The long-term goal of this program is to understand bacterial stress responses in enough detail to eventually design small-molecule inhibitors of stress response as antimicrobial potentiators. The present R21 application describes a basic-science, proof-of-principle project to study a new protein kinase that when inactivated decreases survival of E. coli cells treated with a variety of antimicrobials, hydrogen peroxide, and high temperature. A deficiency of the kinase reduces bacterial survival to quinolone treatment by 10- to 100-fold and causes the bacteriostatic compound chloramphenicol to become bactericidal. It also dramatically lowers the ability of nalidixic acid to induce new resistant mutants. The kinase deficiency is suppressed by deletion of a toxin-antitoxin gene pair thought to contribute both to protection from stress and to bacterial apoptosis. This genetic interaction with toxin-antitoxin systems leads to the hypothesis that the kinase normally limits toxin activity; in the absence of the kinase, toxins kill cells during stressful conditions, thereby enhancing the action of many antimicrobials at the same time. The kinase gene is also implicated in the Cpx envelope protein stress response pathway by having two CpxR binding sites upstream of its promoter region, establishing another link of the kinase to stress responses. Both the genetics and biochemistry of this stress-response kinase will be studied. The upstream regulation of the kinase gene will be studied through effects of mutations in the Cpx and other related two-component regulatory systems, and downstream effects will be studied through genetic interactions with the toxin-antitoxin systems. To obtain a framework for the role of the kinase in stress response networks, gene expression profiling will also be carried out with a variety of stresses in the presence/absence of the kinase activity. The kinase has been purified. As a part of its further characterization, the enzymatic reaction conditions will be optimized, the autophosphorylation site(s) will be determined, and proteins it normally phosphorylates will be identified. The proposed work constitutes an early characterization of regulatory networks involved in bacterial stress response, persistence/tolerance, and apoptosis. Such studies may eventually lead to ways for making many antimicrobials more effective by interfering with bacterial stress responses. ? ? Bacterial resistance, tolerance, and persistence to antimicrobial treatment is a growing threat for our ability to cure infections. Protective genes involved in bacterial stress responses help bacteria evade and survive antimicrobial treatment. These protective stress response networks will be studied with the long-term goal of developing small-molecule inhibitors for antimicrobial enhancement. ? ? ? ?
