Posttranscriptional modifications of bacterial and eukaryotic ribosomes are linked to many human diseases, but the precise role of most modifications remains undefined. Dimethylation of a universally conserved adenine, A2058, in bacterial rRNA causes cross-resistance against all three critically important families of antibiotics (macrolides, lincosamides, and streptogramins (MLS)). A2058 dimethylation occludes MLS from the ribosome, thereby allowing normal protein biosynthesis and bacterial growth. The thirty-five classes of Erm methyltransferases responsible for A2058 dimethylation are invariantly encoded by a two-gene operon preceded by a short ribosome stalling leader sequence. These short stalling peptides considerably vary in size and sequence composition. The functional and evolutionary connections between the stalling sequence and its cognate erm gene are poorly understood. A previous `ribosome stalling' model suggests that macrolide-mediated translational stalling of the leader sequence is required for the upregulation of downstream co-transcribed erm, but clinical surveillance and our data indicate the existence of an alternative pathway. Our unpublished data further show that collateral sensitivity to unrelated antibiotics, reduction in virulence gene expression, accumulation of inactive ribosomes, and loss of in vivo fitness are all part of the trade-offs associated with the A2058 dimethylated ribosome. The exact mechanistic links between these traits are unknown. There is also an unmet need to understand the mechanism by which Erm recognizes and acts on 23S rRNA. This proposal will use a multi-pronged approach consisting of high-precision next-generation sequencing, bacterial genetics, proteomics, comparative genomics, biochemistry and structural biology to address three central questions: What are the underlying mechanisms of the trade-offs conferred by the A2058 dimethylated ribosome? How does the erm operon evolve, and how is the expression of erm regulated? How does Erm find its target substrate RNA? The erm operons are widespread among nosocomial Gram-negative and Gram-positive bacteria, addressing these questions will offer significant mechanistic insight into new antimicrobial strategies tailored to disrupt these biochemical interactions and regulatory pathways.
(Public health relevance) Antimicrobial resistance is a global health threat, predicted to cause ten million deaths and cumulative healthcare costs of $US100 trillion annually by 2050 if no new approaches are developed. This proposal seeks to obtain a mechanistic understanding of the acquisition of multidrug resistance. The results will have the potential to generate innovative strategies to reduce the spread of resistance determinants and restore the efficacy of MLS antibiotics.
