Unit: Toxins and Bacterial Pathogenesis - URA 2172 du CNRS

Director: Michèle MOCK

The virulence mechanisms of Bacillus anthracis, the aetiological agent of anthrax, an extracellular toxinogenic bacterium, are investigated. The structural organisation of the toxins and their role in pathogenesis , and the role of the spore and of the bacilli cell surface structures in the interaction of the pathogen with the host are analysed. An anthrax vaccine composed of protective antigen and inactivated spores has been devised for human use, and the relevant mechanisms of protective immunity are studied.

Spore surface

(Patricia Sylvestre - Evelyne Couture-Tosi - Michèle Mock)

The spore is both the form of persistance in the environment of B. anthracis and the infectant form starting the infection after germination in the host. The exosporium is the most external structure of the spore. Its role in the early interaction with the host and its contribution to immunoprotection are analysed. A glycoprotein termed BclA (for Bacillus collagen-like) has been shown to be the structural component of the filaments located at the surface of the exosporium. BclA contains a central region presenting similarity to mammalian collagen proteins. This collagen region (CLR) consists of GXX collagen-like triplets. The number of GXX repeats varies condiderably between strains and we have shown that this variation is responsible for the length variation of the filament nap covering the exosporium outer layer.

BclA is specific of the spore. It is produced in the mother cell compartment and appeared in the late stages of sporulation and the proteins ExsFA and ExsFB are required for the proper localisation of BclA on the spore surface and for the stability of the exosporium crystalline layers.

The characterization of other exosporium components and proteins required for the localisation and glycosylation of BclA is currently investigated.

Toxins of Bacillus anthracis - Vaccinal strategies and therapeutics

(Pierre Goossens - Ian Glomski - Michèle Mock)

Bacillus anthracis secretes two toxins composed of three proteins: lethal toxin (PA + LF) and edema toxin (PA + EF). PA (protective antigen) is the common component able to bind and deliver EF (edema factor) and LF (lethal factor) into target eukaryotic cells. EF is a calmodulin-dependent adenylate cyclase and LF is a metalloprotease which acts specifically on the MAPKKinases family. The three dimensional structure of PA, EF and LF is known. The three proteins are organised in a functional domain type structure. Specific inhibitors of LF, and EF activities have been obtained, and their potential therapeutic interest against infection is tested in collaborative research projects.

Monoclonal antibodies against PA and LF, inhibiting PA binding to the receptor or inhibiting formation of PA/LF complex have been obtained. They neutralize lethal toxin action in vitro, and protect mice against experimental infection with the toxin producing Sterne strain.

A non-living anthrax vaccine composed of protective antigen and inactivated spores has been devised for human use. The mechanisms of protective immunity induced by this vaccinal composition are currently studied. Additionally, the initial innate immune response to inactivated spores, which aids in the development of adaptive immunity, is investigated. A cutaneous model of infection used in conjunction with bioluminescent bacteria has been developed in mice. This model allows the characterization of the infection locally, at the site of inoculation, and the elucidation of the mechanisms leading to bacterial dissemination to deeper tissues in a living mouse using an ultrasensitive digital camera. Comparison of these events between naive hosts and immunized hosts should highlight hypotheses on the mechanisms controlling infection. Furthermore, in collaboration with the CRSSA, infection models have been worked out to study the efficacy of the vaccine after aerosolized infection. This is part of projects devoted to bioterrorism counter measures.

Beside the analysis of the adaptative response to vaccination, the mechanisms of innate immunity which are possibly involved in the early control of germination and bacterial dissemination are investigated. We have shown in vitro that group IIA phospholipase A2, a component secreted by neutrophils and alveolar macrophages, is highly bactericidal against B. anthracis; its in vivo effect on the infection is now studied. We analyse the interactions in vitro and in vivo between the spores and the dendritic cells (joint project IP/CRSSA). The use of strains mutated in the virulence factors will help to characterise the relative contribution of each toxin on cytokine and chemokine secretion profile, and on the intracellular signalisation pathways.

Vegetative form surface

(Agnès Fouet - Evelyne Couture-Tosi)

The bacilli isolated from animals dying of anthrax are encapsulated. The capsule covers a structural array termed S-layer. Yet, capsule and S-layer can be independently synthesized. The capsule has an anti-phagocytic role. Four proteins, which genes belong to an operon, are necessary for capsule synthesis. One of these, which was recently discovered, is a 47 amino acid peptide with a structural role. A fifth protein, also encoded by that operon, catalyzes the capsule covalent anchoring to the peptidoglycan.

A surface protein anchoring mechanism has been described in many Gram+ pathogens. "Sortases" catalyze the covalent anchoring of proteins harboring an "LPXTG" motif. This mechanism exists in B. anthracis which possesses three sortases and probably 14 "LPXTG" proteins. The repertoire of these sortases is currently being studied by genetic and biochemical approaches. Indeed, whereas sortase A anchors most "LPXTG" proteins, the other two sortases frequently anchor proteins with specific roles, related to particular environmental situations. We have constructed mutants of each of these enzymes. We have identified GamR, the γ phage receptor, and shown that it is an "LPXTG" protein that is anchored by sortase A. The γ phage has long been used to discriminate between B. anthracis and other species of the same group (Bacillus cereus group). The function and potential role during infection of the proteins, which are anchored by the other sortases, will be analyzed.

Genomic and transcriptional analysis

(Maryse Moya-Nilges - Odile Mary-Possot - Agnès Fouet)

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis belong to the B. cereus group. A thorough genetic analysis has shown that these three bacteria are in fact one species.The complete genome sequences of the Ames strain of B. anthracis and of several B. cereus strains are available. The use of recent bioinformatic tools allows us to perform a genome annotation better adapted to a functional analysis. In addition, the transcriptional profiling should define genes which are specifically expressed during bacterial development in the host. Genes under the control of AtxA, a master regulator of toxin, capsule, and surface component suntheses, are analysed under various culture conditions. The study comprises the genes controlled by PagR, a pleiotropic regulator itself belonging to the AtxA regulon. Various statistical analyses of the results are currently developed.

Phylogeny and ecology of B. anthracis

(Patricia Sylvestre - Michèle Mock)

Anthrax is a worldwide mammal zoonose that has considerably regressed in the occidental countries. Nevertheless, it still exists in France where it sporadically appears each year in various regions. Collaborative phylogenetic studies on a French B. anthracis strain collection, provided by AFSSA, has revealed that these strains belong to the two main groups (totalizing more than 80 genotypes) defined world-wide. Moreover, four genotypes are defined uniquely by French strains. The two genotypes GT79 et GT80 are the only members found in the sub-group B2 which is the most common in France, in the mountain regions, but is extremely rare elsewhere in the world. These observations suggest a potential role of environnemental factors on the selection of particular genotypes.

Moreover, the BclA exosporium glycoproteins of the B2 group strains are particularly interesting. In contrast to the other B. anthracis strains which give a BclA pattern with a single glycosylated band, the B2 sub-group strains give a multiple-glycosylated-band pattern.

Gene exchange experiments indicate that the presence of several glycosylated bands is dependent on the genetic background of these strains. This suggests that posttranslational modifications, probably implicated in BclA glycosylation, are responsible for the phenomenom. The characterization of potential functions implicated in these modifications is under investigation.

Keywords: Anthrax, surface, toxins, virulence, spores, vaccine

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