Structure of a mutant ß toxin from Staphylococcus aureus reveals domain swapping and conformational flexibility.

The 3.35 Šresolution 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.

Beta toxin catalyzes formation of nucleoprotein matrix in staphylococcal biofilms.

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.

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab -/- mice.

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.

Structure and biological activities of beta toxin from Staphylococcus aureus.

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.

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides.

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.

Structure of the streptococcal cell wall C5a peptidase.

The structure of a cell surface enzyme from a gram-positive pathogen has been determined to 2-A resolution. Gram-positive pathogens have a thick cell wall to which proteins and carbohydrate are covalently attached. Streptococcal C5a peptidase (SCP), is a highly specific protease and adhesin/invasin. Structural analysis of a 949-residue fragment of the [D130A,S512A] mutant of SCP from group B Streptococcus (S. agalactiae, SCPB) revealed SCPB is composed of five distinct domains. The N-terminal subtilisin-like protease domain has a 134-residue protease-associated domain inserted into a loop between two beta-strands. This domain also contains one of two Arg-Gly-Asp (RGD) sequences found in SCPB. At the C terminus are three fibronectin type III (Fn) domains. The second RGD sequence is located between Fn1 and Fn2. Our analysis suggests that SCP binding to integrins by the RGD motifs may stabilize conformational changes required for substrate binding.

Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis.

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.

Structure of catechol 1,2-dioxygenase from Pseudomonas arvilla.

Catechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the betabeta isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429-440.] that the catechol 1,2-dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the alpha and beta polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (alphaalpha, alphabeta, and betabeta) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203-212) is proposed to be central in communicating between the lipid binding site and the active site.

Biophysical analyses of designed and selected mutants of protocatechuate 3,4-dioxygenase1.

The catechol dioxygenases allow a wide variety of bacteria to use aromatic compounds as carbon sources by catalyzing the key ring-opening step. These enzymes use specifically either catechol or protocatechuate (2,3-dihydroxybenozate) as their substrates; they use a bare metal ion as the sole cofactor. To learn how this family of metalloenzymes functions, a structural analysis of designed and selected mutants of these enzymes has been undertaken. Here we review the results of this analysis on the nonheme ferric iron intradiol dioxygenase protocatechuate 3,4-dioxygenase.

Characterization of a novel staphylococcal enterotoxin-like superantigen, a member of the group V subfamily of pyrogenic toxins.

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.