010-08-31
An optical data latch is formed on a substrate from a pair of optical logic gates in a cross-coupled arrangement in which optical waveguides are used to couple an output of each gate to an photodetector input of the other gate. This provides an optical bi-stability which can be used to store a bit of optical information in the latch. Each optical logic gate, which can be an optical NOT gate (i.e. an optical inverter) or an optical NOR gate, includes a waveguide photodetector electrically connected in series with a waveguide electroabsorption modulator. The optical data latch can be formed on a III-V compound semiconductor substrate (e.g. an InP or GaAs substrate) from III-V compound semiconductor layers. A number of optical data latches can be cascaded to form a clocked optical data shift register.
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Karg, Saskia R; Frey, Alexander D; Kallio, Pauli T
2010-03-01
Plant-specific N-glycosylation, such as the introduction of core alpha1,3-fucose and beta1,2-xylose residues, is a major obstacle to the utilization of plant cell- or plant-derived recombinant therapeutic proteins. The beta1,4-N-acetylglucosaminyltransferase III (GnTIII) introduces a bisecting GlcNAc residue into N-glycans, which exerts a high level of substrate mediated control over subsequent modifications, for example inhibiting mammalian core fucosylation. Based on similar findings in plants, we used Nicotianatabacum BY-2 cells to study the effects of localization and expression levels of GnTIII in the remodeling of the plant N-glycosylation pathway. The N-glycans produced by the cells expressing GnTIII were partially bisected and practically devoid of the paucimannosidic type which is typical for N-glycans produced by wildtype BY-2 suspension cultured cells. The proportion of human-compatible N-glycans devoid of fucose and xylose could be increased from an average of 4% on secreted protein from wildtype cells to as high as 59% in cells expressing chimeric GnTIII, named GnTIII(A.th.) replacing its native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II. The changes in N-glycosylation observed were dependent on the catalytic activity of GnTIII, as the expression of catalytically inactive GnTIII mutants did not show a significant effect on N-glycosylation. Copyright 2010 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Geng, Yu
With the increase of clock speed and wiring density in integrated circuits, inter-chip and intra-chip interconnects through conventional electrical wires encounter increasing difficulties because of the large power loss and bandwidth limitation. Optical interconnects have been proposed as an alternative to copper-based interconnects and are under intense study due to their large data capacity, high data quality and low power consumption. III-V compound semiconductors offer high intrinsic electron mobility, small effective electron mass and direct bandgap, which make this material system advantageous for high-speed optoelectronic devices. The integration of III-V optoelectronic devices on Si substrates will provide the combined advantage of a high level of integration and large volume production of Si-based electronic circuitry with the superior electrical and optical performance of III-V components, paving the way to a new generation of hybrid integrated circuits. In this thesis, the direct heteroepitaxy of photodetectors (PDs) and light emitters using metal-organic chemical vapor deposition for the integration of photonic devices on Si substrates were studied. First we studied the selective-area growth of InP/GaAs on patterned Si substrates for PDs. To overcome the loading effect, a multi-temperature composite growth technique for GaAs was developed. By decreasing various defects such as dislocations and anti-phase domains, the GaAs and InP buffer layers are with good crystalline quality and the PDs show high speed and low dark current performance both at the edge and center of the large growth well. Then the growth and fabrication of GaAs/AlGaAs QW lasers were studied. Ellipsometry was used to calibrate the Al composition of AlGaAs. Thick p and n type AlGaAs with a mirrorlike surface were grown by high V/III ratio and high temperature. The GaAs/AlGaAs broad area QW laser was successfully grown and fabricated on GaAs substrate and showed a pulsed lasing result with a threshold current density of about 800 A/cm2. For the integration of lasers on Si substrate, quantum dot (QD) lasers were studied. A flow-and-stop process of TBA was used to grow InAs QDs with the in-situ monitor EpiRas. QDs with a PL wavelength of ˜1.3 mum were grown on GaAs and Si substrates. To decrease the PL degradation problem caused by the contaminations from AlGaAs, an InGaAs insertion layer was inserted in between the AlGaAs and QDs region. Microdisk and a-Si waveguide lasers are designed and fabricated.
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Hybrid Integration of III-V Solar Microcells for High Efficiency Concentrated Photovoltaic Modules
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tauke-Pedretti, Anna; Cederberg, Jeffery; Cruz-Campa, Jose Luis
The design, fabrication and performance of InGaAs and InGaP/GaAs microcells are presented. These cells are integrated with a Si wafer providing a path for insertion in hybrid concentrated photovoltaic modules. Comparisons are made between bonded cells and cells fabricated on their native wafer. The bonded cells showed no evidence of degradation in spite of the integration process which involved significant processing including the removal of the III-V substrate. Results from a number of hybrid cell configurations were reported. These cells employed integration techniques including wafer level bonding of processed cells and solder bonding of the cells. Lastly, the cells themselvesmore » showed evidence of degradation in spite of the integration process, which involved significant processing including the removal of the III-V substrate.« less
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Hybrid Integration of III-V Solar Microcells for High Efficiency Concentrated Photovoltaic Modules
Tauke-Pedretti, Anna; Cederberg, Jeffery; Cruz-Campa, Jose Luis; ...
2018-03-09
The design, fabrication and performance of InGaAs and InGaP/GaAs microcells are presented. These cells are integrated with a Si wafer providing a path for insertion in hybrid concentrated photovoltaic modules. Comparisons are made between bonded cells and cells fabricated on their native wafer. The bonded cells showed no evidence of degradation in spite of the integration process which involved significant processing including the removal of the III-V substrate. Results from a number of hybrid cell configurations were reported. These cells employed integration techniques including wafer level bonding of processed cells and solder bonding of the cells. Lastly, the cells themselvesmore » showed evidence of degradation in spite of the integration process, which involved significant processing including the removal of the III-V substrate.« less
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Chiang, Chunyi; Karuri, Stella W; Kshatriya, Pradnya P; Schwartz, Jeffrey; Schwarzbauer, Jean E; Karuri, Nancy W
2012-01-10
We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.
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Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner.
Conos, Stephanie A; Chen, Kaiwen W; De Nardo, Dominic; Hara, Hideki; Whitehead, Lachlan; Núñez, Gabriel; Masters, Seth L; Murphy, James M; Schroder, Kate; Vaux, David L; Lawlor, Kate E; Lindqvist, Lisa M; Vince, James E
2017-02-07
Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.
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Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner
Conos, Stephanie A.; Hara, Hideki; Whitehead, Lachlan; Núñez, Gabriel; Masters, Seth L.; Murphy, James M.; Schroder, Kate; Vaux, David L.; Lawlor, Kate E.; Vince, James E.
2017-01-01
Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases. PMID:28096356
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Giraud, Caroline; Bernard, Christophe S.; Calderon, Virginie; Ewald, Friederike; Plésiat, Patrick; Nguyen, Cathy; Grunwald, Didier; Attree, Ina; Jeannot, Katy; Fauvarque, Marie-Odile
2012-01-01
Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections. PMID:23209420
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Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.; ...
2015-07-06
Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.
Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less
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Snyder, Rae Ana; Bell, Caleb B.; Diao, Yinghui; Krebs, Carsten; Bollinger, J. Martin; Solomon, Edward I.
2013-01-01
Myo-inositol oxygenase (MIOX) catalyzes the 4e− oxidation of myo-inositol (MI) to D-glucuronate using a substrate activated Fe(II)Fe(III) site. The biferrous and Fe(II)Fe(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10,000 cm−1, split by ~2,000 cm−1, characteristic of 6 coordinate (6C) Fe(II) sites, indicating that the modest reactivity of the biferrous form toward O2 can be attributed to the saturated coordination of both irons. Upon oxidation to the Fe(II)Fe(III) state, MIOX shows two LF transitions in the ~10,000 cm−1 region, again implying a coordinatively saturated Fe(II) site. Upon MI binding, these split in energy to 5,200 cm−1 and 11,200 cm−1, showing that MI binding causes the Fe(II) to become coordinately unsaturated. VTVH MCD magnetization curves of unbound and MI-bound Fe(II)Fe(III) forms show that upon substrate binding, the isotherms become more nested, requiring that the exchange coupling and ferrous zero field splitting (ZFS) both decrease in magnitude. These results imply that MI binds to the ferric site, weakening the Fe(III)-μ-OH bond and strengthening the Fe(II)-μ-OH bond. This perturbation results in the release of a coordinated water from the Fe(II) that enables its O2 activation. PMID:24066857
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Innovative microchannel plate with reformulation of composition and modification of microstructure
NASA Astrophysics Data System (ADS)
Pan, Jingsheng; Lv, Jingwen; Kesaev, S. A.; Liu, Shulin; Liu, Zhanying; Li, Junguo; Chong, Xiaoqin; Shu, Detan
2009-07-01
The signal-to-noise ratio (SNR) and mean time to failure (MTTF) are two important attributes to describe the performance and operation life of an image intensifier. The presents of the ion barrier film (IBF) in Gen. III image intensifier, which used to suppress MCP's ion feedback, while dramatically improve the MTTF but significantly reduce the SNR, so more completely diminishing the ion poisoning source within the channels of MCP are crucial for improved Gen. III; image intensifier to thinned thickness IBF and achieving this two conflicting attributes promotion simultaneously. This research was originally initiated to develop a MCP with glass composition redesigned specially for GaAs photocathode image intensifier, proved which can be imposed an exceedingly intensive electron bombard degassing but without suffering a fatal gain degrade, and had achieved significantly improved SNR of Gen. III image intensifier but with a short distance to meet the lifetime success, so that our research work step forward to intent upon the restriction of ion poisoning source formation within the MCP substrate, we reformulated the MCP glass composition, and modified the microstructure of this MCP glass substrate though a glass-crystal phase transition during the MCP fabricate heating process, we present an innovative MCP based on a glass-ceramic substrate, with reformulated composition and close-linked network microstructure mix with many of nanometer size crystal grains, provide this MCP with sustainable high gain, lower ion feedback and less outgasing performance, this glass-ceramic MCPs are assembled to Gen. III image intensifiers which results showing promoting both the MTTF and SNR of Gen. III image intensifier.
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Cyclic Di-GMP modulates the disease progression of Erwinia amylovora.
Edmunds, Adam C; Castiblanco, Luisa F; Sundin, George W; Waters, Christopher M
2013-05-01
The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.
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Cyclic Di-GMP Modulates the Disease Progression of Erwinia amylovora
Edmunds, Adam C.; Castiblanco, Luisa F.; Sundin, George W.
2013-01-01
The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis. PMID:23475975
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Brown, I R; Mansfield, J W; Taira, S; Roine, E; Romantschuk, M
2001-03-01
The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.
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Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C. B.; Lakshmipriya, Thangavel; Tang, Thean-Hock
2015-01-01
Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues. PMID:26180812
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Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria.
Majewski, Dorothy D; Worrall, Liam J; Strynadka, Natalie Cj
2018-03-23
The acquisition and evolution of customized and often highly complex secretion systems allows Gram-negative bacteria to efficiently passage large macromolecules across both inner and outer membranes and, in some cases, that of the infected host. Essential to the virulence and ultimate survival of the many pathogenic species that encode them, secretion systems export a wide variety of effector proteins and DNA as well as the downstream extracellular filaments of the secretion apparatus themselves. Although these customized secretion systems differ in their cytosolic and inner membrane components, several commonly rely on the secretin family of giant pores to allow these large substrates to traverse the outer membrane. Recently, several near-atomic resolution cryo-EM secretin structures have unveiled the first insights into the unique structural motifs required for outer membrane localization, assembly, hallmark ultrastable nature, spontaneous membrane insertion, and mechanism of action-including the requisite central gating needed to prevent deleterious passage of periplasmic contents to the extracellular space. Copyright © 2018. Published by Elsevier Ltd.
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Briant, Linford J B; Dodd, Michael S; Chibalina, Margarita V; Rorsman, Nils J G; Johnson, Paul R V; Carmeliet, Peter; Rorsman, Patrik; Knudsen, Jakob G
2018-06-12
Glucagon, the principal hyperglycemic hormone, is secreted from pancreatic islet α cells as part of the counter-regulatory response to hypoglycemia. Hence, secretory output from α cells is under high demand in conditions of low glucose supply. Many tissues oxidize fat as an alternate energy substrate. Here, we show that glucagon secretion in low glucose conditions is maintained by fatty acid metabolism in both mouse and human islets, and that inhibiting this metabolic pathway profoundly decreases glucagon output by depolarizing α cell membrane potential and decreasing action potential amplitude. We demonstrate, by using experimental and computational approaches, that this is not mediated by the K ATP channel, but instead due to reduced operation of the Na + -K + pump. These data suggest that counter-regulatory secretion of glucagon is driven by fatty acid metabolism, and that the Na + -K + pump is an important ATP-dependent regulator of α cell function. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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Sone, Hideyuki; Sasaki, Yuka; Komai, Michio; Toyomizu, Masaaki; Kagawa, Yasuo; Furukawa, Yuji
2004-02-13
Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.
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Juárez-Hernández, Estefania O; Casados-Vázquez, Luz E; Bideshi, Dennis K; Salcedo-Hernández, R; Barboza-Corona, José E
2017-09-01
ChiA74 has modular structure that includes a secretion signal peptide (sp) sequence, and catalytic (CD), chitin insertion (CID), fibronectin type-III (FnIII) and chitin binding (CBD) domains. We described for the first time the existence of a putative CID in ChiA74. Mature ChiA74 lacking its sp sequence (rChiA74Δsp, ∼70kDa) and two truncated versions, rChiA74Δsp-60, rChiA74Δsp-50 lacking, respectively, CBD and CDB-FnIII were produced. rChiA74Δsp and rChiA74Δsp-60 are unstable and were processed to generate stable proteins of ∼50kDa. With colloidal chitin, rChiA74Δsp and rChiA74Δsp-50 had higher activity than rChiA74Δsp-60. rChiA74Δsp showed similar ability to bind chitin than rChiA74Δsp-50. The catalytic efficiencies (kcat/Km) of rChiA74Δsp and rChiA74Δsp-50 were higher, ∼ 21-fold than rChiA74Δsp-60, using chitin as the substrate. Optimal activity was detected at pH 7 and 40°C. Data suggest that the CBD in ChiA74 is important for binding to chitin, but not necessary as the presence of a CID together with the CD in a stable truncated version (i.e. ChiA74Δsp-50) has similar affinity and hydrolytic activity as the mature enzyme. The CID of ChiA74 showed identities of ∼ 55% with CIDs of other chitinases such as those from B. circulans and B. licheniformis, respectively, and conserved residues important for interacting with chitin. Copyright © 2017 Elsevier B.V. All rights reserved.
