Inhibition of HipA to Reduce Multidrug Tolerance in E. coli
The HipBA operon is a bacterial toxin-antitoxin module that plays a crucial role in multidrug tolerance in E. coli. HipA, the toxin, is a kinase that functions by phosphorylating translation factors, inhibiting translation in the cell and inducing a state of dormancy in which cellular processes that are targeted by antibiotics are shut down, allowing the cell to evade antibiotic poisoning. The goal of my research is to identify molecules that bind to HipA and inhibit its kinase activity. HipA autophosphorylates, so its activity can be measured by its phosphorylation. To measure phosphorylation, wild-type HipA is completely dephosphorylated using a phosphatase enzyme, and its phosphorylation after the addition of ATP can be visualized through a ProQ Diamond Phosphoprotein Gel Stain. This assay can be repeated with wild-type HipA in the presence of target molecules that are believed to inhibit its kinase activity. A lack of autophosphorylation in the presence of a molecule indicates that this molecule inhibits HipA and could be a potential molecule of study for the development of an antibiotic.