Contribution of the NADase function of bacterial TIR domain proteins to the inhibition of innate immune responses

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Abstract Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with mutations already known to inhibit NADase activity. We are now using mutant TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing reporter assays to analyze the effects of these mutant TIRs on NF-kB signaling. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity, and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.