In vertebrates, the innate immune system is considered as the first line of defence against microbial invaders. Toll-like receptors (TLRs) are crucial components of the innate immune system that act as pathogen-recognition receptors (PRRs), recognizing various types of pathogen-associated molecular patterns (PAMPs). Toll/interleukin-1 receptor (TIR) domains mediate key molecular interactions in the TLR signaling pathways. TLR signaling requires the dimerization of the intracellular TIR domains to establish an active conformation for the recruitment of cytosolic TIR domain-containing adaptor molecules. Eventually, transcription factors such as the nuclear factor kappa B (NF-κB) mediate gene activation in the nucleus, leading to anti-microbial responses. Several bacteria produce TIR domain-containing proteins to suppress host innate immune responses, in favor of their survival. The TIR domain-containing protein from the pathogenic bacterium Brucella melitensis (TcpB) inhibits dendritic cell maturation and affects microtubule dynamic modulation. TcpB was identified to suppress TLR4- and TLR2-mediated NF-κB activation. Such a mechanism may involve the ubiquitination or degradation of the MyD88 adaptor-like protein (MAL), or the direct interaction with MAL or MyD88 (myeloid differentiation primary response gene 88). However, the exact molecular basis is yet to be characterized.
The objective of this study was to structurally and functionally investigate the Brucella melitensis TIR domain-containing protein TcpB. In light of this, Chapter 3 illustrates the full-length TcpB (TcpB-fl) protein production and the unsuccessful crystallization attempts. Chapter 3 also describes a high-throughput strategy employed to screen for constructs that produce soluble TcpB fragments involving the TIR domain, with a quality and in the quantity suitable for crystallization trials. Two selected TcpB fragments (TcpB70-250 and TcpB120-250) were crystallized. However, only TcpB120-250 (TIR domain) protein produced diffraction quality crystals that led to a successful structure determination using molecular replacement as described in Chapter 4. Chapter 5 describes the structural refinement and analysis of the TcpB120-250 structure. The structure revealed significant differences to mammalian and bacterial TIR domain structures, which may reflect diverse functional specificity. Furthermore, TcpB-fl was identified to interact with TLR4, MyD88, and MAL proteins and only interfered with TLR4:MAL interaction, as assessed by the co-immunoprecipitation approach. This led to the conclusion that TcpB specifically targets MAL-mediated TLR4 signaling. TcpB-fl dimerization was identified in solution using the biophysical technique multi-angle laser scattering (MALS). The dimerization interface observed in the crystal lattice between two TcpB TIR domain monomers was characterized in solution by performing mutagenesis analysis coupled with MALS. Furthermore, TcpB was found to inhibit NF-κB activation using luciferase reporter assays, with residues in the dimer interface found to be important for TcpB inhibitory activity. We also investigated the overall structural environment of TcpB in solution using small-angle X-ray scattering, which gave us an insight into the possible orientation of the TIR and the N-terminal domains during dimerization.
In summary, structural and functional data reported in this thesis have added significant insights towards the understanding of the molecular basis of the suppression of the host innate immune responses by bacterial TIR domain-containing proteins.