Espressione e caratterizzazione di proteine batteriche e mammifere contenenti il dominio TIR nelle cellule di Saccharomyces cerevisiae

Toll-like receptors (TLR) are involved in the innate immune system. They recognize pathogen-associated molecular patterns and trigger signalling pathways culminating in the production of inflammatory mediators. Central to TLR signalling pathway are heterotypic protein-protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in cytoplasmic regions of TLRs and adaptor proteins. There are five TIR-containing adaptor proteins in mammals: myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein including interferon-β (TRIF), MyD88-adaptor like (MAL), TRIF related adaptor molecule (TRAM) and sterile α- and armadillo-motif-containing protein (SARM). In the last few years, bacterial TIR domain-containing proteins (Btps) have been described. In the pathogen Brucella, BtpA acts by inhibiting TLR- and MyD88-specific signalling, so the TLR pathway is blocked and the proinflammatory response cannot be activated. Thus, BtpA suppresses innate immunity and increases Brucella virulence. Phylogenetic studies suggested that the TIR domain of SARM1 is the most closely related to prokaryotic TIR domains and it has been recently discovered that SARM1 and prokaryotic TIR domain are NAD hydrolases. WxxxE is the highly conserved motif responsible for this catalytic activity within the TIR domain. TIR bacterial domain could promote virulence via their NADase activity beyond their ability to evade the host immune system by interfering with TLR signalling, while the NADase activity of SARM1 has been associated with axon degeneration. Based on this background, the aims of this study were, based on heterologous expression in the model yeast S. cerevisiae, i)to study the localization of TRIF, ii)to study localization and toxicity of full length and in TIR domain alone of BtpA, wild type and Glu to Ala mutated variant, and iii)of SARM1. It was necessary to fuse the proteins with GFP in order to observe them under the fluorescence microscope. Plasmids obtained: pAG425-GAL1-EGFP-TRIF, pRS426-GAL1-EGFP-BtpA(E217A), pRS426-GAL1-EGFP-BtpATir(E217A), pRS426-GAL1-EGFP-Sarm1 and pRS426-GAL1-EGFP-Sarm1Tir. TRIF-GFP was partially associated to the yeast Golgi apparatus. Full-length BtpA was less toxic in yeast than its TIR domain alone. This could be due to the different localization of BtpA and BtpA-TIR, as full-length BtpA was sequestered in the nucleus. Glu residue of BtpA of the WxxxE motif was critical for toxicity, as its mutation to Ala abrogated toxicity both in full-length BtpA and in its TIR domain alone. Furthermore BtpA-TIR formed filaments while its E217A mutated version did not, suggesting that loss of the filament shape and toxicity might be related. SARM1 localized as cytoplasmic spots, while its TIR domain alone was in the cytosol but they were not toxic for the yeast cell, in contrast with the Brucella proteins. Our results contribute to establish a yeast model to study the function of TIR domain-containing proteins.