Alexander V. Dmitriev

The Rgg Regulator of Streptococcus pyogenes Influences Utilization of Nonglucose Carbohydrates, Prophage Induction, and Expression of the NAD-Glycohydrolase Virulence Operon

The expression of many virulence-associated genes in Streptococcus pyogenes is controlled in a growth phase-dependent manner. Unlike the model organisms Escherichia coli and Bacillus subtilis, such regulation is apparently not dependent upon alternative sigma factors but appears to rely on complex interactions among several transcriptional regulators, including Rgg. The purpose of this study was to identify changes in gene expression associated with inactivation of the rgg gene in S. pyogenes strain NZ131 (serotype M49). To this end, the transcriptomes of wild-type and rgg mutant strains were analyzed during both the exponential and postexponential phases of growth using Affymetrix NimbleExpress gene chips. Genomewide differences in transcript levels were identified in both phases of growth. Inactivation of rgg disrupted coordinate expression of genes associated with the metabolism of nonglucose carbon sources, such as fructose, mannose, and sucrose. The changes were associated with an inability of the mutant strain to grow using these compounds as the primary carbon source. Bacteriophage transcript levels were also altered in the mutant strain and were associated with decreased induction of at least one prophage. Finally, transcripts encoding virulence factors involved in cytolysin-mediated translocation of NAD-glycohydrolase, including the immunity factor IFS and the cytolysin (streptolysin O [SLO]), were more abundant in the mutant strain, which correlated with the amount of NADase and SLO activities in culture supernatant fluids. The results provide further evidence that Rgg contributes to growth phase-dependent gene regulation in strain NZ131.

Growth phase-associated changes in the transcriptome and proteome of Streptococcus pyogenes

Streptococcus pyogenes is responsible for approximately 500,000 deaths each year worldwide. Many of the associated virulence factors are expressed in a growth phase-dependent manner. To identify growth phase-associated changes in expression on a genomescale, the exponential and stationary phase transcriptomes and proteomes of S. pyogenes strain NZ131 (serotype M49) were compared by using Affymetrix NimbleExpress gene chips and two-dimensional gel electrophoresis. At the transcript level, the expression of 689 genes, representing approximately 40% of the chromosome, differed by twofold or more between the two growth phases. The majority of transcripts that were more abundant in the early-stationary phase encoded proteins involved in energy conversion, transport, and metabolism. At the protein level, an average of 527 and 403 protein spots were detected in the exponential and stationary phases of growth, respectively. Tandem mass spectrometry was used to identify 172 protein spots, 128 of which were growth phase regulated. Enzymes involved in glycolysis and pyruvate metabolism and several stress-responsive proteins were more abundant in the stationary phase of growth. Overall, the results identified growth phase-regulated genes in strain NZ131 and revealed significant post-transcriptional complexity associated with pathogen adaptation to the stationary phase of growth.

Inter- and intraserotypic variation in the Streptococcus pyogenes Rgg regulon.

Human isolates of Streptococcus pyogenes, a Gram-positive bacterium, are characterized by significant genetic and phenotypic variation. The rgg locus, also known as ropB, is a global transcriptional regulator of genes associated with metabolism, stress responses, and virulence in S. pyogenes strain NZ131 (serotype M49). To assess the breadth of the Rgg regulon, the rgg gene was inactivated in three additional strains representing serotypes M1 (strains SF370 and MGAS5005) and M49 (strain CS101). Changes in gene expression were identified in the postexponential phase of growth using Affymetrix NimbleExpress Arrays. The results identified an Rgg core-regulon consisting of speB and adjacent hypothetical protein gene, spy2040, and a variable and strain-specific subregulon, ranging in size from a single gene (spy1793) in strain MGAS5005 to 43 genes in strain SF370. Thus, both interserotypic and intraserotypic variation is characteristic of the Rgg regulon in S. pyogenes.

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 (Rgg103P); in contrast, all other strains encode a serine at this position (Rgg103S). 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 Rgg103S, Rgg103P 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, Rgg103P 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.

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.

Inactivation of DNA-binding response regulator Sak189 abrogates β-antigen expression and affects virulence of Streptococcus agalactiae.

Background Streptococcus agalactiae is able to colonize numerous tissues employing different mechanisms of gene regulation, particularly via two-component regulatory systems. These systems sense the environmental stimuli and regulate expression of the genes including virulence genes. Recently, the novel two-component regulatory system Sak188/Sak189 was identified. In S. agalactiae genome, it was adjacent to the bac gene encoding for β-antigen, an important virulence factor. Methodology/Principal Findings In this study, the sak188 and sak189 genes were inactivated, and the functional role of Sak188/Sak189 two-component system in regulation of the β-antigen expression was investigated. It was demonstrated that both transcription of bac gene and expression of encoded β-antigen were controlled by Sak189 response regulator, but not Sak188 histidine kinase. It was also found that the regulation occurred at transcriptional level. Finally, insertional inactivation of sak189 gene, but not sak188 gene, significantly affected virulent properties of S. agalactiae. Conclusions/Significance Sak189 response regulator is necessary for activation of bac gene transcription. It also controls the virulent properties of S. agalactiae. Given that the primary functional role of Sak188/Sak189 two-component systems is a control of bac gene transcription, this system can be annotated as BgrR/S (bac gene regulatory system).

The Streptococcus pyogenes proteome: maps, virulence factors and vaccine candidates.

Streptococcus pyogenes is an important cause of human morbidity and mortality worldwide. A wealth of genomic information related to this pathogen has facilitated exploration of the proteome, particularly in response to environmental conditions thought to mimic various aspects of pathogenesis. Proteomic approaches are also used to identify immunoreactive proteins for vaccine development and to identify proteins that may induce autoimmunity. These studies have revealed new mechanisms involved in regulating the S. pyogenes proteome, which has opened up new avenues in the study of S. pyogenes pathogenesis. This article describes the methods used, and progress being made towards characterizing the S. pyogenes proteome, including studies seeking to identify potential vaccine candidates.

Adjacent location of thebac gene and two-component regulatory system genes within the putativeStreptococcus agalactiae pathogenicity island

A chromosomal DNA fragment of 8992 bp in size that has not been previously identified inStreptococcus agalactiae, was cloned and sequenced from strain 98-D60C. In particular, this 8992-bp fragment contained genes homologous to the sensor histidine kinase gene and the DNA-binding response-regulator gene ofStreptococcus pneumoniae, andS. agalactiae bac gene. Structural and genetic features of the 8992-bp fragment were highly similar to those specific for bacterial pathogenicity islands. Analysis of epidemiologically unrelatedS. agalactiae strains revealed that this fragment was present only inbac gene-positive strains. The possible origin of the 8992-bp fragment inS. agalactiae and its significance for molecular mechanisms of “bacteria-host” interactions are discussed.

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.

Gene sak0192 of Streptococcus agalactiae contains direct repeats and spacers serving as genetic markers characterizing the strains

A new method of strain differentiation in Streptococcus agalactiae is proposed in this work. It is based on revealing the polymorphism of structure of gene sak0192 encoding a hypothetical protein, which in different strains of Str. agalactiae contains a different number of regularly alternating repeats and spacer sites having sizes of 16 and 44 bp, respectively. The sequences of forward repeats are mostly identical, while spacers are characterized with expressed heterogeneity. In general, the structure of gene sak0192 is similar to one of sites with direct repeats and spacers in genomes of Mycobacterium tuberculosis and Str. pyogenes. The possibility of using polymorphism of sak0192 in both identification of species affiliation and intraspecific differentiation of Str. agalactiae strains is demonstrated.