PROJECT SUMMARY
Pseudomonas aeruginosa is an opportunistic pathogen that can withstand treatment with bactericidal
antibiotics even when lacking identifiable resistance genes. It is thought that these recalcitrant infections are
attributable to phenotypically antibiotic-tolerant cells called persisters. Despite the acknowledged contribution of
P. aeruginosa to chronic and recurrent infections, there is a lack of basic research into cellular mechanisms that
underlie P. aeruginosa antibiotic persistence. My central hypotheses in this research proposal are that the
cellular responses following treatment will govern P. aeruginosa antibiotic persistence and resistance
development (Aim 1), and that increased duration of coexistence with common co-isolate, S. aureus, will
increase P. aeruginosa persistence by priming it in a more stress-tolerant state (Aim 2). This fellowship research
will provide critical insight into persister physiology as well as the opportunity to learn cutting-edge techniques,
analyses, and skills that will prepare me to lead independent research efforts in the future as a dentist-scientist
interested in bacteria of the oral cavity.
In Aim 1, I will investigate how P. aeruginosa persisters reawaken after drug treatment. I will perform RNA-
seq to screen for genes that are differentially expressed between untreated cultures and cells that are viable
after antibiotic treatment. Hits from this screen will be functionally validated by conducting persister assays with
multiple genetic models for the genes of interest, including transcriptional reporters, knockout mutants, and
inducible complementation strains. Significant genes of interest will be tested in biofilm cultures and in host-
mimicking media to add clinical relevance. In Aim 2, I will determine how the duration of co-culture with S. aureus
affects P. aeruginosa persistence and the physiologies of surviving cells. To efficiently passage and assay P.
aeruginosa in co-culture, I am implementing a novel, dual-chambered apparatus that I designed, called the “H-
Cell.” The H-Cell allows dynamic crosstalk between species while maintaining segregated populations for
efficient sampling. I will determine the transcriptomic changes between P. aeruginosa persisters grown in
monoculture or in H-Cell co-culture with S. aureus by RNA-seq. I will validate the hits by testing genetic
constructs. Furthermore, I will test P. aeruginosa persistence in strains that are co-isolated with S. aureus from
clinical sputum samples and thus have co-existed in a host environment.
From my use of multiple bacterial strains, antimicrobials, and culture conditions, I aim to reveal the
generalizability or specificity of P. aeruginosa persistence mechanisms and how they contribute to the
development of antibiotic resistant progeny. Furthermore, I can apply the investigative approach, technical skills,
and tools that I develop throughout this proposal to my future research on bacterial physiology in multispecies
communities of the oral cavity. Altogether, completion of this research can inspire anti-persister strategies to
reduce the burden of recalcitrant infections and their contributions to the broader antibiotic resistance crisis.
