Evelyne Couture-Tosi

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.

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.

Contribution of ExsFA and ExsFB proteins to the localization of BclA on the spore surface and to the stability of the bacillus anthracis exosporium.

Spores of Bacillus anthracis, the etiological agent of anthrax, and the closely related species Bacillus cereus and Bacillus thuringiensis, possess an exosporium, which is the outermost structure surrounding the mature spore. It consists of a paracrystalline basal layer and a hair-like outer layer. To date, the structural contribution of only one exosporium component, the collagen-like glycoprotein BclA, has been described. It is the structural component of the hair-like filaments. Here, we describe two other proteins, ExsFA and ExsFB, which are probably organized in multimeric complexes with other exosporium components, including BclA. Single and double exsF deletion mutants were constructed and analyzed. We found that inactivation of exsF genes affects the BclA content of spores. BclA is produced by all mutants. However, it is partially and totally released after mother cell lysis of the DeltaexsFA and DeltaexsFA DeltaexsFB mutant strains, respectively. Electron microscopy revealed that the exsF mutant spores have defective exosporia. The DeltaexsFA and DeltaexsFA DeltaexsFB spore surfaces are partially and totally devoid of filaments, respectively. Moreover, for all mutants, the crystalline basal layer appeared unstable. This instability revealed the presence of two distinct crystalline arrays that are sloughed off from the spore surface. These results indicate that ExsF proteins are required for the proper localization of BclA on the spore surface and for the stability of the exosporium crystalline layers.

Developmental switch of S-layer protein synthesis in Bacillus anthracis

Adjustment of the synthesis of abundant protein to the requirements of the cell involves processes critical to the minimization of energy expenditure. The regulation of S‐layer genes might be a good model for such processes because expression must be controlled, such that the encoded proteins exactly cover the surface of the bacterium. Bacillus anthracis has two S‐layer genes, sap and eag, encoding the S‐layer proteins Sap and EA1 respectively. We report that the production and surface localization of Sap and EA1 are under developmental control, suggesting that an exponential phase ‘Sap layer’ is subsequently replaced by a stationary phase ‘EA1 layer’. This switch is controlled at the transcriptional level: sap is most certainly transcribed by RNA polymerase containing σA, whereas eag expression depends on σH. More importantly, Sap is required for the temporal control of eag, and EA1 is involved in strict feedback regulation of eag. This control may be direct because both S‐layer proteins bind, in vitro, the eag promoter, specifically suggesting that they might act as transcriptional repressors.

The spore differentiation pathway in the enteric pathogen Clostridium difficile.

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σF in the forespore, and σE in the mother cell control early stages of development and are replaced, at later stages, by σG and σK, respectively. Starting with σF, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σE is partially independent of σF, that σG activity is not dependent on σE, and that the activity of σK does not require σG. We also show that σK is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σF-to-σE, σE-to-σG, and σG-to-σK cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.

Crucial residues in the carboxy-terminal end of C1 inhibitor revealed by pathogenic mutants impaired in secretion or function.

The last exon of the C1-1NH gene was screened for point mutations in 36 unrelated hereditary angioedema patients. Mutations were found in eight patients, predicting changes in the short COOH-terminal region which anchors the reactive site loop on its COOH-terminal side. The effects of each of these mutations were examined in transiently transfected Cos-7 cells. Complete intracellular retention or degradation was observed with substitutions in the COOH-terminal strands 4B or 5B: Leu459-->Pro, Leu459-->Arg, and Pro467-->Arg were all blocked at early stages of intracellular transport, but differences in the immunofluorescence patterns indicated that a significant fraction of the Leu459-->Pro and of the Pro467-->Arg proteins reached a compartment distinct from the endoplasmic reticulum. In line with previous findings with alpha 1-antitrypsin, chain termination within strand 5B resulted in rapid degradation. Mutant Val451-->Met, in strand 1C, and mutant Pro476-->Ser, replacing the invariant proline near the COOH terminus, yielded reduced secretion, but these extracellular proteins were unable to bind the target protease C1s. Presence of low levels of both dysfunctional proteins in patient plasmas defies the conventional classification of C1 inhibitor deficiencies as type I or type II. These data point to a key role of certain residues in the conserved COOH-terminal region of serpins in determining the protein foldings compatible with transport and proper exposure of the reactive site loop.

