Fundamental Studies of ParDE Toxin-Antitoxin Systems for Their Potential to Impact Bacterial Cell Growth

Abstract

The rise of antimicrobial-resistant bacteria, coupled with a shortage of new antibiotics, has threatened treatment of infections as well as modern medical procedures. To counteract with this burgeoning issue, targeting bacterial toxin-antitoxin (TA) systems, which engage in controlling bacterial cell growth under stress conditions, e.g., antibiotics treatment, presents a promising strategy for developing novel antimicrobial agents to combat resistance. TA systems are widely distributed in prokaryotes and the operon is composed of a small bicistronic locus encoding antitoxin and toxin genes. In the case of type II ParDE TA systems, the ParE toxin protein selectively inhibits DNA gyrase, an essential type II bacterial topoisomerase, by stabilizing gyrase-mediated DNA cleavage complex, leading to double strand DNA break. The toxicity of ParE protein can be neutralized by its co-encoded cognate ParD antitoxin protein through direct protein-protein interaction. The work presented in this dissertation aim to provides deeper insights to understand the structural properties of the ParDE TA systems and the impact on bacterial cell growth and resulting influence on mutation driven from ParE-mediated gyrase inhibition. Chapter II of this work focuses on the impact of VcParE toxins induced expression in the native host, Vibrio cholerae (Vc). The results showed that strong expression of VcParE toxins caused cell death, likely arising from extensive DNA break generated by inhibition of gyrase, and overwhelming the repair machinery. However, lower levels of ParE expression could be tolerated, and the induced DNA breaks was able to be repaired, permitting cell survival. As it is known that this type of repair can be error-prone, the result confirmed increases in mutation frequency after lower induction of VcParE toxins. Importantly, however, this did not correlate with decreasing antibiotics susceptibility. Overall these results indicate that VcParE toxins are potent inhibitors of DNA gyrase in bacterial cells, and that damage generated by gyrase inhibition do not lead to increase in antibiotic resistance. Chapter III of this work explore the feasibility to obtain purified ParE toxins following reported methods and developed an optimized purification approach. We tried various expression and purification strategies, including denaturation-refolding of the ParDE complex, in vitro protein synthesis, bulky fusion protein expression and co-transformation with gyrase, to overcome ParE toxicity and obtain sufficient protein for future characterization. Our result showed that co-transformation with gyrase was effective to lessen VcParE1 toxicity and the purified VcParE1 proved to be active in the inhibition of gyrase-mediated supercoiling assay. The approach developed in this chapter enables us to obtain quantitive VcParE1 for future structural studies to further elucidate the structure of ParE-gyrase complex. Chapter IV of this work investigates the intrinsic degradation of the PaParD1 antitoxin, analyzing factors influencing its stability. Our result indicated that the degradation of PaParD1 is affected by temperature, pH value, and concentration, with maximal degradation observed at physiological pH and temperature. Higher concentrations of PaParD1 also increased degradation rate. We also established the effect of ionic strength on PaParD1 dimerization and degradation, with increased degradation rate as the ratio of monomeric PaParD1 increased. While initial hypothesis suggested a glutamine-mediated cleavage mechanism, mutagenesis experiments revealed no direct role for glutamine in PaParD1 degradation. To further identify protein speices appearing in the PaParD1 degradation process, Mass spectrometry was used and identified unexpectedly co-purifying DnaK chaperone which potentially modulate PaParD1 stability. In aggregate, Chapter IV provides optimal conditions for PaParD1 stability for in vitro storage, and propose a dynamic PaParD1 dimerization in varied ionic strength, offering a foundation for future research on TA system regulation. Overall, the study of this dissertation advanced the understanding of ParDE TA systems and their potential for antimicrobial development. ParE toxins were shown to strongly inhibit DNA gyrase, causing DNA damage and cell death, though lower level of expression increased mutation frequency but did not accumulate to become antibiotic resistance. Additionally, the approach of co-transformation with gyrase developed in this dissertation is able to produce decent quantity of active VcParE1, enabling future structural studies for the ParE-gyrase complex. Lastly, investigation of the intrinsic stability of the PaParD1 revealed core conditions affecting its degradation, including temperature, pH, concentration, and ionic strength. The co-purifying DnaK chaperone is also identified and may function in modulating PaParD1 stability. Taken together, the research in this dissertation extend the understanding of ParDE TA system, from the structural to functional properties, providing a foundation for future studies on their regulatory mechanisms as well as potential application in antimicrobial development.