Bacillus anthracis

About: Bacillus anthracis is a research topic. Over the lifetime, 3994 publications have been published within this topic receiving 128122 citations.

Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution

Abstract: Introduction. As part of a structural genomics program, we are determining structures of proteins from the causative agent of anthrax, Bacillus anthracis, a Grampositive spore-forming bacterium. Among our initial candidates for crystallographic analysis are the products of essential genes based on knock-out studies in Bacillus subtilis. The BA3935 gene of B. anthracis (www.tigr.org), annotated as DapA2, encodes a putative protein consisting of 292 amino acid residues with a subunit molecular weight of 31,233 Da. The predicted protein has 60% identity with dihydrodipicolinate synthase (DHDPS or DapA) from B. subtilis and 40% amino acid sequence identity to its orthologue in Escherichia coli. dapA is one of only 271 out of the total of 4118 genes in B. subtilis that are indispensable for growth of the organism on standard laboratory media. 1 DHDPS catalyses the condensation of aspartate semialdehyde and pyruvate and is the first committed step on the pathway to diaminopimelate and L-lysine in prokaryotes, some fungi, and higher plants (Scheme 1). The product released from the E. coli enzyme has been shown to be 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTDPA) rather than L-2,3-dihydrodipicolinic acid (DHDPA); as the name of the enzyme suggests, 2 DHDPA can be formed spontaneously from HTDPA by elimination of water. The steps of aspartate semialdehyde synthesis from aspartate are shared with the biosynthetic pathways leading methionine and threonine. The diaminopimelate/ lysine pathway is thought to be of particular importance in Gram-positive bacteria because diaminopimelate makes up a higher proportion of the dry cell weight than it does in Gram-negative bacteria, a consequence of the thicker cell wall in the former. In Bacilli, the product of the DHDPS reaction is further reduced by dipicolinate synthase to dipicolinate, which makes up 10% of the dry weight of spores. Crystal structures have been determined of DHDPS from E. coli, 3 Nicotiana sylvestris, 4 and most recently from Thermotoga maritima. 5 In this article, we report the crystal structure of DHDPS from B. anthracis (Ba DHDPS), the first structure of a dihydrodipicolinate synthase from a Gram-positive bacterium. The structure of Ba DHDPS was determined by molecular replacement using the coordinate set for the E. coli orthologue (PDB code 1DHP) as the search model. 3 Two structures have been determined to 1.9 and 2.2 A resolution in different orthorhombic crystal forms. Data collection, refinement, and model-building statistics are summarized in Table I. The structures in the two crystal forms are very similar and unless otherwise stated, the commentary here will refer to the structure refined to higher resolution

Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax

Abstract: Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD50) and against an aerosol with 75 LD50 of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax. Bacillus anthracis is a gram-positive, aerobic, facultatively anaerobic, spore-forming, rod-shaped bacterium and is the etiologic agent of anthrax. B. anthracis resides in the soil as a dormant spore that is highly resistant to adverse conditions and can remain viable for years. The spore typically enters herbivores through ingestion; although anthrax is predominantly a disease of herbivores, humans can be infected through incidental exposure during handling of animals or animal products. In humans, the disease may take three forms—cutaneous, gastrointestinal, or pulmonary—depending on the site of entry. The most common human form is cutaneous anthrax, typically caused by spores infecting open wounds or skin abrasions. The mortality of cutaneous anthrax is near 20% if untreated (21). Gastrointestinal anthrax may in some cases extend to neuromeningitidis and generally leads to fatal systemic disease if untreated (5, 21). Naturally acquired pulmonary anthrax is very unusual. However, the mortality of pulmonary anthrax is almost 100% if not treated very early (80). Inhalational anthrax manifests as the rapid development of nonspecific, flulike

EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin

Abstract: The lethality of infection by Bacillus anthracis is largely due to its plasmid-encoded toxins, which consist of a carrier protein, the protective antigen (PA), in combination with either the lethal-factor or edema-factor moiety. During B. anthracis infections, PA secreted by bacteria binds to membrane receptors of susceptible cells, is cleaved proteolytically, attaches to lethal factor or edema factor, undergoes oligomerization and internalization, and transports its toxin partners to acidic endosomes where they are released into the cytosol. To identify specific host functions that mediate these events, we used RNA encoded by a lentivirus-based library of ≈40,000 human ESTs to inactivate chromosomal genes in a human cell population, and we isolated clones that survived PA-dependent toxin-induced death. This phenotypic screen and subsequent analysis identified ARAP3, which is a phosphoinositide-binding protein implicated previously in membrane vesicle trafficking and cytoskeletal organization, as a mammalian host-cell gene that is essential for normal anthrax toxicity. ARAP3 deficiency produced by antisense expression of an ARAP3 EST impaired entry of PA and its bound toxigenic moieties into both human and mouse cells, resulting in reduced toxin sensitivity. Our results demonstrate the usefulness of antisense EST libraries for global chromosomal gene inactivation, establish the practicality of loss-of-function phenotypic screens for the identification of genomic loci required for pathogen effects in mammalian cells, and reveal an important role for ARAP3 in cellular internalization of anthrax toxin.