Early expression of capsule during Bacillus anthracis germination.

Bacillus anthracis is a spore-forming bacterium that produces two major virulence factors, a tripartite toxin with two enzymatic toxic activities and a pseudo-proteic capsule. One of the main described functions of the poly-gamma-d-glutamate capsule is to enable B. anthracis bacilli to escape phagocytosis. Thus, kinetics of expression of the capsule filaments at the surface of the emerging bacillus during germination is an important step for the protection of the nascent bacilli. In this study, through immunofluorescence and electron microscopic approaches, we show the emergence of the capsule through a significant surface of the exosporium in the vast majority of the germinating spores, with co-detection of BclA and capsular material. This suggests that, due to an early capsule expression, the extracellular life of B. anthracis might occur earlier than previously thought, once germination is triggered. This raises the prospect that an anti-capsular vaccine may play a protective role at the initial stage of infection by opsonisation of the nascent encapsulated bacilli before their emergence from the exosporium.

Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

In vivo germination of Bacillus anthracis spores during murine cutaneous infection.

BACKGROUND: Germination is a key step for successful Bacillus anthracis colonization and systemic dissemination. Few data are available on spore germination in vivo, and the necessity of spore and host cell interactions to initiate germination is unclear. METHODS: To investigate the early interactions between B. anthracis spores and cutaneous tissue, spores were inoculated in an intraperitoneal cell-free device in guinea pigs or into the pinna of mice. Germination and bacterial growth were analyzed through colony-forming unit enumeration and electron microscopy. RESULTS: In the guinea pig model, germination occurred in vivo in the absence of cell contact. Similarly, in the mouse ear, germination started within 15 minutes after inoculation, and germinating spores were found in the absence of surrounding cells. Germination was not observed in macrophage-rich draining lymph nodes, liver, and spleen. Edema and lethal toxin production were not required for germination, as a toxin-deficient strain was as effective as a Sterne-like strain. B. anthracis growth was locally controlled for 6 hours. CONCLUSIONS: Spore germination involving no cell interactions can occur in vivo, suggesting that diffusible germinants or other signals appear sufficient. Different host tissues display drastic differences in germination-triggering capacity. Initial control of bacterial growth suggests a therapeutic means to exploit host innate defenses to hinder B. anthracis colonization.

Cell-wall preparation containing poly-γ-D-glutamate covalently linked to peptidoglycan, a straightforward extractable molecule, protects mice against experimental anthrax infection.

Bacillus anthracis is the causative agent of anthrax that is characterized by septicemia and toxemia. Many vaccine strategies were described to counteract anthrax infection. In contrast with veterinary live vaccines, currently human vaccines are acellular with the protective antigen, a toxin component, as the main constituent. However, in animal models this vaccine is less efficient than the live vaccine. In this study, we analyzed the protection afforded by a single extractable surface element. The poly-γ-D-glutamate capsule is covalently linked to the peptidoglycan. A preparation of peptidoglycan-linked poly-γ-D-glutamate (GluPG) was tested for its immunogenicity and its protective effect. GluPG injection, in mice, elicited the production of specific antibodies directed against poly-glutamate and partially protected the animals against lethal challenges with a non-toxinogenic strain. When combined to protective antigen, GluPG immunization conferred full protection against cutaneous anthrax induced with a fully virulent strain.

Fast and sensitive detection of Bacillus anthracis spores by immunoassay.

Bacillus anthracis is one of the most dangerous potential biological weapons, and it is essential to develop a rapid and simple method to detect B. anthracis spores in environmental samples. The immunoassay is a rapid and easy-to-use method for the detection of B. anthracis by means of antibodies directed against surface spore antigens. With this objective in view, we have produced a panel of monoclonal antibodies against B. anthracis and developed colorimetric and electrochemiluminescence (ECL) immunoassays. Using Meso Scale Discovery ECL technology, which is based on electrochemiluminescence (ECL) detection utilizing a sulfo-Tag label that emits light upon electrochemical stimulation (using a dedicated ECL plate reader, an electrical current is placed across the microplate with electrodes integrated into the bottom of the plate, resulting in a series of electrically induced reactions leading to a luminescent signal), a detection limit ranging between 0.3 × 10(3) and 10(3) CFU/ml (i.e., 30 to 100 spores per test), depending on the B. anthracis strain assayed, was achieved. In complex matrices (5 mg/ml of soil or simulated powder), the detection level (without any sample purification or concentration) was never altered more than 3-fold compared with the results obtained in phosphate-buffered saline.

