title: NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.
abstract: A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Onkar
authors.initials: O
authors.lastname: Sharma
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Maghnus
authors.initials: M
authors.lastname: O'Seaghdha
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Jorge J
authors.initials: JJ
authors.lastname: Velarde
authors.affiliation: Division of Infectious Diseases, Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Wessels
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival; D004847:Epithelial Cells / Q000382:microbiology; D005098:Exotoxins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology / Q000382:microbiology; D015603:Keratinocytes / Q000382:microbiology; D008247:Lysosomes / Q000382:microbiology; D009244:NAD+ Nucleosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins; D013290:Streptococcal Infections / Q000276:immunology / Q000382:microbiology*; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000276:immunology / Q000472:pathogenicity / Q000502:physiology; D013301:Streptolysins / Q000378:metabolism*; D014774:Virulence
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; D011994:Recombinant Proteins; D013301:Streptolysins; C030325:anthrax toxin; C025964:streptolysin O; D009244:NAD+ Nucleosidase