Structure of the exosporium and sublayers of spores of the Bacillus cereus family revealed by electron crystallography

We report on the first step in mapping out the spatial location of structural proteins within the exosporium, namely a description of its three‐dimensional architecture. Using electron microscopy and image analysis, we have characterized crystalline fragments from the exosporium of Bacillus cereus, B. thuringiensis and B. anthracis strains and identified up to three distinct crystal types. Type I and type II crystals were examined in three dimensions and shown to form arrays of interlinked crown‐like structures each enclosing a cavity ∼26–34 Å deep with threefold symmetry. The arrays appear to be permeated by tunnels allowing access from one surface to the other, possibly indicating that the exosporium forms a semi‐permeable barrier. The pore size of ∼23–34 Å would allow passage of the endospore germinants, alanine or inosine but not degradative enzymes or antibodies. Thus the structures appear compatible with a protective role for the exosporium. Furthermore the outermost crystalline layer must act as a scaffold for binding the BclA protein that contributes to the ‘hairy nap’ layer. The array of crowns may also act as a matrix for the binding or adsorption of other proteins that have been identified in the exosporium such as GroEL, immune inhibitor A and arginase.

Identification of the Bacillus anthracis γ Phage Receptor

Bacillus anthracis , a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. It belongs to the Bacillus cereus group, which also contains Bacillus cereus and Bacillus thuringiensis . Most B. anthracis strains are sensitive to phage γ, but most B. cereus and B. thuringiensis strains are resistant to the lytic action of phage γ. Here, we report the identification of a protein involved in the bacterial receptor for the γ phage, which we term GamR ( Gam ma phage r eceptor). It is an LPXTG protein (BA3367, BAS3121) and is anchored by the sortase A. A B. anthracis sortase A mutant is not as sensitive as the parental strain nor as the sortase B and sortase C mutants, whereas the GamR mutant is resistant to the lytic action of the phage. Electron microscopy reveals the binding of the phage to the surface of the parental strain and its absence from the GamR mutant. Spontaneous B. anthracis mutants resistant to the phage harbor mutations in the gene encoding the GamR protein. A B. cereus strain that is sensitive to the phage possesses a protein similar (89% identity) to GamR. B. thuringiensis 97-27, a strain which, by sequence analysis, is predicted to harbor a GamR-like protein, is resistant to the phage but nevertheless displays phage binding.

complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.

Structural Analysis and Evidence for Dynamic Emergence of Bacillus anthracis S-Layer Networks

Surface layers (S-layers), which form the outermost layers of many Bacteria and Archaea, consist of protein molecules arranged in two-dimensional crystalline arrays. Bacillus anthracis, a gram-positive, spore-forming bacterium, responsible for anthrax, synthesizes two abundant surface proteins: Sap and EA1. Regulatory studies showed that EA1 and Sap appear sequentially at the surface of the parental strain. Sap and EA1 can form arrays. The structural parameters of S-layers from mutant strains (EA1(-) and Sap(-)) were determined by computer image processing of electron micrographs of negatively stained regular S-layer fragments or deflated whole bacteria. Sap and EA1 projection maps were calculated on a p1 symmetry basis. The unit cell parameters of EA1 were a = 69 A, b = 83 A, and gamma = 106 degrees, while those of Sap were a = 184 A, b = 81 A, and gamma = 84 degrees. Freeze-etching experiments and the analysis of the peripheral regions of the cell suggested that the two S-layers have different settings. We characterized the settings of each network at different growth phases. Our data indicated that the scattered emergence of EA1 destabilizes the Sap S-layer.