Pseudomonas aeruginosa forms biofilms and chronic infections on pulmonary tissue of patients with cystic fibrosis (CF). Longitudinal studies of individual patients indicate that P. aeruginosa strains are often clonal over time, suggesting that bacteria not killed by antibiotics (persister cells) repopulate the biofilms following treatments. Persister cells are metabolically dormant and less susceptible to most antibiotics. However, they are subject to other environmental stresses including oxidative stress from host defensive cells and cell aging. These stresses cause protein misfolding, inactivation, and aggregation. Using laser capture microdissection (LCM) and transcriptomics, we identified ibpA as the most abundant mRNA in the dormant cell fraction of P. aeruginosa biofilms. In Escherichia coli, IbpA/B is responsible for binding misfolded protein aggregates and delivering them to other chaperones and proteases for protein refolding or degradation. The goal of this research is to determine the role of IbpA in maintaining the viability of dormant persister cells in P. aeruginosa biofilms. In this research we will: (i) characterize the spatial and temporal expression patterns of ibpA in P. aeruginosa biofilms. Using green fluorescent protein (GFP) fusions and time-lapse confocal scanning laser microscopy (CSLM) we will characterize the roles of transcriptional and post-transcriptional processes in ibpA expression. GFP fusions will be used to determine if P. aeruginosa ibpA is expressed in all cells or in subset of aged biofilm cells, and if the IbpA protein compartmentalizes misfolded proteins. We will also: (ii) characterize the molecular activities of IbpA and its role in survival of dormant P. aeruginosa biofilm cells. Using ibpA and rpoH deletion mutants, we will determine the role of IbpA in allowing resuscitation of dormant cells exposed to stresses, including aging, oxidizing agents, and antibiotics. Functional enzyme assays will be used to characterize the role of P. aeruginosa IbpA in binding or reactivation of misfolded proteins. Ultimately, we will determine if IbpA may be used as a molecular target in combination with other antibiotic treatments to eliminate the persister cell subpopulations that cause chronic P. aeruginosa pulmonary infections. )
Bacterial infections associated with surfaces, including pulmonary tissue or artificial implant devices, are often resistant to antibiotic treatment. Resistance may be mediated by a dormant cell subpopulation that repopulates the infection following treatment. The goals of this research are to characterize molecular activities of the dormant cell subpopulations of Pseudomonas aeruginosa, a bacterium that causes infections on pulmonary tissue. We will determine the role of a stress response protein that allows the bacteria to survive prolonged dormancy and resuscitate into pulmonary biofilms.
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|Williamson, Kerry S; Richards, Lee A; Perez-Osorio, Ailyn C et al. (2012) Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population. J Bacteriol 194:2062-73|