title: Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.
abstract: β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Alfa
authors.initials: A
authors.lastname: Herrera
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Bao G
authors.initials: BG
authors.lastname: Vu
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Stach
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Merriman
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Horswill
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Wilmara
authors.initials: W
authors.lastname: Salgado-Pabón
authors.affiliation: Department of Microbiology, University of Iowa Carver College of Medicine , Iowa City, Iowa 52242, United States.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schlievert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000009:adverse effects* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D004696:Endocarditis / Q000201:enzymology / Q000209:etiology* / Q000473:pathology; D005260:Female; D006460:Hemolysin Proteins / Q000009:adverse effects* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008025:Ligases / Q000172:deficiency*; D008297:Male; D011487:Protein Conformation; D011817:Rabbits; D018805:Sepsis / Q000201:enzymology / Q000209:etiology* / Q000473:pathology; D013108:Sphingomyelin Phosphodiesterase / Q000009:adverse effects / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D013203:Staphylococcal Infections / Q000150:complications / Q000382:microbiology; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D013108:Sphingomyelin Phosphodiesterase; C029878:hlb protein, Staphylococcus aureus; D008025:Ligases