title: Streptococcal 5'-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion.
abstract: Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 μm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.
authors.affiliation: From the Department of Molecular Medicine and Pathology, School of Medical Sciences, and.
authors.forename: Lisa
authors.initials: L
authors.lastname: Zheng
authors.affiliation: From the Department of Molecular Medicine and Pathology, School of Medical Sciences, and.
authors.forename: Adrina
authors.initials: A
authors.lastname: Khemlani
authors.affiliation: From the Department of Molecular Medicine and Pathology, School of Medical Sciences, and the Maurice Wilkins Center, University of Auckland, Auckland, New Zealand.
authors.forename: Natalie
authors.identifier: http://orcid.org/0000-0002-5480-1714
authors.initials: N
authors.lastname: Lorenz
authors.affiliation: From the Department of Molecular Medicine and Pathology, School of Medical Sciences, and the Maurice Wilkins Center, University of Auckland, Auckland, New Zealand.
authors.forename: Jacelyn M S
authors.identifier: http://orcid.org/0000-0002-9428-1215
authors.initials: JM
authors.lastname: Loh
authors.affiliation: From the Department of Molecular Medicine and Pathology, School of Medical Sciences, and the Maurice Wilkins Center, University of Auckland, Auckland, New Zealand.
authors.forename: Ries J
authors.initials: RJ
authors.lastname: Langley
authors.affiliation: From the Department of Molecular Medicine and Pathology, School of Medical Sciences, and the Maurice Wilkins Center, University of Auckland, Auckland, New Zealand t.proft@auckland.ac.nz.
authors.forename: Thomas
authors.identifier: http://orcid.org/0000-0002-9275-5042
authors.initials: T
authors.lastname: Proft
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D001770:Blood Bactericidal Activity / Q000276:immunology*; D006801:Humans; D057131:Immune Evasion; D013294:Lactococcus lactis / Q000235:genetics / Q000276:immunology; D008264:Macrophages; D050296:Microbial Viability / Q000235:genetics / Q000276:immunology; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000276:immunology*; D013297:Streptococcus pyogenes / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity*; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors; D009699:N-Glycosyl Hydrolases
keywords: Streptococcus pyogenes (S. pyogenes); adenosine; enzyme catalysis; group A streptococcus; immune evasion; model structure; mutation analysis; nucleoside/nucleotide metabolism; virulence factor