Attachment of LcrV fromYersinia pestisat Dual Binding Sites to Human TLR-2 and Human IFN-γ Receptor

Abstract

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.

Interleukin-10 Induction Is an Important Virulence Function of the Yersinia pseudotuberculosis Type III Effector YopM

“…This observation was later linked to upregulation of IL-10 (34). Other groups followed by demonstrating that recombinant LcrV was directly responsible for the induction of IL-10, via a Toll-like receptor 2 (TLR2)-and CD14-mediated mechanism (1,6,47,49). However, different regions of Y. enterocolitica O8 LcrV (amino acids 31 to 49) (47) and Y. pestis LcrV (amino acids 271 to 300) (34,36) were shown to be important for IL-10-inducing activity.…”

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens

“…Radioligand analysis of binding 125 I-labeled type IV collagen to L. crispatus 2029 was performed as previously described [32]. 125 I-labeled type IV collagen (0.1 mCu/lg) samples were prepared with Iodo-Gen (Sigma) and Na 125 I and then separated by chromatography on Sephadex G-25.…”

Attenuated enzootic (pestoides) isolates of Yersinia pestis express active aspartase

“…Nevertheless, enzootic strains are attenuated in many mammalian species, including guinea pigs and primates, but virulent in the rodent Superfamily Muroidea (Anisimov et al, 2004). Although pestoides isolates possess polymorphism in LcrV (Anisimov et al, 2007), a major pCD/pYVencoded regulator, T3SS effector, and virulence factor, it seems unlikely that these differences influence biological activity (Abramov et al, 2007). The purposes of this report are to provide a description of ten typical enzootic isolates and to demonstrate that only three produce functional Zwf while, in stark contrast to epidemic isolates of Y. pestis, all express biologically active AspA.…”