Investigation of a Novel DNase of Streptococcus suis Serotype 2

Abstract

A secreted nuclease, SsnA, was identified in the virulent Streptococcus suis isolate SX332 and subsequently in each of the type strains of capsular serotypes 1 through 9. Screening of 258 porcine clinical isolates from surface (nasal mucosa or palatine tonsil) or internal (joint, brain or other internal organ) locations revealed a significant relationship (P < 0.001) between expression of nuclease and isolation from an internal site. A 3,126-bp gene, ssnA, was identified from a phenotypically nuclease-negative pGh9:ISS1 insertion mutant, and analysis of the predicted SsnA sequence revealed a 35-amino-acid (aa) secretion signal sequence, a 22-aa DNA-binding domain, and a typical gram-positive cell wall sorting motif. A requirement of Ca 2؉ and Mg 2؉ for SsnA activity was determined, and the substrate specificity was found to be for single-and double-stranded linear DNA. Reverse transcription-PCR experiments revealed that ssnA is expressed throughout all stages of S. suis growth, and Western blots with porcine anti-S. suis immune sera against a recombinant, truncated SsnA derivative (rSsnA ⌬ ) confirmed that SsnA is expressed in vivo. Furthermore, anti-rSsnA ⌬ antibodies were sufficient to neutralize SsnA activity. Analyses of subcellular fractions of SX332 and derived mutants, on DNAcontaining polyacrylamide gels and by Western blotting, suggest that SsnA is cell wall located.

Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes

“…Genes encoding this protein (more than 99.4 % identity at the nucleotide level) are present in all S. pyogenes strains for which the whole genomes have been sequenced: M1 (Sumby et al, 2005b), M2 (Beres et al, 2006), M3 (Beres et al, 2002;Nakagawa et al, 2003), M4 (Beres et al, 2006), M5 (Holden et al, 2007), M6 (Banks et al, 2004), M12 (Beres et al, 2006), M18 , M28 (Green et al, 2005) and M49 (McShan et al, 2008). The homologous protein was reported as SsnA (for S. suis nuclease A) of S. suis serotype 2 (Fontaine et al, 2004). We designated the Spy0747 protein as SpnA (for S. pyogenes nuclease A).…”

“…Spy0747 is homologous to the SsnA (S. suis nuclease A) DNase protein of S. suis serotype 2 (Fontaine et al, 2004). The recombinant protein exhibited DNase activity, which was Ca 2+ -and Mg 2+ -dependent like that of SsnA.…”

“…Although we detected the SpnA protein as well as M protein, which is a typical membrane protein and may be released from the streptococcal surface (Herwald et al, 2004), in the culture supernatant in SF370, we detected SpnA also in the insoluble fraction by shotgun proteomic analysis. In addition, SpnA was reported to be anchored to the cell wall (Severin et al, 2007), as was the SpnA homologue SsnA (Fontaine et al, 2004). Furthermore, SpnA contains the LPxTG motif of surface proteins of Gram-positive cocci, and was also predicted to be a membrane protein by the SOSUI program.…”

“…In these comprehensive analyses of culture supernatant proteins, we detected another putative DNase protein, Spy0747, in the culture supernatant of S. pyogenes SF370. The Spy0747 protein is homologous to the SsnA protein, which is a cell-wall-located DNase expressed in Streptococcus suis serotype 2 that requires Ca 2+ and Mg 2+ for its activity (Fontaine et al, 2004). Hence we designated the Spy0747 protein as SpnA.…”

Protective capacities of cell surface-associated proteins of Streptococcus suis mutants deficient in divalent cation-uptake regulators

“…RNA was isolated as described elsewhere (Fontaine et al, 2004), with slight modifications. A 10 ml sample of an S. suis culture in the mid-exponential growth phase (OD 600 0.6) was collected by centrifugation at 8000 g for 10 min.…”