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Bacillus cereusG9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried byBacillus anthracisand encodes the tripartite anthrax toxin genes andatxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle ofB. cereusG9241 using a transcriptomic analysis and by studying spore formation, an important part of theB. anthracislifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37°C in comparison to 25°C. At 37°C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showedB. cereusG9241 sporulated rapidly in comparison to theB. cereussensu stricto type strain ATCC 14579, particularly at 37°C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37°C in comparison to 25°C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant ofB. cereusG9241. This study provides an insight on how the extrachromosomal genetic elements inB. cereusG9241 has an influence in bacterial phenotypes.

Bacillus cereusG9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other: the pleiotropic transcriptional regulator PlcR found in most members of theBacillus cereusgroup but truncated in allBacillus anthracisisolates, and the anthrax toxin regulator AtxA found in allB. anthracisstrains and a fewB. cereussensu stricto strains. Here we report cytotoxic and haemolytic activity of cell freeB. cereusG9241 culture supernatants cultured at 25 °C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37 °C, behaving much like the supernatants generated byB. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25 °C compared to 37 °C. Furthermore, we demonstrate that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37 °C, giving rise to the temperature-dependent haemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on howB. cereusG9241 is able to ‘switch’ betweenB. cereusandB. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA.

Antibiotics are very important for treating bacterial infections in humans, pets, and livestock. However, improper use of antibiotics has resulted in antibiotic resistance, making it difficult to treat some infections. There is an urgent need to discover new antibiotics, but how do we find them? The answer potentially lies with bees and the honey they produce. Bees who feed on unique plants produce honey with high levels of antibiotic activity, due to the presence of antibacterial compounds in the nectar that the bees collect to make honey. In addition to nectar, the bees also collect pollen, which contains a sample of each visited plant’s unique DNA. By studying this DNA, we have identified plants that are sources of the antibacterial compounds found in honey. We are now working to develop new antibiotics from this honey, and we have installed beehives on university buildings to create our own super honey.

Challenges faced by schools include how to make science interesting, relevant and engaging for their pupils. This can perhaps be made more difficult by staff members not having direct experience in many areas of the science they are tasked to teach, and therefore lacking the confidence to teach in these areas (Murphy et al., 2007). Within the higher education sector, there is demand for researchers to actively engage with the local community (Linder and Spear, 2003; Wynne, 2006). Working with eight schools, we co-developed a programme that both highlights academic research and complements the Key Stage 2 national curriculum with the aims of improving science aspirations, knowledge and confidence in Key Stage 2 (Year 6) children, supporting teachers in the delivery of areas of the curriculum identified as challenging, and increasing science communication and engagement within the university. We delivered two separate sessions with all eight primary schools, interacting with approximately three hundred and fifty pupils. Overall, the project had a positive impact on teachers, children and academic staff. Key findings indicated that 92.9 per cent of 348 children surveyed felt that they had learned something new, while 85.7 per cent surveyed felt they were more interested in science, and 14.3 per cent reported no change in their feelings towards science. The School Science Club represents a collaborative engagement project which highlights the requirement of accurate co-development and outcome settings from both the university and school in order to have a positive impact on all those involved. Findings suggested improvements in planning and delivery for future such endeavours.

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p

Whilst various remedial human monoclonal antibodies have been developed to treat the potentially life-threatening systemic complications associated with anthrax infection, an optimal and universally effective administration route has yet to be established. In the later stages of infection when antibody administration by injection is more likely to fail one possible route to improve outcome is via the use of an antibody-bound, adsorbent haemoperfusion device. We report here the development of an adsorbent macroporous polymer column containing immobilised B. anthracis exotoxin-specific antibodies, PANG (a non-glycosylated, version of a plant-produced human monoclonal antibody) and Valortim (a fully human monoclonal N-linked glycosylated antibody), for removal of anthrax protective antigen (PA) from freshly frozen human plasma and human whole blood. In addition, we have demonstrated that continuous extracorporeal blood recirculation through a Valortim-bound haemoperfusion column significantly reduced the blood plasma concentration of anthrax PA over 2 hours using an in vivo PA rat infusion model. This work provides proof-of-concept evidence to support the development of such alternative detoxification platforms.

