HP0953 - hypothetical virulence factor overexpresion and localization during Helicobacter pylori infection of gastric epithelium.

BACKGROUND: The high prevalence and persistence of Helicobacter pylori (H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340 hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor. AIM: To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric (AGS) cells. METHODS: Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0, 3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling. RESULTS: HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells. Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells. Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found; modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage. CONCLUSION: The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.

Diversification of the vacAs1m1 and vacAs2m2 Strains of Helicobacter pylori in Meriones unguiculatus.

The bacterium Helicobacter pylori exhibits great genetic diversity, and the pathogenic roles of its virulence factors have been widely studied. However, the evolutionary dynamics of H. pylori strains during stomach colonization are not well-characterized. Here, we analyzed the microevolutionary dynamics of the toxigenic strain vacAs1m1, the non-toxigenic strain vacAs2m2, and a combination of both strains in an animal model over time. Meriones unguiculatus were inoculated with the following bacteria: group 1-toxigenic strain vacAs1m1/cagA+/cagE+/babA2+; ST181, group 2-non-toxigenic strain vacAs2m2/cagA+/cagE+/babA2+; ST2901, and group 3-both strains. The gerbils were euthanized at different time points (3, 6, 12, and 18 months). In group 1, genetic alterations were observed at 6 and 12 months. With the combination of both strains, group 3 also exhibited genetic alterations at 3 and 18 months; moreover, a chimera, vacA m1-m2, was detected. Additionally, four new sequence types (STs) were reported in the PubMLST database for H. pylori. Synonymous and non-synonymous mutations were analyzed and associated with alterations in amino acids. Microevolutionary analysis of the STs (PHYLOViZ) identified in each group revealed many mutational changes in the toxigenic (vacAs1m1) and non-toxigenic (vacAs2m2) strains. Phylogenetic assessments (eBURST) did not reveal clonal complexes. Our findings indicate that the toxigenic strain, vacAs1m1, and a combination of toxigenic and non-toxigenic strains acquired genetic material by recombination. The allelic combination, vacAs2m1, displayed the best adaptation in the animal model over time, and a chimera, m1-m2, was also identified, which confirmed previous reports.