Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes.

Streptococcus pyogenes adapts to different niches encountered in the human host via the activity of numerous regulatory proteins including the Rgg family of transcriptional regulators. The S. pyogenes chromosome encodes four Rgg paralogues designated Rgg1 (RopB), Rgg2 (MutR), Rgg3, and Rgg4 (ComR). In order to understand the role of the Rgg2 protein in the regulation of metabolic and virulence-associated properties of S. pyogenes, the rgg2 gene was inactivated in the M1 serotype strain SF370. Inactivation of rgg2 increased the growth yield of S. pyogenes in THY broth, increased biofilm formation, and increased production of SIC, which is an important virulence factor that inhibits complement mediated lysis. To identify Rgg2-regulated genes, the transcriptomes of SF370 and the rgg2 mutant strains were compared in the middle-exponential and post-exponential phases of growth. Rgg2 was found to control the expression of dozens of genes primarily in the exponential phase of growth, including genes associated with virulence (sse, scpA, slo, nga, mf-3), DNA transformation, and nucleotide metabolism. Inactivation of rgg2 decreased the ability of S. pyogenes to adhere to epithelial cells. In addition, the mutant strain was more sensitive to killing when incubated with human blood and avirulent in a murine bacteremia model. Finally, inoculation of mice with the avirulent rgg2 mutant of S. pyogenes SF370 conferred complete protection to mice subsequently challenged with the wild-type strain. Restoration of an intact rgg2 gene in mutant strain restored the wild-type phenotypes. Overall, the results demonstrate that Rgg2 is an important regulatory protein in S. pyogenes involved in controlling genes associated with both metabolism and virulence.

Vaccination against the M protein of Streptococcus pyogenes prevents death after influenza virus: S. pyogenes super-infection.

Influenza virus infections are associated with a significant number of illnesses and deaths on an annual basis. Many of the deaths are due to complications from secondary bacterial invaders, including Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pyogenes. The ß-hemolytic bacteria S. pyogenes colonizes both skin and respiratory surfaces, and frequently presents clinically as strep throat or impetigo. However, when these bacteria gain access to normally sterile sites, they can cause deadly diseases including sepsis, necrotizing fasciitis, and pneumonia. We previously developed a model of influenza virus:S. pyogenes super-infection, which we used to demonstrate that vaccination against influenza virus can limit deaths associated with a secondary bacterial infection, but this protection was not complete. In the current study, we evaluated the efficacy of a vaccine that targets the M protein of S. pyogenes to determine whether immunity toward the bacteria alone would allow the host to survive an influenza virus:S. pyogenes super-infection. Our data demonstrate that vaccination against the M protein induces IgG antibodies, in particular those of the IgG1 and IgG2a isotypes, and that these antibodies can interact with macrophages. Ultimately, this vaccine-induced immunity eliminated death within our influenza virus:S. pyogenes super-infection model, despite the fact that all M protein-vaccinated mice showed signs of illness following influenza virus inoculation. These findings identify immunity against bacteria as an important component of protection against influenza virus:bacteria super-infection.

CodY-mediated regulation of Streptococcus pyogenes exoproteins.

BACKGROUND: The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene. RESULTS: We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain. CONCLUSIONS: Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.

Growth phase-dependent modulation of Rgg binding specificity in Streptococcus pyogenes.

Streptococcus pyogenes Rgg is a transcriptional regulator that interacts with the cofactor LacD.1 to control growth phase-dependent expression of genes, including speB, which encodes a secreted cysteine protease. LacD.1 is thought to interact with Rgg when glycolytic intermediates are abundant in a manner that prevents Rgg-mediated activation of speB expression via binding to the promoter region. When the intermediates diminish, LacD.1 dissociates from Rgg and binds to the speB promoter to activate expression. The purpose of this study was to determine if Rgg bound to chromatin during the exponential phase of growth and, if so, to identify the binding sites. Rgg bound to 62 chromosomal sites, as determined by chromatin immunoprecipitation coupled with DNA microarrays. Thirty-eight were within noncoding DNA, including sites upstream of the genes encoding the M protein (M49), serum opacity factor (SOF), fibronectin-binding protein (SfbX49), and a prophage-encoded superantigen, SpeH. Each of these sites contained a promoter that was regulated by Rgg, as determined with transcriptional fusion assays. Purified Rgg also bound to the promoter regions of emm49, sof, and sfbX49 in vitro. Results obtained with a lacD.1 mutant showed that both LacD.1 and Rgg were necessary for the repression of emm49, sof, sfbX49, and speH expression. Overall, the results indicated that the DNA binding specificity of Rgg is responsive to environmental changes in a LacD.1-dependent manner and that Rgg and LacD.1 directly control virulence gene expression in the exponential phase of growth.

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

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.

Inactivation of the group A Streptococcus regulator srv results in chromosome wide reduction of transcript levels, and changes in extracellular levels of Sic and SpeB.

Group A Streptococcus is characterized by the ability to cause a diverse number of human infections including pharyngitis, necrotizing fasciitis, toxic shock syndrome, and acute rheumatic fever, yet the regulation of streptococcal genes involved in disease processes and survival in the host is not completely understood. Genome scale analysis has revealed a complex regulatory network including 13 two-component regulatory systems and more than 100 additional putative regulators, the majority of which remain uncharacterized. Among these is the streptococcal regulator of virulence, Srv, the first Group A Streptococcus member of the Crp/Fnr family of transcriptional regulators. Previous work demonstrated that the loss of srv resulted in a significant decrease in Group A Streptococcus virulence. To begin to define the gene products influenced by Srv, we combined microarray and two-dimensional gel electrophoresis analysis. Loss of srv results in a chromosome wide reduction of gene transcription and changes in the production of the extracellular virulence factors Sic (streptococcal inhibitor of complement) and SpeB (cysteine proteinase). Sic levels are reduced in the srv mutant, whereas the extracellular concentration and activity of SpeB is increased. These data link Srv to the increasingly complex GAS regulatory network.

Rgg influences the expression of multiple regulatory loci to coregulate virulence factor expression in Streptococcus pyogenes.

The human pathogen Streptococcus pyogenes secretes many proteins to the cell wall and extracellular environment that contribute to virulence. Rgg regulates the expression of several exoproteins including a cysteine protease (SPE B), a nuclease (MF-1), a putative nuclease (MF-3), and autolysin. The functional heterogeneity of Rgg-regulated exoproteins and the lack of a conserved regulatory motif in the promoter regions of the genes suggested that Rgg interacts with additional regulatory networks to influence gene expression. DNA microarrays were used to test this hypothesis by comparing genomewide transcript profiles of S. pyogenes NZ131 and isogenic derivative NZ131 rgg during the exponential phase of growth. Transcripts of known and putative virulence-associated genes were more abundant in the rgg mutant, including emm, scpA, orfX, scl1, hasAB, slo, sagA, ska, speH, grab, mac, mf-1, and mf-3. Increased transcription of emm, scpA, and orfX in the rgg mutant was associated with increased production of the corresponding proteins. Differences in the expression of virulence-associated genes were associated with changes in the expression of several regulatory genes, including mga, sagA, csrRS, and fasBCA. The results show that Rgg influences the expression of multiple regulatory networks to coregulate virulence factor expression in S. pyogenes.