Presence of a Prophage Determines Temperature-Dependent Capsule Production in Streptococcus pyogenes

Abstract

A hyaluronic acid capsule is a major virulence factor in the pathogenesis of Streptococcus pyogenes. It acts as an anti-phagocytic agent and adhesin to keratinocytes. The expression of the capsule is primarily regulated at the transcriptional level by the two-component regulatory system CovRS, in which CovR acts as a transcriptional repressor. The covRS genes are frequently mutated in many invasive strains, and a subset of the invasive CovRS mutants does not produce a detectable level of the capsule at 37 °C, but produces a significant amount of the capsule at sub-body temperatures. Here, we report that a prophage has a crucial role in this capsule thermoregulation. Passaging CovR-null strains showing capsule thermoregulation using a lab medium produced spontaneous mutants producing a significant amount of the capsule regardless of incubation temperature and this phenotypic change was caused by curing of a particular prophage. The lab strain HSC5 contains three prophages on the chromosome, and only ΦHSC5.3 was cured in all spontaneous mutants. This result indicates that the prophage ΦHSC5.3 plays a crucial role in capsule thermoregulation, most likely by repressing capsule production at 37 °C.

Keywords: Capsule thermoregulation; CovRS; Hyaluronic acid capsule; Prophages; Streptococcus pyogenes.

Figure 1

Quantitation of the capsule produced by Streptococcus pyogenes strains. The capsule produced by strains was quantitated and compared to that of the background strain CovRIFD incubated at 25 °C. Strains used: CovRIFD (covR in-frame deletion strain), the background strain showing capsule thermoregulation; spontaneous mutants, spontaneously-generated capsule thermoregulation-negative mutants; Tpn-generated L897_07695 mutant, a mutant with a transposon insertion in the L897_07695 gene in CovRIFD; and ΩL897_07695, a strain with a targeted disruption of the L897_07695 gene in CovRIFD. The data are the means and standard deviations of at least three independent experiments. * indicates significance (p < 0.01) for the difference between the amount of capsule production by strains at a given conditions and that by CovRIFD at 37 °C as calculated by the two-tailed paired Student’s t-test.

Figure 2

Whole genome sequencing revealed the deletion of ΦHSC5.3 in spontaneous capsule thermoregulation-negative mutants. The chromosomes of spontaneous capsule thermoregulation mutants were sequenced and compared to that of the wild-type HSC5. Each vertical line in the boxed figures is a pile-up of DNA sequences from Illumina next generation sequencing and indicates the presence of the sequence on the chromosome. The numbers under the boxed figures represent the location on the chromosome. Each horizontal arrow on top of the boxed figures indicates the position of each prophage on the chromosome: 964214–1010974 for ΦHSC5.1, 1130407–1166414 for ΦHSC5.2, and 1454753–1496038 for ΦHSC5.3. Even though ΦHSC5.3 was cured in the mutants, some sequences in ΦHSC5.3 appeared due to the existence of homologous genes in the prophages ΦHSC5.1 and ΦHSC5.2.

Figure 3

PCR assay to determine the presence of prophages on the chromosome of Streptococcus pyogenes strains. (A) Deletion of ΦHSC5.3 in spontaneous capsule thermoregulation mutants. Spontaneously generated capsule thermoregulation mutants producing a large amount of the capsule regardless of incubation temperature were screened after passaging the background strain CovRIFD in THY medium. Control colonies still displaying capsule thermoregulation on the same plates were also selected for this assay. PCR was performed to determine the presence of prophages using the primers to amplify a portion of a gene in ΦHSC5.1 (I), ΦHSC5.2 (II), or ΦHSC5.3 (III), or primers that amplify HSC5 chromosomal DNA only when ΦHSC5.3 was cured (IV). Strains used: WT (wild-type), HSC5; M1–M4 (mutant strains), randomly chosen four spontaneously arisen capsule thermoregulation-negative mutants; C1–C3 (control strains), colonies still showing capsule thermoregulation after passaging; (B) The existence of ΦHSC5.3 in the transposon-generated mutants. Since the excision of ΦHSC5.3 occurs spontaneously and influences capsule thermoregulation, the presence of ΦHSC5.3 in the previous transposon-generated mutants was determined through PCR. The same primers were used as those used in Figure. A. Strains used: T1–T4 (transposon-generated capsule thermoregulation mutants), a transposon insertion occurred in the gene L897_07695 in ΦHSC5.3 (T1), at or near the ribosome binding site of cvfA (T2 and T3, previously described as CovRIFD:TnCvfA1 and CovRIFD:TnCvfA2, respectively [10]), or in the intergenic region (IGR) immediately upstream of the capsule operon (T4).

Figure 4

Prophage integration sites on the chromosome of Streptococcus pyogenes HSC5. (A) Positions of prophages on the S. pyogenes HSC5 genome. The circle represents the S. pyogenes chromosome. ori indicates the chromosomal replication origin for bidirectional DNA synthesis. The positions of the prophages, ΦHSC5.1, ΦHSC5.2, and ΦHSC5.3 on the chromosome are 31.8 min, 37.3 min, and 48 min, respectively. Inverted triangles indicate integration sites of the prophages. L denotes the position of attL next to integrase genes, and R denotes attR next to paratox genes; and (B) prophage integration sites in the HSC5 genome. White arrows indicate ORFs identified by locus tags (L897 numbers) of the S. pyogenes HSC5 genome.