Browsing by Author "Huseby, Medora Jean"

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    Structure and activities of beta toxin: a virulence factor of Staphylococcus aureus
    (2009-06) Huseby, Medora Jean
    Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as to scavenge nutrients. The structure of beta toxin was determined at 2.4 Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show the biological activities of hemolysis and lymphotoxicity are due to the sphingomyelinase activity of the enzyme. The structures of all bacterial neutral sphingomyelinases solved to date reveal a solvent exposed hydrophobic beta hairpin. We examined the role of this beta hairpin in beta toxin virulence. Altering the length but not the content of the beta hairpin attenuates the biological activities associated with beta toxin. The beta hairpin is an important stabilizing structure. X-ray crystallographic analysis of beta hairpin mutants revealed very minimal structural changes. We show for the first time diacylglycerol bound in the beta toxin truncation (275-280) structure near the beta hairpin region. We also show at 1.75 Å resolution Mg2+ and phosphate bound to the F277A P289A structure. Neutral sphingomyelinases belong to the DNase I super-family of proteins (CATH class 3.60). Beta toxin shares the overall fold of DNase I with an RMSD value 3.3 Å over 220 Cαs. Beta toxin does not function as a DNase and instead precipitates nucleic acid. DNA causes beta toxin to non-specifically cross-link to other proteins. Extracellular DNA is a major structural component of the S. aureus biofilm matrix. Here we demonstrate that beta toxin has a profound effect on forming the matrix on which biofilms grow through the nucleic acid-dependent formation of cross-linked beta toxin monomers as well as other proteins. These links, plus the ability to bind eDNA, enables formation of the underlying nucleoprotein matrix essential to establish a biofilm. The goal of this thesis project is to understand the structural foundations for the role of the virulence factor beta toxin in order to understand the biological mechanism that allows S. aureus to successfully invade, colonize, and attack a host.