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

• Published in: Microbiology (Society for General Microbiology) 2010-01, Vol.156 (1), p.184-190
• Main Authors: Hasegawa, Tadao, Minami, Masaaki, Okamoto, Akira, Tatsuno, Ichiro, Isaka, Masanori, Ohta, Michio
• Format: Article
• Language: English
• Subjects: Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Cell Wall - enzymology; Deoxyribonucleases - genetics; Deoxyribonucleases - metabolism; Electrophoresis, Gel, Two-Dimensional; Female; Fundamental and applied biological sciences. Psychology; Gene Knockout Techniques; Mice; Mice, Inbred ICR; Microbiology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Peptide Mapping; Proteomics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Streptococcus pyogenes - enzymology; Streptococcus pyogenes - genetics; Streptococcus pyogenes - pathogenicity; Virulence
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.031955-0

1 Department of Bacteriology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan 2 Department of Bacteriology, Nagoya University Graduate School of Medicine, Nagoya, Japan We investigated culture supernatant proteins from the M1 serotype of Streptococcus pyogenes by two-dimensional gel electrophoresis and peptide mass mapping analysis, and characterized the single protein spots. Among them, we analysed the Spy0747 protein. This protein is homologous to the SsnA protein, a cell-wall-located DNase expressed in Streptococcus suis serotype 2. We designated the Spy0747 protein as SpnA. SpnA protein was also detected in the insoluble fraction of whole-cell lysates using shotgun proteomic analysis, suggesting that SpnA is also located in the cell wall. SpnA was expressed as a glutathione S -transferase-fusion protein in Escherichia coli . We confirmed that the recombinant protein had DNase activity that was dependent on Ca 2+ and Mg 2+ , like SsnA. Blood bactericidal assays and mouse infection model experiments showed that the spnA knockout strain was less virulent than the parental strain, thus suggesting that SpnA could play an important role in virulence. Using PCR, we found that the spnA gene was present in all clinical S. pyogenes strains we examined. Our results, together with a previous report identifying Spy0747 as a surface-associated protein, suggest that SpnA is an important cell-wall-located DNase that is generally produced in S. pyogenes and is involved in virulence. Correspondence Tadao Hasegawa tadaoh{at}med.nagoya-cu.ac.jp Abbreviations: 2-DE, two-dimensional gel electrophoresis; GST, glutathione S -transferase; NET, neutrophil extracellular trap; STSS, streptococcal toxic shock syndrome A supplementary table showing the results of shotgun proteomic analysis is available with the online version of this paper.