Clostridium difficile is a major cause of hospital acquired infection that poses significant diagnostic challenges. In this paper, a new type of resonant microwave applicator is proposed for the liberation of DNA from C. difficile spores via microwave disruption, followed by rapid detection using a sandwich hybridization assay. A split-ring resonator is designed to operate at 2.4 GHz with a 3 mm active gap region that contains an isolate volume of approximately 10 mm 3 , exposed to pulsed microwaves of 12 W rms power. The parallel electric field configuration maximizes the interaction between the microwaves and the sample. In a proof of principle study, in combination with pathogen specific DNA probes, we have used the system to correctly identify virulent strains of C. difficile using magnetic bead extraction of DNA suitable for point-of-care application

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation., Author summary Anthrax and plague are ancient infectious diseases that continue to affect people living in poor, endemic regions and to threaten industrialized nations due to their potential use in biowarfare. Candidate vaccines need improvement to minimize non-desirable effects and increase their efficacy. The purpose of this work was to develop and evaluate a single subunit vaccine capable of conferring protection against Bacillus anthracis and Yersinia pestis. To this end, specific regions from their genome or key protective protein sequences from both microorganisms were combined to obtain either recombinant plasmids or recombinant proteins and tested as vaccine candidates in mice. The recombinant protein MaF2 induced specific antibody responses and afforded full and partial protection against Y. pestis and B. anthracis, respectively. Meanwhile, the DNA vaccine equivalent to MaF2 conferred only partial protection against Y. pestis, which increased when combined with an MaF2 protein boost. MaF2 emerged as a promising dual pathogen recombinant vaccine that warrants further investigation.

Microwaves are a form of non-ionizing radiation composed of electric (E) and magnetic (H) fields and are absorbed by biological tissues with a high water content. Our study investigated the effect of the E field, H field, and a combination of both (E + H) field’s exposure of structurally diverse micro-organisms, at a frequency of 2.45 GHz. We observed that the exposure to a microwave E field of an amplitude of 9.3 kV/m had no significant effect on cell viability; however, it did increase membrane permeability of Mycobacterium smegmatis to propidium iodide and to a range of different sized dextran particles in Escherichia coli, Staphylococcus aureus, Candida albicans, and M. smegmatis. The permeability of propidium iodide was observed in microwave treated cells (M. smegmatis) but not in heat-treated cells. Permeability of 3 kDa sized fluorescently labeled dextrans was observed across all cell types; however, this was found not to be the case for larger 70 kDa dextran particles. In terms of efflux, DNA was detected following E field exposure of M. smegmatis. In contrast, H field exposure had no effect on cell viability and did not contribute to increase cell’s membrane to dextran particles. In conclusion, this study shows that microwave generated E fields can temporarily disrupt membrane integrity without detrimentally impacting on cell viability. This approach has the potential to be developed as a high efficiency electropermeabilization method and as a means of releasing host DNA to support diagnostic applications.

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen’s global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1–3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region.

Tuberculosis is a major global health hazard. The search for new antimycobacterials has focused on such as screening combinational chemistry libraries or designing chemicals to target predefined pockets of essential bacterial proteins. The relative ineffectiveness of these has led to a reappraisal of natural products for new antimycobacterial drug leads. However, progress has been limited, we suggest through a failure in many cases to define the drug target and optimize the hits using this information. We highlight methods of target discovery needed to develop a drug into a candidate for clinical trials. We incorporate these into suggested analysis pipelines which could inform the research strategies to accelerate the development of new drug leads from natural products.

