Friday, September 22, 2023
9/22/2023
Bacteria living in biofilms are less susceptible to antibiotic treatments than free-living planktonic cells. One mechanism for this increased antibiotic tolerance is that biofilms contain physiologically heterogeneous subpopulations of cells, including antibiotic-tolerant dormant bacteria. When bacteria enter a dormant state, they undergo a variety of physiological changes that reduce energy-expensive processes, yet protect the integrity of macromolecules required for resuscitation. Among these physiological changes is inactivation and preservation of ribosomes. The ribosome accessory proteins (RMF and HPF) are hibernation factors that block translation and protect ribosomes from degradation when the cells are dormant. By protecting ribosomes, ribosome hibernation factors enable the cells to resuscitate from dormancy when conditions are favorable, resulting in persistent infections. Ribosome entry into and exit from hibernation are dynamic processes, and biofilm subpopulations have cells with ribosomes in differing states, depending on the local environmental conditions. In the research proposed here, we will characterize the role of ribosome hibernation in the antibiotic tolerance of biofilm bacteria, by characterizing protein-protein interactions between ribosomal proteins and ribosome accessory factors. In preliminary work, we developed a bimolecular fluorescence complementation (BiFC) system that allows real-time fluorescence-based imaging of ribosome dynamics in Pseudomonas aeruginosa biofilm cells. This system allows us to image biofilm bacteria with hibernating ribosomes, and to sort the cells based on their ribosome state. The BiFC system also allows us to identify additional ribosome hibernation factors, such as molecular chaperones, that mediate the loading of hibernation factors. The goals of these studies are to: (i) generate molecular constructs that provide optimal fluorescent reporter signals for bacteria with ribosomes in active and hibernating states. (ii) Use the fluorescent reporter systems to identify and sort active from dormant bacteria over the course of biofilm development, and compare their antibiotic sensitivity profiles, and (iii) identify additional dormancy factors that contribute to the antibiotic tolerance of dormant biofilm cells. Since many commonly used antibiotics target bacterial translation, the ultimate goal of these studies is to identify molecular targets that, when disrupted, enhance biofilm sensitivity to ribosome- targeting antibiotics. Targeting ribosome hibernation factors, or their molecular chaperones, may provide a means to inhibit the viability of dormant bacteria, thereby increasing biofilm sensitivity to antibiotics.
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