Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on pulmonary tissue and on
diabetic ulcer tissue, causing chronic antibiotic-tolerant infections. P. aeruginosa biofilms contain
physiological heterogeneous subpopulations of cells, including dormant cells in the anaerobic or the nutrient
poor biofilm microenvironments. Since most antibiotics target active cells, the dormant cells may survive the
antimicrobial therapies, then repopulate the biofilms when treatments are discontinued. Longitudinal
genomics studies of P. aeruginosa isolated from chronic pulmonary infections of cystic fibrosis (CF) patients
support this mechanism of antibiotic tolerance, since clones of the original founder strains often reemerge
within patients treated with antimicrobial therapies. In order to treat chronic infections, it will be necessary to
prevent resuscitation of the dormant bacteria, in addition to killing the active cells. Here, we propose to identify
the molecular targets that, when inhibited, impair P. aeruginosa resuscitation from starvation-induced
dormancy. In prior work, we characterized the P. aeruginosa ribosomal hibernation promoting factor (HPF),
which is required for protection of a minimum ribosome supply in starved P. aeruginosa cells, and therefore
required for de novo protein synthesis of resuscitating cells. Protection of other macromolecules or cellular
structures, in addition to ribosomes, is also likely required for dormant cells to resuscitate, since dormant cells
are subject to aging, protein oxidation, and protein degradation. In preliminary studies, we identified
additional mutant strains where gene disruptions prevent optimal resuscitation of P. aeruginosa from
starvation. In the research here, we propose to continue to identify and characterize P. aeruginosa dormancy
factors. Homologs to the factors identified here may be used to study dormancy in other bacteria that cause
chronic infections. In this research, we will: (i) Perform a comprehensive transposon screen for mutations that
affect the ability of P. aeruginosa to recover from starvation. The genes of the resulting mutant strains will be
characterized for their effect on maintenance of macromolecular integrity during dormancy. (ii) Characterize
physiological heterogeneity of the candidate mutant strains cultured in biofilms. Since dormant cells have
greater tolerance to antibiotics than active cells, we will differentially label dormant and active cells, and
determine if the gene disruptions affect the ability of antibiotic-treated dormant cells to resuscitate. Ultimately,
we will identify new molecular targets that when inhibited may not have an effect on active cells, but that
inhibit the dormant cells from resuscitation. Therapies that target these dormancy factors may then be used
in combination with traditional antibiotic therapies to help prevent chronic and persistent infections.