While proving highly effective in controlling Anthrax in farm animals all over the world currently attenuated live anthrax vaccines employed in a veterinary context suffer from drawbacks such as residual virulence, short term protection, variation in quality and, most importantly, lack of efficacy if administered simultaneously with antibiotics. These limitations have stimulated the development of non-living component vaccines which induce a broad spectrum immune response capable of targeting both toxaemia (as in the case of PA based vaccines) and bacteraemia. To contribute to this several new approaches were tested in outbred NMRI mice for antibody titres and protectiveness. Plasmids encoding a recombinant toxin derived fusion peptide and a spore surface derived peptide were tested as DNA-vaccines in comparison to their protein counterparts utilising two adjuvant approaches and two DNA-vector backbones. The combination of two plasmids encoding LFD1PAD4-mIPS1 and TPA-BclAD1D3-LAMP1, when delivered by GeneGun, protected 90% of the animals against a lethal challenge with 25LD50 spores of the Ames strain of Bacillus anthracis. Single applications of either antigen component showed significantly lower protection rates, indicating the beneficial interaction between anti-spore and anti-toxin components for an acellular vaccine formulation.

Clostridium difficile is the primary cause of healthcare associated diarrhoea globally and produces spores which are resistant to commonly used biocides and are able persist on contaminated surfaces for months. This study examined the effect of sublethal concentrations of the biocide sodium dichloroisocyanurate (NaDCC) on the viability of spores produced by 21 clinical isolates of C. difficile representing a range of PCR ribotypes. Spores exposed to 500 ppm NaDCC for 10 minutes exhibited between a 4 – 6 log10 reduction in viability which was independent of spore PCR ribotype. The effect of sublethal concentrations of biocide on the surface properties of exosporium positive and negative clinical isolates was determined using a spore adhesion to hydrocarbon assay. These isolates differed markedly in their responses suggesting that exposure to biocide can have a profound effect on hydrophobicity and thus the ability of spores to adhere to surfaces. This raises the intriguing possibility that sublethal exposure to NaDCC could inadvertently promote the spread of the pathogen in healthcare facilities.

A recent application of microwaves in the healthcare area is for the rapid detection of bacterial spores, particularly of clinically significant spores such as Clostridium difficile. Here, we present a working model of C. difficile spore disruption by the action of a 2.45 GHz microwave electric field, independent of overall sample heating. The model shows how inner layers of the spore with the lower complex permittivity values will be subject to higher values of the electric field. The model also shows how the electric effects can be enhanced, focusing the electric field into “hotspots” using “angled” nanoparticles, yielding effective DNA release even at low microwave power levels. The model’s predictions have been validated through experimental results, which show an enhancement of DNA release from spores.

Hostile reconnaissance is vital to successful terrorist activity. Individuals carrying out this activity are likely to experience raised levels of stress and this will manifest itself at biological, physiological, psychological and behavioural levels, providing an opportunity for detection. A field trial was undertaken in an ecologically valid environment measuring variables considered likely to be salient during hostile intent. The parameters examined in the field trial varied in a predictable manner and suggest that stressed individuals secrete a volatile steroid based marker that could form the basis for remote detection. Thus, overall the findings of this research provide a validated model of hostile intent that can be used by other researchers to test interventions aimed at detecting or deterring hostile intent.

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule -ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC50 for RT was 0.08 ± 0.004 ng/mL whereas the IC50 for RT + 100 μM eGCG was 3.02 ± 0.572 ng/mL, indicating that eGCG mediated a significant (p

Aim To determine the contribution of the exosporium, the outer layer of the Bacillus anthracis spore, to soil attachment. Persistence of spores in soil and their ability to infect animals has been linked to a range of factors which include the presence of organic material and calcium (OMC), pH > 6.0, temperatures above 15.5° C and cycles of local flooding which are thought to transport buried spores to the surface. Methods and Results The ability of wild type (exosporium +ve) and sonicated (exosporium -ve) spores to bind to soils which differed in their composition was determined using a flow-through soil column based method. A statistically significant difference (P

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.

