Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface

Novel regulatory small RNAs in Streptococcus pyogenes

Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes

Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface

Capsule production by mucoid invasive <i>S. pyogenes</i> strains at different temperatures.

#<p>Isolates were recovered through Active Bacterial Core surveillance for <i>S. pyogenes</i> during 2007–2010 in the United States. Refer to surveillance reports at <a href="http://www.cdc.gov/abcs/reports-findings/surv-reports.html" target="_blank">http://www.cdc.gov/abcs/reports-findings/surv-reports.html</a>.</p>%<p>New <i>emm</i> subtypes that have not yet been provided <i>emm</i> designations. All <i>emm</i> designations are shown at the downloadable database <a href="ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/tsemm/" target="_blank">ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/tsemm/</a>.</p>*<p>Mucoidity assay was performed with colonies grown on THY (Todd Hewitt Yeast) agar plates.</p><p>− : Basal level capsule production (less than 100 fg/cfu), not observable with visual inspection such as HSC5 or MGAS315 wild type.</p><p>+ : Capsule production of 100–300 fg/cfu, barely detectable with visual inspection.</p><p>++ : Capsule production of 300–500 fg/cfu, clearly detectable with visual inspection but less amount of capsule production than HSC5 CovRS null mutants incubated at 25°C.</p><p>+++ : Capsule production of more than 500 fg/cfu, clearly detectable with visual observation, almost the same amount of capsule production as HSC5 CovRS null mutants incubated at 25°C.</p>**<p>These strains were preselected as being mucoid on blood agar plates by CDC.</p>@<p>Blank cells: Values were not determined.</p

Capsule thermoregulation occurs at a post-transcriptional level.

<p>(A) The ratio of the capsule transcript quantity of the CovR null strain to that of the wild type. <i>S. pyogenes</i> cells were grown on THY agar plates at 37°C overnight (∼18 hrs) and the fully grown colonies were incubated for another six to eight hrs at each temperature (25°C or 37°C). After RNA was extracted from the colonies, the relative amounts of the capsule transcript between strains were measured through real time-RT (reverse transcriptase) PCR. Regardless of the incubation temperatures, the capsule transcript quantity in the CovR⁻ strain was more than 100 times higher than that in the wild type, confirming that the CovRS two-component system regulates the expression of the capsule transcript at the transcriptional level. (B) The ratio of the capsule transcript quantity at 25°C to that at 37°C. Regardless of the incubation temperatures, the capsule transcript quantity in both strains (the wild type and the CovR⁻ strain) was the same, indicating that thermoregulation of capsule production occurs at a post-transcriptional level. The following strains were used: wild type (WT), HSC5; CovR⁻ strain (CovR⁻), CovRIFD.</p

Regulation of capsule production by <i>S. pyogenes</i> strains, HSC5 and MGAS315.

<p>Capsule production by the non-mucoid <i>S. pyogenes</i> strains, HSC5 and MGAS315, is regulated both at the transcriptional and post-transcriptional level. Phosphorylated CovR binds to its binding sites in the capsule promoter region, and represses <i>hasABC</i> transcription <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037367#pone.0037367-Federle2" target="_blank">[45]</a>. A thermoregulatory system regulates the production of capsule synthesis proteins at a post-transcriptional level. Capsule production occurs only when CovR is not bound to the <i>hasABC</i> promoter region and environmental temperature is lower than 37°C.</p

CvfA is required for the thermoregulation of capsule production.

