SummaryThe fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination.

SummaryThe spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage-like RAW264.7 cells and alveolar macrophages. We identi®ed a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXOl. The three predicted proteins (40, 55 and 37 kDa in size) have signi®cant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC ). Primer extension analysis identi®ed a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB±lacZ transcriptional fusion. Expression began 2.5±3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate ef®ciently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerXencoded proteins are involved in the virulence of B. anthracis.

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA؉LF) and edema (PA؉EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live B. anthracis was used to follow the in vivo interaction of LF and PA. We found that the binding of LF to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.

Outbreaks of anthrax zoonose occurred in two regions of France in 1997. Ninety-four animals died, and there were three nonfatal cases in humans. The diagnosis of anthrax was rapidly confirmed by bacteriological and molecular biological methods. The strains ofBacillus anthracis in animal and soil samples were identified by a multiplex PCR assay. They all belonged to the variable-number tandem repeat (VNTR) group (VNTR)3. A penicillin-resistant strain was detected. Nonvirulent bacilli related to B. anthracis, of all VNTR types, were also found in the soil.