This resubmission for a K08 physician-scientist career award is for support to transition to a career of independent investigation in the field of Pseudomonas aeruginosa quorum sensing. I recently transitioned from fellow to Acting Instructor of Pulmonary and Critical Care Medicine at the University of Washington (UW) and am conducting research in the lab of E. Peter Greenberg in the Department of Microbiology. I have the immediate goal of continuing my research into the evolution of quorum sensing that I initiated as a fellow. The Greenberg lab, Pulmonary Division, and the University of Washington all provide outstanding support to me. Dr. Greenberg is an authority on quorum sensing and bacterial pathogenesis, and I will continue to work in his group and its resources as I transition to an independent research career. The Pulmonary Division and the School of Medicine will ensure that, in addition to the funds provided, I have protected time for research and the institutional support necessary to achieve my long-term goal of independent investigation. This includes a mentoring committee composed of physician-scientists and clinicians, in addition to Dr. Greenberg, to ensure my continued progress during the term of this award. I propose the following research in this application: P. aeruginosa is a leading cause of morbidity and mortality in the genetic disease cystic fibrosis (CF). It also causes difficult-to-treat burn and diabetic wound infections, keratitis, and ventilator-associated pneumonia. P. aeruginosa engages in quorum sensing, a cell-density-dependent intercellular signaling mechanism that coordinates group behaviors including production of virulence factors and secreted proteases. P. aeruginosa quorum sensing is mediated in part by the transcription factor LasR. LasR regulates the transcription of hundreds of genes, many of which encode proteins that are involved in the production of secreted products. Such secreted products are "public goods" that are available to an entire community, not only the producing cell. As such, quorum sensing is susceptible to social cheating, wherein individuals mutate LasR so that they gain benefit from public goods without bearing a metabolic cost of production. In the CF lung, the frequency of LasR mutants can be high and these LasR mutants, which evolve de novo in the lung, have been implicated in the pathogenesis and progression of CF-related lung disease. In the laboratory, P. aeruginosa LasR mutants can evolve from the wildtype under conditions where a quorum-sensing-controlled secreted protease is required for growth. LasR mutants benefit from protease production by the wildtype and these LasR mutants achieve a population equilibrium with the wildtype. In my initial experiments, I showed that the emergence of LasR mutants can be prevented by the availability of adenosine, a "private good" and quorum-sensing controlled nutrient. This provides a mechanism for the population to police against deviant behavior. My proposal seeks to build on these initial experiments by answering the following questions: (1) What are the mechanisms by which the wildtype and LasR mutants achieve an equilibrium with one another? Because quorum sensing requires a sufficient density of cooperators, the emergence of LasR mutants should cause population collapse by reducing the number of cooperators below the threshold, but this does not occur;I seek reasons why. (2) Does spatial structure affect the emergence and frequency of LasR mutants? Infectious settings, including the CF lung, are likely to be structured and I propose to study the effects of spatial structure on the ability of LasR mutants to emerge in a population. Finally, (3), it has been proposed that the CF lung is a singular environment that supports the emergence of LasR mutants. I plan to test this hypothesis by collecting 100 or more environmental samples of P. aeruginosa and examining them for lasR mutations. The experiments described in this proposal will advance our knowledge of the evolution of cooperative behavior and give insight into the ecology of the CF lung.
The opportunistic human pathogen Pseudomonas aeruginosa engages in an intercellular signaling process called quorum sensing, which can coordinate bacterial group activities and has been a target for therapeutic intervention in chronic P. aeruginosa infections. In preliminary work, I established a laboratory system in which quorum-sensing mutants emerge from a quorum-sensing intact predecessor over 20 days, and I demonstrated one circumstance in which mutants are suppressed. My project seeks to understand aspects of the evolution of the quorum sensing system, with an eye to manipulation of bacterial populations in chronic infections, particularly those of the disease cystic fibrosis.