The role of VacA in Helicobacter pylori-host cell cholesterol homeostasis

PROJECT SUMMARY (ABSTRACT) Helicobacter pylori is a Gram-negative bacterium that colonizes the stomachs of about half the world’s human population. Although most infected individuals remain asymptomatic, H. pylori infection can lead to adverse health outcomes, including peptic ulcer disease, gastric adenocarcinoma, or gastric lymphoma. H. pylori infection is the primary risk factor for the development of gastric cancer, a leading cause of cancer-related deaths, and has been designated a Group I Carcinogen by the World Health Organization. Differences in health outcomes among H. pylori-infected individuals can be partially attributed to the genetic diversity of H. pylori: certain strains produce virulence factors that are associated with a higher risk of disease development. One of these virulence factors is Vacuolating Cytotoxin A (VacA), a secreted pore-forming toxin. The vacA gene contains highly polymorphic regions that vary among strains. Certain forms of VacA display more activity in cell culture, and H. pylori strains producing these active forms are associated with a higher risk of gastric cancer than strains producing less active forms. Active variants of VacA form channels in cell membranes and induce a range of alterations within cells, including the swelling of late endosomes (vacuolation), mitochondrial dysfunction, and even cell death in certain cell lines. However, the mechanisms by which VacA promotes H. pylori fitness and modulates host cell function remain incompletely understood. My preliminary studies indicate that VacA influences host cell cholesterol metabolism by significantly increasing the transcript abundances of genes involved in cholesterol biosynthesis and altering cellular cholesterol levels. VacA-induced alterations in cellular cholesterol biosynthesis are likely to be relevant in multiple contexts: (i) VacA binds to cholesterol-rich regions of host cell membranes, (ii) H. pylori is a cholesterol auxotroph that can acquire cholesterol from host cells, (iii) dysregulated cholesterol biosynthesis contributes to malignant transformation and gastric cancer risk, and (iv) statin therapy for high cholesterol has been associated with a lower incidence of stomach cancer in humans. I hypothesize that VacA disrupts cholesterol host cell cholesterol homeostasis, and that this process influences H. pylori cholesterol acquisition. The proposed research will address these hypotheses through three specific aims. In Specific Aim 1, I will further define the specific alterations in gastric cell sterol biosynthesis triggered by different forms of VacA using enzymatic assays, liquid-chromatography tandem mass spectrometry (LC-MS/MS), and RT-qPCR. In Specific Aim 2, I will assess the effects of VacA on cholesterol efflux, uptake, and trafficking using LC-MS/MS and confocal imaging assays. In Specific Aim 3, I will determine whether VacA influences H. pylori cholesterol acquisition by comparing the cholesterol uptake of VacA-producing strains and various VacA mutant strains. Taken together, these aims will lead to a better understanding of cellular responses to VacA and will define the consequences of VacA-induced cholesterol dysregulation for the host and bacteria.