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. Anthrax is caused by Bacillus anthracis, a bacterial pathogen that forms spores, dormant bacteria that are highly resistant to destruction. Infections initiate from the introduction of spores into airways or damaged skin, or from the consumption of contaminated food. Within the host, spores germinate, then bacteria secrete toxins that cripple the immune response and sheath themselves in a capsule that prevents them from being phagocytosed. We strove to determine in real space and time where and when spores introduced by these three routes of infection germinate and how bacteria subsequently disseminate in a mouse model. This was achieved through the development of light-emitting B. anthracis that could be tracked inside a living mouse. Contrary to current models, our studies indicated that spores germinated in situ in the skin, the intestines, and the nasal passages without needing to be transported to lymph nodes. Furthermore, bacteria disseminate from initial sites of infection in a similar fashion, first to the draining lymph nodes, then the spleen, and finally the lungs and blood. These findings imply that spore interactions with local sites of entry are critical in the development of systemic disease and that disruption of these interactions may offer new methods of anthrax prevention.

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. Anthrax is caused by the bacterium Bacillus anthracis that affects all mammals worldwide. It emerged more than 10,000 years ago from a Bacillus cereus precursor. In the past decade, B. cereus bacteria were isolated in the USA from anthrax-like pneumonia cases. They harbour one virulence plasmid very similar to the toxin–encoding plasmid of B. anthracis. Recently, an anthrax-like disease in great apes in Africa was caused by emerging B. cereus strains, named B. cereus biovar anthracis. These strains are atypical as they possess both plasmids coding for toxin and capsule similar to those so far found only in B. anthracis. These unusual pathogenic B. cereus are currently neglected. We explored the virulence of these pathogens and their colonisation and dissemination capacity within the murine host. We found that these toxinogenic strains harbour two capsules, the classical B. anthracis capsule and an additional polysaccharidic capsule. This latter capsule confers virulence alone or in combination with toxins. Both capsules are concomitantly expressed, under the control of a common global regulator and host signals. Our results show that acquisition of new genetic information by these B. cereus clearly gives them a selective advantage, favouring their dissemination within infected hosts and the environment.

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-γ in response to FIS. The IL-12 was secreted by CD11b cells, which functioned to induce the production of IFN-γ 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-γ 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.

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-γ- 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.