Bacillus anthracis

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

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

Abstract: Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.

Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein.

Abstract: The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 μg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.

Bacillus anthracis genetics and virulence gene regulation.

Abstract: The Bacillus anthracis genome consists of an approximately 5.3-Mb chromosome and two plasmids, pXO1 (182 kb) and pXO2 (96 kb). Genetic analysis has focused primarily on the structural genes for the anthrax toxin proteins, pagA, lef, and cya, the biosynthetic genes for capsule synthesis, capB, capC, and capA, and a gene associated with depolymerization of capsule, dep. The three toxin genes are located at distinct loci on pXO1, while the cap and dep genes are arranged in an apparent operon on pXO2. Additional genes that may play a role in B. anthracis virulence include the germination operon gerX and the general stress transcription factor sigB. Host-related signals affecting transcription of the toxin and capsule genes include temperature (37°C) and bicarbonate/CO2. The B. anthracis plasmids carry two regulatory genes that share little sequence similarity with regulators in other bacteria. The pXO1-encoded gene atxA positively controls expression of the toxin and capsule genes, and has been implicated in control of other genes of unknown function. atxA mutants are avirulent in mice, and mice infected with atxA-null strains show a decreased immunological response to the toxin proteins. The pXO2-encoded regulator, acpA, shares sequence similarity with atxA. Yet acpA function appears to be restricted to positive control of capsule gene expression. The chromosomal gene abrB, a homologue of a well-characterized B. subtilis transition state regulator, controls growth phase-specific transcription of the toxin genes. Genetic manipulation of B. anthracis can be achieved by using natural means of DNA transfer and by electroporation of recombinant DNAs into B. anthracis. Genetic exchange can occur between B. anthracis strains and between B. anthracis and closely-related species. Although pXO1 and pXO2 are not self-transmissible, these plasmids and others can be transferred by conjugative plasmids originating in B. thuringiensis. Generalized transducing phage that permit inter-species transfer of chromosomal and plasmid DNA have also been described.

Control of Anthrax Toxin Gene Expression by the Transition State Regulator abrB

Abstract: Bacillus anthracis produces the anthrax toxin proteins protective antigen (PA), lethal factor (LF), and edema factor (EF) in a growth phase-dependent manner when cultured in liquid medium. Expression of the toxin genes pagA, lef, and cya peaks in late log phase, and steady-state levels of the toxin proteins are highest during the transition into stationary phase. Here we show that an apparent transition state regulator negatively regulates toxin gene expression. We identified two orthologues of the B. subtilis transition state regulator abrB in the B. anthracis genome: one on the chromosome and one on the 182-kb virulence plasmid pXO1. The orthologue located on the chromosome is predicted to encode a 94-amino-acid protein that is 85% identical to B. subtilis AbrB. The hypothetical protein encoded on pXO1 is 41% identical to B. subtilis AbrB but missing 27 amino acid residues from the amino terminus compared to the B. subtilis protein. Deletion of the pXO1-encoded abrB orthologue did not affect toxin gene expression under the conditions tested. However, a B. anthracis mutant in which the chromosomal abrB gene was deleted expressed pagA earlier and at a higher level than the parent strain. Expression of a transcriptional pagA-lacZ fusion in the abrB mutant was increased up to 20-fold during early exponential growth compared to the parent strain and peaked in mid-exponential rather than late exponential phase. In contrast to the strong effect of abrB on pagA expression, lef-lacZ and cya-lacZ expression during early-log-phase growth was increased only two- to threefold in the abrB null mutant. Western hybridization analysis showed increased PA, LF, and EF synthesis by the mutant. As is true in B. subtilis, the B. anthracis abrB gene is negatively regulated by spo0A. Our findings tie anthrax toxin gene expression to the complex network of postexponential phase adaptive responses that have been well studied in B. subtilis.

Capillary morphogenesis protein-2 is the major receptor mediating lethality of anthrax toxin in vivo.

Abstract: Anthrax toxin, a major virulence factor of Bacillus anthracis, gains entry into target cells by binding to either of 2 von Willebrand factor A domain-containing proteins, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). The wide tissue expression of TEM8 and CMG2 suggest that both receptors could play a role in anthrax pathogenesis. To explore the roles of TEM8 and CMG2 in normal physiology, as well as in anthrax pathogenesis, we generated TEM8- and CMG2-null mice and TEM8/CMG2 double-null mice by deleting TEM8 and CMG2 transmembrane domains. TEM8 and CMG2 were found to be dispensable for mouse development and life, but both are essential in female reproduction in mice. We found that the lethality of anthrax toxin for mice is mostly mediated by CMG2 and that TEM8 plays only a minor role. This is likely because anthrax toxin has approximately 11-fold higher affinity for CMG2 than for TEM8. Finally, the CMG2-null mice are also shown to be highly resistant to B. anthracis spore infection, attesting to the importance of both anthrax toxin and CMG2 in anthrax infections.