Showing papers by "Alison D. O'Brien published in 2007"

Bacillus anthracis exosporium protein BclA affects spore germination, interaction with extracellular matrix proteins, and hydrophobicity.

Abstract: Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ΔbclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ΔbclA and wild-type spores. The ΔbclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.

Recombinant Exosporium Protein BclA of Bacillus anthracis Is Effective as a Booster for Mice Primed with Suboptimal Amounts of Protective Antigen

Abstract: Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protection to only 1 of 10 animals. However, all mice that received suboptimal amounts of recombinant PA and that then received rBclA 2 weeks later survived spore challenge. Additionally, anti-rBclA IgG, compared to anti-PA IgG, promoted a sevenfold-greater uptake of opsonized spores by mouse macrophages and markedly decreased intramacrophage spore germination. Since BclA has some sequence similarity to human collagen, we also tested the extent of binding of anti-rBclA antibodies to human collagen types I, III, and V and found no discernible cross-reactivity. Taken together, these results support the concept of rBclA as being a safe and effective boost for a PA-primed individual against anthrax and further suggest that such rBclA-enhanced protection occurs by the induction of spore-opsonizing and germination-inhibiting antibodies.

Rapid Detection of Shiga Toxin-Producing Escherichia coli by Optical Immunoassay

Abstract: In a multi-health center study, a new rapid optical immunoassay (OIA) for the detection of Shiga toxin types 1 and 2, the BioStar OIA SHIGATOX kit (Inverness Medical Professional Diagnostics, Inc.), was used to prospectively screen 742 fresh fecal samples for Shiga toxins in parallel with the Premier enterohemorrhagic Escherichia coli (EHEC) kit (Meridian BioScience, Inc.) with and without enrichment of the specimens by incubation in MacConkey broth. Additionally, 85 previously tested frozen fecal samples were assessed as described above. All positive immunoassay results were confirmed by the Vero cell cytotoxicity assay. A further modification of the screening procedure was evaluated on 470 of the prospectively screened specimens. Swabs of growth from conventionally plated stool culture media were subjected to the OIA SHIGATOX, and results were compared with those obtained with the Premier EHEC kit following broth enrichment. Overall, the OIA SHIGATOX kit was significantly more sensitive than the Premier EHEC kit on fresh direct stool specimens (sensitivities, 96.8% and 83.9%, respectively; P < 0.05). The two assays performed equally well with each other on frozen and broth-enriched samples. The colony sweep method used in conjunction with the OIA kit was somewhat more effective at detection of Shiga toxins from growth on agar than the overnight broth enrichment procedure used with the Premier EHEC assay (sensitivities, 100% and 92%, respectively; P < 0.09). Overall, the OIA SHIGATOX kit provided rapid, easy-to-interpret results and was highly effective at detection of Shiga toxin-producing E. coli in fecal samples and overnight cultures.

Two domains of cytotoxic necrotizing factor type 1 bind the cellular receptor, laminin receptor precursor protein.

Abstract: Cytotoxic necrotizing factor type 1 (CNF1) and CNF2 are highly homologous toxins that are produced by certain pathogenic strains of Escherichia coli. These 1,014-amino-acid toxins catalyze the deamidation of a specific glutamine residue in RhoA, Rac1, and Cdc42 and consist of a putative N-terminal binding domain, a transmembrane region, and a C-terminal catalytic domain. To define the regions of CNF1 that are responsible for binding of the toxin to its cellular receptor, the laminin receptor precursor protein (LRP), a series of CNF1 truncated toxins were characterized and assessed for toxin binding. In particular, three truncated toxins, ΔN63, ΔN545, and ΔC469, retained conformational integrity and in vitro enzymatic activity and were immunologically reactive against a panel of anti-CNF1 monoclonal antibodies (MAbs). Based on a comparison of these truncated toxins with wild-type CNF1 and CNF2 in LRP and HEp-2 cell binding assays and in MAb and LRP competitive binding inhibition assays and based on the results of confocal microscopy, we concluded that CNF1 contains two major binding regions: one located within the N terminus, which contained amino acids 135 to 164, and one which resided in the C terminus and included amino acids 683 to 730. The data further indicate that CNF1 can bind to an additional receptor(s) on HEp-2 cells and that LRP can also serve as a cellular receptor for CNF2.

