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Attachment of LcrV from Yersinia pestis at Dual Binding Sites to Human TLR-2 and Human IFN-γ Receptor

Vyacheslav M. Abramov,^‡ Valentin S. Khlebnikov,^‡ Anatoly M. Vasiliev,^‡ Igor V. Kosarev,^‡ Raisa N. Vasilenko,^‡ Nataly L. Kulikova,^‡ Anna V. Khodyakova,^‡ Valentin I. Evstigneev,^‡ Vladimir N. Uversky,^‡ Vladimir L. Motin,^# Georgy B. Smirnov,^§ and Robert R. Brubaker*^¶

Institute of Immunological Engineering, 142380 Lyubuchany, Russia, Departments of Pathology/Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas 77555, The Gamaleya Institute of Epidemiology and Microbiology, 123098 Moscow, Russia, and Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824

J. Proteome Res., 2007, 6 (6), pp 2222–2231

DOI: 10.1021/pr070036r

Publication Date (Web): April 19, 2007

‡

Institute of Immunological Engineering.

University of Texas Medical Branch.

The Gamaleya Institute of Epidemiology and Microbiology.

Correspondence should be addressed to: Robert R. Brubaker, Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-6463. Fax: (517) 353-8957. E-mail: brubake3@msu.edu.

Michigan State University.

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-γ or a synthetic C-terminal fragment (hIFN-γ₁₃₂_-₁₄₃). The latter, but not mouse IFN-γ (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-α in human target cells. The ability of LcrV to utilize human IFN-γ (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.