The innate immune system is responsible for initiating the host immune response to infection. The study of microbial virulence has uncovered numerous mechanisms for microbes to evade innate immune detection. In contrast, relatively little is understood about the strategies employed by the host to prevent microbes from evading innate immunity. The NAIP innate immune receptors provide an intriguing case study to investigate these strategies. In mice, Naip has undergone gene duplication and drift, recombination, and pseudogenization1, all of which can be signatures of a co-evolutionary arms race with targeted pathogens2. This duplication and specialization has allowed NAIP paralogs to recognize several distinct bacterial proteins: NAIP5 binds bacterial flagellin (FlaA), and NAIP2 detects the inner rod protein (PrgJ) of the pathogen-associated type III secretion system (T3SS)3,4.

I sought to address how gene duplication and drift enabled functional specialization by first defining which NAIP domains bind to bacterial ligands. I analyzed a panel of chimeric proteins, in which homologous domains of NAIP5 and NAIP2 were swapped, to determine which domains conferred the ability to recognize FlaA or PrgJ. A long-standing expectation in the field was that the auto-inhibitory C-terminal leucine-rich repeat (LRR) domain mediates ligand binding. Surprisingly, I found that the LRR was dispensable for ligand specificity. Instead, ligand recognition was mediated by several alpha-helical domains in the center of the protein. Strikingly, these domains are specifically evolving under positive selection, in which non-synonymous mutations are repeatedly selected to provide altered ligand binding surfaces. Separation of sensing and auto-inhibition functions into different domains may allow NAIPs to sample ligand recognition-altering mutations without disrupting steady-state auto-inhibition.

These data suggested that NAIPs are engaged in a co-evolutionary arms race with bacteria over innate immune detection. However, bacterial ligands can evolve much more rapidly than mammalian NAIPs. To determine how NAIPs can successfully compete in such an arms race, I conducted alanine scanning screens of FlaA and PrgJ to comprehensively identify the ligand motifs recognized by NAIPs. Both NAIP5 and NAIP2 recognized multiple conserved surfaces, near the N- and C-termini, of their respective ligands. This multi-surface recognition strategy conferred NAIPs with robust detection of their bacterial ligands, as single point mutations in any recognition motif did not affect NAIP recognition. Rather, bacterial immune evasion required simultaneous mutation of multiple recognition motifs. However, highly mutated ligands that escaped immune detection also lost their native function, suggesting that multi-surface recognition serves to constrain bacterial immune evasion.

To verify these biochemical results, we have determined the cryo-EM structure of NAIP5 bound to FlaA, an event which triggers oligomerization with the adapter protein NLRC4 into a large (>1 MDa) signaling complex. The structure reveals direct contacts between the NAIP5 ligand recognition domains and both recognition surfaces of FlaA. The extensive and largely hydrophobic contacts between NAIP5 and FlaA are consistent with a lack of “hot spot” binding sites and likely contribute to the robust recognition of NAIP5 for FlaA single point mutants. Additionally, our structure reveals how binding to FlaA triggers a conformational change in NAIP5 to expose its oligomerization surface, allowing the recruitment of NLRC4. The polymerization of NLRC45 and subsequent recruitment of the signaling effector, CASPASE-1, illustrates the switch-like mechanism by which the detection of a single ligand monomer is amplified into oligomerization-induced signaling.

Collectively, this dissertation elucidates the biochemical mechanism of NAIP innate immune detection of bacterial ligands. Furthermore, it has provided surprising insights into strategies employed by innate immune receptors to compete with bacteria in an evolutionary arms race over host defense.