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Broadly Protective Shigella Vaccine Based on Type III Secretion Apparatus Proteins
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
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Trantas, Emmanouil A.; Licciardello, Grazia; Almeida, Nalvo F.; Witek, Kamil; Strano, Cinzia P.; Duxbury, Zane; Ververidis, Filippos; Goumas, Dimitrios E.; Jones, Jonathan D. G.; Guttman, David S.; Catara, Vittoria; Sarris, Panagiotis F.
2015-01-01
The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for genes that encode proteins involved in commercially important chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of type III secretion system and known type III effector-encoding genes from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes. Genome-mining also revealed the presence of gene clusters for biosynthesis of siderophores, polyketides, non-ribosomal peptides, and hydrogen cyanide. A highly conserved quorum sensing system was detected in all strains, although species specific differences were observed. Our study provides the basis for in-depth investigations regarding the molecular mechanisms underlying virulence strategies in the battle between plants and microbes. PMID:26300874
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Casselli, Timothy; Lynch, Tarah; Southward, Carolyn M.; Jones, Bryan W.; DeVinney, Rebekah
2008-01-01
Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis; however, its virulence mechanisms are not well understood. The identification of type III secreted proteins has provided candidate virulence factors whose functions are still being elucidated. Genotypic strain variability contributes a level of complexity to understanding the role of different virulence factors. The ability of V. parahaemolyticus to inhibit Rho family GTPases and cause cytoskeletal disruption was examined with HeLa cells. After HeLa cells were infected, intracellular Rho activation was inhibited in response to external stimuli. In vitro activation of Rho, Rac, and Cdc42 isolated from infected HeLa cell lysates was also inhibited, indicating that the bacteria were specifically targeting GTPase activation. The inhibition of Rho family GTPase activation was retained for clinical and environmental isolates of V. parahaemolyticus and was dependent on a functional chromosome I type III secretion system (CI-T3SS). GTPase inhibition was independent of hemolytic toxin genotype and the chromasome II (CII)-T3SS. Rho inhibition was accompanied by a shift in the total actin pool to its monomeric form. These phenotypes were abrogated in a mutant strain lacking the CI-T3S effector Vp1686, suggesting that the inhibiting actin polymerization may be a downstream effect of Vp1686-dependent GTPase inhibition. Although Vp1686 has been previously characterized as a potential virulence factor in macrophages, our findings reveal an effect on cultured HeLa cells. The ability to inhibit Rho family GTPases independently of the CII-T3SS and the hemolytic toxins may provide insight into the mechanisms of virulence used by strains lacking these virulence factors. PMID:18347050
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Mead, Ben; Logan, Ann; Berry, Martin; Leadbeater, Wendy; Scheven, Ben A
2013-11-15
To investigate the potential therapeutic benefit of intravitreally implanted dental pulp stem cells (DPSCs) on axotomized adult rat retinal ganglion cells (RGCs) using in vitro and in vivo neural injury models. Conditioned media collected from cultured rat DPSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were assayed for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) secretion using ELISA. DPSCs or BMSCs were cocultured with retinal cells, with or without Fc-TrK inhibitors, in a Transwell system, and the number of surviving βIII-tubulin⁺ retinal cells and length/number of βIII-tubulin⁺ neurites were quantified. For the in vivo study, DPSCs or BMSCs were transplanted into the vitreous body of the eye after a surgically induced optic nerve crush injury. At 7, 14, and 21 days postlesion (dpl), optical coherence tomography (OCT) was used to measure the retinal nerve fiber layer thickness as a measure of axonal atrophy. At 21 dpl, numbers of Brn-3a⁺ RGCs in parasagittal retinal sections and growth-associated protein-43⁺ axons in longitudinal optic nerve sections were quantified as measures of RGC survival and axon regeneration, respectively. Both DPSCs and BMSCs secreted NGF, BDNF, and NT-3, with DPSCs secreting significantly higher titers of NGF and BDNF than BMSCs. DPSCs, and to a lesser extent BMSCs, promoted statistically significant survival and neuritogenesis/axogenesis of βIII-tubulin⁺ retinal cells in vitro and in vivo where the effects were abolished after TrK receptor blockade. Intravitreal transplants of DPSCs promoted significant neurotrophin-mediated RGC survival and axon regeneration after optic nerve injury.
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Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you
2014-01-01
The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism. PMID:24675748
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Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you
2014-01-01
The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9 ≤ X ≤ 25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.
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NASA Astrophysics Data System (ADS)
Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying
2015-02-01
Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing. Electronic supplementary information (ESI) available: Sequences of RNA and DNA used in this study, relationship of the proposed three modes, CRET mechanism of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein system, calculation of the surface coverage of hairpin probe I-1 on MGO, control experiment, comparison between different modes for microRNA detection, and advantages of the proposed strategy. See DOI: 10.1039/c4nr06603k
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Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.
Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William
2005-01-01
Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.
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Johnson, Tanya L.; Waack, Ursula; Smith, Sara; Mobley, Harry
2015-01-01
ABSTRACT Gram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates of Acinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons. Despite this unique genetic organization, we show here that the A. baumannii T2S system is functional. Deletion of gspD or gspE in A. baumannii ATCC 17978 results in loss of secretion of LipA, a lipase that breaks down long-chain fatty acids. Due to a lack of extracellular lipase, the gspD mutant, the gspE mutant, and a lipA deletion strain are incapable of growth on long-chain fatty acids as a sole source of carbon, while their growth characteristics are indistinguishable from those of the wild-type strain in nutrient-rich broth. Genetic inactivation of the T2S system and its substrate, LipA, also has a negative impact on in vivo fitness in a neutropenic murine model for bacteremia. Both the gspD and lipA mutants are outcompeted by the wild-type strain as judged by their reduced numbers in spleen and liver following intravenous coinoculation. Collectively, our findings suggest that the T2S system plays a hitherto-unrecognized role in in vivo survival of A. baumannii by transporting a lipase that may contribute to fatty acid metabolism. IMPORTANCE Infections by multidrug-resistant Acinetobacter baumannii are a growing health concern worldwide, underscoring the need for a better understanding of the molecular mechanisms by which this pathogen causes disease. In this study, we demonstrated that A. baumannii expresses a functional type II secretion (T2S) system that is responsible for secretion of LipA, an extracellular lipase required for utilization of exogenously added lipids. The T2S system and the secreted lipase support in vivo colonization and thus contribute to the pathogenic potential of A. baumannii. PMID:26668261
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Influence of stress in GaN crystals grown by HVPE on MOCVD-GaN/6H-SiC substrate
Zhang, Lei; Yu, Jiaoxian; Hao, Xiaopeng; Wu, Yongzhong; Dai, Yuanbin; Shao, Yongliang; Zhang, Haodong; Tian, Yuan
2014-01-01
GaN crystals without cracks were successfully grown on a MOCVD-GaN/6H-SiC (MGS) substrate with a low V/III ratio of 20 at initial growth. With a high V/III ratio of 80 at initial growth, opaque GaN polycrystals were obtained. The structural analysis and optical characterization reveal that stress has a great influence on the growth of the epitaxial films. An atomic level model is used to explain these phenomena during crystal growth. It is found that atomic mobility is retarded by compressive stress and enhanced by tensile stress. PMID:24569601
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Bulk single crystal ternary substrates for a thermophotovoltaic energy conversion system
Charache, Greg W.; Baldasaro, Paul F.; Nichols, Greg J.
1998-01-01
A thermophotovoltaic energy conversion device and a method for making the device. The device includes a substrate formed from a bulk single crystal material having a bandgap (E.sub.g) of 0.4 eV
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Bulk single crystal ternary substrates for a thermophotovoltaic energy conversion system
Charache, G.W.; Baldasaro, P.F.; Nichols, G.J.
1998-06-23
A thermophotovoltaic energy conversion device and a method for making the device are disclosed. The device includes a substrate formed from a bulk single crystal material having a bandgap (E{sub g}) of 0.4 eV < E{sub g} < 0.7 eV and an emitter fabricated on the substrate formed from one of a p-type or an n-type material. Another thermophotovoltaic energy conversion device includes a host substrate formed from a bulk single crystal material and lattice-matched ternary or quaternary III-V semiconductor active layers. 12 figs.
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Yao, Liang-Zi; Crisostomo, Christian P.; Yeh, Chun-Chen; ...
2015-11-05
We have carried out systematic first-principles electronic structure computations of growth of ultrathin films of compounds of group III (B, Al, In, Ga, and Tl) with group V (N, P, As, Sb, and Bi) elements on Si(111) substrate, including effects of hydrogenation. Two bilayers (BLs) of AlBi, InBi, GaBi, TlAs, and TlSb are found to support a topological phase over a wide range of strains, in addition to BBi, TlN, and TlBi which can be driven into the nontrivial phase via strain. A large band gap of 134 meV is identified in hydrogenated 2 BL film of InBi. One andmore » two BL films of GaBi and 2 BL films of InBi and TlAs on Si(111) surface possess nontrivial phases with a band gap as large as 121 meV in the case of 2 BL film of GaBi. Persistence of the nontrivial phase upon hydrogenations in the III-V thin films suggests that these films are suitable for growing on various substrates.« less
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Role of Disproportionation in the Dissolution of Mn from Lithium Manganate Spinel
Benedek, Roy
2017-09-18
Dissolution of Mn from lithium-manganese spinel has hindered its commercialization as a cathode material in Li-ion batteries. Disproportionation of near-surface Mn(III), in the presence of acid, has been widely thought to result in dissolved divalent Mn. To what extent stray acidic water in the cell (as opposed to the organic electrolyte) acts as the solvent for Mn ions has not been established. Simulations by Leung show that a small displacement of trivalent Mn from its equilibrium site at an LiMn 2O 4 (001)/ ethylene carbonate interface leads to its reduction to Mn(II). In the present work, Thermodynamic Integration is performed,more » based on first-principles molecular dynamics simulations within the Blue-Moon ensemble, for the detachment of Mn(III) ions at the LiMn 2O 4 (001)/water interface. The results show that reduction of Mn(III) to Mn(II) occurs also in the case of an aqueous interface. The simulations were performed for both neutral and acidic water (in the presence of HF), with the coordination number of the dissolving Mn ion with substrate oxygen ions taken as the reaction coordinate. The simulations indicate that an F - ion strongly binds to a surface Mn(III) ion, and weakens its adhesion to the substrate. Owing to this weakening, a surface Mn-F complex traverses regions of phase space at room temperature where disproportionation becomes energetically favorable. Although this disproportionation occurs close to the substrate, where the Mn coordination number is only slightly lowered from its equilibrium value, we argue that the likelihood of reattachment after disproportionation is small (Leung arrived at a similar interpretation in the case of the LiMn 2O 4 (001)/ EC interface). We suggest that the critical role of F - in promoting dissolution is to weaken the Mn binding to the substrate so as to enable disproportionation. The partially detached MnF complex may then undergo additional interaction with the solvent to form, e.g., MnF 2, which would enable transport away from the substrate. In conclusion, the EPR measurements by Shilina et al. which appear to show Mn(III) as the predominant solvated species are discussed.« less
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Pearson, Jaclyn S; Riedmaier, Patrice; Marchès, Olivier; Frankel, Gad; Hartland, Elizabeth L
2011-01-01
Many bacterial pathogens utilize a type III secretion system (T3SS) to inject virulence effector proteins into host cells during infection. Previously, we found that enteropathogenic Escherichia coli (EPEC) uses the type III effector, NleE, to block the inflammatory response by inhibiting IκB degradation and nuclear translocation of the p65 subunit of NF-κB. Here we screened further effectors with unknown function for their capacity to prevent p65 nuclear translocation. We observed that ectopic expression of GFP–NleC in HeLa cells led to the degradation of p65. Delivery of NleC by the T3SS of EPEC also induced degradation of p65 in infected cells as well as other NF-κB components, c-Rel and p50. Recombinant His6-NleC induced p65 and p50 cleavage in HeLa cell lysates and mutation of a consensus zinc metalloprotease motif, HEIIH, abrogated NleC proteolytic activity. NleC inhibited IL-8 production during prolonged EPEC infection of HeLa cells in a protease activity-dependent manner. A double nleE/nleC mutant was further impaired for its ability to inhibit IL-8 secretion than either a single nleE or a single nleC mutant. We conclude that NleC is a type III effector protease that degrades NF-κB thereby contributing the arsenal of bacterial effectors that inhibit innate immune activation. PMID:21306441
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Triplett, Lindsay R; Wedemeyer, William J; Sundin, George W
2010-09-01
The structure of DspF, a type III secretion system (T3SS) chaperone required for virulence of the fruit tree pathogen Erwinia amylovora, was modeled based on predicted structural homology to characterized T3SS chaperones. This model guided the selection of 11 amino acid residues that were individually mutated to alanine via site-directed mutagenesis. Each mutant was assessed for its effect on virulence complementation, dimerization and interaction with the N-terminal chaperone-binding site of DspE. Four amino acid residues were identified that did not complement the virulence defect of a dspF knockout mutant, and three of these residues were required for interaction with the N-terminus of DspE. This study supports the significance of the predicted beta-sheet helix-binding groove in DspF chaperone function. Copyright 2010 Elsevier Masson SAS. All rights reserved.
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Amino acid residues 196-225 of LcrV represent a plague protective epitope.
Quenee, Lauriane E; Berube, Bryan J; Segal, Joshua; Elli, Derek; Ciletti, Nancy A; Anderson, Deborah; Schneewind, Olaf
2010-02-17
LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196-225 were necessary and sufficient for MAb BA5 binding. Compared to full-length LcrV, a variant lacking its residues 196-225 retained the ability of eliciting plague protection. These results identify LcrV residues 196-225 as a linear epitope that is recognized by the murine immune system to confer plague protection. Copyright (c) 2009 Elsevier Ltd. All rights reserved.
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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.
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The genome sequence of the facultative intracellular pathogen Brucella melitensis.
