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Consequences of a sortase A mutation in Streptococcus gordonii

Angela H. Nobbs^1,,

Reka M. Vajna^1,

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 (V288srtA^–, DL1srtA^–) were constructed in S. gordonii V288 (wtV288) and DL1 (wtDL1). When compared to wtV288, the V288srtA^– mutant showed decreased biofilm formation on polystyrene, and reduced binding to immobilized purified salivary agglutinin (BIAcore analysis). The wtV288 and V288srtA^– strains were similar in ultrastructure, but immunogold-labelled SspA/SspB surface expression was reduced on the V288srtA^– mutant. DL1srtA^– was also complemented to obtain DL1srtA⁺. From the wild-type strains (wtV288, wtDL1), srtA^– mutants (V288srtA^–, DL1srtA^–), and the complemented mutant (DL1srtA⁺), 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 (V288srtA^–, DL1srtA^–), but restored to wild-type levels in DL1srtA⁺. 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.

These authors contributed equally to this work.

Present address: Novartis Vaccines, Centro Ricerche, Via Fiorentina 1, 53100 Siena, Italy.