RESUMEN
The 3.35â Å resolution crystal structure of a mutant form of the staphylococcal sphingomyelinase ß toxin in which a conserved hydrophobic ß-hairpin has been deleted is reported. It is shown that this mutation induces domain swapping of a C-terminal ß-strand, leading to the formation of dimers linked by a conformationally flexible hinge region. Eight dimers are seen in the asymmetric unit, exhibiting a broad spectrum of conformations trapped in place by intermolecular contacts within the crystal lattice. Furthermore, the 16 monomers within each asymmetric unit exhibit a remarkable heterogeneity in thermal factors, which can be accounted for by the varying degrees to which each monomer interacts with other molecules in the crystal. This structure provides a unique example of the challenges associated with crystallographic study of flexible proteins.
Asunto(s)
Toxinas Bacterianas/química , Staphylococcus aureus/química , Toxinas Bacterianas/genética , Cristalografía por Rayos X , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus aureus/genéticaRESUMEN
Biofilms are surface-associated communities of microbes encompassed by an extracellular matrix. It is estimated that 80% of all bacterial infections involve biofilm formation, but the structure and regulation of biofilms are incompletely understood. Extracellular DNA (eDNA) is a major structural component in many biofilms of the pathogenic bacterium Staphylococcus aureus, but its role is enigmatic. Here, we demonstrate that beta toxin, a neutral sphingomyelinase and a virulence factor of S. aureus, forms covalent cross-links to itself in the presence of DNA (we refer to this as biofilm ligase activity, independent of sphingomyelinase activity) producing an insoluble nucleoprotein matrix in vitro. Furthermore, we show that beta toxin strongly stimulates biofilm formation in vivo as demonstrated by a role in causation of infectious endocarditis in a rabbit model. Together, these results suggest that beta toxin cross-linking in the presence of eDNA assists in forming the skeletal framework upon which staphylococcal biofilms are established.
Asunto(s)
Toxinas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas Hemolisinas/metabolismo , Nucleoproteínas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Staphylococcus/crecimiento & desarrollo , Animales , Catálisis , ADN Bacteriano , Endocarditis , Conejos , Staphylococcus/patogenicidadRESUMEN
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 scavenge nutrients. The structure of beta toxin was determined at 2.4-A resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human 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 that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.