Evolution of pleiotropic consequences via mucoid phage resistance in escherichia coli populations

The National Institute of General Medical Sciences (NIGMS) awarded University of Wisconsin-Milwaukee $572,553 under the Biomedical Research and Research Training program (CFDA 93.859) for a three-year project (September 1, 2025 through August 31, 2028) investigating pleiotropic consequences of mucoid phage resistance in Escherichia coli populations. The research will deliver fundamental scientific knowledge on how bacterial resistance to bacteriophages develops through exopolysaccharide overproduction and the associated trade-offs this mechanism creates for bacterial virulence and antibiotic susceptibility. The project addresses a critical gap in understanding molecular mechanisms of pleiotropy—where genetic changes produce multiple phenotypic effects—with potential applications for developing phage-based therapeutic alternatives to antibiotics.

The research will be conducted through three specific aims: identifying how bacterial host strain impacts evolution of mucoid-based phage resistance; characterizing pleiotropic effects between mucoidy, antibiotic resistance, and bacterial motility; and testing how environmental spatial structure and antibiotic presence influence mucoidy evolution. The ultimate goal is to establish evolutionary principles for using bacteriophages to not only eliminate target pathogenic bacteria but also "steer" bacterial populations toward less virulent phenotypes through pleiotropic mutations, thereby advancing phage therapy as a viable medical countermeasure to antibiotic resistance.

Abstract

Pleiotropy, which occurs when a genetic change alters two or more phenotypes, can be observed during pathogen evolution when resistance to one treatment results in sensitivity to another treatment. Prior research suggests that bacteriophage-based alternatives to antibiotics can be more effective when phage resistance comes at a cost to bacterial virulence, but the molecular mechanisms of pleiotropy are often uncharacterized. One common phage-resistance mechanism, mucoidy, occurs through over-production of exopolysaccharide, creating a physical barrier between the cell and phage. As a virulence factor for many pathogens, mucoidy is of interest to the medical applications of phages as well as the study of naturally occurring phages. The objective of this application is to test predictions about the pleiotropic side effects of mucoid-based resistance to a candidate therapeutic phage. The hypothesis is that the evolution of mucoidy will be constrained based on genetic and environmental conditions. The rationale is that resistance to phages reliably arises by evolution, but that different phage-resistance mutations incur different costs in different environments, as shown throughout the preliminary data. The long-term goal is to apply evolutionary principles to use phages to not only to kill target host bacteria, but to also evolutionarily 'steer' bacterial populations towards a less pathogenic state via pleiotropic mutations. The research objective will be met through three specific aims: (1) identify how the host strain impacts the evolution of mucoidy-based phage resistance. (2) characterize the pleiotropic effects between mucoidy, antibiotic resistance, and motility. (3) test how environmental spatial structure and antibiotic presence impact the evolution of mucoidy. The proposed work is innovative because IT will: (a) leverage details about the molecular genetics of mucoid phage resistance to generate hypotheses about evolution. Preliminary data details the mucoid mutations through the e. Coli RCS phosphorelay, which also regulates other virulence factors. (b) assess the potential for mucoidy to be a barrier or asset to phage therapy in phage U136B. Phage U136B relies on the tolc antibiotic efflux pump protein as a receptor, driving a pleiotropic tradeoff between antibiotic and phage resistance. (c) test how bacterial genetic background influences the evolution of phage resistance. The applied significance is understanding the balance between the challenges and opportunities of phage resistance, which is crucial to the responsible use of phage therapeutics. The fundamental significance is understanding how bacterial evolution is constrained by pleiotropy in different genetic and environmental contexts. This project significantly impacts undergraduate research by providing excellent mentorship, supplies, and salaries for six undergraduate researchers. The PI has a highly established record of undergraduate mentorship, CO-authorship, awards, research-based pedagogy, and focused one-on-one mentorship. The proposal presents substantial preliminary data collected by undergraduates, and undergraduates will be involved in all proposed work.