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Olaf Schneewind - One of the best experts on this subject based on the ideXlab platform.
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Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis.
mBio, 2017Co-Authors: Anthony Mitchell, Derek Elli, Christina Tam, Patrick Osei-owusu, Farbod Fazlollahi, Kym F. Faull, Thomas M Charlton, Olaf SchneewindAbstract: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.IMPORTANCEYersinia 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.
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Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis
American Society for Microbiology, 2017Co-Authors: Anthony Mitchell, Olaf Schneewind, Derek Elli, Christina Tam, Thomas Charlton, Patrick Osei-owusu, Farbod Fazlollahi, Kym F. Faull, Samuel I. MillerAbstract: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
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Polymorphisms in the LcrV Gene of Yersinia enterocolitica and Their Effect on Plague Protective Immunity
2016Co-Authors: Nathan C. Miller, Lauriane E. Quenee, Derek Elli, Nancy Ciletti, Olaf SchneewindAbstract: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 Yer-sinia enterocolitica strains and suggested that LcrV polymorphisms may provide for escape from LcrV-mediated plague immu-nity. We show here that polyclonal or monoclonal antibodies raised against Y. pestis KIMD27 LcrV (LcrVD27) bind LcrV from Y. enterocoliticaO:9 strainW22703 (LcrVW22703) or O:8 strainWA-314 (LcrVWA-314) but are otherwise unable to block type III in-jection by Y. enterocolitica strains. Replacing the LcrV gene on the pCD1 virulence plasmid of Y. pestis KIMD27 with either LcrVW22703 or LcrVWA-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. LcrVD27-specific antibodies blocked type III injection by Y. pestis expressing LcrVW22703 or LcrVWA-314 and protected mice against intravenous lethal plague challenge with these strains. Thus, al-though antibodies raised against LcrVD27 are unable to block the type III injection of Y. enterocolitica strains, expression of LcrVW22703 or LcrVWA-314 in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intra-venous challenge model. Three Yersinia species—Yersinia enterocolitica, Yersinia pestis,and Yersinia pseudotuberculosis—are pathogenic to humans (13). All three species use virulence plasmid-encoded type III se
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LcrV Mutants That Abolish Yersinia Type III Injectisome Function
Journal of bacteriology, 2012Co-Authors: Katherine Given Ligtenberg, Nathan C. Miller, Gregory V. Plano, Anthony Mitchell, Olaf SchneewindAbstract: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.
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Polymorphisms in the LcrV Gene of Yersinia enterocolitica and Their Effect on Plague Protective Immunity
Infection and immunity, 2012Co-Authors: Nathan C. Miller, Lauriane E. Quenee, Derek Elli, Nancy Ciletti, Olaf SchneewindAbstract:ABSTRACT 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 (LcrVD27) bind LcrV from Y. enterocolitica O:9 strain W22703 (LcrVW22703) or O:8 strain WA-314 (LcrVWA-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 LcrVW22703 or LcrVWA-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. LcrVD27-specific antibodies blocked type III injection by Y. pestis expressing LcrVW22703 or LcrVWA-314 and protected mice against intravenous lethal plague challenge with these strains. Thus, although antibodies raised against LcrVD27 are unable to block the type III injection of Y. enterocolitica strains, expression of LcrVW22703 or LcrVWA-314 in Y. pestis did not allow these strains to escape LcrV-mediated plague protective immunity in the intravenous challenge model.
Matthew L. Nilles - One of the best experts on this subject based on the ideXlab platform.
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Structure-Function Analysis of the C-Terminal Domain of LcrV from Yersinia pestis
Journal of bacteriology, 2007Co-Authors: Mohamad A. Hamad, Matthew L. NillesAbstract:LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.
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Roles of YopN, LcrG and LcrV in controlling Yops secretion by Yersinia pestis.
Advances in experimental medicine and biology, 2007Co-Authors: Mohamad A. Hamad, Matthew L. NillesAbstract:Control of Yops secretion in pathogenic Yersinia is achieved at several levels. These levels likely include transcriptional, post-transcriptional, translational and secretional controls. Secretion control appears to be mediated by two pathways. One pathway involves YopN and proteins that interact with YopN. The second pathway consists of LcrG and its interaction with LcrV. LcrV is a postive regulator of Yops secretion that exerts control over Yops secretion by negating the secretion blocking role of LcrG. However, the intersection of these two control pathways is not understood. Recent work has allowed the development of a speculative model that brings YopN-mediated and LcrG-LcrV-mediated control together in the context of the ability of the needle complex to respond to Ca 2+ .
