Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting

Cutibacterium acnes (C. acnes) is a gram positive bacteria and a member of the human skin microbiome. Despite being the most abundant skin commensal, it has also been associated with common inflammatory disorders such as acne vulgaris. The availability of its complete genome sequence has enabled the identification of three putative methyltransferases, some of them potentially belonging to restriction-modification (RM) systems which protect the host of invading DNA. However, little is known on whether these systems remain functional in the different C. acnes strains. Using Oxford Nanopore Technologies (ONT) sequencing, we analyzed the methylome of six representative C. acnes strains and their epigenetic imprints on phage DNA. We detected the presence of a 6mA modification at a defined DNA consensus sequence in one of these C. acnes strains. Recombinant expression of this R-M system confirmed its methylation activity on plasmid DNA. Additionally, we performed whole cell lysate restriction assays, confirming the implication of this modification to the restriction activity of the strain. We measured defensive properties of methylated DNA. We observed resistance of phage infection with non-methylated phages and showed its change of susceptibility by knocking out the R-M system in KPA171202. We show how by modulating the host producer of phages we can change phage's genetic imprinting and change killing specificity. Finally, we demonstrate how we can produce phages which can selectively kill acne-prone strains while protecting probiotic ones.