Acquired CRISPR spacers and rhamnose-glucose polysaccharide defects confer resistance toStreptococcus mutansphage ɸAPCM01

Streptococcus mutansis a major cause of dental caries worldwide. Targeted therapeutic strategies to eradicateS. mutansinclude oral phage rinses. In this study, we investigated how phage resistance develops inS. mutans. As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel CRISPR spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide (RGP) biosynthetic genes, three of which also acquired spacers. Only thergpmutants exhibited defects in phage absorption, supporting the role of these cell surface glycans as the phage receptor. Mutations inrgpFand the newly identified genergpXled to severe cell division defects and impaired biofilm formation, the latter of which shared by thergpDmutant. Thus,rgpmutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defense mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.