The innate immune system can detect the presence of microbial infection by employing germline-encoded receptors specific for conserved microbial ligands. For example, the extracellular presence of the bacterial protein flagellin is sensed by Toll-like receptor 5, leading to activation of the transcription factor NF-κB, cytokine expression, and immune responses. In addition, certain flagellated bacterial pathogens, also appear to be sensed by an inflammasome. The inflammasome is a cytosolic molecular complex that contains a host Nod-like protein, Ipaf, and triggers caspase-1 and macrophage death. We used the intracellular pathogen Legionella pneumophila as a model intracellular bacterium to study the activation of the Ipaf inflammasome. Macrophage resistance to Legionella growth is entirely dependent upon the host expression of Ipaf and the bacterial expression of flagellin making it a useful tool to probe this pathway.

Using a retroviral expression vector, we found that the cytosolic expression of bacterial flagellin, in the absence of other bacterial contaminants and virulence factors was sufficient to trigger macrophage cell death. Through the use of genetic knock-out macrophages we found that this cell death is dependent upon caspase-1 and Ipaf. Taken together these results indicate that flagellin expressed in the cytosol is sufficient to activate the Ipaf inflammasome. We then mapped the region of flagellin required to activate Ipaf to the highly conserved C-terminus of flagellin. This finding indicates that the region of flagellin recognized by Ipaf is distinct from the region recognized by Toll-like receptor 5. Indeed, when the residues recognized by Toll-like receptor 5 are mutated the flagellin is still capable of activating Ipaf. The minimal region of flagellin that is required for sensing by Ipaf is the C-terminal 35 amino acids including three conserved C-terminal leucine residues that are critcal for sensing. Interestingly, activation of the inflammasome in response to this minimal peptide also requires the host protein Naip5, a unique member of the Nod-like and inhibitor of apoptosis (IAP) gene families.

Consistent with this result, the restriction of L. pneumophila growth within macrophages is dependent upon both Naip5 and Ipaf. However, Naip5 was dispensable for the response to other Ipaf-dependent stimuli including retroviral transduction of full-length flagellin. Activation of caspase-1 in response to S. typhimurium infection is also only partially dependent on Naip5. By expressing Salmonella flagellin in L. pneumophila we were able to show that it is not the type of flagellin, but rather the mode of delivery that results in a Naip5-dependent or -independent response. Indeed, Salmonella expresses an Ipaf activator, prgJ, that is Naip5 independent, explaining why some of the inflammasome activation in response to Salmonella is Naip5 independent. We have also determined that the N-terminus of flagellin plays a critical role in determining the differential roles of Naip5 and Ipaf in flagellin-recognition. Our results provide a molecular framework for understanding the cytosolic recognition of flagellin by host cells.