There has been a long history of defining T cell epitopes to track viral immunity and to design rational vaccines, yet few data of this type exist for bacterial infections. Bacillus anthracis, the causative agent of anthrax, is both an endemic pathogen in many regions and a potential biological warfare threat. T cell immunity in naturally infected anthrax patients has not previously been characterized, which is surprising given concern about the ability of anthrax toxins to subvert or ablate adaptive immunity. We investigated CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax. Responses to B. anthracis protective Ag and lethal factor (LF) were investigated at the protein, domain, and epitope level. Several years after antibiotic-treated anthrax infection, strong T cell memory was detectable, with no evidence of the expected impairment in specific immunity. Although serological responses to existing anthrax vaccines focus primarily on protective Ag, the major target of T cell immunity in infected individuals and anthrax-vaccinated donors was LF, notably domain IV. Some of these anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns. Of specific CD4 T cell epitopes targeted within LF domain IV, one is preferentially seen in the context of bacterial infection, as opposed to vaccination, suggesting that studies of this type will be important in understanding how the human immune system confronts serious bacterial infection.

Previous reports suggested that lethal toxin (LT)-induced caspase-1 activity and/or IL-1β accounted for Bacillus anthracis (BA) infection lethality. In contrast, we now report that caspase-1-mediated IL-1β expression in response to BA spores is required for anti-BA host defenses. Caspase-1–/– and IL-1β–/– mice are more susceptible than wild-type (WT) mice to lethal BA infection, are less able to kill BA both in vivo and in vitro, and addition of rIL-1β to macrophages from these mice restored killing in vitro. Non-germinating BA spores induced caspase-1 activity, IL-1β and nitric oxide, by which BA are killed in WT but not in caspase-1–/– mice, suggesting that the spore itself stimulated inflammatory responses. While spores induced IL-1β in LT-susceptible and -resistant macrophages, LT induced IL-1β only in LT-susceptible macrophages. Cooperation between MyD88-dependent and -independent signaling pathways was required for spore-induced, but not LT-induced, IL-1β. While both spores and LT induced caspase-1 activity and IL-1β, LT did not induce IL-1β mRNA, and spores did not induce cell death. Thus different components of the same bacterium each induce IL-1β by distinct signaling pathways. Whereas the spore-induced IL-1β limits BA infection, LT-induced IL-1β enables BA to escape host defenses. Supporting Information for this article is available at www.wiley-vch.de/contents/jc_2040/2008/38141_s.pdf

The use of a combination of low-cost technologies to both extract and detect anthrax DNA from spores and vegetative cells in two steps within 1 min is described. In a cavity, microwave energy is highly focused using thin-film aluminum "bow-tie" structures, to extract DNA from whole spores within 20 s. The detection of the released DNA, from less than 1000 vegetative cells, without additional preprocessing steps is accomplished in an additional 30 s by employing the microwave-accelerated metal-enhanced fluorescence technique. The new platform technology presented here is a highly attractive alternative method for DNA extraction and the fast detection of gram-positive bacteria and potentially other pathogenic species and cells as well.

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called 'cryptic' or 'subdominant' epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes.

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy forBacillus anthracisinfection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] ofB. anthracisSterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected withB. anthracisSterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in activated macrophages is key to its germination and survival. In a previous publication, we discovered that exposure of primary murine macrophages to B. anthracis endospores upregulated NOS 2 concomitant with an ·NO-dependent bactericidal response. Since NOS 2 also generates O 2 · − , experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO − ) from the reaction of ·NO with O 2 · − and if so, was ONOO − microbicidal toward B. anthracis . Our findings suggest that ONOO − was formed upon macrophage infection by B. anthracis endospores; however, ONOO − does not appear to exhibit microbicidal activity toward this bacterium. In contrast, the exosporium of B. anthracis , which exhibits arginase activity, protected B. anthracis from macrophage-mediated killing by decreasing ·NO levels in the macrophage. Thus, the ability of B. anthracis to subvert ·NO production has important implications in the control of B. anthracis -induced infection.

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50100 μg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.

Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim®), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim® attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim® antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p

Individuals trying to conceal knowledge from interrogators are likely to experience raised\ud levels of stress that can manifest itself across biological, physiological, psychological and\ud behavioural factors, providing an opportunity for detection. Using established research\ud paradigms an innovative scalable interrogation was designed in which participants were\ud given a ‘token’ that represented information they had to conceal from interviewers. A control\ud group did not receive a token and therefore did not have to deceive the investigators. The\ud aim of this investigation was to examine differences between deceivers and truth-tellers\ud across the four factors by collecting data for cortisol levels, sweat samples, heart-rate,\ud respiration, skin temperature, subjective stress ratings and video and audio recordings. The\ud results provided an integrated understanding of responses to interrogation by those actively\ud concealing information and those acting innocently. Of particular importance, the results also\ud suggest, for the first time in an interrogation setting, that stressed individuals may secrete a\ud volatile steroid based marker that could be used for stand-off detection. The findings are\ud discussed in relation to developing a scalable interrogation protocol for future research in this\ud area.

The spore-forming, gram-positive bacterium Bacillus anthracis , the causative agent of anthrax, has achieved notoriety due to its use as a bioterror agent. In the environment, B. anthracis exists as a dormant endospore. Upon infection, germination of endospores occurs during their internalization within the phagocyte, and the ability to survive exposure to antibacterial killing mechanisms, such as O 2 ·− , NO · , and H 2 O 2 , is a key initial event in the infective process. Macrophages generate NO · from the oxidative metabolism of l -arginine, using an isoform of nitric oxide synthase (NOS 2). Exposure of murine macrophages (RAW264.7 cells) to B. anthracis endospores up-regulated the expression of NOS 2 12 h after exposure, and production of NO · was comparable to that achieved following other bacterial infections. Spore-killing assays demonstrated a NO · -dependent bactericidal response that was significantly decreased in the presence of the NOS 2 inhibitor l - N 6 -(1-iminoethyl)lysine and in l -arginine-depleted media. Interestingly, we also found that B. anthracis bacilli and endospores exhibited arginase activity, possibly competing with host NOS 2 for its substrate, l -arginine. As macrophage-generated NO · is an important pathway in microbial killing, the ability of endospores of B. anthracis to regulate production of this free radical has important implications in the control of B. anthracis -mediated infection.

The unpredictable nature of bio-terrorism compels us to develop medical countermeasures that will enable authorities to treat individuals exposed to agents such as anthrax. We report the feasibility of producing a protective, human-derived, monoclonal antibody directed against the protective antigen (PA) of Bacillus anthracis in plants. This was achieved by transient expression using agroinfiltration of Nicotiana benthamiana plants. The resulting antibody was able to neutralize toxin activity in vitro and in vivo at a comparable level to that seen for its hybridoma-produced counterpart.

DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development. DNA vaccination against anthrax is an active area of research showing promising results at present, which in the short-term and in the future could form the basis for new advances in multi-agent vaccine development. The anthrax 'model' constitutes an important experimental system for genetic immunisation technology development.

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.

Anthrax is a disease caused by a Gram-positive, aerobic, spore-forming bacillus called Bacillus anthracis. This bacterium primarily infects animals and also humans, sometimes with fatal consequence...

The protective antigen (PA) of Bacillus anthracis and the V antigen of Yersinia pestis are potent immunogens and candidate vaccine sub-units. When plasmid DNA encoding either PA or V antigen was used to immunise the Balb/c mouse, a low serum IgG titre was detected (log (10)1.0 or less) which was slightly increased by boosting with plasmid DNA. However, when mice immunised with plasmid DNA were later boosted with the respective recombinant protein, a significant increase in titre (up to 100-fold) was observed. Mice primed with a combination of each plasmid and boosted with a combination of the recombinant proteins, were fully protected (6/6) against challenge with Y. pestis. This compared favourably with mice primed only with plasmid DNA encoding the V antigen and boosted with rV, which were partially protected (3/6) against homologous challenge or with mice primed and boosted with plasmid DNA encoding the V antigen which were poorly protected (1/6). Combined immunisation with the two plasmid DNA constructs followed by boosting with a combination of the encoded recombinant proteins enhanced the protective immune response to Y. pestis compared with priming only with plasmid DNA encoding the V antigen and boosting with rV. This enhancement may be due to the effect of CpG motifs known to be present in the plasmid DNA construct encoding PA.