Roles of Sortase in Surface Expression of the Major Protein Adhesin P1, Saliva-Induced Aggregation and Adherence, and Cariogenicity of Streptococcus mutans

Abstract

Sortase is a newly discovered transpeptidase that covalently links LPXTGX-containing surface proteins to the gram-positive bacterial cell wall. In this study, the sortase gene (srtA) was isolated from Streptococcus mutans NG8 by PCR. The gene encoded a 246-amino-acid protein, including a 40-amino-acid signal peptide. The srtA gene was insertionally inactivated by a tetracycline resistance cassette. P1, a major surface protein adhesin previously shown to anchor to the peptidoglycan by the LPXTGX motif, was secreted into the culture medium by the srtA mutant. In contrast, the wild-type P1 remained cell wall associated. Complementation of the mutant with srtA restored the P1 surface expression phenotype. P1 produced by the mutant, but not that produced by the wild type and the srtA-complemented mutant, was recognized by an antibody raised against the hydrophobic domain and charged tail C terminal to the LPXTGX motif. These results suggest that the failure to anchor P1 to the cell wall is due to the lack of cleavage of P1 at the LPXTGX motif. The srtA mutant was markedly less hydrophobic than the wild type and the complemented mutant. The srtA mutant failed to aggregate in the presence of saliva or salivary agglutinin and adhered poorly to saliva-or salivary agglutinincoated hydroxylapatite. In rats, the srtA mutant colonized the teeth poorly when sucrose was absent. When sucrose was present, the srtA mutant colonized the teeth but less effectively and induced significantly less caries (P < 0.05) than the wild-type strain. In conclusion, the sortase enzyme in S. mutans is responsible for anchoring P1 to the cell surface and plays a role in modulating the surface properties and cariogenicity of S. mutans.The major cell surface protein P1, also known as antigen I/II (ca. 185 kDa), from Streptococcus mutans is an adhesin that interacts with a high-molecular-weight salivary agglutinin and has been implicated in the adherence of the bacterium to the tooth surface (16). Sequence analysis of the P1 gene reveals common features at the C terminus of the protein, as found in several hundreds of surface proteins from many gram-positive coccal bacteria (17). These features include a hydrophilic (wall-associated) region, a highly conserved hexapeptide LPXTGX, a hydrophobic (membrane-spanning) domain, and a charged tail (22,23).In our previous studies, we have demonstrated the requirement of the C-terminal domains in P1 surface localization in S. mutans (10, 11). Site-directed mutagenesis analysis showed that the Thr residue within the LPXTGX motif plays a crucial role in enzymatic cleavage and anchoring of P1 to the cell wall (15).A transpeptidase termed sortase (SrtA) was identified initially in Staphylococcus aureus (27) and recently in Streptococcus gordonii (5), Streptococcus suis (20), Streptococcus pyogenes (2), and Listeria monocytogenes (4, 9). The enzyme is responsible for covalently anchoring protein A to the cell wall (27) and plays a role the pathogenesis of S. aureus (12, 18). Recently, the role of SrtA in virulence...

Association of polymorphisms in the carbonic anhydrase 6 gene with salivary buffer capacity, dental plaque pH, and caries index in children aged 7–9 years

“…25,26 It is worth mentioning that children had the main meals at school (breakfast, snack, and lunch), which partially reduce the effects of variations in diet composition. It is known that bicarbonate in saliva diffuses into dental plaque and combines with H þ to form carbonic acid.…”

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

“…This cation, present in millimolar amounts in host tissues, activates sortase activity eightfold (84). Analysis of the sortase NMR spectra in the presence and absence of calcium revealed that Glu 105 , Glu 108 , and Asp 108 side chains of the ␤3-␤4 loop interact with the cation. In contrast, the ␤6-␤7 loop forms a flap that is disordered in the absence of calcium (131,227).…”

Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii

“…Cell surface proteins containing the conserved peptide motif (LPXTG) are cleaved between the threonine and glycine residues, and are covalently attached to cell wall peptidoglycan by the cysteine protease-transpeptidase sortase A (SrtA) (see Paterson & Mitchell, 2004 for a review). In streptococci, inactivation of the sortase A gene (srtA) results in a failure to cross-link LPXTG cell wallassociated proteins, including b-galactosidase and neuraminidase A from Streptococcus pneumoniae (Kharat & Tomasz, 2003) and P1 antigen from Streptococcus mutans (Lee & Boran, 2003), to the cell wall. In a recent study , SspA and SspB proteins were recovered in elevated amounts from the medium in srtA 2 mutants of S. gordonii DL1, reflecting reduced cross-linking to the cell surface.…”