Molecular tools from bacteriophages: A structural and functional characterisation of the BREX bacteriophage resistance system

Abstract

The interminable arms race between bacteriophages (phages) and their bacterial hosts has produced an abundance of phage defence system modalities. Phage-bacteria interactions have provided a great number of the molecular biology tools routinely applied in laboratories around the world. Additionally, the re-emergence of phage therapy provides a plausible solution to the rise of multi-drug resistant bacteria. Wide-scale use of phage therapy will require a thorough understanding of mechanisms by which bacteria resist phage infection. Recently, systematic approaches to defence system discovery have unearthed a plethora of new systems and attempts to characterise defence system functions and mechanisms have fallen behind. This study aimed to provide valuable insight into the mechanisms of BREX phage resistance systems through structural and functional characterisation of a type I BREX system from Salmonella Typhimurium strain D23580.

Through assaying the Salmonella D23580 system against the Durham Phage Collection alongside type I BREX systems from Escherichia coli and Escherichia fergusonii, it was shown that phage defence varies between systems against a given phage in a manner which does not correlate with the number of recognition motifs within respective phage genomes. Further, phages appear to encode mechanisms of inhibiting species specific BREX systems. Next, analysis of gene deletions demonstrated essential genes for host methylation and phage defence, again showing variation from similar studies in the literature. Unusually, deletion of brxL elicited an increase in phage defence by several orders of magnitude. To provide further insight into the function of individual components, the structure of the methyltransferase, PglX, was solved to a resolution of 3.4 Å. PglX displays distinct N and C-terminal domains joined by a central hinge, with conserved methyltransferase regions. To shed light on mechanisms of phage escape from BREX systems, the structure of PglX bound to the BREX inhibitor, Ocr, was also solved to 3.5 Å. Ocr binds along the C-terminal domain of PglX and provides insight on potential DNA binding positions. Finally, PglX was rationally mutated to alter the BREX recognition motif, both changing host methylation patterns and allowing defence against a previously resistant phage. As such, PglX is the sole specificity factor of BREX defence, despite other components encoding DNA binding functionalities. Mutations in PglX in nature would allow rapid retargeting of BREX defence against new phage threats. Together, these results will guide further studies into BREX systems towards understanding the molecular mechanisms of phage defence.