DelVecchio, Vito G; Kapatral, Vinayak; Redkar, Rajendra J; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross
2002-01-08
Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.
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The genome sequence of the facultative intracellular pathogen Brucella melitensis
DelVecchio, Vito G.; Kapatral, Vinayak; Redkar, Rajendra J.; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H.; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross
2002-01-01
Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other α-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti. PMID:11756688
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Structure of a bacterial type III secretion system in contact with a host membrane in situ.
Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R; Hayward, Richard D
2015-12-11
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.
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Amino acid residues 196–225 of LcrV represent a plague protective epitope
Quenee, Lauriane E.; Berube, Bryan J.; Segal, Joshua; Elli, Derek; Ciletti, Nancy A.; Anderson, Deborah; Schneewind, Olaf
2010-01-01
LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196–225 were necessary and sufficient for MAb-BA5 binding. Compared to full length LcrV, a variant lacking its residues 196–225 retained the ability of eliciting plague protection. These results identify LcrV residues 196–225 as a linear epitope that is recognized by the murine immune system to confer plague protection. PMID:20005318
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Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants.
Aung, Kyaw; Xin, Xiufang; Mecey, Christy; He, Sheng Yang
2017-01-01
Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector systems are a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors that target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellular localization of bacterial effector proteins in host cells could provide substantial clues to understanding the molecular and cellular basis of the virulence activities of effector proteins. In this chapter, we present methods for transient or stable expression of bacterial effector proteins in tobacco and/or Arabidopsis thaliana for live cell imaging as well as confirming the subcellular localization in plants using fluorescent organelle markers or chemical treatment.
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Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar
2015-01-01
Objective To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Methods and Design Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20–22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Results Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Conclusions Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols. PMID:26401848
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Hals, Ingrid K; Bruerberg, Simon Gustafson; Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar
2015-01-01
To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of β-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.
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Reeve, Wayne; van Berkum, Peter; Ardley, Julie; ...
2017-03-04
Bradyrhizobium elkanii USDA 76 T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76 T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76 T are described, together with its genome sequence information and annotation. The 9,484,767 bpmore » high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76 T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76 T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76 T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76 T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76 T to introduce various effector molecules into this host to enable nodulation.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Reeve, Wayne; van Berkum, Peter; Ardley, Julie
Bradyrhizobium elkanii USDA 76 T (INSCD = ARAG00000000), the type strain for Bradyrhizobium elkanii, is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Glycine max (L. Merr) grown in the USA. Because of its significance as a microsymbiont of this economically important legume, B. elkanii USDA 76 T was selected as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria sequencing project. Here the symbiotic abilities of B. elkanii USDA 76 T are described, together with its genome sequence information and annotation. The 9,484,767 bpmore » high-quality draft genome is arranged in 2 scaffolds of 25 contigs, containing 9060 protein-coding genes and 91 RNA-only encoding genes. The B. elkanii USDA 76 T genome contains a low GC content region with symbiotic nod and fix genes, indicating the presence of a symbiotic island integration. A comparison of five B. elkanii genomes that formed a clique revealed that 356 of the 9060 protein coding genes of USDA 76 T were unique, including 22 genes of an intact resident prophage. A conserved set of 7556 genes were also identified for this species, including genes encoding a general secretion pathway as well as type II, III, IV and VI secretion system proteins. The type III secretion system has previously been characterized as a host determinant for Rj and/or rj soybean cultivars. Here we show that the USDA 76 T genome contains genes encoding all the type III secretion system components, including a translocon complex protein NopX required for the introduction of effector proteins into host cells. While many bradyrhizobial strains are unable to nodulate the soybean cultivar Clark (rj1), USDA 76 T was able to elicit nodules on Clark (rj1), although in reduced numbers, when plants were grown in Leonard jars containing sand or vermiculite. In these conditions, we postulate that the presence of NopX allows USDA 76 T to introduce various effector molecules into this host to enable nodulation.« less
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III-nitride core–shell nanorod array on quartz substrates
Bae, Si-Young; Min, Jung-Wook; Hwang, Hyeong-Yong; Lekhal, Kaddour; Lee, Ho-Jun; Jho, Young-Dahl; Lee, Dong-Seon; Lee, Yong-Tak; Ikarashi, Nobuyuki; Honda, Yoshio; Amano, Hiroshi
2017-01-01
We report the fabrication of near-vertically elongated GaN nanorods on quartz substrates. To control the preferred orientation and length of individual GaN nanorods, we combined molecular beam epitaxy (MBE) with pulsed-mode metal–organic chemical vapor deposition (MOCVD). The MBE-grown buffer layer was composed of GaN nanograins exhibiting an ordered surface and preferred orientation along the surface normal direction. Position-controlled growth of the GaN nanorods was achieved by selective-area growth using MOCVD. Simultaneously, the GaN nanorods were elongated by the pulsed-mode growth. The microstructural and optical properties of both GaN nanorods and InGaN/GaN core–shell nanorods were then investigated. The nanorods were highly crystalline and the core–shell structures exhibited optical emission properties, indicating the feasibility of fabricating III-nitride nano-optoelectronic devices on amorphous substrates. PMID:28345641
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Material growth and characterization for solid state devices
NASA Technical Reports Server (NTRS)
Collis, Ward J.; Abul-Fadl, Ali; Iyer, Shanthi
1988-01-01
During the period of this research grant, the process of liquid phase electroepitaxy (LPEE) was used to grow ternary and quaternary alloy III-V semiconductor thin films. Selective area growth of InGaAs was performed on InP substrates using a patterned sputtered quartz or spin-on glass layer. The etch back and growth characteristics with respect to substrate orientation were investigated. The etch back behavior is somewhat different from wet chemical etching with respect to the sidewall profiles which are observed. LPEE was also employed to grow epitaxial layers of InGaAsP alloys on InP substrates. The behavior of Mn as an acceptor dopant was investigated with low temperature Hall coefficient and photoluminescence measurements. A metal-organic vapor phase epitaxy system was partially complete within the grant period. This atmospheric pressure system will be used to deposit III-V compound and alloy semiconductor layers in future research efforts.
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Failure Mechanisms for III-Nitride HEMT Devices
2013-11-19
rf plasma-assisted molecular beam epitaxy on freestanding GaN substrates, J. Cryst. Growth 380, 14-17 (2013). ii) Conference presentations (Invited...1 eFinal Report – AOARD Grant FA-2386-11-1-4107 Failure Mechanisms for III-nitride HEMT devices 19 November 2013 Principal Investigators: Martha...aspects of III-nitride HEMT materials and devices. Energy-filtered imaging of unstressed and stressed Ni/Au-gated AlGaN/GaN HEMTs indicated that
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Yordem, Burcu K.; Conte, Sarah S.; Ma, Jian Feng; Yokosho, Kengo; Vasques, Kenneth A.; Gopalsamy, Srinivasa N.; Walker, Elsbeth L.
2011-01-01
Background and Aims Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency. Methods The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography. Key Results Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated. Conclusions PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis. PMID:21831857
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Nakasone, Noboru; Ogura, Yasunori; Higa, Naomi; Toma, Claudia; Koizumi, Yukiko; Takaesu, Giichi; Suzuki, Toshihiko; Yamashiro, Tetsu
2018-05-23
We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections. © 2018 The Societies and John Wiley & Sons Australia, Ltd.
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Proximal tubular secretion of creatinine by organic cation transporter OCT2 in cancer patients.
Ciarimboli, Giuliano; Lancaster, Cynthia S; Schlatter, Eberhard; Franke, Ryan M; Sprowl, Jason A; Pavenstädt, Hermann; Massmann, Vivian; Guckel, Denise; Mathijssen, Ron H J; Yang, Wenjian; Pui, Ching-Hon; Relling, Mary V; Herrmann, Edwin; Sparreboom, Alex
2012-02-15
Knowledge of transporters responsible for the renal secretion of creatinine is key to a proper interpretation of serum creatinine and/or creatinine clearance as markers of renal function in cancer patients receiving chemotherapeutic agents. Creatinine transport was studied in transfected HEK293 cells in vitro and in wild-type mice and age-matched organic cation transporter 1 and 2-deficient [Oct1/2(-/-)] mice ex vivo and in vivo. Clinical pharmacogenetic and transport inhibition studies were done in two separate cohorts of cancer patients. Compared with wild-type mice, creatinine clearance was significantly impaired in Oct1/2(-/-) mice. Furthermore, creatinine inhibited organic cation transport in freshly isolated proximal tubules from wild-type mice and humans, but not in those from Oct1/2(-/-) mice. In a genetic association analysis (n = 590), several polymorphisms around the OCT2/SLC22A2 gene locus, including rs2504954 (P = 0.000873), were significantly associated with age-adjusted creatinine levels. Furthermore, in cancer patients (n = 68), the OCT2 substrate cisplatin caused an acute elevation of serum creatinine (P = 0.0083), consistent with inhibition of an elimination pathway. Collectively, this study shows that OCT2 plays a decisive role in the renal secretion of creatinine. This process can be inhibited by OCT2 substrates, which impair the usefulness of creatinine as a marker of renal function. ©2012 AACR.
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Disruption of protein-tyrosine phosphatase 1B expression in the pancreas affects β-cell function.
Liu, Siming; Xi, Yannan; Bettaieb, Ahmed; Matsuo, Kosuke; Matsuo, Izumi; Kulkarni, Rohit N; Haj, Fawaz G
2014-09-01
Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and energy balance. However, the role of PTP1B in pancreatic endocrine function remains largely unknown. To investigate the metabolic role of pancreatic PTP1B, we generated mice with pancreas PTP1B deletion (panc-PTP1B KO). Mice were fed regular chow or a high-fat diet, and metabolic parameters, insulin secretion and glucose tolerance were determined. On regular chow, panc-PTP1B KO and control mice exhibited comparable glucose tolerance whereas aged panc-PTP1B KO exhibited mild glucose intolerance. Furthermore, high-fat feeding promoted earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion in panc-PTP1B KO mice. The secretory defect in glucose-stimulated insulin secretion was recapitulated in primary islets ex vivo, suggesting that the effects were likely cell-autonomous. At the molecular level, PTP1B deficiency in vivo enhanced basal and glucose-stimulated tyrosyl phosphorylation of EphA5 in islets. Consistently, PTP1B overexpression in the glucose-responsive MIN6 β-cell line attenuated EphA5 tyrosyl phosphorylation, and substrate trapping identified EphA5 as a PTP1B substrate. In summary, these studies identify a novel role for PTP1B in pancreatic endocrine function.
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Ennouri, Habiba; d'Abzac, Paul; Hakil, Florence; Branchu, Priscilla; Naïtali, Murielle; Lomenech, Anne-Marie; Oueslati, Ridha; Desbrières, Jacques; Sivadon, Pierre; Grimaud, Régis
2017-01-01
The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.
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A Mechanistic Investigation of the Gold(III)-Catalyzed Hydrofurylation of C-C Multiple Bonds.
Hossein Bagi, Amin; Khaledi, Yousef; Ghari, Hossein; Arndt, Sebastian; Hashmi, A Stephen K; Yates, Brian F; Ariafard, Alireza
2016-11-09
The gold-catalyzed direct functionalization of aromatic C-H bonds has attracted interest for constructing organic compounds which have application in pharmaceuticals, agrochemicals, and other important fields. In the literature, two major mechanisms have been proposed for these catalytic reactions: inner-sphere syn-addition and outer-sphere anti-addition (Friedel-Crafts-type mechanism). In this article, the AuCl 3 -catalyzed hydrofurylation of allenyl ketone, vinyl ketone, ketone, and alcohol substrates is investigated with the aid of density functional theory calculations, and it is found that the corresponding functionalizations are best rationalized in terms of a novel mechanism called "concerted electrophilic ipso-substitution" (CEIS) in which the gold(III)-furyl σ-bond produced by furan auration acts as a nucleophile and attacks the protonated substrate via an outer-sphere mechanism. This unprecedented mechanism needs to be considered as an alternative plausible pathway for gold(III)-catalyzed arene functionalization reactions in future studies.
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Point, Vanessa; Bénarouche, Anaïs; Jemel, Ikram; Parsiegla, Goetz; Lambeau, Gérard; Carrière, Frédéric; Cavalier, Jean-François
2013-01-01
Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance. Copyright © 2012 Elsevier Masson SAS. All rights reserved.
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Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability
Rücker, Nadine; Billig, Sandra; Bücker, René; Jahn, Dieter
2015-01-01
ABSTRACT Mycobacterium tuberculosis persists inside granulomas in the human lung. Analysis of the metabolic composition of granulomas from guinea pigs revealed that one of the organic acids accumulating in the course of infection is acetate (B. S. Somashekar, A. G. Amin, C. D. Rithner, J. Troudt, R. Basaraba, A. Izzo, D. C. Crick, and D. Chatterjee, J Proteome Res 10:4186–4195, 2011, doi:http://dx.doi.org/10.1021/pr2003352), which might result either from metabolism of the pathogen or might be provided by the host itself. Our studies characterize a metabolic pathway by which M. tuberculosis generates acetate in the cause of fatty acid catabolism. The acetate formation depends on the enzymatic activities of Pta and AckA. Using actyl coenzyme A (acetyl-CoA) as a substrate, acetyl-phosphate is generated and finally dephosphorylated to acetate, which is secreted into the medium. Knockout mutants lacking either the pta or ackA gene showed significantly reduced acetate production when grown on fatty acids. This effect is even more pronounced when the glyoxylate shunt is blocked, resulting in higher acetate levels released to the medium. The secretion of acetate was followed by an assimilation of the metabolite when other carbon substrates became limiting. Our data indicate that during acetate assimilation, the Pta-AckA pathway acts in concert with another enzymatic reaction, namely, the acetyl-CoA synthetase (Acs) reaction. Thus, acetate metabolism might possess a dual function, mediating an overflow reaction to release excess carbon units and resumption of acetate as a carbon substrate. IMPORTANCE During infection, host-derived lipid components present the major carbon source at the infection site. β-Oxidation of fatty acids results in the formation of acetyl-CoA. In this study, we demonstrate that consumption of fatty acids by Mycobacterium tuberculosis activates an overflow mechanism, causing the pathogen to release excess carbon intermediates as acetate. The Pta-AckA pathway mediating acetate formation proved to be reversible, enabling M. tuberculosis to reutilize the previously secreted acetate as a carbon substrate for metabolism. PMID:26216844
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Hakobyan, Shoghik; Rzhepishevska, Olena; Björn, Erik; Boily, Jean-François; Ramstedt, Madeleine
2016-07-01
Development of antibiotic resistance in bacteria causes major challenges for our society and has prompted a great need for new and alternative treatment methods for infection. One promising approach is to target bacterial virulence using for example salicylidene acylhydrazides (hydrazones). Hydrazones coordinate metal ions such as Fe(III) and Ga(III) through a five-membered and a six-membered chelation ring. One suggested mode of action is via restricting bacterial Fe uptake. Thus, it was hypothesized that the chelating strength of these substances could be used to predict their biological activity on bacterial cells. This was investigated by comparing Ga chelation strength of two hydrazone complexes, as well as bacterial Ga uptake, biofilm formation, and virulence in the form of production and secretion of a toxin (ExoS) by Pseudomonas aeruginosa. Equilibrium constants for deprotonation and Ga(III) binding of the hydrazone N'-(5-chloro-2-hydroxy-3-methylbenzylidene)-2,4-dihydroxybenzhydrazide (ME0329), with anti-virulence effect against P. aeruginosa, were determined and compared to bacterial siderophores and the previously described Ga(III) 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (Ga-ME0163) and Ga-citrate complexes. In comparison with these two complexes, it was shown that the uptake of Ga(III) was higher from the Ga-ME0329 complex. The results further show that the Ga-ME0329 complex reduced ExoS expression and secretion to a higher extent than Ga-citrate, Ga-ME0163 or the non-coordinated hydrazone. However, the effect against biofilm formation by P. aeruginosa, by the ME0329 complex, was similar to Ga-citrate and lower than what has been reported for Ga-ME0163. Copyright © 2016. Published by Elsevier Inc.
