Identification of Rgg-regulated exoproteins of Streptococcus pyogenes

Abstract

Streptococcus pyogenes secretes many proteins that influence host-pathogen interactions. Despite their importance, relatively little is known about the regulation of these proteins. The rgg gene (also known as ropB) is required for the expression of streptococcal erythrogenic toxin B (SPE B), an extracellular cysteine protease that contributes to virulence. Proteomics was used to determine if rgg regulates the expression of additional exoproteins. Exponential- and stationary-phase culture supernatant proteins made by S. pyogenes NZ131 rgg and NZ131 speB were separated by two-dimensional electrophoresis. Differences were identified in supernatant proteins from both exponential- and stationary-phase cultures, although considerably more differences were detected among stationary-phase supernatant proteins. Forty-two proteins were identified by peptide fingerprinting with matrix-assisted laser desorption mass spectrometry. Mitogenic factor, DNA entry nuclease (open reading frame [ORF 226]), and ORF 953, which has no known function, were more abundant in the culture supernatants of the rgg mutant compared to the speB mutant. ClpB, lysozyme, and autolysin were detected in the culture supernatant of the speB mutant but not the rgg mutant. To determine if Rgg affected protein expression at the transcriptional level, real-time (TaqMan) reverse transcription (RT)-PCR was used to quantitate Rgg-regulated transcripts from NZ131 wild-type and speB and rgg mutant strains. The results obtained with RT-PCR correlated with the proteomic data. We conclude that Rgg regulates the transcription of several genes expressed primarily during the stationary phase of growth.

FIG. 1

Coomassie colloidal blue-stained 2-DE gels of supernatant proteins from exponential-phase cultures of NZ131 speB (A) and NZ131 rgg (B). Proteins identified by peptide mass fingerprinting are summarized in Table 3. Diamonds around selected proteins are used to orient the gels to each other. The migration of molecular mass standards is indicated. The gels (oriented with acidic proteins to the left) are representative of two independent experiments.

FIG. 2

Coomassie colloidal blue-stained 2-DE gels of supernatant proteins from stationary-phase cultures of NZ131 speB (A) and NZ131 rgg (B). Proteins identified by peptide mass fingerprinting are summarized in Table 2. Diamonds around selected proteins are used to orient the gels to each other. The migration of molecular mass standards is indicated. The gels (oriented with acidic proteins to the left) are representative of two independent experiments.

FIG. 3

Relative quantities of Rgg-regulated gene transcripts assessed by TaqMan assays. cDNA detected from stationary-phase cultures of NZ131 wild-type (wt), speB, and rgg strains was quantified for mf, the DNA entry nuclease gene (orf226), orf953, the autolysin gene (orf1669), and clpB (orf204). The cDNA values were normalized to the quantity of gyrA cDNA in each sample. The experiments were repeated using at least two independently isolated RNA preparations, and representative results are shown.

FIG. 4

Relative quantities of rgg cDNA in the exponential and stationary phases of growth assessed by TaqMan assays. The amount of rgg cDNA was determined and normalized to the amount of gyrA cDNA following reverse transcription of total RNA isolated from NZ131 wild-type (wt) and speB strains. The results shown are representative of those obtained with two independently isolated RNA preparations.