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Amino acid substitutions in LcrV at putative sites of interaction with Toll-like receptor 2 do not affect the virulence of Yersinia pestis.

ABSTRACT: LcrV, a component of the type III secretion system (T3SS) translocon in Yersinia pestis, has been concerned in suppressing inflammation through Toll-like receptor 2 (TLR2) by inducing expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Previous studies have reported that LcrV aa E33, E34, K42 and/or E204 and E205 were important for interactions with TLR2 in vitro. While, recently there have been conflicting reports doubting this interaction and its importance in vivo. To further investigate the role of these residues, we replaced the wild-type lcrV gene on the pCD1Ap virulence plasmid of Y. pestis with lcrV2345 gene, which encodes a mutant protein by substituting all five of the amino acid residues with glutamine. The characteristics of the wild-type LcrV and mutant LcrV2345 were evaluated in tissue culture and mice. When purified protein was incubated with HEK293 cells synthesizing human TLR2 with or without CD14, LcrV2345 induced higher levels of IL-8 than wild-type LcrV, indicating that the LcrV2345 was not impaired in its ability to interact with TLR2. LcrV2345 stimulated higher levels of tumor necrosis factor-alpha (TNF-?) production than LcrV in J774A.1 cells, while neither protein elicited significant levels of IL-10. We also found there was no statistically significant difference in virulence between strains with wild-type LcrV and with mutated LcrV2345 administered by either subcutaneous or intranasal route in mice. Additionally, there were no discernible differences in survival kinetics. Serum levels of cytokines, such as IL-10 and TNF-?, bacterial burden, and the extent of organ inflammation were also indistinguishable in both strains. Our data confirm that immunomodulation mediated by LcrV/TLR2 interactions does not play a significant role in the pathogenicity of Y. pestis.

SUBMITTER: Sun W

PROVIDER: S-EPMC3470733 | biostudies-literature | 2012 Nov-Dec

REPOSITORIES: biostudies-literature

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Amino acid substitutions in LcrV at putative sites of interaction with Toll-like receptor 2 do not affect the virulence of Yersinia pestis.

Sun Wei W Curtiss Roy R

Microbial pathogenesis 20120724 5-6

LcrV, a component of the type III secretion system (T3SS) translocon in Yersinia pestis, has been concerned in suppressing inflammation through Toll-like receptor 2 (TLR2) by inducing expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Previous studies have reported that LcrV aa E33, E34, K42 and/or E204 and E205 were important for interactions with TLR2 in vitro. While, recently there have been conflicting reports doubting this interaction and its importance in vivo. To further ...[more]

PMID: 22841961

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Project description:BACKGROUND:Yersinia pestis, the etiological pathogen of plague, is capable of repressing the immune response of white blood cells to evade phagocytosis. The V-antigen (LcrV) was found to be involved in this process by binding to human Toll-like Receptor 2 (TLR2). The detailed mechanism behind this LcrV and TLR2 mediated immune response repression, however, is yet to be fully elucidated due to the lack of structural information. RESULTS:In this work, with protein structure modelling, we were able to construct a structure model of the heterotetramer of Y. pestis LcrV and human TLR2. Molecular dynamics simulation suggests the stability of this structure in aquatic environment. The LcrV model has a dumbbell-like structure with two globule domains (G1 at N-terminus and G2 away from membrane) connected with a coiled-coil linker (CCL) domain. The two horseshoe-shape TLR2 subunits form a V-shape structure, are not in direct contact with each other, and are held together by the LcrV homodimer. In this structure model, both the G1 and CCL domains are involved in the formation of LcrV homodimer, while all three domains are involved in LcrV-TLR2 binding. A mechanistic model was proposed based on this heterotetrameric structure model: The LcrV homodimer separates the TLR2 subunits to inhibit the dimerization of TLR2 and subsequent signal transfer for immune response; while LcrV could also inhibit the formation of heterodimers of TLR2 with other TLRs, and leads to immune response repression. CONCLUSIONS:A heterotetrameric structure of Y. pestis LcrV and human TLR2 was modelled in this work. Analysis of this modelled structure showed its stability in aquatic environments and the role of LcrV domains and residues in protein-protein interaction. A mechanistic model for the role of LcrV in Y. pestis pathogenesis is raised based on this heterotetrameric structure model. This work provides a hypothesis of LcrV function, with which further experimental validation may elucidate the role of LcrV in human immune response repression.

Dataset Information

Amino acid substitutions in LcrV at putative sites of interaction with Toll-like receptor 2 do not affect the virulence of Yersinia pestis.

Publications

Amino acid substitutions in LcrV at putative sites of interaction with Toll-like receptor 2 do not affect the virulence of Yersinia pestis.

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