Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens.

BACKGROUND: New and improved antimicrobial countermeasures are urgently needed to counteract increased resistance to existing antimicrobial treatments and to combat currently untreatable or new emerging infectious diseases. We demonstrate that computational comparative genomics, together with experimental screening, can identify potential generic (i.e., conserved across multiple pathogen species) and novel virulence-associated genes that may serve as targets for broad-spectrum countermeasures. RESULTS: Using phylogenetic profiles of protein clusters from completed microbial genome sequences, we identified seventeen protein candidates that are common to diverse human pathogens and absent or uncommon in non-pathogens. Mutants of 13 of these candidates were successfully generated in Yersinia pseudotuberculosis and the potential role of the proteins in virulence was assayed in an animal model. Six candidate proteins are suggested to be involved in the virulence of Y. pseudotuberculosis, none of which have previously been implicated in the virulence of Y. pseudotuberculosis and three have no record of involvement in the virulence of any bacteria. CONCLUSION: This work demonstrates a strategy for the identification of potential virulence factors that are conserved across a number of human pathogenic bacterial species, confirming the usefulness of this tool.

Analysis of protection afforded by a Clostridium perfringens alpha-toxoid against heterologous clostridial phospholipases C.

The major virulence determinant in clostridial myonecrosis caused by Clostridium perfringens is a phospholipase C (PLC), the alpha-toxin. Previously, mice have been protected against challenge with heterologous alpha-toxin or Clostridium perfringens spores by immunisation with the C-domain (known as Cpa(247-370) or alpha-toxoid) of the alpha-toxin. In this study, we have determined the ability of the alpha-toxoid to protect against the lethal effects of a divergent C. perfringens alpha-toxin and against the PLCs of C. absonum or C. bifermentans, species which have been isolated from cases of clostridial myonecrosis. Protection against the C. perfringens alpha-toxin variant, the C. absonum alpha-toxin or the C. bifermentans PLC was elicited by immunisation with the alpha-toxoid in vivo.

Analysis of known bacterial protein vaccine antigens reveals biased physical properties and amino acid composition.

Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location.

Structural and functional role of threonine 112 in a superantigen Staphylococcus aureus enterotoxin B.

Bacterial superantigens are potent T-cell stimulatory protein molecules produced by Staphylococcus aureus and Streptococcus pyogenes. Their superantigenic activity can be attributed to their ability to cross-link major histocompatibility complex class II molecules with T-cell receptors (TCRs) to form a tri-molecular complex. Each superantigen is known to interact with a specific V(beta) element of TCR. Staphylococcal enterotoxin B (SEB, a superantigen), a primary cause of food poisoning, is also responsible for a significant percentage of non-menstrual associated toxic shock syndrome in patients with a variety of staphylococcal infections. Structural studies have elucidated a binding cavity on the toxin molecule essential for TCR binding. To understand the crucial residues involved in binding, mutagenesis analysis was performed. Our analysis suggest that mutation of a conserved residue Thr(112) to Ser (T112S) in the binding cavity induces a selective reduction in the affinity for binding one TCR V(beta) family and can be attributed to the structural differences in the native and mutant toxins. We present a detailed comparison of the mutant structure determined at 2.0 A with the previously reported native SEB and SEB-TCR V(beta) complex structures.