Michèle Mock

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence

ABSTRACT Bacillus anthracis, Bacillus cereus, andBacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes thatB. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

Germination of Bacillus anthracis spores within alveolar macrophages

The 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.

Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNγ-induced release of NO and TNFα

The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in animals and cause macrophage lysis. LF is a zinc‐endopeptidase which cleaves two mitogen‐activated proten kinase kinases (MAPKKs), Mek1 and Mek2, within the cytosol. Here, we show that also MKK3, another dual‐specificity kinase that phosphorylates and activates p38 MAP kinase, is cleaved by LF in macrophages. No direct correlation between LF‐induced cell death and cleavage of these MAPKKs was found in macrophage cell lines and primary peritoneal cells exhibiting different sensitivity to LF. However, we present the first evidence that sublytic doses of LF cleave Meks and cause a substantial reduction in the production of NO and tumour necrosis factor‐α induced by lipopolysaccharide/interferonγ. We suggest that this effect of LF is relevant during the first stages of B. anthracis infection, when a reduction of the inflammatory response would permit growth and diffusion of the bacterium.

A collagen‐like surface glycoprotein is a structural component of the Bacillus anthracis exosporium

Bacillus anthracis , the aetiological agent of anthrax, is a Gram‐positive spore‐forming bacterium. The exosporium is the outermost integument surrounding the mature spore. Here, we describe the purification and the characterization of an immunodominant protein of the spore surface. This protein was abundant, glycosylated and part of the exosporium. The amino‐terminal sequence was determined and the corresponding gene was identified. It encodes a protein of 382 amino acid residues, the central part of which contains a region of GXX motifs presenting similarity to mammalian collagen proteins. Thus, this collagen‐like surface protein was named BclA (for Bacillus c ollagen‐ l ike protein of anthracis ). BclA was absent from vegetative cells; it was detected only in spores and sporulating cells. A potential promoter, dependent on the sigma factor σ K , which is required for a variety of events late in sporulation, was found upstream from the bclA gene. A bclA deletion mutant was constructed and analysed. Electron microscopy studies showed that BclA is a structural component of the filaments covering the outer layer of the exosporium.

Bacterial SLH domain proteins are non‐covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation

Several bacterial proteins are non‐covalently anchored to the cell surface via an S‐layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non‐covalent interaction between the SLH domain and peptidoglycan‐associated polymers. Here we report the characterization of a two‐gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH‐containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan‐associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH‐containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Fate of germinated Bacillus anthracis spores in primary murine macrophages

We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7. We found that the lethal toxin and the oedema toxin are germ‐associated factors that are essential for the survival of the vegetative form in host cells. We also found that pX02 is not involved in this complex pathogenic process. By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage. Our data strongly suggest that the B. anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages. These results contributed to reveal the strategies used by B. anthracis to survive within the host and to reach the external medium where they proliferate.

Polymorphism in the Collagen-Like Region of the Bacillus anthracis BclA Protein Leads to Variation in Exosporium Filament Length

We recently identified a Bacillus anthracis glycoprotein which is a structural constituent of the exosporium filaments (P. Sylvestre, E. Couture-Tosi, and M. Mock, Mol. Microbiol. 45:169-178, 2002). This Bacillus collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets. Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P. Le Flèche et al., BMC Microbiol. 1:2, 2001), maps within the open reading frame encoding BclA. The bclA gene in 11 B. anthracis strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames bclA gene sequence. The amino- and carboxy-terminal sequences surrounding the CLR are conserved. The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)(5)GDTGTT, named the BclA repeat. The length of the filament on the spore surface differed between the strains. We exchanged the bclA gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis. The length of the BclA CLR is responsible for the variation in filament length.

The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence

Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a trans‐activator of anthrax toxin synthesis. Here we show that transcription of atx A does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atx A‐null mutant in which the atx A‐coding sequence on pXO1 is replaced with an ωkm‐2 cassette. Transcription of all three toxin genes is decreased in the absence of atx A. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5′ ends mapping to P1 are decreased in the atx A‐null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the Pi site is sufficient for atx A‐mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5′ ends mapping to these sites are not detected in the afxA‐null mutant. The atx A‐null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atx A‐null mutant‐infected mice. These data suggest that the atx A gene product also regulates toxin gene expression during infection.

Anthrax Spores Make an Essential Contribution to Vaccine Efficacy

ABSTRACT Anthrax is caused by Bacillus anthracis, a gram-positive spore-forming bacterium. Septicemia and toxemia rapidly lead to death in infected mammal hosts. Currently used acellular vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, in experimental animals such vaccines are less protective than live attenuated strains. Here we demonstrate that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA elicits total protection against challenge with virulent B. anthracis strains in mice and guinea pigs. The toxin-neutralizing activities of sera from mice immunized with PA alone or PA plus FIS were similar, suggesting that the protection conferred by PA plus FIS was not only a consequence of the humoral response to PA. A PA-deficient challenge strain was constructed, and its virulence was due solely to its multiplication. Immunization with FIS alone was sufficient to protect mice partially, and guinea pigs totally, against infection with this strain. This suggests that spore antigens contribute to protection. Guinea pigs and mice had very different susceptibilities to infection with the nontoxigenic strain, highlighting the importance of verifying the pertinence of animal models for evaluating anthrax vaccines.

Anthrax Edema Toxin Cooperates with Lethal Toxin to Impair Cytokine Secretion during Infection of Dendritic Cells

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-α, whereas LT targets IL-10 and TNF-α production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-γ secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.