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The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB-MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88(DD)-MyD88(DD), MyD88(DD)-MyD88(TIR) and MyD88(DD)-MyD88 interactions but not MyD88-MyD88 or MyD88(TIR)-MyD88(TIR) interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD-TIR domain interface., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Toll/interleukin-1 like receptors are evolutionarily conserved proteins in eukaryotes that play crucial role in pathogen recognition and innate immune responses. Brucella are facultative intracellular bacterial pathogens causing brucellosis in animal and human hosts. Brucella behave as a stealthy pathogen by evading the immune recognition or suppressing the TLR signaling cascades. Brucella encode a TIR domain containing protein, TcpB, which suppresses NF-kappaB activation as well as pro-inflammatory cytokine secretion mediated by TLR2 and TLR4 receptors. TcpB targets the TIRAP mediated pathway to suppress TLR signaling. With the objective of detailed characterization, we have over expressed and purified TcpB from Brucella melitensis in native condition. The purified protein exhibited lipid-binding properties and cell permeability. NF-kappaB inhibition property of endogenous TcpB has also been demonstrated. The data provide insight into the mechanism of action of TcpB in the intracellular niche of Brucella., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Human brucellosis is characterized by the presence of both acute inflammatory episodes and chronic inflammation with granuloma formation. On this basis, the proinflammatory effects of smooth lipopolysaccharide of Brucella (S-LPS) were addressed and compared to those of LPS from Escherichia coli. For this purpose, the induction of cyclooxygenase-2 (COX-2), the production of the chemokine monocyte chemoattractant protein-1 (MCP-1), and the activation of the nuclear factor kappa B (NF-kappa B) were studied. S-LPS was found to induce both COX-2 expression and MCP-1 production; however, the potency of E. coli LPS exceeded that of Brucella S-LPS by some orders of magnitude. However, at concentrations above 1 microg/ml, all of the LPS produced comparable effects, including their ability to activate the NF-kappa B system. These observations help explain the inflammatory events associated with Brucella infection and the ability of Brucella to produce monocyte recruitment and granuloma formation.