The role of type IV CRISPR-Cas systems in plasmid warfare and their application as a novel therapeutic

Klebsiella pneumoniae is an opportunistic pathogen and multidrug-resistant (MDR) strains have become common in most of their extra-intestinal infections. This is worrisome because they increasingly acquire resistance to last-resort antibiotics1,2. MDR is mediated by conjugative plasmids encoding for antibiotic-resistance (ABR) determinants, which can transfer horizontally between neighbouring bacteria. Repurposed CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems are a promising novel approach to combat ABR pathogens3,4. The plasmid-encoded type IV CRISPR-Cas systems are not well understood but predicted to be involved in inter-plasmid competition5. To investigate the dynamics of type IV-driven inter-plasmid competition, I will employ in vitro and in vivo experiments, bioinformatic analyses, and mathematical modelling. Objective 1 will be the first study quantifying the contribution of type IV CRISPR-Cas systems to plasmid dynamics in the native host K. pneumoniae. Further, in Objective 2, I propose to harness type IV CRISPR-Cas to restore antimicrobial susceptibility of MDR K. pneumoniae. I will employ common molecular methods to design a type IV CRISPR-Cas-based tool to target genes encoding for carbapenemase. Efficacy will be screened in clinical MDR K. pneumoniae and in a murine model with a multi-species gut community. While an in-depth understanding of the mechanism and dynamics of type IV CRISPR-Cas-driven inter-plasmid competition is essential in itself, it is also crucial for its repurposing to a reliable tool to restore carbapenem susceptibility in MDR K. pneumoniae