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Dissecting the Structure of LcrV from Yersinia pestis, a Truly Unique Virulence Protein
Structure (London England : 1993), 2004Co-Authors: Matthew L. NillesAbstract:Abstract Last month in the February issue, Structure published the crystal structure of LcrV from Yersinia pestis , the infectious agent that causes plague. LcrV activity is essential for disease, and the structure of this protein provides a valuable tool to dissect the nature of its pathogenicity.
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Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface
BMC Microbiology, 2002Co-Authors: Jyl S. Matson, Matthew L. NillesAbstract:Background Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis . Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein. Results In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis . A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking. Conclusion Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.
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Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface
BMC microbiology, 2002Co-Authors: Jyl S. Matson, Matthew L. NillesAbstract:Background Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.
Susan C. Straley - One of the best experts on this subject based on the ideXlab platform.
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Yersinia pestis YadC: a novel vaccine candidate against plague.
Advances in experimental medicine and biology, 2007Co-Authors: Brian S. Murphy, Susan C. Straley, Beth A. Garvy, Christine R. WulfAbstract:Current subunit vaccines provide partial protection against pneumonic plague if the infecting Y. pestis strain is encapsulated (F1 - ). Here we describe YadC, a novel Y. pestis outer membrane protein that provides partial protection against a F1 - Y. pestis strain. Swiss-Webster mice were immunized subcutaneously with glutathione S-transferase (GST) or His 6 -tagged (HT) purified fusion proteins (GST-YadC 137-409 or HT-LcrV) or buffer emulsified with Alhydrogel. Intravenous challenge with 1 x 10 4 F1 - Δpgm Y. pestis C099-3015 revealed no protection for those mice immunized with GST-Alhydrogel alone, full protection for HT-LcrV-immunized mice, and partial protection for GST-YadC 137-409 -immunized mice. Similarly, C57BL/6 mice were immunized with GST-YadC 137-409 , HT-LcrV, or GST all with Alhydrogel adjuvant. After intranasal challenge with 3 x 10 3 Fl Y. pestis C099-3015, 87% of GST-YadC 137-409 -immunized mice survived pneumonic plague. This is compared to the GST control group (0 surviving mice) and the LcrV-immunized group where 50% survived the challenge. This protection was correlated with a predominantly IgGI response in LcrV-immunized mice and an IgG1/IgG3 antibody response in YadC-immunized mice. Additionally, we report the cytokine response from HT-LcrV- and GST-YadC 137-409 -stimulated peripherally derived macrophages. YadC-stimulated cells demonstrated a predominant pro-inflammatory cytokine production. This mixed Th1/Th2 response suggests that YadC's protection may involve a different adaptive immune response than the LcrV protein that currently is part of plague vaccines.
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Anti-LcrV antibody inhibits delivery of Yops by Yersinia pestis KIM5 by directly promoting phagocytosis.
Infection and immunity, 2005Co-Authors: Clarissa Cowan, Alexander V. Philipovskiy, Christine R. Wulff-strobel, Susan C. StraleyAbstract:LcrV of Yersinia pestis is a major protective antigen proposed for inclusion in subunit plague vaccines. One way that anti-LcrV antibody is thought to protect is by inhibiting the delivery of toxins called Yops to host cells. The present study characterizes the relation between this inhibition and the phagocytosis of the bacteria. J774A.1 cells were infected with Y. pestis KIM5 in the presence of a protective polyclonal anti-LcrV antibody or a nonprotective polyclonal anti-YopM antibody, and delivery of YopH and YopE into the cytoplasm was assayed by immunoblotting. The ability to inhibit the delivery of these Yops depended upon having antibody bound to the cell surface; blocking conditions that prevented the binding of antibody to Fc receptors prevented the inhibition of Yop delivery. Anti-LcrV antibody also promoted phagocytosis of the yersiniae, whereas F(ab′)2 fragments did not. Further, anti-LcrV antibody could not inhibit the delivery of Yops into cells that were unable to phagocytose due to the presence of cytochalasin D. However, Yops were produced only by extracellular yersiniae. We hypothesize that anti-LcrV antibody does not directly inhibit Yop delivery but instead causes phagocytosis, with consequent inhibition of Yop protein production in the intracellular yersiniae. The prophagocytic effect of anti-LcrV antibody extended to mouse polymorphonuclear neutrophils (PMNs) in vitro, and PMNs were shown to be critical for protection: when PMNs in mice were ablated, the mice lost all ability to be protected by anti-LcrV antibody.
