Mechanisms of reverse transcriptase mediated phage resistance in Enterobacteriaceae

PROJECT SUMMARY Intestinal colonization by multidrug-resistant (MDR) Enterobacteriaceae represents an urgent public health threat, promoting the dissemination of antimicrobial resistance mechanisms and leading to severe and often lethal infections. Multiple avenues of protection exist in the gut to defend against potentially pathogenic organisms, with bacteriophages constituting a crucial node of control over bacterial invasion and expansion. However, given that bacteria encode a variety of defense pathways to ward off phage predation, the activity of phage defense systems may serve an essential role in modulating the colonization potential and pathogenicity of intestinal microbes. The investigation of phage defense mechanisms and their roles in bacterial pathogenesis is therefore critical for understanding the proliferation of MDR Enterobacteriaceae in the context of the intestinal microbiome. Additionally, detailed study of phage defense systems has the potential to guide the engineering of phages as alternatives to antibiotic treatment, an emerging strategy that is already showing considerable promise in the treatment of MDR infections. A group of reverse transcriptase (RT) genes that is broadly represented in Enterobacteriaceae has recently been discovered to function in phage defense. However, the mechanistic link between their RNA- templated DNA synthesis activity and antiphage immunity remains puzzling. This proposal aims to investigate the functions of defense-associated RTs (DRTs) in phage defense, and evaluate their contributions to the pathogenicity of intestinal microbes. Aim 1 will focus on the development of an unbiased experimental approach for discovering the in vivo RNA substrates and cDNA products of RTs, and will apply this approach to study diverse DRT systems. Aim 2 will utilize biochemical and genetics assays to determine the minimal components underlying DRT activity. Aim 3 will leverage a combination of genetic screening and in vivo bacterial pathogenesis modeling to investigate how dynamic interactions between phages and DRTs modulate the pathogenicity of Enterobacteriaceae. In addition to illuminating mechanistic features of a compelling new group of phage defense systems, and linking the activity of these systems to colonization and proliferation of pathogenic bacteria, this work has the potential to guide future strategies for the prevention and treatment of antibiotic-resistant infections. This fellowship will support the candidate’s training in the MD/PhD program at Columbia University, which includes thesis work in the laboratory of Dr. Samuel Sternberg and the remainder of medical training in the Vagelos College of Physicians and Surgeons. The combination of rigorous scientific training, clinical exposure, and career development guidance outlined in the proposal will prepare the candidate for a successful career as an independent investigator and physician-scientist.