Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting

Abstract

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of six representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

Putative pseudolysogeny-dependent phage gene implicated in the superinfection resistance of Cutibacterium acnes

“…Our work, along with previous research, demonstrates that there are likely multiple evolved mechanisms governing C. acnes resistance to phage infection, including the CRISPR-Cas system, restrictionmodification systems, and SIE mechanisms [11,59,60,62,63] . An understanding of the mechanism(s) by which this resistance may be produced in bacteria that were previously capable of being lysed by phage may give rise to the exploitation of the phenomenon in the fine-tuning of phage-based therapeutics.…”

“…Ideally, characterization of this protein would be confirmed via studies in which recombineered phage strains that do not possess gp41 (i.e., protein knockouts) are isolated as described above, and C. acnes strains are infected with them to observe for the SIE phenotype, including strains of C. acnes preeminently engineered to contain gp41. However, cloning via electroporation in C. acnes bacteria is currently not possible [62] . Alternatively, escape mutant studies may be reperformed on a larger scale, potentially with concomitant use of a mutagen such as what was done in the study conducted by Leavitt et al (2023), to try to induce mutations that localize to gp41 as an alternative to inducing targeted mutants, e.g., with the use of CRISPR-Cas systems [19,59,60] .…”