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YopJ Family Effectors Promote Bacterial Infection through a Unique Acetyltransferase Activity.
Ma, Ka-Wai; Ma, Wenbo
2016-12-01
Gram-negative bacterial pathogens rely on the type III secretion system to inject virulence proteins into host cells. These type III secreted "effector" proteins directly manipulate cellular processes to cause disease. Although the effector repertoires in different bacterial species are highly variable, the Yersinia outer protein J (YopJ) effector family is unique in that its members are produced by diverse animal and plant pathogens as well as a nonpathogenic microsymbiont. All YopJ family effectors share a conserved catalytic triad that is identical to that of the C55 family of cysteine proteases. However, an accumulating body of evidence demonstrates that many YopJ effectors modify their target proteins in hosts by acetylating specific serine, threonine, and/or lysine residues. This unique acetyltransferase activity allows the YopJ family effectors to affect the function and/or stability of their targets, thereby dampening innate immunity. Here, we summarize the current understanding of this prevalent and evolutionarily conserved type III effector family by describing their enzymatic activities and virulence functions in animals and plants. In particular, the molecular mechanisms by which representative YopJ family effectors subvert host immunity through posttranslational modification of their target proteins are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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An, Yi; Wang, Jiawei; Li, Chen; Leier, André; Marquez-Lago, Tatiana; Wilksch, Jonathan; Zhang, Yang; Webb, Geoffrey I; Song, Jiangning; Lithgow, Trevor
2018-01-01
Bacterial effector proteins secreted by various protein secretion systems play crucial roles in host-pathogen interactions. In this context, computational tools capable of accurately predicting effector proteins of the various types of bacterial secretion systems are highly desirable. Existing computational approaches use different machine learning (ML) techniques and heterogeneous features derived from protein sequences and/or structural information. These predictors differ not only in terms of the used ML methods but also with respect to the used curated data sets, the features selection and their prediction performance. Here, we provide a comprehensive survey and benchmarking of currently available tools for the prediction of effector proteins of bacterial types III, IV and VI secretion systems (T3SS, T4SS and T6SS, respectively). We review core algorithms, feature selection techniques, tool availability and applicability and evaluate the prediction performance based on carefully curated independent test data sets. In an effort to improve predictive performance, we constructed three ensemble models based on ML algorithms by integrating the output of all individual predictors reviewed. Our benchmarks demonstrate that these ensemble models outperform all the reviewed tools for the prediction of effector proteins of T3SS and T4SS. The webserver of the proposed ensemble methods for T3SS and T4SS effector protein prediction is freely available at http://tbooster.erc.monash.edu/index.jsp. We anticipate that this survey will serve as a useful guide for interested users and that the new ensemble predictors will stimulate research into host-pathogen relationships and inspiration for the development of new bioinformatics tools for predicting effector proteins of T3SS, T4SS and T6SS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Heine, Shannon J.; Diaz-McNair, Jovita; Andar, Abhay U.; Drachenberg, Cinthia B.; van de Verg, Lillian; Walker, Richard; Picking, Wendy L.; Pasetti, Marcela F.
2014-01-01
Shigella is one of the leading pathogens contributing to the vast pediatric diarrheal disease burden in low-income countries. No licensed vaccine is available and the existing candidates are only partially effective and serotype-specific. Shigella type III secretion system proteins IpaB and IpaD, which are conserved across Shigella spp., are candidates for a broadly protective, subunit-based vaccine. Herein, we investigated the immunogenicity and protective efficacy of IpaB and IpaD administered intradermally (i.d.) with a double-mutant of the E. coli heat-labile enterotoxin (dmLT) adjuvant using microneedles. Different dosage levels of IpaB and IpaD with or without dmLT were tested in mice. Vaccine delivery into the dermis, recruitment of neutrophils, macrophages, dendritic cells (DC) and Langerhans cells (LC), and colocalization of vaccine antigens within skin-activated antigen presenting cells (APC) was demonstrated through histology and immunofluorescence microscopy. Ag-loaded neutrophils, macrophages, DC and LC remained in the tissue at least one week. IpaB, IpaD and dmLT-specific serum IgG and IgG secreting cells were produced following i.d. immunization. The protective efficacy was 70% against S. flexneri and 50% against S. sonnei. Similar results were obtained when the vaccine was administered intranasally, with the i.d. route requiring 25-40 times lower doses. Distinctively, IgG was detected in mucosal secretions; sIgA as well as mucosal and systemic IgA antibody secreting cells (ASC) were seemingly absent. Vaccine-induced T cells produced IFN-γ, IL-2, TNF-α, IL-17, IL-4, IL-5 and IL-10. These results demonstrate the potential of i.d. vaccination with IpaB and IpaD to prevent Shigella infection and support further studies in humans. PMID:24453241
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Litou, Chara; Vertzoni, Maria; Xu, Wei; Kesisoglou, Filippos; Reppas, Christos
2017-06-01
To propose media for simulating the intragastric environment under reduced gastric acid secretion in the fasted state at three levels of simulation of the gastric environment and evaluate their usefulness in evaluating the intragastric dissolution of salts of weak bases. To evaluate the importance of bicarbonate buffer in biorelevant in vitro dissolution testing when using Level II biorelevant media simulating the environment in the fasted upper small intestine, regardless of gastric acid secretions. Media for simulating the hypochlorhydric and achlorhydric conditions in stomach were proposed using phosphates, maleates and bicarbonates buffers. The impact of bicarbonates in Level II biorelevant media simulating the environment in upper small intestine was evaluated so that pH and bulk buffer capacity were maintained. Dissolution data were collected using two model compounds, pioglitazone hydrochloride and semifumarate cocrystal of Compound B, and the mini-paddle dissolution apparatus in biorelevant media and in human aspirates. Simulated gastric fluids proposed in this study were in line with pH, buffer capacity, pepsin content, total bile salt/lecithin content and osmolality of the fasted stomach under partial and under complete inhibition of gastric acid secretion. Fluids simulating the conditions under partial inhibition of acid secretion were useful in simulating concentrations of both model compounds in gastric aspirates. Bicarbonates in Level III biorelevant gastric media and in Level II biorelevant media simulating the composition in the upper intestinal lumen did not improve simulation of concentrations in human aspirates. Level III biorelevant media for simulating the intragastric environment under hypochlorhydric conditions were proposed and their usefulness in the evaluation of concentrations of two model salts of weak bases in gastric aspirates was shown. Level II biorelevant media for simulating the environment in upper intestinal lumen led to underestimation of concentrations in aspirates, even when bicarbonate buffer was used. Copyright © 2017 Elsevier B.V. All rights reserved.
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Inorganic arsenic impairs differentiation and functions of human dendritic cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Macoch, Mélinda; Morzadec, Claudie; Fardel, Olivier
2013-01-15
Experimental studies have demonstrated that the antileukemic trivalent inorganic arsenic prevents the development of severe pro-inflammatory diseases mediated by excessive Th1 and Th17 cell responses. Differentiation of Th1 and Th17 subsets is mainly regulated by interleukins (ILs) secreted from dendritic cells (DCs) and the ability of inorganic arsenic to impair interferon-γ and IL-17 secretion by interfering with the physiology of DCs is unknown. In the present study, we demonstrate that high concentrations of sodium arsenite (As(III), 1–2 μM) clinically achievable in plasma of arsenic-treated patients, block differentiation of human peripheral blood monocytes into immature DCs (iDCs) by inducing their necrosis.more » Differentiation of monocytes in the presence of non-cytotoxic concentrations of As(III) (0.1 to 0.5 μM) only slightly impacts endocytotic activity of iDCs or expression of co-stimulatory molecules in cells activated with lipopolysaccharide. However, this differentiation in the presence of As(III) strongly represses secretion of IL-12p70 and IL-23, two major regulators of Th1 and Th17 activities, from iDCs stimulated with different toll-like receptor (TLR) agonists in metalloid-free medium. Such As(III)-exposed DCs also exhibit reduced mRNA levels of IL12A and/or IL12B genes when activated with TLR agonists. Finally, differentiation of monocytes with non-cytotoxic concentrations of As(III) subsequently reduces the ability of activated DCs to stimulate the release of interferon-γ and IL-17 from Th cells. In conclusion, our results demonstrate that clinically relevant concentrations of inorganic arsenic markedly impair in vitro differentiation and functions of DCs, which may contribute to the putative beneficial effects of the metalloid towards inflammatory autoimmune diseases. Highlights: ► Inorganic arsenic impairs differentiation and functions of human dendritic cells (DCs) ► Arsenite (> 1 μM) blocks differentiation of dendritic cells by inducing necrosis ► Arsenite (0.1 to 0.5 μM) slightly reduces endocytotic activity of immature DCs ► Arsenite (0.1 to 0.5 μM) represses expression of IL-12p70 and IL-23 in activated DCs ► Arsenite (0.1 to 0.5 μM) reduces the ability of DCs to activate human T lymphocytes.« less
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Characterization of NopP, a Type III Secreted Effector of Rhizobium sp. Strain NGR234
Ausmees, Nora; Kobayashi, Hajime; Deakin, William J.; Marie, Corinne; Krishnan, Hari B.; Broughton, William J.; Perret, Xavier
2004-01-01
The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect. PMID:15231809
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Cuevas, M E; Callard, G
1992-10-01
Using the dogfish shark (Squalus acanthias) testis model in which different germ cell stages are topographically separated, we previously observed that steroidogenic enzyme activities vary qualitatively and quantitatively during spermatogenesis. To determine whether these data, obtained by radiolabeled tracer analysis of testicular microsomes, accurately predict steroid secretion by intact cultured spermatocysts (germ cell/Sertoli cell units), steroids were radioimmunoassayed directly in spent medium. Steroid output in basal medium was low, but after addition of 25-hydroxycholesterol (25-OH chol, 60 microM), large amounts of progestins (P) and testosterone (T) accumulated. Dehydroepiandrosterone (DHEA), androstenedione (A), and estradiol (E2) were low or nondetectable in the presence or absence of 25-OH chol; however, addition of T (1 microM) as substrate elevated E2 above assay limits. T and P contents of media increased progressively over a 24-hr culture period (Day 1). Replacement and analysis of spent media at 24-hr intervals indicated that secretion was continuous up to 6 days after seeding, although secretory rates (T plus P) and T/P ratios changed with time in culture. Spermatocysts in premeiotic (PrM), meiotic (M), and post-meiotic (PoM) stages all secreted T and P; however, absolute values, T/P ratios, and stage-dependent patterns varied from one experiment to another with no obvious seasonal pattern. Also steroid secretion by staged cysts did not consistently agree with predictions based on steroidogenic enzyme activities measured in earlier cell-free assays, whether or not 25-OH chol was present. This discrepancy was not explained by stage-related patterns of steroid secretion vs retention or by addition of putative in vivo regulators. Agents that elevated P and/or T were dibutyryl cyclic AMP, 1-isobutyl-3-methylxanthine (IBMX), and forskolin, whereas ventral lobe extract and Ca2+ ionophore (A23817) were inhibitory. An autoregulatory feedback loop was indicated by a negative relationship between cyst concentration per well and steroid secretion rate. In contrast to T and P, E2 secretion in the presence of substrate (T, 1 microM) was as predicted from previously determined patterns of aromatase activity (M > PrM > PoM) and increased in response to IBMX. Added [3H]P, [3H]T and [3H]E2 all were extensively metabolized to polar products and P metabolism was stage related (PoM > M > PrM). These data indicate that analysis of free P, T, and E2 in static cyst cultures may underestimate true steroidogenic potential. Assuming further characterization of the system, we conclude that cultured spermatocysts from dogfish testis have potential for studying the multifactorial regulation of steroid synthesis and secretion stage-by-stage during spermatogenesis.
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Solution Structure of Homology Region (HR) Domain of Type II Secretion System*
Gu, Shuang; Kelly, Geoff; Wang, Xiaohui; Frenkiel, Tom; Shevchik, Vladimir E.; Pickersgill, Richard W.
2012-01-01
The type II secretion system of Gram-negative bacteria is important for bacterial pathogenesis and survival; it is composed of 12 mostly multimeric core proteins, which build a sophisticated secretion machine spanning both bacterial membranes. OutC is the core component of the inner membrane subcomplex thought to be involved in both recognition of substrate and interaction with the outer membrane secretin OutD. Here, we report the solution structure of the HR domain of OutC and explore its interaction with the secretin. The HR domain adopts a β-sandwich-like fold consisting of two β-sheets each composed of three anti-parallel β-strands. This structure is strikingly similar to the periplasmic region of PilP, an inner membrane lipoprotein from the type IV pilus system highlighting the common evolutionary origin of these two systems and showing that all the core components of the type II secretion system have a structural or sequence ortholog within the type IV pili system. The HR domain is shown to interact with the N0 domain of the secretin. The importance of this interaction is explored in the context of the functional secretion system. PMID:22253442
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An expert-based model for selecting the most suitable substrate material type for antenna circuits
NASA Astrophysics Data System (ADS)
AL-Oqla, Faris M.; Omar, Amjad A.
