Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. against invading pathogens [1]. The TLRs contain an extracellular domain comprised of a leucine-rich repeat linked via a single transmembrane region to an intracellular Toll/interleukin 1 receptor (TIR). Central to both the initiation and propagation of TLR signalling are heterotypic TIR-TIR interactions involving the TLRs and cytosolic adaptor proteins. There are four TIR containing TLR adaptor proteins involved in upregulation of the innate immune response Myeloid differentiation factor 88 (MyD88) TIR domain containing adaptor protein inducing interferon β (TRIF) MyD88-adaptor like Tal1 (MAL also known as TIRAP) and TRIF-related adaptor molecule (TRAM) [2]. MAL and TRAM are bridging adaptors mediating recruitment of MyD88 and TRIF respectively to active TLRs although both MyD88 and TRIF GTx-024 can interact directly with some TLRs [3]. This in turn is thought to cause association of other proteins crucial in TLR signalling into a multi-protein complex called a Supramolecular Organizing Centre (SMOC) [4]. The SMOC propagates downstream signalling leading to activation of the NFκB transcription factor and thus production of proinflammatory cytokines and type I interferons central to the host response against infection. A fifth TIR domain containing protein Sterile α and armadillo-motif containing protein (SARM) GTx-024 has been shown to be a negative regulator of the TLR system [5 6 SARM is likely to be a part of the normal homeostatic regulation of the TLR signalling system although its precise mechanism of action remains unclear [7]. SARM has also been shown to associate with cytoskeletal structures [8] and regulate microtubule stability via tubulin acetylation [9]. TIR domain proteins (Tdps) have also been identified in a range of microbes [10] including a number of pathogenic bacterial species [11-14]. Several of these proteins have roles in virulence [11-13] and there is substantial evidence that they are involved in subversion of the innate immune response [14-16]. In most cases it appears that the bacterial Tdp domains function to interfere with the heterotypic TIR-TIR interactions essential for initiation and propagation of the TLR signalling pathway [17]. To this GTx-024 end the bacterial Tdps appear to act as molecular mimics. This is illustrated by the fact that TIR domains present in bacterial Tdps have core structures very similar to those of mammalian TIR domains [15 18 For example the structure of the TIR domain of TcpB shows root mean square deviation (RMSD) values of 2.5-3.0 ? for the TIR domain structures of human MyD88 MAL and TLR2 [15 19 The functionally important BB loop named for connecting the strand βB and helix αB adopts GTx-024 similar conformations in the two bacterial TIR domain structures solved however this loop adopts markedly different conformations in the mammalian TIR protein structures [15]. The amino acid residues in the BB loop have been shown to play important roles in the normal functioning of the TLR signalling pathway [20-23] and also in the inhibitory function of bacterial Tdps [14 16 A Tdp has previously been identified in [10] the causative agent of anthrax. Expression of the Tdp gene in is upregulated 2.3 fold in mouse macrophages between 1-2 h post-infection [24] a possible indication that the protein is functionally related to virulence. spores typically infect mammals via inhalation and are subsequently subjected to phagocytosis by macrophages whereupon they germinate. However the mechanisms regulating intracellular development and how the bacteria resist lysosomal degradation inside the cell are not fully understood. Hu and colleagues have previously shown that cultured primary mouse macrophages efficiently kill both anthrax spores and vegetative bacteria within 4 h of infection [25] making the process behind initiation of infection unclear. In light of previous research [17] we speculated that this protein (denoted BaTdp in this manuscript equivalent to BA_4098 in Ames) may be involved in the evasion of the host immune response through negative regulation of the TLR signalling pathway. However in addition to the production of inflammatory cytokines.