Acquired CRISPR spacers and rhamnose-glucose polysaccharide defects confer resistance to Streptococcus mutans phage ɸAPCM01

<i>Streptococcus mutans</i> is commonly associated with the development of dental caries worldwide. Due to their specificity for <i>S. mutans</i>, phage represents a promising avenue for future targeted therapeutic strategies. In this study, we investigated how phage resistance develops in <i>S. mutans</i>. 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 clustered regularly interspaced short palindromic repeats (CRIPSR) spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide biosynthetic genes, three of which also acquired spacers. Only the <i>rgp</i> mutants exhibited defects in phage adsorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in <i>rgpF</i> and the newly identified gene <i>rgpX</i> led to severe cell division defects and impaired biofilm formation, the latter of which was also shared by an <i>rgpD</i> mutant. Thus, <i>rgp</i> mutations 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 defence mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.