Carlos Detres

Carlos Detres

PI: Timothy Cover, MD , Department of Medicine

Defining amino acids required for oligomerization of Helicobacter pylori VacA toxin

Helicobacter pylori is a Gram-negative bacterium that lives in the mucus layer overlying the gastric mucosa of humans. H. pylori secretes a pore-forming toxin known as VacA. VacA can cause an assortment of alterations in human cells, the most prominent of which is vacuolation of gastric epithelial cells. The formation of VacA channels in cell membranes is dependent on the ability of VacA to assemble into oligomeric structures. The laboratory recently conducted experiments in which VacA oligomers were treated with a cross-linking reagent, and cross-linked peptides were then identified by mass spectrometry. These experiments allowed the identification of candidate residues localized at interfaces believed to mediate VacA protein-protein interactions. To test the hypothesis that these residues are required for VacA oligomerization, I am performing site-directed mutagenesis to produce mutant forms of the toxin in which the residues of interest are altered. After completing the mutagenesis, I will perform Western blotting to verify that the mutant VacA proteins are produced. I will purify the mutant toxins as well as wild-type VacA, and then will compare functional properties of these proteins. Assays will include a cell vacuolation assay, as well as biochemical and electron microscopic analyses to evaluate oligomer formation. This research will provide important new insights into the molecular mechanisms by which VacA causes alterations in human cells.