A naturally occurring Rgg variant in serotype M3 Streptococcus pyogenes does not activate speB expression due to altered specificity of DNA binding

Abstract

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.

FIG. 1.

Rgg is expressed in strain MGAS315. (A) Diagram of Rgg indicating the position of the N-terminal helix-turn-helix (HTH) motif and the amino acid variation at position 103. (B) Cytoplasmic proteins isolated from wild-type MGAS315 (wt) (lane 1) and an isogenic rgg mutant derivative (lane 2) analyzed by immunoblotting using antibodies to Rgg.

FIG. 2.

Rgg_103S variant activates SpeB production in strain MGAS315. (A) Strains were stab inoculated into casein agar plates and incubated for 48 h anaerobically. Translucent zones surrounding the stab sites indicate SpeB caseinolytic activity. (B) Culture supernatant proteins analyzed by immunoblotting using antisera to SpeB.

FIG. 3.

DNA binding ability of the Rgg_103S and Rgg_103P variants as assessed by electrophoretic mobility shift assays. (A) Lanes 1 to 9 contained 5.0 fmol of speB DNA fragment. Lanes 2 to 5 contained different amounts of Rgg_103S, and lanes 6 to 9 contained different amounts of Rgg_103P. Lanes 5 and 9 also contained 1.0 pmol of unlabeled speB DNA fragment (Cold probe). (B) Lanes 1 to 9 contained 3.3 fmol of a norA DNA fragment. Lanes 2 to 5 contained different amounts of Rgg_103S, and lanes 6 to 9 contained different amounts of Rgg_103P. Lanes 5 and 9 also contained 1.0 pmol of an unlabeled norA DNA fragment. (C) Alignment of the Rgg binding site for speB with the predicted binding site in the putative promoter region of norA.

FIG. 4.

Rgg_103P variant influences growth in CDM. Strains were cultured with (A) THY broth or (B) CDM containing 2% glucose as the primary energy source. Growth was measured by determining the absorbance at 600 nm at various time points. The data are the means and standard errors of the means from three independent experiments.