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 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
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
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.
University of Minnesota Ph.D. dissertation. June 2009. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Douglas Ohlendorf. 1 computer file (PDF); x, 116 pages.
Huseby, Medora Jean.
Structure and activities of beta toxin: a virulence factor of Staphylococcus aureus.
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