Unlocking the Potential of Bacterial ParE Toxins: Developing a Blueprint for Co-Opting Molecular Time Bombs That Impact Bacterial Cell Survival

We are proposing a new way to control bacterial growth, including genetically resistant as well as phenotypically persistent bacteria, by manipulating chromosomally encoded bacterial "time bombs" called toxin-antitoxin (TA) systems. This idea is directly responsive to the "Area of Encouragement" identified as "Antimicrobial Resistance," for the "Development of novel and/or innovative interventions to prevent the spread of or treat infections from multidrug-resistant organisms, focused on hardware-associated infections and biofilms." As emerging infections and increases in resistance make the need for antibacterials more pressing, it is increasingly evident that our homeostatic balance and health also depend on bacteria. This revelation then further constricts antibacterial approaches to try to minimize impact on beneficial "good" bacteria. Antibacterial discovery has long relied on directed serendipity via screening of natural products and libraries to identify inhibitors and their corresponding bacterial targets. Currently the most fruitful approaches are dominated by derivatization of existing antibacterials; these activities are absolutely required for short-term defenses against infection. However, longer-term approaches that rely on new and unique strategies are badly needed, especially as emerging resistance is outpacing antibacterial development. TA systems are a non-secreted component of a bacterial cell's intrinsic physiologic response. These are protein pairs used to tailor bacterial physiology towards either death (a "time bomb") or survival, depending on the cellular target of the toxin, in effect acting as resiliency factors. An accepted idea in the field is to potentially co-opt TA systems for health purposes. This idea remains unfeasible because of a lack of fundamental knowledge on how to leverage TA systems as tools. The current proposal, however, is focused not on translation-inhibiting toxins but instead on the ParE subtypes. These toxins have qualities that make them uniquely useful in this approach: in their ability to mediate detrimental DNA degradation to the expressing bacterial cells and their widespread presence in different Gram-negative bacteria of concern. We are proposing to co-opt these ParE toxins to directly cause death to only the specific type of bacteria in which they are found, an advantageous narrow-spectrum approach. These types of ParE toxins are present in bacteria of significant concern to human health and will be the focus of our investigations: P. aeruginosa, V. cholera, M. tuberculosis, and Burkholderia sp. Of specific interest to the funder, these pathogens have a directly negative impact on military personnel in field environments and when dealing with wounds, including biofilm formation, that can occur in non-optimal treatment conditions. Hypothesis: That the presence of ParE toxins within a bacterial cell imparts (1) an increased mutagenic potential that at a native concentrations contributes to emerging antibiotic resistance, and that (2) increasing ParE toxin activity can significantly weaken the bacterial cell's ability to survive, and this effect will be additive or synergistic with existing antibiotic regimens. To assess this hypothesis, the following is proposed: Specific Aims: (1) Determine the spectrum of ParE activity in native hosts by measuring viability, accumulation of mutations, and antibiotic susceptibility as a function of induced ParE toxin expression. (2) Increase ParE availability in vivo as proof of concept of a therapeutic approach by engineering each targeted species' ParD antitoxin degradation model system in an E. coli host. The outcomes of this project will be (1) understanding fundamental mechanism potentially contributing to rise of resistance, providing a window for potential intervention, and (2) demonstrating proof of concept of co-opting this mechanism into a novel treatment that by definition will be specific for a given bacterial species. Innovation: This project is directly responsive to the pressing need for alternative antibacterial strategies. The demonstration that ParE toxins can be co-opted will be transformative in multiple fields, including microbial physiology, therapeutic development, and the wider TA community. The outcomes will offer (1) very high specificity to a single pathogen, (2) versatility in providing a means to re-sensitize "tolerant" metabolic states to current treatments in a potentiating approach, (3) will provide insight into a potential fundamental mechanism of genetic resistance through error-prone repair after low dose toxin-induced DNA damage, and (4) targeting of the antitoxin is predicted to be less prone to resistance because of the need to maintain a productive pairing between cognate toxins and antitoxins. Less