2015-06-01
Quality and properties of microwave circuits depend on all the circuit components. One of these components is the substrate. The process of substrate material selection is a decision-making problem that involves multicriteria with objectives that are diverse and conflicting. The aim of this work was to select the most suitable substrate material type to be used in antennas in the microwave frequency range that gives best performance and reliability of the substrate. For this purpose, a model was built to ease the decision-making that includes hierarchical alternatives and criteria. The substrate material type options considered were limited to fiberglass-reinforced epoxy laminates (FR4 εr = 4.8), aluminium (III) oxide (alumina εr = 9.6), gallium arsenide III-V compound (GaAs εr = 12.8) and PTFE composites reinforced with glass microfibers (Duroid εr = 2.2-2.3). To assist in building the model and making decisions, the analytical hierarchy process (AHP) was used. The decision-making process revealed that alumina substrate material type was the most suitable choice for the antennas in the microwave frequency range that yields best performance and reliability. In addition, both the size of the circuit and the loss tangent of the substrates were found to be the most contributing subfactors in the antenna circuit specifications criterion. Experimental assessments were conducted utilising The Expert Choice™ software. The judgments were tested and found to be precise, consistent and justifiable, and the marginal inconsistency values were found to be very narrow. A sensitivity analysis was also presented to demonstrate the confidence in the drawn conclusions.
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novel crystalline substrates for growth of III-V Nitride semiconductors for solid-state lighting applications. Since then he has been involved in various projects involving the growth of III-V semiconductor .; Romero, M.; and Lee, M.J. "Metamorphic GaAsP buffers for growth of wide-bandgap InGaP solar cells
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Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase
Monroe, Nicole; Han, Han; Shen, Peter S; Sundquist, Wesley I; Hill, Christopher P
2017-01-01
Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases. DOI: http://dx.doi.org/10.7554/eLife.24487.001 PMID:28379137
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Structural control of In2Se3 polycrystalline thin films by molecular beam epitaxy
NASA Astrophysics Data System (ADS)
Okamoto, T.; Nakada, Y.; Aoki, T.; Takaba, Y.; Yamada, A.; Konagai, M.
2006-09-01
Structural control of In2Se3 polycrystalline thin films was attempted by molecular beam epitaxy (MBE) technique. In2Se3 polycrystalline films were obtained on glass substrates at substrate temperatures above 400 °C. VI/III ratio greatly affected crystal structure of In2Se3 polycrystalline films. Mixtures of -In2Se3 and γ-In2Se3 were obtained at VI/III ratios greater than 20, and layered InSe polycrystalline films were formed at VI/III ratios below 1. γ-In2Se3 polycrystalline thin films without α-phase were successfully deposited with VI/III ratios in a range of 2 to 4. Photocurrent spectra of the γ-In2Se3 polycrystalline films showed an abrupt increase at approximately 1.9 eV, which almost corresponds with the reported bandgap of γ-In2Se3. Dark conductivity and photoconductivity measured under solar simulator light (AM 1.5, 100 mW/cm2) were approximately 10-9 and 10-5 S/cm in the γ-In2Se3 polycrystalline thin films, respectively.
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Kim, Sang Bok; Jayaraman, Ashwin; Chua, Danny; Davis, Luke M; Zheng, Shao-Liang; Zhao, Xizhu; Lee, Sunghwan; Gordon, Roy G
2018-06-05
Indium oxide is a major component of many technologically important thin films, most notably the transparent conductor indium tin oxide (ITO). Despite being pyrophoric, homoleptic indium(III) alkyls do not allow atomic layer deposition (ALD) of In 2 O 3 using water as a co-precursor at substrate temperatures below 200 °C. Several alternative indium sources have been developed, but none allows ALD at lower temperatures except in the presence of oxidants such as O 2 or O 3 , which are not compatible with some substrates or alloying processes. We have synthesized a new indium precursor, tris(N,N'-diisopropylformamidinato)indium(III), compound 1, which allows ALD of pure, carbon-free In 2 O 3 films using H 2 O as the only co-reactant, on substrates in the temperature range 150-275 °C. In contrast, replacing just the H of the anionic iPrNC(H)NiPr ligand with a methyl group (affording the known tris(N,N'-diisopropylacetamidinato)indium(III), compound 2) results in a considerably higher and narrower ALD window in the analogous reaction with H 2 O (225-300 °C). Kinetic studies demonstrate that a higher rate of surface reactions in both parts of the ALD cycle gives rise to this difference in the ALD windows. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Influence of organic substrates on the kinetics of bacterial As(III) oxidation
NASA Astrophysics Data System (ADS)
Lescure, T.; Joulian, C.; Bauda, P.; Hénault, C.; Battaglia-Brunet, F.
2012-04-01
Soil microflora plays a major role on the behavior of metals and metalloids. Arsenic speciation, in particular, is related to the activity of bacteria able to oxidize, reduce or methylate this element, and determines mobility, bioavailability and toxicity of As. Arsenite (AsIII) is more toxic and more mobile than arsenate (AsV). Bacterial As(III)-oxidation tends to reduce the toxicity of arsenic in soils and the risk of transfer toward underlying aquifers, that would affect the quality of water resources. Previous results suggest that organic matter may affect kinetics or efficiency of bacterial As(III)-oxidation in presence of oxygen, thus in conventional physico-chemical conditions of a surface soil. Different hypothesis can be proposed to explain the influence of organic matter on As(III) oxidation. Arsenic is a potential energy source for bacteria. The presence of easily biodegradable organic matter may inhibit the As(III) oxidation process because bacteria would first metabolize these more energetic substrates. A second hypothesis would be that, in presence of organic matter, the Ars system involved in bacterial resistance to arsenic would be more active and would compete with the Aio system of arsenite oxidation, decreasing the global As(III) oxidation rate. In addition, organic matter influences the solubility of iron oxides which often act as the main pitfalls of arsenic in soils. The concentration and nature of organic matter could therefore have a significant influence on the bioavailability of arsenic and hence on its environmental impact. The influence of organic matter on biological As(III) oxidation has not yet been determined in natural soils. In this context, soil amendment with organic matter during operations of phytostabilization or, considering diffuse pollutions, through agricultural practices, may affect the mobility and bio-availability of the toxic metalloid. The objective of the present project is to quantify the influence of organic matter on the bacterial speciation of arsenic in contaminated soils. Moreover, the biogeochemical consequences of this phenomenon on the mobility and ecotoxicity of this metalloid will be studied. The first task of this program is the precise and systematic investigation of the influence of different types and concentrations of organic matters on the activity of As(III)-oxidizing pure strains. Influence of aspartate, succinate (simple substrates) and yeast extract (complex substrate) on As(III)-oxidation kinetics has been studied. For each experiment, the bacterial growth and the expression of genes involved in the speciation of arsenic, i.e. aio and ars genes, has been monitored. A direct perspective of this work will be to perform experiments with humic and fulvic acids (complex organic matter commonly found in soils), and with water-extracted organic matter from polluted soils. Then the As(III)-oxidation activity of bacterial communities extracted from contaminated soils will be followed. These assays should allow the screening of conditions which will be applied in subsequent experiments with several real contaminated soils, including a former mining site, impacted industrial sites, and a forest soil heavily contaminated after arsenical ammunitions storage. This work is co-funded by BRGM and ADEME (convention TEZ 11-16).
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Neil, Chelsea W; Lee, Byeongdu; Jun, Young-Shin
2014-10-21
Iron(III) (hydr)oxides play an important role in the geochemical cycling of contaminants in natural and engineered aquatic systems. The ability of iron(III) (hydr)oxides to immobilize contaminants can be related to whether the precipitates form heterogeneously (e.g., at mineral surfaces) or homogeneously in solution. Utilizing grazing incidence small-angle X-ray scattering (GISAXS), we studied heterogeneous iron(III) (hydr)oxide nucleation and growth on quartz substrates for systems containing arsenate and phosphate anions. For the iron(III) only system, the radius of gyration (Rg) of heterogeneously formed precipitates grew from 1.5 to 2.5 (± 1.0) nm within 1 h. For the system containing 10(-5) M arsenate, Rg grew from 3.6 to 6.1 (± 0.5) nm, and for the system containing 10(-5) M phosphate, Rg grew from 2.0 to 4.0 (± 0.2) nm. While the systems containing these oxyanions had more growth, the system containing only iron(III) had the most nucleation events on substrates. Ex situ analyses of homogeneously and heterogeneously formed precipitates indicated that precipitates in the arsenate system had the highest water content and that oxyanions may bridge iron(III) hydroxide polymeric embryos to form a structure similar to ferric arsenate or ferric phosphate. These new findings are important because differences in nucleation and growth rates and particle sizes will impact the number of available reactive sites and the reactivity of newly formed particles toward aqueous contaminants.
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Pandelia, Maria E; Li, Ning; Nørgaard, Hanne; Warui, Douglas M; Rajakovich, Lauren J; Chang, Wei-Chen; Booker, Squire J; Krebs, Carsten; Bollinger, J Martin
2013-10-23
Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or monounsaturated C(n) fatty aldehydes to formate and the corresponding C(n-1) alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 cosubstrate to the oxidation state of water and incorporates one O-atom from O2 into the formate coproduct. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2(III/III) complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2(III/III) center with resolved subsites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t(1/2) ~ 400 s at 5 °C) to produce a very modest yield of formate (<0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazinium methylsulfate ((MeO)PMS) to yield product, albeit at only ~50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or heterodinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2(III/III)-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1-C2 radical fragmentation and formation of the alk(a/e)ne and formate products.
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Plant-bacterial pathogen interactions mediated by type III effectors.
Feng, Feng; Zhou, Jian-Min
2012-08-01
Effectors secreted by the bacterial type III system play a central role in the interaction between Gram-negative bacterial pathogens and their host plants. Recent advances in the effector studies have helped cementing several key concepts concerning bacterial pathogenesis, plant immunity, and plant-pathogen co-evolution. Type III effectors use a variety of biochemical mechanisms to target specific host proteins or DNA for pathogenesis. The identifications of their host targets led to the identification of novel components of plant innate immune system. Key modules of plant immune signaling pathways such as immune receptor complexes and MAPK cascades have emerged as a major battle ground for host-pathogen adaptation. These modules are attacked by multiple type III effectors, and some components of these modules have evolved to actively sense the effectors and trigger immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Structure and biophysics of type III secretion in bacteria.
Chatterjee, Srirupa; Chaudhury, Sukanya; McShan, Andrew C; Kaur, Kawaljit; De Guzman, Roberto N
2013-04-16
Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.
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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.
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Toward a III-V Multijunction Space Cell Technology on Si
NASA Technical Reports Server (NTRS)
Ringel, S. A.; Lueck, M. R.; Andre, C. L.; Fitzgerald, E. A.; Wilt, D. M.; Scheiman, D.
2007-01-01
High efficiency compound semiconductor solar cells grown on Si substrates are of growing interest in the photovoltaics community for both terrestrial and space applications. As a potential substrate for III-V compound photovoltaics, Si has many advantages over traditional Ge and GaAs substrates that include higher thermal conductivity, lower weight, lower material costs, and the potential to leverage the extensive manufacturing base of the Si industry. Such a technology that would retain high solar conversion efficiency at reduced weight and cost would result in space solar cells that simultaneously possess high specific power (W/kg) and high power density (W/m2). For terrestrial solar cells this would result in high efficiency III-V concentrators with improved thermal conductivity, reduced cost, and via the use of SiGe graded interlayers as active component layers the possibility of integrating low bandgap sub-cells that could provide for extremely high conversion efficiency.1 In addition to photovoltaics, there has been an historical interest in III-V/Si integration to provide optical interconnects in Si electronics, which has become of even greater relevance recently due to impending bottlenecks in CMOS based circuitry. As a result, numerous strategies to integrate GaAs with Si have been explored with the primary issue being the approx.4% lattice mismatch between GaAs and Si. Among these efforts, relaxed, compositionally-graded SiGe buffer layers where the substrate lattice constant is effectively tuned from Si to that of Ge so that a close lattice match to subsequent GaAs overlayers have shown great promise. With this approach, threading dislocation densities (TDDs) of approx.1 x 10(exp 6)/sq cm have been uniformly achieved in relaxed Ge layers on Si,5 leading to GaAs on Si with minority carrier lifetimes greater than 10 ns,6 GaAs single junction solar cells on Si with efficiencies greater than 18%,7 InGaAs CW laser diodes on Si,8 and room temperature GaInP red laser diodes on Si.9 Here we report on the first high performance dual junction GaInP/GaAs solar cells grown on Si using this promising SiGe engineered substrate approach.
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Morrell-Falvey, Jennifer L.; Elkins, James G.; Wang, Zhi-Wu
2015-05-30
This study took advantage of resorufin cellobioside as a fluorescent substrate to determine the distribution of cellulase activity in cellulosic biomass fermentation systems. Cellulolytic biofilms were found to express nearly four orders greater cellulase activity compared to planktonic cultures of Clostridium thermocellum and Caldicellulosiruptor obsidiansis, which can be primarily attributed to the high cell concentration and surface attachment. The formation of biofilms results in cellulases being secreted close to their substrates, which appears to be an energetically favorable stategy for insoluble substrate utilization. For the same reason, cellulases should be closely associated with the surfaces of suspended cell in solublemore » substrate-fed culture, which has been verified with cellobiose-fed cultures of C. thermocellum and C. obsidiansis. This study addressed the importance of cellulase activity distribution in cellulosic biomass fermentation, and provided theoretical foundation for the leading role of biofilm in cellulose degradation. System optimization and reactor designs that promote biofilmformation in cellulosic biomass hydrolysismay promise an improved cellulosic biofuel process.« less
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Barta, Michael L.; Dickenson, Nicholas E.; Patil, Mrinalini; Keightley, Andrew; Wyckoff, Gerald J.; Picking, William D.; Picking, Wendy L.; Geisbrecht, Brian V.
