Date Thesis Awarded

5-2001

Document Type

Honors Thesis

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Dr. Mark Forsyth

Committee Members

Dr. Shanta Hinton

Dr. Diane Shakes

Dr. Ryan Fletcher

Abstract

Helicobacter pylori is a gram-negative bacterium that colonizes the gastric mucosa of about 50% of the world’s human population. H. pylori utilizes a diverse variety of outer membrane proteins for survival, colonization, and adherence to host human gastric epithelial cells in human stomach. Outer membrane proteins involved in adhesion, referred to as adhesins, are critical for bacterial survival and pathogenesis. Previous studies have shown that contact with host human gastric epithelial cells alters the transcription levels of several H. pylori genes. However, whether or not contact induces alterations in transcription and the mechanisms through which H. pylori senses contact with gastric epithelial cells are unknown. The present study investigates changes in the gene expression of several outer membrane proteins induced by contact with human gastric adenocarcinoma (AGS) cells, and the possible role that two-component systems (TCS) play in this sensing contact. We showed that H. pylori cells that adhered to AGS cells possessed increased oipA transcript levels compared to non-adherent H. pylori cells. In addition, AGS attachment assays revealed that attachment to AGS cells was significantly higher in H. pylori strains possessing a phase-on allele of oipA compared to an isogenic strain that had oipA experimentally turned phase off. ELISA assays revealed that H. pylori producing a functional OipA protein and containing the cag Pathogenicity Island (cagPAI) were positively correlated with both higher levels of attachment and higher levels of IL-8 production by AGS cells, suggesting that OipA interacts with the cagPAI to induce a pro-inflammatory reaction. Additional experiments revealed that contact with AGS cells upregulated babA mRNA levels in adherent H. pylori and repressed sabA mRNA levels in nonadherent H. pylori. Subsequent experiments using conditioned media possessing AGS cell secretions showed a significant decrease in sabA mRNA levels in free-swimming H. pylori when compared to H. pylori incubated in unconditioned media. We hypothesize that sabA repression by AGS cell secretions aids in the separation of the two populations of H. pylori that occur in the stomachs infected individuals (adherent and non-adherent, motile H. pylori cells). Finally, we showed that mRNA levels of babA, encoding a well characterized outer membrane protein adhesin, are significantly reduced when the histidine kinases of the three known two-component systems are knocked out. Furthermore, we found no statistical difference between the mRNA level of babA in naïve H. pylori cells and the triple histidine kinase knock out mutation strain. and that two-component systems have a role in sensing contact with host gastric epithelial cells. This data suggests that contact dependent changes in babA expression is regulated via a two-component system. Our data also suggests that oipA and sabA are not regulated by two-component systems in a contact dependent manner. This indicates that two-component systems have a role in mediating contact dependent alterations in the gene for at least one H. pylori outer membrane protein.

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