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Antibody against V Antigen Prevents Yop-Dependent Growth of Yersinia pestis
Infection and immunity, 2005Co-Authors: Alexander V. Philipovskiy, Clarissa Cowan, Christine R. Wulff-strobel, Sandra H. Burnett, Edward J. Kerschen, Donald A. Cohen, Alan M. Kaplan, Susan C. StraleyAbstract:The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10−/− mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.
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virulence role of v antigen of yersinia pestis at the bacterial surface
Infection and Immunity, 1999Co-Authors: Kenneth A. Fields, Matthew L. Nilles, Clarissa Cowan, Susan C. StraleyAbstract:Yersinia pestis, the etiologic agent of plague, secretes a set of environmentally regulated, plasmid pCD1-encoded virulence proteins termed Yops and V antigen (LcrV) by a type III secretion mechanism (Ysc). LcrV is a multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. In this study, we constructed an in-frame deletion within lcrG (DeltalcrG3) to further analyze the requirement of LcrV in Yop targeting. We confirmed the essentiality of LcrV and found that LcrG may have a facilitative role, perhaps by promoting efficient secretion of LcrV. We also constructed mutants of LcrV expressing LcrV truncated at the N or C terminus. Both the N and C termini of LcrV were required for the secretion of LcrV into the medium and targeting of Yops. LcrV was detected in punctate zones on the surface of fixed Y. pestis by laser-scanning confocal microscopy, and this localization required a functional Ysc. However, the truncated LcrV proteins were not found on the bacterial surface. Finally, we tested the ability of LcrV-specific Fab antibody fragments or full-length antibody to interfere with Yop targeting and found no interference, even though this antibody protects mice against plague. These results indicate that LcrV may function in Yop targeting at the extracellular surface of yersiniae and that the protective efficacy of LcrV-specific antibodies can be manifested without blocking Yop targeting.
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LcrV of Yersinia pestis enters infected eukaryotic cells by a virulence plasmid-independent mechanism.
Infection and immunity, 1999Co-Authors: Kenneth A. Fields, Susan C. StraleyAbstract:Yersinia pestis is the causative agent of bubonic plague and possesses a set of plasmid-encoded, secretable virulence proteins termed LcrV and Yops which are essential for survival in mammalian hosts. Yops and LcrV are secreted by a type III mechanism (Ysc), and Yops are unidirectionally targeted into the cytosol of associated eukaryotic cells in a tissue culture infection model. LcrV is required for Yops targeting, and recent findings have revealed that it can localize to the bacterial surface; however, its fate in this infection model has not been investigated in detail. In this study, we compared the localization of LcrV to that of the targeted proteins YopE and YopM by immunoblot analysis of fractions of Yersinia-infected HeLa cultures or by laser-scanning confocal microscopy of infected monolayers. Both LcrV and YopE were secreted by contact-activated, extracellularly localized yersiniae and were targeted to the HeLa cell cytosol. Although a significant amount of LcrV partitioned to the culture medium (unlike YopE), this extracellular pool of LcrV was not the source of the LcrV that entered HeLa cells. Unlike targeting of YopE and YopM, targeting of LcrV occurred in the absence of a functional Ysc apparatus and other virulence plasmid (pCD1)-expressed proteins. However, the Ysc is necessary for LcrV to be released into the medium, and our recent work has shown that localization of LcrV on the bacterial surface requires the Ysc. These results indicate that two mechanisms exist for the secretion of LcrV by Y. pestis, both of which are activated by contact with eukaryotic cells. LcrV secreted by the Ysc reaches the bacterial surface and the surrounding medium, whereas the second is a novel, Ysc-independent pathway which results in localization of LcrV in the cytosol of infected cells but not the surrounding medium.
Guy R. Cornelis - One of the best experts on this subject based on the ideXlab platform.
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function and molecular architecture of the yersinia injectisome tip complex
Molecular Microbiology, 2007Co-Authors: Petr Broz, Catherine A Mueller, Shirley A Muller, Ansgar Philippsen, Isabel Sorg, Andreas Engel, Guy R. CornelisAbstract:By quantitative immunoblot analyses and scanning transmission electron microscopy (STEM), we determined that the needle of the Yersinia enterocolitica E40 injectisome consists of 139 +/- 19 YscF subunits and that the tip complex is formed by three to five LcrV monomers. A pentamer represented the best fit for an atomic model of this complex. The N-terminal globular domain of LcrV forms the base of the tip complex, while the central globular domain forms the head. Hybrids between LcrV and its orthologues PcrV (Pseudomonas aeruginosa) or AcrV (Aeromonas salmonicida) were engineered and recombinant Y. enterocolitica expressing the different hybrids were tested for their capacity to form the translocation pore by a haemolysis assay. There was a good correlation between haemolysis, insertion of YopB into erythrocyte membranes and interaction between YopB and the N-terminal globular domain of the tip complex subunit. Hence, the base of the tip complex appears to be critical for the functional insertion of YopB into the host cell membrane.
