Inactivation of the srtA gene in Streptococcus gordonii inhibits cell wall anchoring of surface proteins and decreases in vitro and in vivo adhesion

Abstract

The srtA gene product, SrtA, has been shown to be required for cell wall anchoring of protein A as well as virulence in the pathogenic bacterium Staphylococcus aureus. There are five major mechanisms for displaying proteins at the surface of gram-positive bacteria (P. Cossart and R. Jonquieres, Proc. Natl. Acad. Sci. USA 97:5013-5015, 2000). However, since many of the known surface proteins of gram-positive bacteria are believed to be exported and anchored via the sortase pathway, it was of interest to determine if srtA plays a similar role in other gram-positive bacteria. To that end, the srtA gene in the human oral commensal organism Streptococcus gordonii was insertionally inactivated. The srtA mutant S. gordonii exhibited a marked reduction in quantity of a specific anchored surface protein. Furthermore, the srtA mutant had reduced binding to immobilized human fibronectin and had a decreased ability to colonize the oral mucosa of mice. Taken together, these results suggest that the activity of SrtA plays an important role in the biology of nonpathogenic as well as pathogenic gram-positive cocci.

FIG. 1

Schematic representation of the plasmid p891:SgsrtA recombining into the S. gordonii chromosome within the srtA locus, resulting in strains SP-06 and SP-09. Primers TB253, TB256, TB264, and TB265 are shown in the location and orientation of their binding.

FIG. 2

Competition ELISA with S. gordonii recombinant strains expressing surface-anchored M protein versus purified M6 protein. The graph shows percent inhibition of binding of MAb 10F5 to E. coli M6 protein by decreasing concentrations of cells. The strains used are the recombinant M protein-expressing strain GP1223, its isogenic srtA knockout SP-09, and wild-type SP204(1-1), which was used as a negative control. Undil., undiluted. Error bars, standard errors.

FIG. 3

Fn binding assay shown as relative fluorescence units (RFU) of bound FITC-labeled bacteria versus cell dilution. Adherence assays were carried out in 96-well plates coated with purified Fn. The strains used are as indicated in the inserted legend. Both knockout mutants, SP-06 and SP-09, had significantly reduced binding to fibronectin. Each point represents a mean ± 1 standard error (error bars) (P = 0.01).

FIG. 4

Colonization of mice with S. gordonii strains. Groups of 20 mice were inoculated orally and intranasally with S. gordonii srtA⁺ and srtA mutant strains. Colonization was monitored weekly by swabing the oral cavity of the mice and plating on blood agar plates with appropriate additives. CFUs were determined after incubating the plates for 2 days. The strains used are as indicated in the inserted legend. Error bars, standard deviations.