Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

Abstract

dStreptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.

A streptolysin S homologue is essential for β-haemolytic Streptococcus constellatus subsp. constellatus cytotoxicity

“…Each gene locus in the sag operon homologues was conserved between b-SAA strain NCTC10713 T and PGS, except for the duplication of sagA (Tabata et al, 2013). Although the order of each gene in the sag operon homologues was conserved strictly, as expected from the findings in b-SAA, each gene sequence in the SCC operon was well conserved amongst AGS but showed lower identity to the gene sequence of PGS (Table 2).…”

“…Previously, we reported that b-SAA strains contained a novel sag operon homologue composed of 10 ORFs (sagA1, sagA2 and sagB-sagI) and that the novel SLS homologues encoded in the operon, designated SagA1 and SagA2, were responsible for the b-haemolytic activity of b-SAA strains (Tabata et al, 2013). However, the sag operon homologue of SCC (designated sag SCC ) was composed of nine ORFs (sagA-sagI), similar to the typical sag operon of S. pyogenes and other PGS (Table 2).…”

“…The purified fragments were ligated using DNA Ligation Mighty Mix (TaKaRa) and used as the template in a nested PCR with the SCC_up_operon_nest-Fw and SCC_operon_down_nest-Bw primer set ( Table 1). The resulting amplicon was purified and used for transformation induced by competence-stimulating peptide (CSP) as described previously (Tabata et al, 2013). The construction of the desired control mutant was confirmed by DNA sequence analysis of a target region fragment amplified by PrimeSTAR HS DNA Polymerase.…”

“…The sequence of the constructed plasmid was confirmed and the plasmid was used for complementation in the sagA SCC deletion mutant induced by CSP. The complementation mutant was screened as described previously (Tabata et al, 2013).…”

“…The construction of the deletion mutants derived from strain W277 was achieved by double-crossing-over homologous recombination, according to a previously described method (Tabata et al, 2013). The control mutant, which only contained the erythromycin resistance gene cassette (erm) oppositely inserted upstream of the sagA SCC promoter region, was constructed by amplifying (PCR) each component of the fragment using the following primer sets (see Table 1) and templates: (i) SCC_up_operon-Fw and SCC_up_operon-Bw(PstI) and W277 genomic DNA (for amplification of the former half of the sagA SCC upstream region), (ii) SCC_up_operon-Fw(BamHI) and SCC_sagB-Bw and W277 genomic DNA (for amplification of the latter half of the sagA SCC upstream region), and (iii) erm-Fw(BamHI) and erm-Bw(PstI) and the genomic DNA of the S. intermedius strain UNS38 ily knockout mutant (Sukeno et al, 2005) (amplified erm).…”