Pseudomonas aeruginosa is a major cause of opportunistic infections in immunocompromised people. P. aeruginosa uses a cell-cell signaling mechanism called quorum sensing (QS) to regulate virulence functions and cooperative behaviors. QS in P. aeruginosa is regulated by two transcription factors, LasR and RhlR. These proteins control the production of extracellular proteases, “public goods” that benefit the entire population. These public goods create an incentive for individuals to cheat by availing themselves of the proteases without incurring their production cost. In fact, when P. aeruginosa is grown in media that requires QS for growth, cheaters emerge in the population and invariably carry mutations in lasR. We were intrigued by the clinical isolate E94, in which we observed the rapid emergence of cheating. E94 contains an inactive LasR via a transposon insertion. Transposons are DNA segments that move between genomic locations. E94 contains dozens of transposable elements. We hypothesized that transposon movement facilitates the adaptability of the E94 genome. I evolved cheaters from wild-type E94 in casein media, which requires QS for growth. I identified cheaters using a phenotypic assay. I then grew cheaters in non-selective media and identified revertant, protease-producing colonies. I used PCR analysis of the cheaters and their protease-producing progeny to determine if the transposon remains inside the lasR gene. I found no transposon movement from lasR upon cheater reversion to a protease-producing phenotype. We also determined that cheaters display no rhlR activity. We then hypothesized that cheating in E94 occurs by disruption of RhlR QS. We did not find mutations in rhlR itself and are performing whole-genome sequencing to identify other genetic targets. Understanding the mechanism of cheating and reversion in E94 gives us insight into the evolution of cooperation and conflict in populations and, potentially, a non-antibiotic approach to controlling bacterial populations a variety of settings.

The dramatic increase in antibiotic-resistant Pseudomonas aeruginosa infections makes it necessary to find new approaches for treatment. P. aeruginosa employs a communication system called quorum sensing (QS) that uses density-dependent accumulation of small diffusible molecules to mediate the production of factors that benefit the entire bacterial community; creating a potential drug target. P. aeruginosa has two QS systems: las and rhl. A previous analysis of the laboratory strain PAO1 revealed that mutations in the las system result in individuals that act as social cheaters that reap the benefits of communally produced goods without expending energy on their production. lasR mutants have a fitness advantage when cultured in a medium requiring the activity of the community goods for survival. I set out to better understand the rhl QS system. I was interested in determining if ΔrhlR mutants act as social cheaters in co-culture with rhlR-competent strains. To make that determination, I screened 12 clinical isolates from the Early Pseudomonas Infection Control (EPIC) study for the ability of their isogenic ΔrhlR mutants to grow in QS and non-QS selective media and their ability to persist in coculture with the parent strain. I also competed these strains against isogenic ΔrhlR mutants by growing them in coculture and using flow cytometry to determine the relative final frequencies of the parent strain and ΔrhlR mutants. Finally, I aimed to determine if rhlR mutants arise spontaneously from the parent strains in a long-term growth experiment by sequencing and functional analysis of the mutants. My preliminary work demonstrates that a small subset of the ΔrhlR mutants dramatically increase relative to concentrations of EPIC strains in coculture and mutations in rhlR do arise in vitro; however, their exact functional effects are still to be determined.  Understanding ΔrhlR cheater dynamics may provide therapeutic targets for antibiotic-resistant P. aeruginosa infections.