Defining the role of the Brucella TIR-NAD hydrolase activity in immunosuppression and pathogenesis

PROJECT SUMMARY Toll/interleukin-1 receptor (TIR) domains, conserved from archaea to mammals, mediate innate immune signaling. Multiple bacterial pathogens use secreted TIR domain-containing effectors to modulate hostpathogen interactions and sabotage host immunity. Recently, bacterial TIR domains were discovered to have nicotinamide adenine dinucleotide (NAO+) hydrolase activity, depleting host cell NAO- during infection. Since NAO- levels regulate metabolism, cell signaling and immune function, bacterial TIR-mediated NAO- hydrolysis is potentially highly relevant for infectious success. However, the role of TIR-NAOase activity in bacterial pathogenesis remains unknown. This proposal will address this knowledge gap, focusing on Bruce/la species that are highly pathogenic for humans as a model system. We will test the overall hypothesis that TIRmediated NAO' hydrolysis directly contributes to bacterial virulence. Aim 1 will determine if TIR-NAO' hydrolysis contributes to the immunosuppressive properties of Bruce/la TIR domain-containing effectors BtpA/BtpB by assessing the inhibition of host cell NF-KB activation and inflammatory cytokines in infected primary macrophages and dendritic cells. This aim will also localize the BtpA/B effectors in infected cells and determine if the TIR-NAOase activity contributes to the targeting of host TLR-adaptor complexes. Aim 2 will establish the role of the BtpA/BtpB TIR-NAOase activities in vivo. Using TIR-NAOase catalytic mutants, splenic inflammation will be quantified in a standard BALB/c murine model of brucellosis. Additionally, the contribution of NAOase activity to virulence will be assessed in a newly developed, completely penetrant, and clinically scorable SKG model of Bruce/la-induced spondyloarthritis, the most common chronic complication of human brucellosis. This project is innovative as it will establish the relevance ofTIR-mediated NAO' depletion in the context of mammalian infection, both in vitro and in vivo. Moreover, this work will validate a new model of Bruce/la-induced spondyloarthritis and its use for dissecting the bacterial mechanisms implicated in chronic brucellosis, for which no model is currently available. The proposed work is highly significant and aligns with the mission of the NIH in that it will define the mechanisms of host immune evasion by this important family of bacterial secreted effectors, potentially resulting in a paradigm shift in our understanding of how bacterial TIR domains contribute to host-pathogen interactions and pathogenesis. The results generated will have a broad impact given the number of bacteria associated with human infectious diseases containing TIR-effector proteins with NADase activity. Finally, targeting TIR-NADase activity may offer a promising new therapeutic approach for infectious diseases.