Mechanisms and Structures of Pseudomonas aeruginosa hibernating ribosomes - Pseudomonas aeruginosa is an opportunistic pathogen that causes persistent biofilm infections. Biofilms are difficult to treat with antibiotics, in part, because biofilms contain heterogeneous subpopulations of cells, including dormant bacteria in zones that are depleted in oxygen or nutrients. The dormant bacteria are not sensitive to antibiotics that target active metabolism. When bacteria enter dormancy, they undergo metabolic reprogramming to reduce energy expensive processes, but preserve essential macromolecules, including ribosomes, which are maintained in an inactive state. Signaling of bacterial growth arrest is mediated, in part, by the stringent response, which in turn controls the expression and activity of dormancy factors. Accessory ribosomal factors that interact directly with ribosomes in nutrient depleted cells include hibernation promoting factor (HPF) and ribosome modulation factor (RMF). These factors bind ribosomes in the mRNA channel, block translation, and protect the rRNA against degradation by ribonucleases. Recent evidence and our preliminary studies suggest that RMF and HPF are not the sole factors required for ribosome hibernation. Entry of ribosomes into a hibernating state is likely mediated by members of the G3E family of GTPases, by ribosome silencing factor (RsfS), and by sequestration to the inner membrane by YqjD-like membrane proteins. Therefore, in P. aeruginosa, signaling of entry into hibernation is complex and requires yet uncharacterized accessory proteins. The goals of this work are to determine the structure and mechanisms by which these hibernation factors bind ribosomes and induce dormancy in P. aeruginosa. We will use a combination of in vivo approaches and structural studies to identify interactions between accessory proteins and ribosomes. For these studies, we will: (i) use in vivo protein-protein interaction approaches to identify the roles of the cell cycle and defined mutations on interactions of hibernation factors with ribosomes. We will also characterize the physiological responses of starved cells that are impaired in ribosome hibernation. (ii) We will use cryo-electron microscopy and single particle analysis to determine the structures of these specialized hibernation factors in complex with ribosomes at near atomic-level resolution. (iii) We will use support from National Cryo-Electron Tomography Centers for in situ 3D studies of ribosome structure and heterogeneity in starved P. aeruginosa cells. The results from this work will allow us to understand the structures, cellular distribution, and heterogeneity of hibernating ribosomes at high resolution as they occur in vivo. Combined, the structural and in vivo studies described here will provide models for ribosome hibernation in P. aeruginosa and other ESKAPE pathogens, while setting the stage for structure-based drug discovery to target dormant bacteria.
