Inactivated whole-cell bacterial vaccines: current status and novel strategies.

Inactivated bacterial whole-cell vaccines have been the most widely studied prophylactic treatment for infectious diseases. They offer an economical, and potentially safe, effective means of preventing disease. The disadvantages of these vaccines have been that parenteral administration, while effective in some instances, may have caused adverse reactions in vaccinees, while oral administration often required high doses and resulted in short-term immunity. More recent studies describing new approaches for improving antigenicity of inactivated whole-cell vaccines and the enhancement of immune responses to oral immunization offer great hope for improving the efficacy of these agents. Promising whole cell vaccines include those against Vibrio cholerae, enterotoxigenic Escherichia coli, and more recently Campylobacter jejuni.

Development of a Dietary Exposure Potential Model for evaluating dietary exposure to chemical residues in food.

The Dietary Exposure Potential Model (DEPM) is a computer-based model developed for estimating dietary exposure to chemical residues in food. The DEPM is based on food consumption data from the 1987-1988 Nationwide Food Consumption Survey (NFCS) administered by the United States Department of Agriculture (USDA) and on residue data from government-sponsored monitoring programs. Foods reported in the NFCS were categorized into exposure core foods (ECFs). A computer program for DOS-based personal computers was developed to link consumption of the ECFs with residue values observed in the foods. The data files utilized by the DEPM were designed in dBASE IV with FoxPro for Windows applications programs for queries and reporting. The program calculates exposure estimates for categories of core foods, such as grain dishes, fruits, or vegetables; for individual core foods, such as wheat and apple combination dishes; and for individual foods, such as apples or carrots. The program, residue summary databases, and core food consumption database permit the analyst to evaluate potential exposure of several population groups to various chemicals via the diet. The DEPM is not intended for risk assessments, but is a suitable tool for identifying data gaps and establishing priorities for research, and for identifying potentially significant foods for human exposure monitoring.

Functional analysis of the Pseudomonas aeruginosa autoinducer PAI.

A series of structural analogs of the Pseudomonas aeruginosa autoinducer [PAI, N-3-oxo-dodecanoyl homoserine lactone] were obtained and tested for their ability to act as autoinducers in stimulating the expression of the gene for elastase (lasB) by measuring beta-galactosidase production from a lasB-lacZ gene fusion in the presence of the transcriptional activator LasR. The data suggest that the length of the acyl side chain of the autoinducer molecule is the most critical factor for activity. Replacement of the ring O by S in the homoserine lactone moiety can be tolerated. Tritium-labelled PAI ([3H]PAI) was synthesized and used to demonstrate the association of [3H]PAI with cells overexpressing LasR. The PAI analogs were also tested for their ability to compete with [3H]PAI for binding of LasR. Results from the competition assays suggest that once again the length of the acyl side chain appears to be crucial for antagonist activity. The presence of the 3-oxo moiety also plays a significant role in binding since analogs which lacked this moiety were much less effective in blocking binding of [3H]PAI. All analogs demonstrating competition with PAI in binding to LasR also exhibited the ability to activate lasB expression, suggesting that they are functional analogs of PAI.

Structure-based design of novel, urea-containing FKBP12 inhibitors.

The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.

Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.

Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.

In Pseudomonas aeruginosa the LasR protein is required for activation of lasB and several other virulence genes. A diffusible signal molecule, the P. aeruginosa autoinducer (PAI), produced by the bacterial cell and released into the growth medium, is required for activity of LasR. By cloning a lasB::lacZ fusion and a lasR gene under control of the lac promoter in Escherichia coli, we have developed a quantitative bioassay for PAI. We have used this assay to follow the purification of PAI from cell-free culture supernatant fluids in which P. aeruginosa or E. coli containing the P. aeruginosa gene required for autoinducer synthesis, lasI, had been grown. Chemical analyses indicated the purified material was 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)dodecanamide. To confirm this assignment, the compound was synthesized and the synthetic compound was shown to have chemical and biological properties identical to those of PAI purified from culture supernatant fluids. The elucidation of the PAI structure suggests therapeutic approaches toward control of P. aeruginosa infections.

Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y.

Clostridium difficile causes pseudomembranous colitis in humans. The enterotoxin (i.e., toxin A) from this organism is believed to be responsible for the initial intestinal pathology associated with this disease. Previous work shows that this toxin binds to carbohydrates that contain Gal alpha 1-3Gal beta 1-4GlcNAc. However, this carbohydrate is not present on normal human cells. Therefore, this study was undertaken to identify potential receptors for toxin A that do exist on human intestinal epithelium. Using enzyme-linked immunosorbent assay, affinity chromatography, and altered migration in an electric field, we assayed the binding of toxin A to purified carbohydrates and glycoproteins. We found that toxin A bound to the carbohydrate antigens designated I, X, and Y. Each of these carbohydrates exist on the intestinal epithelium of humans.

Toxin A of Clostridium difficile is a potent cytotoxin.

Clostridium difficile is the cause of antibiotic-associated colitis in humans. The organism produces toxin A, which is generally known as the enterotoxin, and toxin B, which is known as the cytotoxin. Toxin A has been reported to have slight cytotoxic activity; in this study we show that cell lines (F9, OTF9-63, and P19) which express a carbohydrate to which toxin A binds are more sensitive to the toxin. These cell lines can be used as research tools for determining concentrations of biologically active toxin A and should also prove useful for studies of the mechanism of action of the toxin.