Flagellar assembly factor FliW2 de-represses Helicobacter pylori FlaA-mediated motility by allosteric obstruction of global regulator CsrA

Helicobacter pylori colonizes the human stomach as a dominant member of the gastric microbiota and is a causative agent of gastric infections that may lead to the development of gastroduodenal diseases and gastric cancer. Unlike Gammaproteobacteria, which are mostly found in intestinal niches, H. pylori’s gastric milieu constitutes a uniquely hostile ecological niche. Enabling it to survive in the stomach, H. pylori constitutively expresses flagellar biosynthesis, chemotaxis, and motility as essential housekeeping functions in vivo. Previous studies have reported that the inactivation of csrA, the gene encoding for carbon storage regulator A, affects the flagellar motility, biofilm formation, and in vivo colonization of H. pylori virulence. CsrA regulates the expression of downstream genes via mechanisms that operate posttranscriptionally and translationally. Although CsrA is a critical determinant of gene regulation and flagellar biosynthesis in H. pylori, the modulation of CsrA is uncertain. In this study, we attempted to understand whether H. pylori CsrA activity is regulated by protein antagonism and how this mechanism affects H. pylori motility. We employed a comparative genomic analysis to identify two fliW homologs (jhp1081-encoded FliW1 and jhp1291-encoded FliW2) from the H. pylori J99 genome. Characterization of the isogenic fliW2 mutant strain by phenotypic and biochemical analyses demonstrated the crucial role of FliW2 in flagellar motility through a direct interaction between FliW2 and CsrA. Using a machine learning approach to predict FliW2-CsrA binding domains and an in vivo bacterial two-hybrid system, our investigation showed that the C-terminal region of CsrA is critical for the CsrA-FliW2 interaction that may indirectly occlude CsrA’s RNA binding. Our data points to novel regulatory roles that the H. pylori flagellar assembly factor FliW2 has in obstructing CsrA activity and antagonizing CsrA’s blocking of flaA mRNA processing and translation. Our findings reveal a new regulatory mechanism of flagellar motility in H. pylori.