Helicobacter pylori cheV1 mutants recover semisolid agar migration due to loss of a previously uncharacterized Type IV filament membrane alignment complex homolog.

The bacterial chemotaxis system is a well-understood signaling pathway that promotes bacterial success. Chemotaxis systems comprise chemoreceptors and the CheA kinase, linked by CheW or CheV scaffold proteins. Scaffold proteins provide connections between chemoreceptors and CheA and also between chemoreceptors to create macromolecular arrays. Chemotaxis is required for host colonization by many microbes, including the stomach pathogen Helicobacter pylori. This bacterium builds chemoreceptor-CheA contacts with two distinct scaffold proteins, CheW and CheV1. H. pylori cheW or cheV1 deletion mutants both lose chemoreceptor array formation, but show differing semisolid agar chemotaxis assay behaviors: ∆cheW mutants exhibit total migration failure, whereas ∆cheV1::cat mutants display a 50% reduction. On investigating these varied responses, we found that both mutants initially struggle with migration. However, over time, ∆cheV1::cat mutants develop a stable, enhanced migration capability, termed "migration-able" (Mig+). Whole-genome sequencing analysis of four distinct ∆cheV1::cat Mig+ strains identified single-nucleotide polymorphisms (SNPs) in hpg27_252 (hp0273) that were predicted to truncate the encoded protein. Computational analysis of the hpg27_252-encoded protein revealed it encoded a hypothetical protein that was a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori lacks Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM. Deleting hpg27_252 in the ∆cheV1::cat or wild type strain resulted in enhanced migration in semisolid agar. Our study thus reveals that while cheV1 mutants initially have significant migration defects, they can recover the migration ability through genetic suppressors, highlighting a complex regulatory mechanism in bacterial migration. IMPORTANCE: Chemotactic motility, present in over half of bacteria, depends on chemotaxis signaling systems comprising receptors, kinases, and scaffold proteins. In Helicobacter pylori, a stomach pathogen, chemotaxis is crucial for colonization, with CheV1 and CheW as key scaffold proteins. While both scaffolds are essential for building chemoreceptor complexes, their roles vary in other assays. Our research reexamines cheV1 mutants' behavior in semisolid agar, a standard chemotaxis test. Initially, cheV1 mutants exhibited defects similar to those of cheW mutants, but they evolved genetic suppressors that enhanced migration. These suppressors involve mutations in a previously uncharacterized gene, unknown in motility behavior. Our findings highlight the significant chemotaxis defects in cheV1 mutants and identify new elements influencing bacterial motility.

Discovery of Type IV filament membrane alignment complex homologs in H. pylori that promote soft-agar migration.

The stomach pathogen Helicobacter pylori utilizes two scaffold proteins, CheW and CheV1, to build critical chemotaxis arrays. Chemotaxis helps bacteria establish and maintain infection. Mutants lacking either of these chemotaxis proteins have different soft agar phenotypes: deletion of cheW creates non-chemotactic strains, while deletion of cheV1 results in 50% loss of chemotaxis. In this work, we characterized the cheV1 deletion mutant phenotype in detail. cheV1 deletion mutants had poor soft-agar migration initially, but regained migration ability over time. This improved bacterial migration was stable, suggesting a genetic suppressor phenotype, termed Che+. Whole-genome sequencing analysis of four distinct cheV1 Che+ strains revealed single nucleotide polymorphisms (SNPs) in a common gene, HPG27_252 (HP0273). These SNPs were predicted to truncate the encoded protein. To confirm the role of HPG27_252 in the cheV1 phenotype, we created a targeted deletion of HPG27_252 and found that loss of HPG27_252 enhanced soft-agar migration. HPG27_252 and CheV1 appear to interact directly, based on bacterial two-hybrid analysis. HPG27_252 is predicted to encode a 179 amino acid, 21 kDa protein annotated as a hypothetical protein. Computational analysis revealed this protein to be a remote homolog of the PilO Type IV filament membrane alignment complex protein. Although H. pylori is not known to possess Type IV filaments, our analysis showed it retains an operon of genes for homologs of PilO, PilN, and PilM, but does not possess other Type IV pili genes. Our data suggest the PilO homolog plays a role in regulating H. pylori chemotaxis and motility, suggesting new ideas about evolutionary steps for controlling migration through semi-solid media.