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CcpA-mediated repression of streptolysin S expression and virulence in the group A streptococcus.

ABSTRACT: 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 DeltaccpA 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 DeltaccpA 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.

SUBMITTER: Kinkel TL

PROVIDER: S-EPMC2493232 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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CcpA-mediated repression of streptolysin S expression and virulence in the group A streptococcus.

Kinkel Traci L TL McIver Kevin S KS

Infection and immunity 20080519 8

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 DeltaccpA mutant exhibited a dramatic "hypervirulent" phenotype compared to the parental MGAS5005 ...[more]

PMID: 18490461

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Project description:UnlabelledStreptolysin O is a potent pore-forming toxin produced by group A Streptococcus. The aims of the present study were to dissect the relative contributions of different structural domains of the protein to hemolytic activity, to obtain a detoxified form of streptolysin O amenable to human vaccine formulation, and to investigate the role of streptolysin O-specific antibodies in protection against group A Streptococcus infection. On the basis of in silico structural predictions, we introduced two amino acid substitutions, one in the proline-rich domain 1 and the other in the conserved undecapeptide loop in domain 4. The resulting streptolysin O derivative showed no toxicity, was highly impaired in binding to eukaryotic cells, and was unable to form organized oligomeric structures on the cell surface. However, it was fully capable of conferring consistent protection in a murine model of group A Streptococcus infection. When we engineered a streptococcal strain to express the double-mutated streptolysin O, a drastic reduction in virulence as well as a diminished capacity to kill immune cells recruited at the infection site was observed. Furthermore, when mice immunized with the toxoid were challenged with the wild-type and mutant strains, protection only against the wild-type strain, not against the strain expressing the double-mutated streptolysin O, was obtained. We conclude that protection occurs by antibody-mediated neutralization of active toxin.ImportanceWe present a novel example of structural design of a vaccine antigen optimized for human vaccine use. Having previously demonstrated that immunization of mice with streptolysin O elicits a protective immune response against infection with group A Streptococcus strains of different serotypes, we developed in this study a double-mutated nontoxic derivative that represents a novel tool for the development of protective vaccine formulations against this important human pathogen. Furthermore, the innovative construction of an isogenic strain expressing a functionally inactive toxin and its use in infection and opsonophagocytosis experiments allowed us to investigate the mechanism by which streptolysin O mediates protection against group A Streptococcus. Finally, the ability of this toxin to directly attack and kill host immune cells during infection was studied in an air pouch model, which allowed parallel quantification of cellular recruitment, vitality, and cytokine release at the infection site.

Dataset Information

CcpA-mediated repression of streptolysin S expression and virulence in the group A streptococcus.

Publications

CcpA-mediated repression of streptolysin S expression and virulence in the group A streptococcus.

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