FapA extends the flagellar cage-dependent network into the cytoplasm to control surface-associated behaviors in Helicobacter pylori
Flagellar systems enable many host-associated bacteria to colonize successfully. One such pathogen is Helicobacter pylori, which assembles an elaborate, high-torque flagellar motor that has a wide C-ring, 18 stator complexes, and is surrounded by a cage that includes homologs of the type IV pilus proteins PilM, PilN, and PilO. PilMNO act to repress soft-agar migration while promoting cell aggregation, microcolony, and biofilm formation, but the pathway linking the flagellar cage to these behaviors is unknown. Here we identify FapA, a member of the flagellar assembly protein A (FapA) family, as a component of this pathway. Deletion of fapA increased soft-agar migration and reduced microcolony and biofilm formation, similar to deletion of pilMNO. Epistasis analysis placed FapA downstream of the PilMNO flagellar cage. Using fluorescent fusions and protein-protein interaction experiments, FapA was found to localize to the cell poles, the sites of flagellar motor placement, and interact with the cytoplasmic PilM component of the cage. Indeed, cryo-electron tomography experiments detected cytoplasmic density near PilM that was lost in the {Delta}fapA deletion mutant. Phylogenetic analysis suggests that FapA proteins are more widespread than previously appreciated, being found in multiple bacterial phyla including Campylobacterota, Bacillota, Spirochaetota, Desulfobacterota, Thermotogota, Synergistota, and Pseudomonadota. Together, these findings support that the H. pylori flagellar cage has additional components that reside in the cytoplasm but similarly function to limit motility and activate a sessile/biofilm lifestyle. Given the widespread nature of FapA, our results suggest that many other bacteria might employ FapA homologs for similar purposes.