Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The Delta rgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the Delta rgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF. The Delta rgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the Delta rgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans.
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