Toll/interleukin-1 (TIR) domains play critical roles in immune responses in diverse organisms. Bacterial TIR-containing proteins mediate anti-phage immunity and act as virulence factors during host infection. Plant and bacteria TIR-domains typically possess enzymatic activity where they hydrolyse NAD+, leading to production of small molecules which active downstream signalling pathways, or cell arrest through NAD+ exhaustion. Here, we focus on a TIR domain from Vibrio parahaemolyticus (VpTIR) and another from Mariprofundus ferrooxydans (MfTIR). Biochemical assays reveal that both TIRs possess NADase activity but MfTIR showed markedly slower catalytic activity. Previously, the NAD+ analogue, 3AD, stabilized the Acinetobacter baumannii TIR domain in an active filamentous state, enabling cryo-EM to resolve key interfaces and the ligand-binding site. Same approach was applied to capture the ligand-bound state of VpTIR. Current cryo-EM reconstructions suggest a potentially novel TIR domains assemblies. Ongoing refinements aim to produce higher-resolution maps for detailed characterization. Additionally, a study showed that plant TIR-domains can bind nucleic acids, leading to alternative filamentous assemblies, along with 2’,3’-cAMP/GMP synthesis. We probed each bacterial TIR’s capacity for DNA interaction and cyclic nucleotide production. Our results showed VpTIR binds with plasmids. Currently we are optimising cryo-EM workflows to uncover binding details, as bacteria TIRs lack the DNA-binding motif observed in plant TIR:dsDNA complexes. This may represent a new function of bacteria TIR domains.
Integrating structural, enzymatic, and ligand-binding analyses, this work broadens our understanding of bacterial TIR domains and sheds light on the evolutionary links to TIR-driven plant and mammalian immune signaling.