Adhesion capacity of Streptococcus gordonii– effects of an srtA mutation

Sortase A (SrtA) is a transpeptidase required for cell-wall anchoring of surface proteins in Gram positive bacteria. srtA negative mutants are known to show changes in surface properties and to have a reduced ability to colonize the oral cavity. While expression of some cell-surface proteins is likely to be reduced, compensatory up-regulation of others may occur in the srtA- mutants.

Objective: To determine the effect of srtA deletion on adhesion of Streptococcus gordonii to oral surfaces and the expression of cell-surface proteins.

Methods: The srtA- mutant was constructed in S. gordonii DL1(Challis). To test adhesion capacity, bacteria were incubated with immortalized human oral keratinocytes grown in mini flow-cells or with saliva-coated flow-cell surfaces. Numbers of adhered bacteria were assessed after staining and confocal microscopy. Whole cell extracts or fractionated proteins from wild-type or srtA- mutants were subjected to 2-dimensional electrophoresis (2DE) and gels stained with silver for protein analysis.

Results: Wild-type DL1 bound well to the surface of keratinocytes (mean = 9 bacteria/cell) after 4 hours of incubation. Inactivation of the srtA gene resulted in almost complete abolition of binding with, on average, less than one bacterium per keratinocyte. A similar pattern was seen for adhesion to a conditioning film (saliva) on glass surfaces. The wild-type DL1 bound avidly (40,000 cells/mm²), while the srtA- mutant bound much less well (200 cells/mm²). Comparative 2DE analysis of total protein expression suggested that the cell-surface proteome in the mutant differed from the wild-type.

Conclusions: These data suggest that expression of SrtA is critical in determining binding ability both to saliva-coated surfaces and keratinocytes in the oral cavity. The reduction in adhesive capacity reflects global changes in the cell-surface proteome of the srtA- mutant of S. gordonii.

Supported by The Swedish Dental Society, The Knowledge Foundation, Sweden, and NIH/NIDCR R01DE08590.