Elucidating physiology of dormant bacteria to combat antibiotic persistence

Harvard Medical School, Boston MA

Investigators

Linked publications & trials

Abstract

PROJECT SUMMARY The vast majority of antibiotics are ineffective at killing non-growing bacteria and it is estimated that 50% of antibiotic tolerance cases are due to phenotypic ‘persistence’ rather than genetic resistance. Common examples of recurrent infections include urinary tract infections of pathogenic E. coli—the most common bacterial infection in women in developed countries—latent tuberculosis, and biofilm-forming bacteria, like the P. aeruginosa infections in cystic fibrosis patients. To develop new strategies to combat recurring infections and persistence, we need a better understanding of the physiology of non-growing bacteria. My laboratory aims to uncover the fundamental biophysical mechanisms that sustain bacterial survival under non-growing conditions and growth when nutrients are abundant. Our approaches will also reveal targetable vulnerabilities of non-growing bacteria by identifying essential survival processes in both a model organism (E. coli) and important pathogens (E. coli, P. aeruginosa, C. difficile). In the last cycle of this award, we discovered an essential process for starvation survival of Gram-negative bacteria. We found that bacteria spend 90% of their energy budget in starvation to maintain their cytoplasm in a contracted state (called plasmolysis), which requires active export of salt ions. By tracking thousands of individual bacteria during starvation over several days, we were able the capture the cell death process of E. coli in starvation with time-lapse microscopy for the first time, confirming the critical role of plasmolysis on a single-cell level. Our work also revealed a crucial role of the cell envelope and permeability, as well as dozens of proteins that are critical for starvation survival. In this cycle, we will address the key question how bacteria regulate the transition into and out of plasmolysis, the state critical for their survival, and identify the molecular players that achieve this transition. Surprisingly, very little is known about how bacteria control their cytoplasmic osmotic pressure (turgor) as a function of growth rate. Our lab has recently proposed counterions of ribosomal RNA as the central generator and regulator of turgor pressure. Therefore, changes in ribosomal RNA could account for growth-rate dependent changes of turgor pressure and degradation of ribosomal RNA may enable plasmolysis in starvation. To address these questions, we will characterize transitions into and out of starvation in wildtype and mutant strains. Using mass spectrometry, we will measure time courses of all major cellular osmolytes including metabolites and salt ions. Using innovative microscopy techniques like Normalized Raman Imaging and Quantitative Phase Microscopy in combination with microfluidics, we will measure cellular biomass density, as well as macromolecular composition during transitions into and out of plasmolysis and at different timepoints into starvation. Combining these data with our existing proteomics analysis, we will identify pathways, processes and genes that are important for starvation survival and characterize corresponding knockout strains. These insights will then be synthesized in a quantitative and predictive mathematical model. This project will key reveal vulnerabilities of non-growing bacteria that could be exploited by novel antibiotics, as well as assays that could be used to identify compounds targeting these vulnerabilities. Given that most antibiotic screens have been done on growing bacteria and that essential physiological processes in non-growing cells are different, it is likely that many drug targets in non-growing bacteria have been missed.