Noninvasive imaging technologies reveal edema toxin as a key virulence factor in anthrax.

Powerful noninvasive imaging technologies enable real-time tracking of pathogen-host interactions in vivo, giving access to previously elusive events. We visualized the interactions between wild-type Bacillus anthracis and its host during a spore infection through bioluminescence imaging coupled with histology. We show that edema toxin plays a central role in virulence in guinea pigs and during inhalational infection in mice. Edema toxin (ET), but not lethal toxin (LT), markedly modified the patterns of bacterial dissemination leading, to apparent direct dissemination to the spleen and provoking apoptosis of lymphoid cells. Each toxin alone provoked particular histological lesions in the spleen. When ET and LT are produced together during infection, a specific temporal pattern of lesion developed, with early lesions typical of LT, followed at a later stage by lesions typical of ET. Our study provides new insights into the complex spatial and temporal effects of B. anthracis toxins in the infected host, suggesting a greater role than previously suspected for ET in anthrax and suggesting that therapeutic targeting of ET contributes to protection.

Extended and global phylogenetic view of the Bacillus cereus group population by combination of MLST, AFLP, and MLEE genotyping data.

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database (http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing (Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.

Carbohydrate metabolism differences between subgroup A1 and B2 strains of Bacillus anthracis as assessed by comparative genomics and functional genetics.

In France, Bacillus anthracis subgroup B2 strains do not metabolize starch or glycogen but can use gluconate, whereas subgroup A1 strains show the inverse pattern. Functional genetic analysis revealed that mutations in the amyS and gntK genes encoding an alpha-amylase and a gluconate kinase, respectively, were responsible for these phenotypes.

Efficacy of a vaccine based on protective antigen and killed spores against experimental inhalational anthrax.

Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD(50)) and against an aerosol with 75 LD(50) of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.

Inhaled non-capsulated Bacillus anthracis in A/J mice: nasopharynx and alveolar space as dual portals of entry, delayed dissemination, and specific organ targeting.

Bacillus anthracis virulence is dependent on toxins and capsule. Encapsulation is associated with dissemination. We hypothesized that eliminating capsule would modify the portal of entry and the spread of bacteria. Using a bioluminescent model of inhalational anthrax, we demonstrated that aerosolized spores of a capsule-deficient strain administered at moderate doses initiated infection in the nasopharynx. Dissemination beyond the nasopharynx was delayed for at least 24h and then targeted the kidneys. Interestingly, high intranasal doses led to spore germination in the alveoli. We conclude that eliminating capsule while maintaining toxin production alters dissemination, but allows infection initiation in the lungs.

Bacillus anthracis: balancing innocent research with dual-use potential.

Anthrax Euronet, a Coordination Action of the EU 6th Framework Programme, was designed to strengthen networking activities between anthrax research groups in Europe and to harmonise protocols for testing anthrax vaccines and therapeutics. Inevitably, the project also addressed aspects of the current political issues of biosecurity and dual-use research, i.e. research into agents of important diseases of man, livestock or agriculture that could be used as agents of bioterrorism. This review provides a comprehensive overview of the biology of Bacillus anthracis, of the pathogenesis, epidemiology and diagnosis of anthrax, as well as vaccine and therapeutic intervention strategies. The proposed requirement for a code of conduct for working with dual-use agents such as the anthrax bacillus is also discussed.

Noncapsulated toxinogenic Bacillus anthracis presents a specific growth and dissemination pattern in naive and protective antigen-immune mice.

Bacillus anthracis is a spore-forming bacterium that causes anthrax. B. anthracis has three major virulence factors, namely, lethal toxin, edema toxin, and a poly-gamma-D-glutamic acid capsule. The toxins modulate host immune responses, and the capsule inhibits phagocytosis. With the goal of increasing safety, decreasing security concerns, and taking advantage of mammalian genetic tools and reagents, mouse models of B. anthracis infection have been developed using attenuated bacteria that produce toxins but no capsule. While these models have been useful in studying both toxinogenic infections and antitoxin vaccine efficacy, we questioned whether eliminating the capsule changed bacterial growth and dissemination characteristics. Thus, the progression of infection by toxinogenic noncapsulated B. anthracis was analyzed and compared to that by previously reported nontoxinogenic capsulated bacteria, using in vivo bioluminescence imaging. The influence of immunization with the toxin component protective antigen (PA) on the development of infection was also examined. The toxinogenic noncapsulated bacteria were initially confined to the cutaneous site of infection. Bacteria then progressed to the draining lymph nodes and, finally, late in the infection, to the lungs, kidneys, and frequently the gastrointestinal tract. There was minimal colonization of the spleen. PA immunization reduced bacterial growth from the outset and limited infection to the site of inoculation. These in vivo observations show that dissemination by toxinogenic noncapsulated strains differs markedly from that by nontoxinogenic capsulated strains. Additionally, PA immunization counters bacterial growth and dissemination in vivo from the onset of infection.

