Consequences of a sortase A mutation in Streptococcus gordonii

1 Department of Diagnostic and Biological Sciences, School of Dentistry, Medical School, University of Minnesota, Minneapolis, MN 55455, USA 2 Department of Genetics, Cell Biology and Development, Medical School, University of Minnesota, Minneapolis, MN 55455, USA 3 Department of Oral Biology, Joint Health Science Center, University of Florida, Gainesville, FL 32611, USA 4 Mucosal and Vaccine Research Center, Minneapolis VA Medical Center, Minneapolis, MN 55417, USA Correspondence Mark C. Herzberg mcherzb{at}umn.edu Sortase A (SrtA) is required for cell-wall anchoring of LPXTG-containing Gram-positive surface proteins. It was hypothesized, therefore, that disruption of the srtA gene would alter surface anchoring and functions of target LPXTG motif-bearing SspA and SspB proteins of Streptococcus gordonii . Mutant strains in srtA (V288 srtA – , DL1 srtA – ) were constructed in S. gordonii V288 (wtV288) and DL1 (wtDL1). When compared to wtV288, the V288 srtA – mutant showed decreased biofilm formation on polystyrene, and reduced binding to immobilized purified salivary agglutinin (BIAcore analysis). The wtV288 and V288 srtA – strains were similar in ultrastructure, but immunogold-labelled SspA/SspB surface expression was reduced on the V288 srtA – mutant. DL1 srtA – was also complemented to obtain DL1 srtA + . From the wild-type strains (wtV288, wtDL1), srtA – mutants (V288 srtA – , DL1 srtA – ), and the complemented mutant (DL1 srtA + ), cytoplasmic, cell-wall and released extracellular protein fractions were isolated. Each fraction was analysed by SDS-PAGE and immunoblotting with anti-P1. Spent medium from srtA – mutant cells contained over-represented proteins, including SspA/SspB (P1 antigen). Mutants showed less P1 on the cell surface than wild-types, as estimated using whole-cell ELISA, and no P1 appeared in the cytoplasmic fractions. Expression of several adhesin genes ( sspA / B , cshA / B , fbpA ) was generally upregulated in the mutants (V288 srtA – , DL1 srtA – ), but restored to wild-type levels in DL1 srtA + . These data therefore imply that in addition to its role in processing LPXTG-containing adhesins, sortase A has the novel function of contributing to transcriptional regulation of adhesin gene expression. Abbreviations: DL1 srtA – , sortase A negative mutant in Streptococcus gordonii DL1 wild-type; DL1 srtA + , complemented DL1 srtA – ; P1, antigen class I/II surface protein of Streptococcus mutans ; RU, resonance units; V288 srtA – , sortas