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.

The major virulence determinant in clostridial myonecrosis caused by Clostridium perfringens is a phospholipase C (PLC), the α-toxin. Previously, mice have been protected against challenge with heterologous α-toxin or Clostridium perfringens spores by immunisation with the C-domain (known as Cpa247–370 or α-toxoid) of the α-toxin. In this study, we have determined the ability of the α-toxoid to protect against the lethal effects of a divergent C. perfringens α-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 α-toxin variant, the C. absonum α-toxin or the C. bifermentans PLC was elicited by immunisation with the α-toxoid in vivo.

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.

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 11 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 nonmenstrual 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 Angstrom with the previously reported native SEB and SEB-TCR V-beta complex structures.

Differences in the biological properties of the Clostridium perfringens phospholipase C (α‐toxin) and the C. bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy‐terminal domains. Three residues in the carboxy‐terminal domain of α‐toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively). We have characterised D269Y, Y331L and F334I variant forms of α‐toxin. Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity. Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp.