Unit: Toxins and Bacterial Pathogenesis - URA 2172 du CNRS

Director: Michèle MOCK

The virulence mechanisms of B. anthracis, the aetiological agent of anthrax, an extracellular toxinogenic bacterium, are investigated. The structural organisation, the toxins and their role in pathogenesis ; 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 - Michèle Mock - Maryse Moya-Nilges)

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 (CLR) presenting similarity to mammalian collagen proteins. The CLR consists of GXX triplets including a large proportion of GPT triplets. Analysis of bclA gene from different strains of B. anthracis revealed that the size of the gene varies considerably. Consequently, the corresponding BclA proteins vary in size. This polymorphism is restricted to the CLR that contains variable numbers of GXX triplets. Spores from all strains are covered by filaments; however, the length of the filaments differs between strains. Gene exchange experiments showed that the length of the BclA CLR is directly responsible for the variation in filament length.

Toxins of Bacillus anthracis - Vaccinal strategies and therapeutics(Ian Glomski - Pierre Goossens - Michèle Mock - Alex Piris Gimenez)

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 activity have been obtained, and their potential therapeutic interest against infection is tested in a collaborative research project.

B. anthracis strains producing the PA protein mutated in the different functional domains have been constructed. Study of their in vivo properties indicates a correlation between the steps of cellular mode of action of toxins on the cells and the pathogenesis. Moreover, a strain isogenic to the vaccinal veterinary Sterne strain, producing genetically detoxified LF and EF, confers protection against anthrax lethal challenge. 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. A cutaneous model of infection has been developed in mice to characterize the infection locally, at the site of inoculation and to elucidate the mechanisms leading to the dissemination to draining lymph node and to invasion of the host. Comparison of these events between naive host and immunized host should highlight hypotheses on the mechanisms controlling infection. Additionnally, in collaboration with the CRESSA, 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. The phospholipase A2, type IIA, secreted by alveolar macrophages, exerts a bactericidal activity against germinated spores and capsulated bacilli of B. anthracis.

In vivo, the germination of the spore can take place in the phagolysosome of macrophages. This study is extended to the analysis of spores of various mutant strains of B. anthracis with putative host cells (macrophages, dendritic cells).

Vegetative form surface(Sophie Bonnot - Thomas Candela - Sophie Davison - Agnès Fouet - Odile Mary-Possot)

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. Two abundant proteins can compose an S-layer. Their synthesis is medium and growth phase-dependent. Therefore, at any given time, one protein predominates, that constitutes the S-layer. In vivo, it seems that only one is synthesized. The S-layer proteins have, in their N-terminal region, a domain that is sufficient for their anchoring to a modified peptidoglycan associated polysaccharide. This non-covalent anchoring mechanism is found in many bacterial species. Inside the B. cereus group, the presence of an S-layer is limited to pathogenic strains.

The capsule has an anti-phagocytic role. At least three proteins, which genes belong to an operon, are necessary for capsule synthesis. A fourth protein, also encoded by that operon, degrades the capsule excess. This protein also catalyzes the capsule covalent anchoring to the peptidoglycan. A common regulator controls the synthesis of the toxin components, the S-layer components and the capsule biosynthetic proteins.

Phylogeny and ecology of B. anthracis (Agnès Fouet - Michèle Mock - Patricia Sylvestre)

B. anthracis, B. cereus and B. thuringiensis belong to the B. cereus group. A thorough biomolecular study implying many B. cereus strains has shown that, based on genetic evidence, these three bacteria are in fact one species. PlcR, a transcriptional regulator of many stationnary phase genes (PlcR regulon) is present and functional in all B. cereus and B. thuringiensis strains tested. In B. anthracis, where the plcR gene is interrupted, the regulon is nevertheless present and the introduction of the B. thuringiensis plcR gene induces its expression. PlcR counter-selection is due to the fact that, in presence of a B. anthracis master regulator, it inhibits sporulation, stage necessary to the bacterium persistence in the environment.

The biomolecular epidemiology of anthrax is only emerging. Although this mammal zoonose has regressed, 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 molecular approach used in this work allows an identification of the origin of B. anthracis strains. These studies are to be developed in the program of Anthrax National Reference Center (CNR), created in 2002.

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

Activity Reports 2003 - Institut Pasteur

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