Patient dependency in emergency care: Do we have the nurses we need?

Abstract: Aims and objectives. To use the previously validated Jones Dependency Tool to establish current patterns in the dependency of patients attending the Emergency Department of an inner city teaching hospital, thereby informing present and future decisions regarding nursing numbers and grade mix. Background. Nursing salaries are the largest single expenditure in the National Health Service, but the number and grade of nursing staff are often determined by historical or arbitrary measures. Staffing calculations based only on the number of patients fail to take account of dependency and, hence, the actual nursing workload. Design and methods. The Jones Dependency Tool, a validated measure of Emergency Department patient dependency, was used prospectively to collect data on all patients attending an adult Emergency Department over a two-week period. The primary outcome measure was patient dependency assessed on arrival and departure and at four and eight hours if the patient was still in the Emergency Department. Data were analysed using descriptive statistics and chi-squared testing. Results. Data were collected on 48% of all patient attendances; 10% of patients were in the high- or total-dependency groups on arrival. Patients tended to become less dependent during their Emergency Department stay and analysis of those individuals in whom dependency increased was valuable. The proportion of high and total-dependency patients did not vary by day of the week, but was significantly increased overnight. This emphasizes the weakness of basing staffing levels on patient numbers alone. Conclusion. Patient dependency is one of the essential determinants of nursing grade mix and with further work and adaptation the Jones Dependency Tool can be used to predict workload, resource use and the optimal staffing levels that will provide safe and effective patient care. Relevance to clinical practice. Dependency can be readily and repeatedly assessed and we recommend this approach to other Emergency Departments.

Shiga toxoid chimeric proteins

Abstract: A chimeric Shiga toxoid according to the invention contains an enzymatically- inactivated StxA subunit and a native StxB subunit. This hybrid Shiga toxoid induces the production of broadly cross-reactive species of antibodies against Shiga toxin following immunization. The StxA subunit is modified so that it is enzymatically inactive. The invention thus encompasses the Shiga toxoid or fragments thereof and the nucleic acid sequence of the Shiga toxoid or fragments thereof. The invention further encompasses the production of a Shiga toxoid, the production of antibodies using the Shiga toxoid and methods of productions, and an immunogenic composition containing the Shiga toxoid.

Shiga Toxins (Stxs): Multifaceted Pathogenicity Determinants

Abstract: The Shiga toxins (Stxs) (also known as Vero toxins and formerly called Shiga-like toxins) constitute a family of toxins that features Stx from Shigella dysenteriae type 1 as the prototype. Among bacterial toxins, the Stxs are particularly potent poisons that are estimated to be as toxic per unit of weight as tetanus toxin and second only to botulinum toxin in potency. The major difference among the Stxs is reflected in the division of the Stx family into two groups, Stx/Stx1 and Stx2, based on the capacity of antitoxin to neutralize the cytotoxic activity of each member of the homologous but not the heterologous group. The Stx-producing Escherichia coli (STEC) bacteria grow within the colon and elaborate Stx1 and/or Stx2. The toxin crosses the intestinal barrier and enters the bloodstream. Once in the bloodstream, the toxin may bind to polymorphonuclear cells and circulate. The major target organ of the Stxs is the kidney, although sites within the central nervous system may also be affected. The interaction of Stx and bacterial lipopolysaccharide with cells of the immune system and locally within the kidney may lead to increased cytokine and chemokine expression. Some epidemiological evidence suggests that STEC strains that produce Stx2 cause more severe disease than STEC strains that make Stx1 or both Stx1 and Stx2.