2012-01-01
Many pathogenic Gram-negative bacteria utilize type III secretion systems (T3SS) to alter the normal functions of target cells. Shigella flexneri uses its T3SS to invade human intestinal cells to cause bacillary dysentery (shigellosis) which is responsible for over one million deaths per year. The Shigella type III secretion apparatus (T3SA) is comprised of a basal body spanning both bacterial membranes and an exposed oligomeric needle. Host altering effectors are secreted through this energized unidirectional conduit to promote bacterial invasion. The active needle tip complex of S. flexneri is composed of a tip protein, IpaD, and two pore-forming translocators, IpaB and IpaC. While the atomic structure of IpaD has been elucidated and studied, structural data on the hydrophobic translocators from the T3SS family remain elusive. We present here the crystal structures of a protease-stable fragment identified within the N-terminal regions of IpaB from S. flexneri and SipB from Salmonella enterica serovar Typhimurium determined at 2.1 Å and 2.8 Å limiting resolution, respectively. These newly identified domains are comprised of extended length (114 Å in IpaB and 71 Å in SipB) coiled-coil motifs that display a high degree of structural homology to one another despite the fact that they share only 21% sequence identity. Further structural comparisons also reveal substantial similarity to the coiled-coil regions of pore-forming proteins from other Gram-negative pathogens, notably colicin Ia. This suggests that these mechanistically-separate and functionally-distinct membrane-targeting proteins may have diverged from a common ancestor during the course of pathogen-specific evolutionary events. PMID:22321794
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Potassium transport of Salmonella is important for type III secretion and pathogenesis
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
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USDA-ARS?s Scientific Manuscript database
Vibrio parahaemolyticus is a gram-negative bacterium that inhabits coastal and marine environments. Thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and the type III secretion system are considered the potential virulent factors of pathogenic V. parahaemolyticus. The frequency of str...
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USDA-ARS?s Scientific Manuscript database
Background: Critical to the development of Salmonellosis in humans is the interaction of the bacterium with the epithelial lining of the gastrointestinal tract. Traditional scientific reasoning held type III secretion system (T3SS) as the virulence factor responsible for bacterial invasion. In this ...
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Purification and Characteriztion of the Type III Secretion System Protein from Burkholderia mallei
2013-08-01
official Department of the Army position unless so designated by other authorizing documents. REPORT DOCUMENTATION PAGE Form Approved OMB No...HP column), the procedure was performed in an automated mode using the following four steps: eluting through a HisTrap crude FF column, desalting
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The rise of the undead:Pseudokinases as mediators of effector-triggered immunity
USDA-ARS?s Scientific Manuscript database
Pathogens use effector proteins to suppress host immunity and promote infection. However, plants can recognize specific effectors and mount an effector-triggered immune response that suppresses pathogen growth. The YopJ/HopZ family of type III secreted effector proteins is broadly distributed in bac...
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Molecular mechanism of substrate recognition and transport by the AtSWEET13 sugar transporter.
Han, Lei; Zhu, Yongping; Liu, Min; Zhou, Ye; Lu, Guangyuan; Lan, Lan; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C
2017-09-19
Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 from Arabidopsis thaliana (AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2'-deoxycytidine 5'-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.
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Ribierre, J C; Ruseckas, A; Staton, S V; Knights, K; Cumpstey, N; Burn, P L; Samuel, I D W
2016-02-07
We study the influence of the film thickness on the time-resolved phosphorescence and the luminescence quantum yield of fac-tris(2-phenylpyridyl)iridium(iii) [Ir(ppy)3]-cored dendrimers deposited on dielectric substrates. A correlation is observed between the surface quenching velocity and the quenching rate by intermolecular interactions in the bulk film, which suggests that both processes are controlled by dipole-dipole interactions between Ir(ppy)3 complexes at the core of the dendrimers. It is also found that the surface quenching velocity decreases as the refractive index of the substrate is increased. This can be explained by partial screening of dipole-dipole interactions by the dielectric environment.
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Bacsi, Attila; Woodberry, Mitchell; Widger, William; Papaconstantinou, John; Mitra, Sankar; Peterson, Johnny W.; Boldogh, Istvan
2011-01-01
3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2•− generation in mitochondria respiring on the complex I substrates pyruvate + malate, an effect fully inhibited by rotenone. Antimycin A increased O2•− production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2•− production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2•− formation driven with the complex II substrate succinate. At 0.6 μM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2•− formation; however, at 40 μM myxothiazol (which completely inhibits both complexes I and III) eliminated O2•− production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2•− from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II. PMID:17011837
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Physical characterization of the cloned protease III gene from Escherichia coli K-12.
Dykstra, C C; Kushner, S R
1985-09-01
Analysis of the cloned protease III gene (ptr) from Escherichia coli K-12 has demonstrated that in addition to the previously characterized 110,000-Mr protease III protein, a second 50,000-Mr polypeptide (p50) is derived from the amino-terminal end of the coding sequence. The p50 polypeptide is found predominantly in the periplasmic space along with protease III, but does not proteolytically degrade insulin, a substrate for protease III. p50 does not appear to originate from autolysis of the larger protein. Protease III is not essential for normal cell growth since deletion of the structural gene causes no observed alterations in the phenotypic properties of the bacteria. A 30-fold overproduction of protease III does not affect cell viability. A simple new purification method for protease III is described.
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Bio-production of Baccatin III, an Important Precursor of Paclitaxel by a Cost-Effective Approach.
Lin, Shu-Ling; Wei, Tao; Lin, Jun-Fang; Guo, Li-Qiong; Wu, Guang-Pei; Wei, Jun-Bin; Huang, Jia-Jun; Ouyang, Ping-Lan
2018-07-01
Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.
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NASA Astrophysics Data System (ADS)
Deng, Gaoqiang; Zhang, Yuantao; Yu, Ye; Yan, Long; Li, Pengchong; Han, Xu; Chen, Liang; Zhao, Degang; Du, Guotong
2018-04-01
In this paper, N-polar GaN films with different V/III ratios were grown on vicinal C-face SiC substrates by metalorganic chemical vapor deposition. During the growth of N-polar GaN film, the V/III ratio was controlled by adjusting the molar flow rate of ammonia while keeping the trimethylgallium flow rate unchanged. The influence of the V/III ratio on the surface morphology of N-polar GaN film has been studied. We find that the surface root mean square roughness of N-polar GaN film over an area of 20 × 20 μm2 can be reduced from 8.13 to 2.78 nm by optimization of the V/III ratio. Then, using the same growth conditions, N-polar InGaN/GaN multiple quantum wells (MQWs) light-emitting diodes (LEDs) were grown on the rough and the smooth N-polar GaN templates, respectively. Compared with the LED grown on the rough N-polar GaN template, dramatically improved interface sharpness and luminescence uniformity of the InGaN/GaN MQWs are achieved for the LED grown on the smooth N-polar GaN template.
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Investigation of Hexagonal Ferrite Film Growth Techniques for Millimeter-Wave Systems Applications.
1987-03-15
hexaferrite LPE layer on CoGa 204 substrate. Some areas of the film exhibit a terraced structure .................................... 32 Figure 17 (a) Layer...49 G), which is the standard substrate for yttrium iron garnet epitaxial film growth. The dielectric constant is slightly larger than values for...Details are given ’.- in Appendices III a:d IV. 4.1 INTIODUCTION In LPE , a small lattice mismatch between film and substrate is .r..r ial tc gro.w good
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Mukherjee, Kakoli; Chattopadhyay, Naibedya
2016-10-01
Osteoporosis is a metabolic bone disease that is characterized by heightened state of bone resorption accompanied by diminished bone formation, leading to a reduction of bone mineral density (BMD) and deterioration of bone quality, thus increasing the risk of developing fractures. Molecular insight into bone biology identified cathepsin K (CatK) as a novel therapeutic target. CatK is a lysosomal cysteine protease secreted by activated osteoclasts during bone resorption, whose primary substrate is type I collagen, the major component of organic bone matrix. Available anti-resorptive drugs affect osteoclast survival and influence both resorption and formation of bone. CatK inhibitors are distinct from the existing anti-resorptives as they only target the resorption process itself without impairing osteoclast differentiation and do not interfere with bone formation. An inhibitor of CatK, odanacatib, robustly increased both trabecular and cortical BMD in postmenopausal osteoporosis patients. The phase III fracture prevention trial with odanacatib ended early due to good efficacy and a favorable benefit/risk profile, thus, enhancing the opportunity for CatK as a pharmacological target for osteoporosis. So far, all the inhibitors that reached to the stage of clinical trial targeted active site of CatK to abrogate the entire proteolytic activity of the enzyme in addition to the desired blockage of excessive elastin and collagen degradation, and could thus pose safety concerns with long term use. Identification of selective exosite inhibitors that inhibit CatK's elastase and/or collagenase activity but do not affect the hydrolysis of other physiologically relevant substrates of CatK would be an improved strategy to inhibit this enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.
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Substrate temperature constrains recruitment and trail following behavior in ants.
van Oudenhove, Louise; Boulay, Raphaël; Lenoir, Alain; Bernstein, Carlos; Cerda, Xim
2012-06-01
In many ant species, foragers use pheromones to communicate the location of resources to nestmates. Mass-recruiting species deposit long-lasting anonymous chemical trails, while group-recruiting species use temporary chemical trails. We studied how high temperature influenced the foraging behavior of a mass-recruiting species (Tapinoma nigerrimum) and a group-recruiting species (Aphaenogaster senilis) through pheromone decay. First, under controlled laboratory conditions, we examined the effect of temperature on the trail pheromone of both species. A substrate, simulating soil, marked with gaster extract was heated for 10 min. at 25°, 35°, 45°, or 55 °C and offered to workers in a choice test. Heating gaster extract reduced the trail following behavior of the mass-recruiters significantly more than that of the group-recruiters. Second, analyses of the chemicals present on the substrate indicated that most T. nigerrimum gaster secretions vanished at 25 °C, and only iridodials persisted up to 55 °C. By contrast, A. senilis secretions were less volatile and resisted better to elevated temperatures to some extent. However, at 55 °C, the only chemicals that persisted were nonadecene and nonadecane. Overall, our results suggest that the foraging behavior of the group-recruiting species A. senilis is less affected by pheromone evaporation than that of the mass-recruiting species T. nigerrimum. This group-recruiting species might, thus, be particularly adapted to environments with fluctuating temperatures.
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A study of overproduction and enhanced secretion of enzymes. Quarterly report
DOE Office of Scientific and Technical Information (OSTI.GOV)
Dashek, W.V.
1993-09-01
Wood decay within forests, a significant renewable photosynthetic energy resource, is caused primarily by Basidiomycetous fungi, e.g., white rot fungi. These organisms possess the ability to degrade lignin, cellulose and hemicellulose, the main organic polymers of wood. In the case of the white rot fungi, e.g., Coriolus versicolor, the capacity results from the fungus` ability to elaborate extracellular cellulolytic and ligninolytic enzymes. With regard to the latter, at least one of the enzymes, polyphenol oxidase (PPO) appears within a defined growth medium. This proposal focuses on the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. There are twomore » major sections to the proposal: (1) overproduction of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electro microscopical techniques and (2) the biochemical/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO enzymes.« less
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The FapF amyloid secretion transporter possesses an atypical asymmetric coiled coil.
Rouse, Sarah L; Stylianou, Fisentzos; Grace Wu, H Y; Berry, Jamie-Lee; Sewell, Lee; Morgan, R Marc L; Sauerwein, Andrea C; Matthews, Steve
2018-06-07
Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits, the fibres of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialised outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8 Å resolution. This domain forms a novel asymmetric trimeric coiled-coil that possesses a single buried tyrosine residue as well as a extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail. Copyright © 2018. Published by Elsevier Ltd.
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MBE growth technology for high quality strained III-V layers
NASA Technical Reports Server (NTRS)
Grunthaner, Frank J. (Inventor); Liu, John K. (Inventor); Hancock, Bruce R. (Inventor)
1990-01-01
The III-V films are grown on large automatically perfect terraces of III-V substrates which have a different lattice constant, with temperature and Group III and V arrival rates chosen to give a Group III element stable surface. The growth is pulsed to inhibit Group III metal accumulation of low temperature, and to permit the film to relax to equilibrium. The method of the invention: (1) minimizes starting step density on sample surface; (2) deposits InAs and GaAs using an interrupted growth mode (0.25 to 2 monolayers at a time); (3) maintains the instantaneous surface stoichiometry during growth (As-stable for GaAs, In-stable for InAs); and (4) uses time-resolved RHEED to achieve aspects (1) through (3).
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Chong, Euming; Dysart, Kevin C; Chidekel, Aaron; Locke, Robert; Shaffer, Thomas H; Miller, Thomas L
2009-04-01
Mechanical ventilation (MV) of the neonatal airway alters mechanical properties and activates tissue-modeling pathways. Heat shock protein (HSP70) is a marker of tissue injury and modulates inflammation, which may influence subsequent pulmonary tissue modeling by matrix metalloproteinases (MMPs). HSP70 secretion is up-regulated in MV airway tissues and associated with changes in airway elasticity and secretion of MMPs. Proximal tracheal segments were isolated in 13 newborn lambs and were either MV for 4 h or SHAM. At baseline and hourly, tracheal segments were flushed and tracheal elasticity was determined. Tracheal wash fluid was assayed for HSP70 by ELISA and for MMPs by substrate zymography. HSP70 secretion increased from baseline to a peak at 1 h in both groups (p < 0.01), greater in the MV group (p < 0.05), and returned to baseline values by 2 h. This response was in contrast to the progressive decrease in tracheal elasticity (p < 0.05). The HSP70 elevation pattern was noted in MMP-2, but beyond 1 h, MMP-2 returned to baseline values in MV group but remained elevated in SHAM (p < 0.05). HSP70 secretion is associated with the degree of biophysical tracheal injury as well as the time course of MMP-2 secretion by tracheal tissues.
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Zylbersztejn, Ana Madeira Brito; de Morais, Carlos Gustavo Vieira; Lima, Ana Karina Castro; Souza, Joyce Eliza de Oliveira; Lopes, Angela Hampshire; Da-Silva, Sílvia Amaral Gonçalves; Silva-Neto, Mário Alberto Cardoso; Dutra, Patrícia Maria Lourenço
2015-01-01
CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.
