CcpA-Mediated Repression of Streptolysin S Expression and Virulence in the Group A Streptococcus▿†

CcpA is the global mediator of carbon catabolite repression (CCR) in gram-positive bacteria, and growing evidence from several pathogens, including the group A streptococcus (GAS), suggests that CcpA plays an important role in virulence gene regulation. In this study, a deletion of ccpA in an invasive M1 GAS strain was used to test the contribution of CcpA to pathogenesis in mice. Surprisingly, the ΔccpA mutant exhibited a dramatic “hypervirulent” phenotype compared to the parental MGAS5005 strain, reflected as increased lethality in a model of systemic infection (intraperitoneal administration) and larger lesion size in a model of skin infection (subcutaneous administration). Expression of ccpA in trans from its native promoter was able to complement both phenotypes, suggesting that CcpA acts to repress virulence in GAS. To identify the CcpA-regulated gene(s) involved, a transcriptome analysis was performed on mid-logarithmic-phase cells grown in rich medium. CcpA was found to primarily repress 6% of the GAS genome (124 genes), including genes involved in sugar metabolism, transcriptional regulation, and virulence. Notably, the entire sag operon necessary for streptolysin S (SLS) production was under CcpA-mediated CCR, as was SLS hemolytic activity. Purified CcpA-His bound specifically to a cre within sagAp, demonstrating direct repression of the operon. Finally, SLS activity is required for the increased virulence of a ΔccpA mutant during systemic infection but did not affect virulence in a wild-type background. Thus, CcpA acts to repress SLS activity and virulence during systemic infection in mice, revealing an important link between carbon metabolism and GAS pathogenesis

The Catabolite Control Protein CcpA Binds to Pmga and Influences Expression of the Virulence Regulator Mga in the Group A Streptococcus▿ †

Carbon catabolite repression (CCR) allows bacteria to alter metabolism in response to the availability of specific sugar sources, and increasing evidence suggests that CCR is involved in regulating virulence gene expression in many pathogens. A scan of the M1 SF370 group A streptococcus (GAS) genome using a Bacillus subtilis consensus identified a number of potential catabolite-responsive elements (cre) important for binding by the catabolite control protein A (CcpA), a mediator of CCR in gram-positive bacteria. Intriguingly, a putative cre was identified in the promoter region of mga upstream of its distal P1 start of transcription. Electrophoretic mobility shift assays showed that a His-CcpA fusion protein was capable of binding specifically to the cre in Pmga in vitro. Deletion analysis of Pmga using single-copy Pmga-gusA reporter strains found that Pmga P1 and its upstream cre were not required for normal autoregulated mga expression from Pmga P2 as long as Mga was produced from its native locus. In fact, the Pmga P1 region appeared to show a negative influence on Pmga P2 in these studies. However, deletion of the cre at the native Pmga resulted in a reduction of total mga transcripts as determined by real-time reverse transcription-PCR, supporting a role for CcpA in initial expression. Furthermore, normal transcriptional initiation from the Pmga P1 start site alone was dependent on the presence of the cre. Importantly, inactivation of ccpA in the M6 GAS strain JRS4 resulted in a reduction in Pmga expression and Mga protein levels in late-logarithmic-phase cell growth. These data support a role for CcpA in the early activation of the mga promoter and establish a link between CCR and Mga regulation in the GAS