The great phage escape: Activating and escaping lactococcal antiphage systems

In recent years, the number of newly discovered systems that bacteria use to combat bacteriophages is increasing at an impressive rate. To obtain mechanistic insights into several antiphage systems identified in previous studies, we isolated 66 phage escape mutants which had become insensitive to 13 distinct, plasmid-encoded lactococcal phage resistance systems (i.e. Rhea, Kamadhenu, Rugutis, Audmula, PARIS, type II CBASS, Septu, AbiA, AbiB, AbiD/F, AbiG, AbiJ, AbiP). Genome analysis of these phage escape mutants identified a total of 15 mutated genes. Six of the encoded proteins appear to activate specific antiphage systems. Furthermore, AbiA escape mutants were found to be insensitive to AbiJ, while distinct antiphage systems (AbiG and AbiP) were observed to be activated by a major phage tail protein, indicating mechanistic commonalities. PARIS homologues encoded by members of different bacterial genera appear to share similar sensing mechanisms, whereas our data indicate mechanistic differences between Septu homologues from different genera. Based on our escape mutant sequence analysis, previously predicted domains, and experimental data using the purified endolysin of phage c2, we propose that Audmula modifies the cell wall of the host bacterium, delaying cell lysis and release of progeny phages, protecting the host cell by a heretofore unknown mode of action. The obtained advances in our understanding of lactococcal antiphage mechanisms provide fundamental insights into phage–host interactions, which undoubtedly benefits the dairy industry but may also be useful for biotechnological or biomedical applications.