<p>A) Transposon insertion sites in the transposon-generated CvfA⁻ mutants. The nucleotide sequence in front of the start codon of <i>cvfA</i> is shown. The horizontal arrow represents 5′- side of the <i>cvfA</i> gene. The putative −10 promoter element and transcription start site (+1) are indicated in bold. Each transposon insertion site in the transposon-generated CvfA⁻ strains was shown with a vertical arrowhead: transposon insertion between G and T in the CovRIFD:TnCvfA1 strain and between A and C in the CovRIFD:TnCvfA2 strain. These transposon insertions eliminated thermoregulation of capsule production. B) The transposon-generated CvfA⁻ mutant regained the phenotype of capsule thermoregulation upon <i>cis</i>-complementation. The transposon insertions that prevent the expression of <i>cvfA</i> abolished the capsule thermoregulation of the CovR<i>⁻</i> background strain; the transposon-generated CvfA⁻ strain overproduced capsule regardless of the incubation temperatures. The introduction of an intact copy of <i>cvfA</i> into the chromosome of the transposon-generated CvfA⁻ strain (<i>cis</i>-complementation) restored capsule thermoregulation. C) The transposon-generated CvfA⁻ mutation did not influence the capsule gene transcription even at 25°C. The transposon-generated CvfA mutant was grown on THY agar plates at 37°C overnight (∼18 hrs) and the fully-grown colonies were incubated for another six to eight hrs at each temperature (25°C or 37°C). The quantity of the capsule transcript in the strain was then measured with real time-RT (reverse transcriptase) PCR and compared to that in the CovR⁻ background strain. The following strains were used: CovR⁻ strain (CovR⁻), CovRIFD; CvfA⁻ CovR⁻ strain (CovR⁻CvfA⁻), CovRIFD:TnCvfA1; CvfA complemented strain of CovRIFD:TnCvfA1 (CovR⁻CvfA⁻pCvfA), CovRIFD:TnCvfA1Comp.</p

Expression of the capsule genes <i>in trans</i> under a heterologous promoter also exhibits capsule thermoregulation.

<p>The <i>hasABC</i> genes were expressed <i>in trans</i> under the <i>rofA</i> promoter on pHasABC and transferred to <i>S. pyogenes</i> HSC5 strains whose expression of chromosomal capsule genes was blocked by disrupting the first transcribed capsule gene, <i>hasA</i>. Those complemented strains with pHasABC exhibited the same phenotype of capsule thermoregulation as the strains whose capsule genes were expressed under the native promoter on the chromosome. The following strains were used: CovR⁻ strain (CovR⁻), CovRIFD; chromosomal capsule gene knock-out strain (HasABC⁻), ΩHasA; <i>in trans</i> capsule gene complemented strain (HasABC⁻(pHasABC)), ΩHasA(pHasABC); CovR⁻ HasABC⁻ strain (CovR⁻ HasABC⁻); CovRIFD:ΩHasA; CovR⁻ HasABC⁻ strain complemented with pHasABC (CovR⁻ HasABC⁻ (pHasABC)), CovRIFD:ΩHasA(pHasABC); CovR⁻ CvfA⁻ HasABC⁻ strain (CvfA⁻ CovR⁻ HasABC⁻), CovRIFD:TnCvfA1:ΩHasA; CovR⁻ CvfA⁻ HasABC⁻ strain complemented with pHasABC (CvfA⁻ CovR⁻ HasABC⁻ (pHasABC)), CovRIFD:TnCvfA1:ΩHasA(pHasABC).</p

Alterations in the <i>covRS</i> sequence of strains exhibiting capsule thermoregulation.^#

#<p>The CovRS sequences of CDC lab isolates were compared to the CovRS sequences of 11 genome-sequenced strains publically available at the NCBI website, <a href="http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi" target="_blank">http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi</a>.</p>*<p>Nucleotide sequence level; nt, nucleotide.</p>%<p>Protein sequence level; aa, amino acid.</p>a<p>Deletion of a thymine out of 7 consecutive thymines.</p>b<p>Duplication of 11 bps of TCTGCATTTTC.</p>c<p>Deletion of 5 bps of AAAGA out of AAAGAAAAGA.</p>d<p>Duplication of 11 bps of TTTTCTCTGCC.</p

The amount of capsule produced by <i>S. pyogenes</i> strains at different temperatures.

*<p>ND: Not Determined.</p>%<p>fg/cfu, femtogram per colony forming unit. Capsule quantitation was performed in triplicate for each sample and at least twice per each strain. The values shown here are average ± standard error.</p