Showing papers by "Agnès Fouet published in 2013"

Distinct functions of polysaccharide deacetylases in cell shape, neutral polysaccharide synthesis and virulence of Bacillus anthracis

Abstract: Summary Peptidoglycan deacetylases (PGNG-dacs) belong to the Carbohydrate Esterase Family 4 (CE4) and have been described as required for bacterial evasion to lysozyme and innate immune responses. Interestingly, there is an unusual occurrence of 10 putative polysaccharide deacetylases, including five PGNG-dacs, in the Bacillus sp. genomes, especially B. cereus and B. anthracis. To elucidate the physiological role of these multiple deacetylases, we employed genetic analysis and protein localization studies of five putative PGNG-dacs from B. anthracis as well as biochemical analysis of their corresponding homologues from B. cereus. Our data confirm that three enzymes are PGNG-dacs. While BA1977, associated with lateral peptidoglycan synthesis, is a bona fide peptidoglycan deacetylase involved in resistance to host lysozyme and required for full virulence, BA1961 and BA3679 participate in the biogenesis of the peptidoglycan during both elongation and cell division. Furthermore, two enzymes are important for neutral polysaccharide attachment to PG and consequently anchoring of S-layer proteins (BA5436) and for polysaccharide modification (BA2944). Our results provide novel and fundamental insights into the function of polysaccharide deacetylases in a major bioterrorism agent.

Identification of CodY Targets in Bacillus anthracis by Genome-Wide In Vitro Binding Analysis

Abstract: In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.

In vivo germination of Bacillus anthracis spores during murine cutaneous infection

Abstract: BACKGROUND: Germination is a key step for successful Bacillus anthracis colonization and systemic dissemination. Few data are available on spore germination in vivo, and the necessity of spore and host cell interactions to initiate germination is unclear. METHODS: To investigate the early interactions between B. anthracis spores and cutaneous tissue, spores were inoculated in an intraperitoneal cell-free device in guinea pigs or into the pinna of mice. Germination and bacterial growth were analyzed through colony-forming unit enumeration and electron microscopy. RESULTS: In the guinea pig model, germination occurred in vivo in the absence of cell contact. Similarly, in the mouse ear, germination started within 15 minutes after inoculation, and germinating spores were found in the absence of surrounding cells. Germination was not observed in macrophage-rich draining lymph nodes, liver, and spleen. Edema and lethal toxin production were not required for germination, as a toxin-deficient strain was as effective as a Sterne-like strain. B. anthracis growth was locally controlled for 6 hours. CONCLUSIONS: Spore germination involving no cell interactions can occur in vivo, suggesting that diffusible germinants or other signals appear sufficient. Different host tissues display drastic differences in germination-triggering capacity. Initial control of bacterial growth suggests a therapeutic means to exploit host innate defenses to hinder B. anthracis colonization.