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de Groot, A; Koster, M; Gérard-Vincent, M; Gerritse, G; Lazdunski, A; Tommassen, J; Filloux, A
2001-02-01
Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).
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Prospects of III-nitride optoelectronics grown on Si.
Zhu, D; Wallis, D J; Humphreys, C J
2013-10-01
The use of III-nitride-based light-emitting diodes (LEDs) is now widespread in applications such as indicator lamps, display panels, backlighting for liquid-crystal display TVs and computer screens, traffic lights, etc. To meet the huge market demand and lower the manufacturing cost, the LED industry is moving fast from 2 inch to 4 inch and recently to 6 inch wafer sizes. Although Al2O3 (sapphire) and SiC remain the dominant substrate materials for the epitaxy of nitride LEDs, the use of large Si substrates attracts great interest because Si wafers are readily available in large diameters at low cost. In addition, such wafers are compatible with existing processing lines for 6 inch and larger wafers commonly used in the electronics industry. During the last decade, much exciting progress has been achieved in improving the performance of GaN-on-Si devices. In this contribution, the status and prospects of III-nitride optoelectronics grown on Si substrates are reviewed. The issues involved in the growth of GaN-based LED structures on Si and possible solutions are outlined, together with a brief introduction to some novel in situ and ex situ monitoring/characterization tools, which are especially useful for the growth of GaN-on-Si structures.
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Towards large size substrates for III-V co-integration made by direct wafer bonding on Si
NASA Astrophysics Data System (ADS)
Daix, N.; Uccelli, E.; Czornomaz, L.; Caimi, D.; Rossel, C.; Sousa, M.; Siegwart, H.; Marchiori, C.; Hartmann, J. M.; Shiu, K.-T.; Cheng, C.-W.; Krishnan, M.; Lofaro, M.; Kobayashi, M.; Sadana, D.; Fompeyrine, J.
2014-08-01
We report the first demonstration of 200 mm InGaAs-on-insulator (InGaAs-o-I) fabricated by the direct wafer bonding technique with a donor wafer made of III-V heteroepitaxial structure grown on 200 mm silicon wafer. The measured threading dislocation density of the In0.53Ga0.47As (InGaAs) active layer is equal to 3.5 × 109 cm-2, and it does not degrade after the bonding and the layer transfer steps. The surface roughness of the InGaAs layer can be improved by chemical-mechanical-polishing step, reaching values as low as 0.4 nm root-mean-square. The electron Hall mobility in 450 nm thick InGaAs-o-I layer reaches values of up to 6000 cm2/Vs, and working pseudo-MOS transistors are demonstrated with an extracted electron mobility in the range of 2000-3000 cm2/Vs. Finally, the fabrication of an InGaAs-o-I substrate with the active layer as thin as 90 nm is achieved with a Buried Oxide of 50 nm. These results open the way to very large scale production of III-V-o-I advanced substrates for future CMOS technology nodes.
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Arakawa, Takatoshi; Kawano, Yoshiaki; Kataoka, Shingo; Katayama, Yoko; Kamiya, Nobuo; Yohda, Masafumi; Odaka, Masafumi
2007-03-09
Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.
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Doucet, Sébastien; Soussignan, Robert; Sagot, Paul; Schaal, Benoist
2009-01-01
Background The communicative meaning of human areolae for newborn infants was examined here in directly exposing 3-day old neonates to the secretion from the areolar glands of Montgomery donated by non related, non familiar lactating women. Methodology/Principal Findings The effect of the areolar stimulus on the infants' behavior and autonomic nervous system was compared to that of seven reference stimuli originating either from human or non human mammalian sources, or from an arbitrarily-chosen artificial odorant. The odor of the native areolar secretion intensified more than all other stimuli the infants' inspiratory activity and appetitive oral responses. These responses appeared to develop independently from direct experience with the breast or milk. Conclusion/Significance Areolar secretions from lactating women are especially salient to human newborns. Volatile compounds carried in these substrates are thus in a position to play a key role in establishing behavioral and physiological processes pertaining to milk transfer and production, and, hence, to survival and to the early engagement of attachment and bonding. PMID:19851461
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Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice
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
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Method for making graded I-III-VI.sub.2 semiconductors and solar cell obtained thereby
Devaney, Walter E.
1987-08-04
Improved cell photovoltaic conversion efficiencies are obtained by the simultaneous elemental reactive evaporation process of Mickelsen and Chen for making semiconductors by closer control of the evaporation rates and substrate temperature during formation of the near contact, bulk, and near junction regions of a graded I-III-VI.sub.2, thin film, semiconductor, such as CuInSe.sub.2 /(Zn,Cd)S or another I-III-VI.sub.2 /II-VI heterojunction.
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Integration of Indium Phosphide Based Devices with Flexible Substrates
NASA Astrophysics Data System (ADS)
Chen, Wayne Huai
2011-12-01
Flexible substrates have many advantages in applications where bendability, space, or weight play important roles or where rigid circuits are undesirable. However, conventional flexible thin film transistors are typically characterized as having low carrier mobility as compared to devices used in the electronics industry. This is in part due to the limited temperature tolerance of plastic flexible substrates, which commonly reduces the highest processing temperature to below 200°C. Common approaches of implementation include low temperature deposition of organic, amorphous, or polycrystalline semiconductors, all of which result in carrier mobility well below 100 cm2V -1s-1. High quality, single crystalline III-V semiconductors such as indium phosphide (InP), on the other hand, have carrier mobility well over 1000 cm 2V-1s-1 at room temperature, depending on carrier concentration. Recently, the ion-cut process has been used in conjunction with wafer bonding to integrate thin layers of III-V material onto silicon for optoelectronic applications. This approach has the advantage of high scalability, reusability of the initial III-V substrate, and the ability to tailor the location (depth) of the layer splitting. However, the transferred substrate usually suffers from hydrogen implantation damage. This dissertation demonstrates a new approach to enable integration of InP with various substrates, called the double-flip transfer process. The process combines ion-cutting with adhesive bonding. The problem of hydrogen implantation was overcome by patterned ion-cut transfer. In this type of transfer, areas of interest are shielded from implantation but still transferred by surrounding implanted regions. We found that patterned ion-cut transfer is strongly dependent upon crystal orientation and that using cleavage-plane oriented donors can be beneficial in transferring large areas of high quality semiconductor material. InP-based devices were fabricated to demonstrate the transfer process and test functionality following transfer. Passive devices (photodetectors) as well as active transistors were transferred and fabricated on various substrates. The transferred device layers were either implanted through with a blanket implant or protected with an ion-mask during implantation. Results demonstrate the viability of the double-flip ion-cut process in achieving very high electron mobility (˜2800 cm2V-1s-1) transistors on plastic flexible substrates.
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Precise Nanoelectronics with Adatom Chains
NASA Technical Reports Server (NTRS)
Yamada, Toshishige
1999-01-01
Adatom chains on an atomically regulated substrate will be building components in future precise nanoelectronics. Adatoms need to be secured with chemical bonding, but then electronic isolation between the adatom and substrate systems is not guaranteed. A one-dimensional model shows that good isolation with existence of surface states is expected on an s-p crossing substrate such as Si, Ge, or GaAs, reflecting the bulk nature of the substrate. Isolation is better if adatoms are electronically similar to the substrate atoms, and can be manipulated by hydrogenation. Chain structures with group IV adatoms with two chemical bonds, or group III adatoms with one chemical bond, are semiconducting, reflecting the surface nature of the substrate. These structures are unintentionally doped due to the charge transfer across the chemical bonds. Physical properties of adatom chains have to be determined for the unified adatom-substrate system.
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Lai, Liying; Dai, Jiao; Tang, Huanyu; Zhang, Shouming; Wu, Chunyan; Qiu, Wancen; Lu, Chengping; Yao, Huochun; Fan, Hongjie; Wu, Zongfu
2017-06-01
Streptococcus suis (SS), an important pathogen for pigs, is not only considered as a zoonotic agent for humans, but is also recognized as a major reservoir of antimicrobial resistance contributing to the spread of resistance genes to other pathogenic Streptococcus species. In addition to serotype 2 (SS2), serotype 9 (SS9) is another prevalent serotype isolated from diseased pigs. Although many SS strains have been sequenced, the complete genome of a non-SS2 virulent strain has been unavailable to date. Here, we report the complete genome of GZ0565, a virulent strain of SS9, isolated from a pig with meningitis. Comparative genomic analysis revealed five new putative virulence or antimicrobial resistance-associated genes in strain GZ0565 but not in SS2 virulent strains. These five genes encode a putative triacylglycerol lipase, a TipAS antibiotic-recognition domain protein, a putative TetR family transcriptional repressor, a protein containing a LPXTG domain and a G5 domain, and a type VII secretion system (T7SS) putative substrate (EsxA), respectively. Western blot analysis showed that strain GZ0565 can secrete EsxA. We generated an esxA deletion mutant and showed that EsxA contributes to SS virulence in a mouse infection model. Additionally, the antibiotic resistance gene vanZ SS was identified and expression of vanZ SS conferred resistance to teicoplanin and dalbavancin in Streptococcus agalactiae. We believe this is the first experimental demonstration of the existence of the T7SS putative substrate EsxA and its contribution to bacterial virulence in SS. Together, our results contribute to further understanding of the virulence and antimicrobial resistance characteristics of SS. Copyright © 2017 Elsevier B.V. All rights reserved.
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Methods of increasing secretion of polypeptides having biological activity
Merino, Sandra
2014-05-27
The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.
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Methods of increasing secretion of polypeptides having biological activity
Merino, Sandra
2014-10-28
The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.
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Methods of increasing secretion of polypeptides having biological activity
Merino, Sandra
2015-04-14
The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.
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Methods of increasing secretion of polypeptides having biological activity
Merino, Sandra
2013-10-01
The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.
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An immunohistochemical study of the endocrine pancreas in raptors.
Palmieri, C; Shivaprasad, H L
2014-12-01
The cytoarchitecture of the endocrine pancreas of 10 raptors (golden eagles, peregrine falcons, Saker falcon, turkey vultures, red-tailed hawk and unspecified falcon) was examined by immunohistochemistry. Three islet types were identified: type A mixed islets composed mainly by glucagon (A)-secreting cells, type B mixed islets with predominantly insulin (B)-secreting cell component and type M mixed islets (type M) consisting of variable number of glucagon-, insulin- and somatostatin (D)-secreting cells. The latter were further characterized into Type I, II or III according to the cell distribution of the three cell types. A and D cells were also randomly scattered within the exocrine pancreas. The results of this study suggest that the classical concept in birds of a segregation of A and B cells in well-defined and distinct islets is not applicable in raptors, reflecting an evolutionary adaptation to different dietary habits and variation in developmental mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Pang, Xiuhua; Samten, Buka; Cao, Guangxiang; Wang, Xisheng; Tvinnereim, Amy R; Chen, Xiu-Lan; Howard, Susan T
2013-01-01
The ESX-1 secretion system exports the immunomodulatory protein ESAT-6 and other proteins important in the pathogenesis of Mycobacterium tuberculosis. Components and substrates of ESX-1 are encoded at several loci, but the regulation of the encoding genes is only partially understood. In this study, we investigated the role of the MprAB two-component system in the regulation of ESX-1 activity. We determined that MprAB directly regulates the espA gene cluster, a locus necessary for ESX-1 function. Transcript mapping determined that the five genes in the cluster form an operon with two transcriptional start points, and several MprA binding sites were detected in the espA promoter. Expression analyses and promoter constructs indicated that MprAB represses the espA operon. However, the MprAB mutant Rv-D981 secreted lower levels of EspA, ESAT-6, and the ESX-1 substrate EspB than control strains. Secretion of CFP10, which is normally cosecreted with ESAT-6, was similar in Rv-D981 and control strains, further demonstrating aberrant ESX-1 activity in the mutant. ESAT-6 induces proinflammatory cytokines, and macrophages infected with Rv-D981 elicited lower levels of interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α), consistent with the reduced levels of ESAT-6. These findings indicate that MprAB modulates ESX-1 function and reveal a new role for MprAB in host-pathogen interactions.
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Kennedy, George M.; Hooley, Gwendolyn C.; Champion, Matthew M.; Mba Medie, Felix
2014-01-01
EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence. PMID:24610712
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Andrade, Gabriela Rabelo; de Araújo, João Locke Ferreira; Nakamura Filho, Arnaldo; Guañabens, Anna Carolina Paganini; Carvalho, Marcela David de; Cardoso, Antônio Valadão
2015-09-01
In this study we characterized the surface morphology and ultrastructure of the foot of the golden mussel, Limnoperna fortunei (Dunker, 1857), relating its characteristics to the attaching mechanisms of this mollusk. The observation of the foot of this bivalve reveals the presence of micro-scaled cilia with a unique shape, which has a narrowing at its end. This characteristic was associated to the capacity for underwater adhesion to substrates through the employment of van der Waals forces, resembling the adhesion phenomenon of the gecko foot. The temporary attachment during locomotion by means of the foot to substrates was observed to be strong even on smooth surfaces, like glass, or hydrophobic waxy surfaces. Comparing TEM and light microscopy results it was possible to associate the mucous secretions and secreting cells found along the tissues to the production of the byssus inside the groove on the ventral portion of the foot. Not only our experiments, but also the state of the art allowed us to discard the involvement of secretions produced in the foot of the mussel to the temporary adhesion. Through SEM images it was possible to build a virtual three-dimensional model where total foot surface was measured for the estimated calculation of van der Waals forces. Also, some theoretical analysis and considerations have been made concerning the characteristics of the functional surface of L. fortunei foot. Copyright © 2015 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Harrell, Permila C.; McCawley, Lisa J.; Fingleton, Barbara
2005-02-15
Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment asmore » compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7{sup HIGH}-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium.« less
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Proximal Tubular Secretion of Creatinine by Organic Cation Transporter OCT2 in Cancer Patients
Ciarimboli, Giuliano; Lancaster, Cynthia S.; Schlatter, Eberhard; Franke, Ryan M.; Sprowl, Jason A.; Pavenstädt, Hermann; Massmann, Vivian; Guckel, Denise; Mathijssen, Ron H. J.; Yang, Wenjian; Pui, Ching-Hon; Relling, Mary V.; Herrmann, Edwin; Sparreboom, Alex
2012-01-01
Purpose Knowledge of transporters responsible for the renal secretion of creatinine is key to a proper interpretation of serum creatinine and/or creatinine clearance as markers of renal function in cancer patients receiving chemotherapeutic agents. Experimental Design Creatinine transport was studied in transfected HEK293 cells in vitro and in wildtype mice and age-matched organic cation transporter 1 and 2-deficient [Oct1/2(−/−)] mice ex vivo and in vivo. Clinical pharmacogenetic and transport inhibition studies were done in two separate cohorts of cancer patients. Results Compared to wildtype mice, creatinine clearance was significantly impaired in Oct1/2(−/−) mice. Furthermore, creatinine inhibited organic cation transport in freshly-isolated proximal tubules from wild-type mice and humans, but not in those from Oct1/2(−/−) mice. In a genetic-association analysis (n=590), several polymorphisms around the OCT2/SLC22A2 gene locus, including rs2504954 (P=0.000873), were significantly associated with age-adjusted creatinine levels. Furthermore, in cancer patients (n=68), the OCT2 substrate cisplatin caused an acute elevation of serum creatinine (P=0.0083), consistent with inhibition of an elimination pathway. Conclusions Collectively, this study shows that OCT2 plays a decisive role in the renal secretion of creatinine. This process can be inhibited by OCT2 substrates, which impair the usefulness of creatinine as a marker of renal function. PMID:22223530
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Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression
Chaffin, W. Lajean; López-Ribot, José Luis; Casanova, Manuel; Gozalbo, Daniel; Martínez, José P.
