Investigation_of_Helicobacter_pylori_virulence_factors_through_targeting_gene_inactivation

The aim is to conduct whole genome sequencing to identify point mutations in individual H. pylori clones to discover important gastric colonization factors. While analyzing the spatial distribution of the GFP- and tdTomato-expressing isogenic strains in co-infections, we discovered that the two isolates are equally fit and identical in every manner except for differences in distribution in the gastric glands. In particular, we have consistently found that the GFP strain dominates in the transition zone glands in comparison to the tdTomato strain. We propose to conduct whole genome sequencing to identify genetic differences that could explain these differences in gastric gland distribution. In addition, we isolated multiple tdTomato clones from the initial transformation reaction and competed these with the GFP strain as co-infections. We discovered that some tdTomato clones either outcompeted or were outcompeted by the GFP strain. We propose that whole genome sequencing onthese tdTomato clones will help us discover novel factors important for fitness during gastric colonization . We also aim to conduct in vivo competition experiments between WT and mutants in important virulence factors. We previously described an H. pylori chemotaxis regulatory protein ChePep that is important for the ability of the bacteria to colonize the gastric glands. The ChePep deletion mutant colonizes mice to similar levels as WT and is found in the surface mucus, but does not colonize the gastric antral glands. These results suggest that sensing and responding to host environmental signals via chemotaxis play important roles in gland colonization. Furthermore, in competition, WT H. pylori completely outcompete the ChePep mutant, suggesting that gland colonization confers an important fitness advantage. We have generated differentially fluorescent mutant strains in ChePep, the chemoreceptors, and several other important H. pylori virulence factors.We are currently competing these mutants against differentially marked WT bacteria in co-infections to examine their fate and distribution in the gastric glands. This has never been done for any virulence factor in H. pylori, and has the potential to shed light on the roles of important virulence factors in fitness and gland colonization. Therefore, we alsopropose to conduct whole genome sequencing on the different mutants that were generated to determine whether other secondary mutations have occurred in these strains that could affect bacterial fitness or localization.