Importance of Post-translational modifications in Streptococcus mutans pathophysiology

University Of Florida, Gainesville FL

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Abstract

Abstract Dental caries affects 2.5 billion people worldwide. Streptococcus mutans is an important pathogen in dental caries due to its biofilm lifestyle, and acidogenic and aciduric nature. In addition, expression of the collagen binding protein Cnm, present in 20% of the strains, by S. mutans is intimately associated with severe caries, systemic infections and auto-immune disorders. Our mass-spectrometry studies have demonstrated that the threonine-rich repeat (TRR) domain of S. mutans Cnm, with >70 threonine residues, undergoes extensive O- glycosylation with N-acetylhexosamines (HexNAc2). We also found that cnm is co-transcribed with a downstream gene encoding a putative GT-A type glycosyltransferase, named pgfS (protein glycosyltransferase of streptococci). We identified three genes immediately downstream of pgfS, namely pgfM1, pgfE and pgfM2, that also contribute to Cnm modification. Importantly, the pgf genes are part of the S. mutans core genome and our findings suggest that we have uncovered an O-glycosylation pathway dedicated to modifying multiple carbohydrate binding adhesins. Surprisingly, we also detected phosphate modifications in the TRR domain of Cnm in pgfS and pgfM2 mutants, and also in low levels in wildtype Cnm. Based on structural homology comparisons and extensive preliminary data, we hypothesize that post-translational modifications significantly contribute to the complexity of S. mutans proteome, playing an important role in S. mutans pathobiology. Specifically, O-linked protein glycosylation of surface proteins in S. mutans proceeds through a series of lipid-linked glycan intermediates and protein kinase modifications. This is important since this same lipid is essential for the biosynthesis of both peptidoglycan and the surface associated rhamnose- glucose polysaccharide (RGP) and therefore regulatory mechanisms must be in place to control its distribution among all three pathways (adhesin glycosylation, and peptidoglycan and RGP biosynthesis). In aim 1 we propose to Characterize the enzymatic function of PgfS, PgfM1 and PgfM2 and localize the cellular orientation of the active sites. In aim 2, we will determine O-glycosylation of other glycan binding surface proteins by the Pgf machinery. Lastly, in aim 3 we will test the impact of the interplay between glycosylation and phosphorylation on S. mutans fitness and pathophysiology. Our goal is to fill major gaps in knowledge on how cell wall perturbations through anchoring of surface glycoproteins and RGP impact S. mutans pathophysiology. Moreover, in this basic science proposal, using S. mutans as a model organism, our findings will likely reveal novel mechanisms of post-translational modifications that could have broader impact on our understanding of cell envelope homeostasis in Gram-positive bacteria.