Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS)

Abstract

Background: Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS.

Results: We found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species.

Conclusion: Genome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS.

Figure 1

Circular representation of the genome of S. dysgalactiae subsp. Equisimilis strain GGS_124. Circle 1 (outermost circle) indicates the distance from the putative origin of replication. Circle 2 shows annotated CDS encoded on the forward (light blue) and reverse (yellow) chromosomal strands, respectively. The rRNA genes (pink), tRNA genes (blue), and tmRNA gene (black) are shown in circle 3. Prophage (green) and ISs (orange) genes are shown in circle 4. CRISPR (red) is shown in circle 5. Circle 6 (innermost circle) shows the G+C content with greater and less than average (0.40) in purple and brown, respectively.

Figure 2

Genome rearrangement maps of S. dysgalactiae subsp. equisimilis GGS_124 with five species in the pyogenic group. Sequences were aligned from the predicted replication origin of each genome. The colored bars separating each genome (red and green) represent similarity matches identified by in silico Molecular Cloning. Links shown in green match in the same orientation, while those in red match in the reverse orientation. Prophages are highlighted as pale blue boxes.

Figure 3

Venn diagram of gene content comparison among S. dysgalactiae subsp. equisimilis GGS_124 (SDSE), S. pyogenes MGAS315 (GAS) and SESZ MGCS10565. The inferred proteomes of SDSE, GAS, and SESZ were compared in a pairwise manner with their translated genomes by in silico Molecular Cloning and are presented as a Venn diagram. The numbers of products for each section are color coded to match the respective genomes. Genes showing more than 40% identity were considered homologues.

Figure 4

Prophage elements and the surrounding gene arrangements of GGS_124 and GAS. The organization of the genes located in the putative prophage regions found in GGS_124 and their insertion points in the genome were compared with those of GAS. Colored boxes between genes indicate level of similarity at the amino acid level (red, ≥90%; orange, 89%-80%; green, 79%-70%).

Figure 5

CRISPR/Cas system structures found in SDSE isolates. The CRISPR structures of SDSE isolates were analyzed by direct sequencing of PCR-amplified CRISPR regions. The repeat-spacer regions (CRISPR) are enlarged. Open circles marked "R" are direct repeats, whereas the square boxes indicate respective spacers with numbers. GGS_124 contained only CRISPR1, whereas some of the other strains also contained CRISPR2 (Table 2).

Figure 6

Organization of genes encoding pilus-like structure proteins in S. dysgalactiae subsp. equisimilis GGS_124. The organizations of genes encoding pilus-like structure proteins in GGS_124 were compared with those in GAS and SESZ. Colored boxes between genes indicate similarity at the amino acid level (red, ≥90%; orange, 90%-80%; yellow-green, 79%-70%; green, 69%-60%; purple, 59%-40%; gray, <40%).

Figure 7

Putative virulence factors and posited virulence function of SDSE. Cell-surface proteins, extracellular secreted proteins, metal transporters, and the two-component regulator CsrR/CsrS, which affect the expression of approximately 10% of all genes in a GAS strain [78,79], are shown. The putative virulence factors on the cell surface of SDSE are adhesins, including M protein, fibronectin binding protein (Fbp), collagen binding protein (Cbp), laminin binding protein (Lbp), and pullulanase (Pul) [80]. Factors that protect bacteria from the host immune system are shown, including cell envelope proteinase A (CepA), which cleaves within the interleukin-8 (IL-8) C-terminal α-helix [38]; immunoglobulin G binding protein (GB) [49]; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and C5a peptidase (Scp) [46]. Putative pathogenic roles of the virulence factors of SDSE, including hyaluronidase (Hyl), streptokinase (Ska), extracellular nuclease and streptodornase (DNase), which digest neutrophil extracellular traps (NETs) released from dead neutrophils [50], and the pore-forming proteins, such as hemolysin (Hly), streptolysin S (SLS), streptolysin O (SLO), and NAD glycohydrolase (NADase), are indicated. FtsABCD and HtsABC are ferrichrome transporters and MtsABC is a metal transporter. Black arrows show protein secretion, red arrows show expression of genes regulated by CsrR, blue arrows show protein attachment to the extracellular matrix, brown arrows show metal transport from the extracellular environment into the cell, and purple arrows show degradation of extracellular matrix by secreted Hyl or Ska. The factors marked with a Stop sign, which are major virulence factors of GAS, do not function in SDSE.