Global reprogramming of virulence and AMR in Pseudomonas aeruginosa by mutations in FusA1

It is now widely accepted that the "golden age" of antibiotics has passed, and that antimicrobial resistance (also known as "AMR") is on the rise. One extremely useful tool in the fight to understand AMR has been whole genome sequencing, in which the entire genetic "blueprint" of an organism can be elucidated. Using whole genome sequencing, researchers have found that mutations in certain genes are strongly associated with AMR. FusA1 is one such gene, and mutations in fusA1 are now recognized as being responsible for high level resistance to an important class of antibiotics called aminoglycosides. More worryingly, these mutations are particularly prevalent in an organism called Pseudomonas aeruginosa (hereafter, PA), which is ubiquitous in the built environment and is a major cause of potentially life threatening infections, especially in people who are less well able to fight such infections. The problem is that we currently have little idea about why mutations in fusA1 lead to AMR, and without this understanding, there is little we can do about this. FusA1 encodes a protein involved in making other proteins; a process called "translation". Here, mini-factories known as ribosomes "translate" the information coded in a molecule called messenger RNA (which itself, is copied from the DNA blueprint) to make all the proteins needed in the cell. The function of FusA1 is to help the ribosomes to "drop off" the RNA once they have made each new protein, or if they encounter a "stall signal". Ribosomal pausing at most stall sites is usually easily overcome if the FusA1 is functioning normally. However, based on our preliminary experiments, we suspect that the ability of mutant forms of FusA1 to facilitate this "ribosome recycling" reaction may be altered, thereby changing the dynamics of translation. For example, if dissociation of ribosomes from key "stall sites" or stop signals is even slightly impaired, ribosomes will start to queue-up at such sites, affecting the translation of the protein encoded on the messenger RNA. If that protein was itself associated (either directly or indirectly) with aminoglycoside resistance, this would provide a tangible link between the mutation in fusA1 and the AMR phenotype - a hypothesis that we are very keen on exploring using state-of-the-art approaches called RNA-seq, Ribo-seq and ChIP-seq. We also suspect that mutations in fusA1 might alter its ability to bind to other molecules in the cell, including other proteins or non-messenger RNA. Cutting-edge technological developments mean that we can now investigate these hypotheses directly using specialized "proteomic" approaches called TurboID and "OOPS", respectively. To increase the probability of success, these approaches will be carried out in collaboration with world leaders in their respective disciplines. By the end of this project, we will have a clear idea about how mutations in fusA1 lead to aminoglycoside resistance. This "mechanistic" understanding will be critical if we want to find better ways of combatting AMR, or of better predicting the AMR phenotype of a strain based on its whole genome sequence (an approach that, with ever-cheaper and faster sequencing, is likely to become widespread in the clinic in the near future). Excitingly, our preliminary data indicate that in principle, it is possible to reverse the AMR associated with fusA1 mutations, offering a line-of-sight - albeit, beyond the scope of the current proposal - towards resensitizing resistant PA to aminoglycoside antibiotics.