The role of the epitranscriptome in bacterial biofilms and phenotypic antibiotic resistance
Many bacterial pathogens respond to the stresses of growth, infection and antibiotic treatment with a genetically-programmed entry into a slowly- or non-replicative state accompanied by the formation of a biofilm or, in the case of mycobacteria, a granuloma. The bacteria in this state are typically resistant or tolerant to a broad range of antibiotics (i.e., persistent), with the bacteria reverting to a drug-sensitive state upon removal of the stress. This persistent state is a major form of antimicrobial resistance (AMR) and is also genetically programmed, but we know exceptionally little about the mechanisms driving persistence. Here we propose to explore the role of RNA modifications – the epitranscriptome –in phenotypic resistance and in the formation of biofilms, focusing initially on two clinically important biofilm-forming pathogens: Entercoccus faecalis (Ef) and Pseudomonas aeruginosa (Pa).
This project has two objectives. One is to test the hypothesis that Efand Parespond to biofilm-inducing stresses by translationally-controlled phenotypic remodeling. This involves stress-specific reprogramming of dozens of modified nucleosides in tRNAs and rRNAs, which leads to selective translation of codon-biased transcripts for families of genes critical to forming a biofilm and becoming persistent. This work entails genomic analysis of codon usage patterns and LC-MS analysis of tRNA modifications and proteins in Ef and Pa in planktonic and biofilm growth states, using biofilm models and Ef strains developed in Prof. Kline’s lab. We are particularly interested in translational regulation of the variety of drug efflux pump families (e.g., Mex and OprM), which have highly biased codon usage patterns and are likely coordinately regulated by tRNA modifications. The second objective is to define the role of rRNA modifications in drug resistance and biofilm formation, such as the erythromycin resistance methyltransferases (Erm) that site-specifically methylate 23S rRNA to cause drug resistance. This work involves the same approaches and tools used for tRNA analysis.