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YscU recognizes translocators as export substrates of the Yersinia injectisome.
The EMBO journal, 2007Co-Authors: Isabel Sorg, Petr Broz, Shirley A Muller, Andreas Engel, Stefanie Wagner, Marlise Amstutz, Yvonne Lussi, Guy R. CornelisAbstract:YscU is an essential component of the export apparatus of the Yersinia injectisome. It consists of an N-terminal transmembrane domain and a long cytoplasmic C-terminal domain, which undergoes auto-cleavage at a NPTH site. Substitutions N263A and P264A prevented cleavage of YscU and abolished export of LcrV, YopB and YopD but not of Yop effectors. As a consequence, yscU(N263A) mutant bacteria made needles without the LcrV tip complex and they could not form translocation pores. The graft of the export signal of the effector YopE, at the N-terminus of LcrV, restored LcrV export and assembly of the tip complex. Thus, YscU cleavage is required to acquire the conformation allowing recognition of translocators, which represent an individual category of substrates in the hierarchy of export. In addition, yscU(N263A) mutant bacteria exported reduced amounts of the YscP ruler and made longer needles. Increasing YscP export resulted in needles with normal size, depending on the length of the ruler. Hence, the effect of the yscU(N263A) mutation on needle length was the consequence of a reduced YscP export.
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The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles.
Science (New York N.Y.), 2005Co-Authors: Catherine A Mueller, Petr Broz, Shirley A Muller, Isabel Sorg, Andreas Engel, Philippe Ringler, Françoise Erne-brand, Marina Kuhn, Guy R. CornelisAbstract:Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.
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Genetic analysis of the formation of the Ysc-Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN.
Microbial Pathogenesis, 2003Co-Authors: Marie-noëlle Marenne, Laure Journet, Luís Jaime Mota, Guy R. CornelisAbstract:The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A LcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of LcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.
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Genetic analysis of the formation of the Ysc–Yop translocation pore in macrophages by Yersinia enterocolitica: role of LcrV, YscF and YopN
Microbial pathogenesis, 2003Co-Authors: Marie-noëlle Marenne, Laure Journet, Luís Jaime Mota, Guy R. CornelisAbstract:The Ysc-Yop type III secretion (TTS) system allows extracellular Yersinia bacteria, adhering to eukaryotic target cells, to inject Yop effector proteins in the cytosol of these cells. The secretion apparatus, called the injectisome, ends up with a needle-like structure made of YscF. YopN, one of the proteins secreted by the injectisome is thought to act as a plug. YopB, YopD and LcrV, three other proteins secreted by the injectisome and called 'translocators' form a pore allowing translocation of the Yop effectors across the target cell plasma membrane. Here, we tested the role of LcrV, YscF and YopN in the formation of this pore in macrophages by monitoring the release of the low-molecular-weight fluorescent dye BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, 623Da) and of the high-molecular-weight lactate dehydrogenase (LDH, 135 kDa). BCECF is released through the translocation pore itself provided no Yop effector is trafficking through the channel. In contrast, LDH is released by the osmotic lysis of the target cell that occurs after pore formation. This release is reduced by the GAP activity of YopE. In order to study the role of LcrV, one has to circumvent the regulatory effect of LcrV on the synthesis of YopB and YopD. We observed here that this regulatory role of LcrV is lost in a yopQ mutant and hence we studied the role of LcrV in a yopQ mutant background. A LcrV, yopQ double mutant was deficient in pore formation while able to produce YopB and YopD. Pore formation was restored by the introduction of LcrV(+) but not yopQ(+) confirming that LcrV itself is directly required for pore formation. Bacteria secreting only YopB, YopD and LcrV could form pores, showing that YopB, YopD and LcrV are sufficient for pore formation provided they are secreted by the same bacterium. LcrV is not involved in secretion of YopB and YopD as suggested previously. Bacteria producing normal Ysc injectisomes, including the YscF needle but no translocators did not form pores, indicating that the needle is not sufficient by itself for pore formation, as was also suggested. yopN mutant bacteria formed needles and released BCECF even if they secreted the effectors. This observation suggests that many translocation pores are not filled in the absence of YopN and thus that YopN might form a link between the needle and the pore, guiding the effectors.