1998-01-01
The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis. PMID:9529890
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Granato, Marcela Queiroz; Massapust, Priscila de Araújo; Rozental, Sonia; Alviano, Celuta Sales; dos Santos, André Luis Souza; Kneipp, Lucimar Ferreira
2015-04-01
Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis.
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Targeting bacterial secretion systems: benefits of disarmament in the microcosm.
Baron, Christian; Coombes, Brian
2007-03-01
Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections.
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Hong, Young Joon; Kim, Yong-Jin; Jeon, Jong-Myeong; Kim, Miyoung; Choi, Jun Hee; Baik, Chan Wook; Kim, Sun Il; Park, Sung Soo; Kim, Jong Min; Yi, Gyu-Chul
2011-05-20
We report on the fabrication of high-quality GaN on soda-lime glass substrates, heretofore precluded by both the intolerance of soda-lime glass to the high temperatures required for III-nitride growth and the lack of an epitaxial relationship with amorphous glass. The difficulties were circumvented by heteroepitaxial coating of GaN on ZnO nanorods via a local microheating method. Metal-organic chemical vapor deposition of ZnO nanorods and GaN layers using the microheater arrays produced high-quality GaN/ZnO coaxial nanorod heterostructures at only the desired regions on the soda-lime glass substrates. High-resolution transmission electron microscopy examination of the coaxial nanorod heterostructures indicated the formation of an abrupt, semicoherent interface. Photoluminescence and cathodoluminescence spectroscopy was also applied to confirm the high optical quality of the coaxial nanorod heterostructures. Mg-doped GaN/ZnO coaxial nanorod heterostructure arrays, whose GaN shell layers were grown with various different magnesocene flow rates, were further investigated by using photoluminescence spectroscopy for the p-type doping characteristics. The suggested method for fabrication of III-nitrides on glass substrates signifies potentials for low-cost and large-size optoelectronic device applications.
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Multi-junction solar cell device
Friedman, Daniel J.; Geisz, John F.
2007-12-18
A multi-junction solar cell device (10) is provided. The multi-junction solar cell device (10) comprises either two or three active solar cells connected in series in a monolithic structure. The multi-junction device (10) comprises a bottom active cell (20) having a single-crystal silicon substrate base and an emitter layer (23). The multi-junction device (10) further comprises one or two subsequent active cells each having a base layer (32) and an emitter layer (23) with interconnecting tunnel junctions between each active cell. At least one layer that forms each of the top and middle active cells is composed of a single-crystal III-V semiconductor alloy that is substantially lattice-matched to the silicon substrate (22). The polarity of the active p-n junction cells is either p-on-n or n-on-p. The present invention further includes a method for substantially lattice matching single-crystal III-V semiconductor layers with the silicon substrate (22) by including boron and/or nitrogen in the chemical structure of these layers.
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Thermodynamic analysis of vapor-phase epitaxial growth of GaAsN on Ge
NASA Astrophysics Data System (ADS)
Kawano, Jun; Kangawa, Yoshihiro; Ito, Tomonori; Kakimoto, Koichi; Koukitu, Akinori
2012-03-01
In this paper, we use thermodynamic analysis to determine how the nitrogen (N) ratio in the source gases affects the solid composition of coherently grown GaAs1-xNx(x˜0.03). The source gases for Ga, As, and N are trimethylgallium ((CH3)3Ga), arsine (AsH3), and ammonia (NH3), respectively. The growth occurs on a Ge substrate, and the analysis includes the stress from the substrate-crystal lattice mismatch. Calculation results indicate that to have just a few percent N incorporation into the grown solid, the V/III ratio in the source gases should be several thousands and the input-gas partial-pressure ratio NH3/(NH3+AsH3) should exceed 0.99. We also find that the lattice mismatch stress from the Ge substrate increases the V/III source-gas ratio required for stable growth, whereas an increase in input Ga partial pressure ratio has the opposite effect.
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Gürlebeck, Doreen; Jahn, Simone; Gürlebeck, Norman; Szczesny, Robert; Szurek, Boris; Hahn, Simone; Hause, Gerd; Bonas, Ulla
2009-03-01
Xanthomonas campestris pv. vesicatoria secretes at least 20 effector proteins through the type III secretion system directly into plant cells. In this study, we uncovered virulence activities of the effector proteins AvrBs1, AvrBs3 and AvrBs4 using Agrobacterium-mediated transient expression of the corresponding genes in Nicotiana benthamiana, followed by microscopic analyses. We showed that, in addition to the nuclear-localized AvrBs3, the effector AvrBs1, which localizes to the plant cell cytoplasm, also induces a morphological change in mesophyll cells. Comparative analyses revealed that avrBs3-expressing plant cells contain highly active nuclei. Furthermore, plant cells expressing avrBs3 or avrBs1 show a decrease in the starch content in chloroplasts and an increased number of vesicles, indicating an enlargement of the central vacuole and the cell wall. Both AvrBs1 and AvrBs3 cause an increased ion efflux when expressed in N. benthamiana. By contrast, expression of the avrBs3 homologue avrBs4 leads to large catalase crystals in peroxisomes, suggesting a possible virulence function of AvrBs4 in the suppression of the plant defence responses. Taken together, our data show that microscopic inspection can uncover subtle and novel virulence activities of type III effector proteins.
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Sinorhizobium fredii USDA257 Translocates NopP into Vigna unguiculata Root Nodules
USDA-ARS?s Scientific Manuscript database
Type III secretion systems (T3SSs), which are found in many Gram-negative bacterial pathogens, inject virulence proteins directly into host cells during infection. T3SSs are also present in some strains of rhizobia, bacteria that form symbiotic associations with legumes and fix nitrogen in speciali...
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USDA-ARS?s Scientific Manuscript database
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...
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The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation
USDA-ARS?s Scientific Manuscript database
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote formation of “pedestals” in the tissue beneath the adherent bacteria. Secreted proteins are key playe...
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Gazi, Anastasia D; Charova, Spyridoula; Aivaliotis, Michalis; Panopoulos, Nicholas J; Kokkinidis, Michael
2015-01-01
Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence
Connolly, James P. R.; Gabrielsen, Mads; Goldstone, Robert J.; Grinter, Rhys; Wang, Dai; Cogdell, Richard J.; Walker, Daniel; Smith, David G. E.; Roe, Andrew J.
2016-01-01
The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to “sense” levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host. PMID:26727373
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Alexander, Kristen L; Mejia, Camilo A; Jordan, Clinton; Nelson, Michael B; Howell, Brian M; Jones, Cameron M; Reynolds, Paul R; Arroyo, Juan A
2016-02-01
Receptor for advanced glycation end products (RAGE) is a receptor implicated in the modulation of inflammation. Inflammation has been associated with pregnancy pathologies including preeclampsia (PE), intrauterine growth restriction (IUGR), and gestational diabetes mellitus (GDM). Our objective was to examine placental RAGE expression in PE, IUGR, and GDM complications. Human placental tissues were obtained for RAGE determination using Q-PCR, immunohistochemistry, and Western blot. Invasive trophoblast cells were cultured and treated with AGES for RAGE activation studies. Compared to control placenta, we observed: (i) decreased RAGE gene expression during GDM, (ii) increased RAGE protein in the PE placenta, and (iii) decreased RAGE protein in the IUGR placenta. In trophoblast cells exposed AGEs, we observed: (i) decreased trophoblast invasion, (ii) increased c-Jun N-terminal kinases (JNK) and Extracellular signal-regulated kinases (ERK), and (iii) increased TNF-α and IL-1β secretion. We conclude that placental RAGE is activated during PE and that RAGE-mediated inflammation in the trophoblast involves increased pro-inflammatory cytokine secretion. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Deane, Janet E.; Cordes, Frank S.; Roversi, Pietro; Johnson, Steven; Kenjale, Roma; Picking, William D.; Picking, Wendy L.; Lea, Susan M.; Blocker, Ariel
2006-01-01
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 MxiHCΔ5 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 Å3 Da−1 for two molecules per asymmetric unit, corresponding to a solvent content of 33%. PMID:16511329
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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 needle from the Shigella flexneri type III secretion system (TTSS), has been overexpressed and purified. Crystals were grown of native and selenomethionine-labelled MxiH(CDelta5) and diffraction data were collected to 1.9 A resolution. The crystals belong to space group C2, with unit-cell parameters a = 183.4, b = 28.1, c = 27.8 A, beta = 96.5 degrees. 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 A3 Da(-1) for two molecules per asymmetric unit, corresponding to a solvent content of 33%.
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Bile salt receptor complex activates a pathogenic type III secretion system
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 structure ofmore » 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
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Ferrari, Cibele Santos; Amaral, Fernanda Plucani; Bueno, Jessica Cavalheiro Ferreira; Scariot, Mirella Christine; Valentim-Neto, Pedro Alexandre; Arisi, Ana Carolina Maisonnave
2014-11-01
Several molecular tools have been used to clarify the basis of plant-bacteria interaction; however, the mechanism behind the association is still unclear. In this study, we used a proteomic approach to investigate the root proteome of Zea mays (cv. DKB240) inoculated with Herbaspirillum seropedicae strain SmR1 grown in vitro and harvested 7 days after inoculation. Eighteen differentially accumulated proteins were observed in root samples, ten of which were identified by MALDI-TOF mass spectrometry peptide mass fingerprint. Among the identified proteins, we observed three proteins present exclusively in inoculated root samples and six upregulated proteins and one downregulated protein relative to control. Differentially expressed maize proteins were identified as hypothetical protein ZEAMMB73_483204, hypothetical protein ZEAMMB73_269466, and tubulin beta-7 chain. The following were identified as H. seropedicae proteins: peroxiredoxin protein, EF-Tu elongation factor protein, cation transport ATPase, NADPH:quinone oxidoreductase, dinitrogenase reductase, and type III secretion ATP synthase. Our results presented the first evidence of type III secretion ATP synthase expression during H. seropedicae-maize root interaction.
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NASA Astrophysics Data System (ADS)
Demers, Jean-Philippe; Habenstein, Birgit; Loquet, Antoine; Kumar Vasa, Suresh; Giller, Karin; Becker, Stefan; Baker, David; Lange, Adam; Sgourakis, Nikolaos G.
2014-09-01
We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.
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Berasaín, P; Goñi, F; McGonigle, S; Dowd, A; Dalton, J P; Frangione, B; Carmona, C
1997-02-01
The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.
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The relative hydrolytic reactivities of pyrophosphites and pyrophosphates.
Mistry, Dharmit; Powles, Nicholas
2013-09-14
The pH-rate profiles for the hydrolysis of pyrophosphate (PP(V)) and pyrophosphite (PP(III), pyro-di-H-phosphonate) are a complex function of pH, reflecting the different ionic species and their relative reactivities. PP(III) is more reactive than PP(V) at all pHs and only PP(III) shows a hydroxide-ion reaction at high pH, so it is 10(10)-fold more reactive than PP(V) in 0.1 M NaOH. The pK(a2) of PP(III) ~0.44, so the dominant species at pH's > 1 is the di-anion PP(III)(2-). Although there is no observable (NMR or ITC) binding of Mg(2+) to the PP(III) di-anion there is a modest increase in the rate of hydrolysis of PP(III) by Mg(2+). PP(III) is neither a substrate nor an inhibitor of pyrophosphatase, the enzyme that efficiently catalyses the hydrolysis of PP(V).
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Efficient production and secretion of oxaloacetate from Halomonas sp. KM-1 under aerobic conditions.
Hannya, Asuka; Nishimura, Taku; Matsushita, Isao; Tsubota, Jun; Kawata, Yoshikazu
2017-11-21
The alkaliphilic, halophilic bacterium Halomonas sp. KM-1 can utilize glucose for the intracellular storage of the bioplastic poly-(R)-3-hydroxybutyric acid (PHB) and extracellular secretion of pyruvate under aerobic conditions. In this study, we investigated the effects of sodium chloride concentration on PHB accumulation and pyruvate secretion in the KM-1 strain and, unexpectedly, observed that oxaloacetate, an important intermediate chemical in the TCA cycle, glycogenesis, and aspartic acid biosynthesis, was secreted. We then further analyzed oxaloacetate productivity after changing the sodium chloride additive concentration, additive time-shift, and culture temperature. In 42-h batch-cultivation experiments, we found that wild-type Halomonas sp. KM-1 secreted 39.0 g/L oxaloacetate at a rate of 0.93 g/(L h). The halophilic bacteria Halomonas has already gained attention for industrial chemical-production processes owing to its unique properties, such as contamination-free culture conditions and a tolerance for high substrate concentrations. Moreover, no commercial scale oxaloacetate production was previously reported to result from bacterial fermentation. Oxaloacetate is an important intermediate chemical in biosynthesis and is used as a health food based on its role in energy synthesis. Thus, these data provided important insights into the production of oxaloacetate and other derivative chemicals using this strain.