Primary involvement of pharynx and peyer's patch in inhalational and intestinal anthrax.

Bacillus anthracis causes three forms of anthrax: inhalational, gastrointestinal, and cutaneous. Anthrax is characterized by both toxemia, which is caused by secretion of immunomodulating toxins (lethal toxin and edema toxin), and septicemia, which is associated with bacterial encapsulation. Here we report that, contrary to the current view of B. anthracis pathogenesis, B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues. Furthermore, we found that Peyer's patches in the mouse intestine are the primary site of bacterial growth after intragastric inoculation, thus establishing an animal model of gastrointestinal anthrax. All routes of infection progressed to the draining lymph nodes, spleen, lungs, and ultimately the blood. These discoveries were made possible through the development of a novel dynamic mouse model of B. anthracis infection using bioluminescent non-toxinogenic capsulated bacteria that can be visualized within the mouse in real-time, and demonstrate the value of in vivo imaging in the analysis of B. anthracis infection. Our data imply that previously unrecognized portals of bacterial entry demand more intensive investigation, and will significantly transform the current perception of inhalational, gastrointestinal, and cutaneous B. anthracis pathogenesis.

Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis.

Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-gamma, and Ab-deficient (muMT(-/-)) or IFN-gamma-insensitive (IFN-gammaR(-/-)) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-gamma-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.

Murine splenocytes produce inflammatory cytokines in a MyD88-dependent response to Bacillus anthracis spores.

Bacillus anthracis is a sporulating Gram-positive bacterium that causes the disease anthrax. The highly stable spore is the infectious form of the bacterium that first interacts with the prospective host, and thus the interaction between the host and spore is vital to the development of disease. We focused our study on the response of murine splenocytes to the B. anthracis spore by using paraformaldehyde-inactivated spores (FIS), a treatment that prevents germination and production of products associated with vegetative bacilli. We found that murine splenocytes produce IL-12 and IFN-gamma in response to FIS. The IL-12 was secreted by CD11b cells, which functioned to induce the production of IFN-gamma by CD49b (DX5) NK cells. The production of these cytokines by splenocytes was not dependent on TLR2, TLR4, TLR9, Nod1, or Nod2; however, it was dependent on the signalling adapter protein MyD88. Unlike splenocytes, Nod1- and Nod2-transfected HEK cells were activated by FIS. Both IL-12 and IFN-gamma secretion were inhibited by treatment with B. anthracis lethal toxin. These observations suggest that the innate immune system recognizes spores with a MyD88-dependent receptor (or receptors) and responds by secreting inflammatory cytokines, which may ultimately aid in resisting infection.

[Anthrax revisited: Bacillus anthracis toxins as novel actors of immune escape?]. / La maladie du charbon revue: les toxines de Bacillus anthracis comme nouveaux acteurs de l'evasion immunitaire de l'agent pathogène?

The recent bioterrorist attacks have stressed the need of a better knowledge of Bacillus anthracis infection pathophysiology. We present here the increasing interests of B. anthracis studies in term of bio-defense, the main pathogen characteristics, the main clinical features of inhalational anthrax (the pulmonary form of the disease), and recent aspects of its physiopathology. Next, we address the main results concerning the toxin effects on immune system through impairing the dendritic cell functions, and we analyze the singular role of anthrax toxins in immune evasion.

Regulatory networks for virulence and persistence of Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive sporulating bacterium. Its life-cycle can be divided schematically into two phases: multiplication in the mammalian host and persistence in the soil. A central regulator AtxA interferes with expression of more than 70 genes in vitro and an undefined number ex vivo. The exact molecular mechanism of action of AtxA is unknown, but the involvement of cascades of relay regulators has been described. Other regulators have also been implicated in the regulatory networks; these are mainly transition state regulators, which have been studied in other Bacillus species. They contribute to the regulation of expression of virulence- and persistence-factor genes, and to the regulation of atxA itself.