Jim Hill - One of the best experts on this subject based on the ideXlab platform.
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Direct Neutralization of Type III Effector Translocation by the Variable Region of a Monoclonal Antibody to Yersinia pestis LcrV
Clinical and vaccine immunology : CVI, 2014Co-Authors: Maya I. Ivanov, Jim Hill, James B. BliskaAbstract:Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersiniaouter proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if antiLcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab=) 2, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.
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tnfα and ifnγ contribute to f1 LcrV targeted immune defense in mouse models of fully virulent pneumonic plague
Vaccine, 2010Co-Authors: Jr Shiuan Lin, Jim Hill, James B. Bliska, Steven Park, David S. Perlin, Jeffrey J Adamovicz, Christopher K Cote, Kei Amemiya, Stephen T SmileyAbstract:Immunization with the Yersinia pestis F1 and LcrV proteins improves survival in mouse and non-human primate models of pneumonic plague. F1- and LcrV-specific antibodies contribute to protection, however, the mechanisms of antibody-mediated defense are incompletely understood and serum antibody titers do not suffice as quantitative correlates of protection. Previously we demonstrated roles for tumor necrosis factor-alpha (TNFα) and gamma-interferon (IFNγ) during defense against conditionally attenuated pigmentation (pgm) locus-negative Y. pestis. Here, using intranasal challenge with fully virulent pgm-positive Y. pestis strain CO92, we demonstrate that neutralizing TNFα and IFNγ interferes with the capacity of therapeutically administered F1- or LcrV-specific antibody to reduce bacterial burden and increase survival. Moreover, using Y. pestis strain CO92 in an aerosol challenge model, we demonstrate that neutralizing TNFα and IFNγ interferes with protection conferred by immunization with recombinant F1-LcrV fusion protein vaccine (p<0.0005). These findings establish that TNFα and IFNγ contribute to protection mediated by pneumonic plague countermeasures targeting F1 and LcrV, and suggest that an individual's capacity to produce these cytokines in response to Y. pestis challenge will be an important co-determinant of antibody-mediated defense against pneumonic plague.
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TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague
Vaccine, 2010Co-Authors: Shiuan Lin, Jim Hill, James B. Bliska, Steven Park, David S. Perlin, Jeffrey J Adamovicz, Christopher K Cote, Kei Amemiya, Stephen T SmileyAbstract:Immunization with the Yersinia pestis F1 and LcrV proteins improves survival in mouse and non-human primate models of pneumonic plague. F1- and LcrV-specific antibodies contribute to protection, however, the mechanisms of antibody-mediated defense are incompletely understood and serum antibody titers do not suffice as quantitative correlates of protection. Previously we demonstrated roles for tumor necrosis factor-alpha (TNFα) and gamma-interferon (IFNγ) during defense against conditionally attenuated pigmentation (pgm) locus-negative Y. pestis. Here, using intranasal challenge with fully virulent pgm-positive Y. pestis strain CO92, we demonstrate that neutralizing TNFα and IFNγ interferes with the capacity of therapeutically administered F1- or LcrV-specific antibody to reduce bacterial burden and increase survival. Moreover, using Y. pestis strain CO92 in an aerosol challenge model, we demonstrate that neutralizing TNFα and IFNγ interferes with protection conferred by immunization with recombinant F1-LcrV fusion protein vaccine (p
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Yersinia pestis can bypass protective antibodies to LcrV and activation with gamma interferon to survive and induce apoptosis in murine macrophages.
Clinical and vaccine immunology : CVI, 2009Co-Authors: Betty L. Noel, Jim Hill, Sarit Lilo, Daniel Capurso, James B. BliskaAbstract:Yersinia pestis, the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naive or gamma interferon (IFN-γ)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis-infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-γ and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-γ impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-γ. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-γ-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naive or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-γ to survive and induce apoptosis in murine macrophages.
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The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis.
Microbes and infection, 2007Co-Authors: Dagmar Reithmeier-rost, Jim Hill, Stephen J. Elvin, Diane Williamson, Svea Dittmann, Annika Schmid, Gottfried Wilharm, Andreas SingAbstract:Abstract Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2−/− mice subcutaneously with Y. pestis GB. While TLR2−/− mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-α, IFN-γ and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.