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To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin [beta]1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to [alpha]4[beta]1- and [alpha]5[beta]]1-integrin complexes, leading to their conjoint endocytosis, and also interacts--weakly relative to CMG2 but comparably to TEM8--with purified [alpha]5[beta]1 complex in vitro. Monoclonal antibody directed against [beta]1-integrin or its a integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid--which interferes with CD44-mediated integrin activation--had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin ( CD44 | microarray | osteopontin | CMG2 | lethal factor doi/ 10.1073/pnas.1010145107

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal E-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines. unfolding pathway | transition state structure | mechanical unfolding doi/10.1073/pnas.0905880106

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. edema toxin | lethal toxin | tumor endothelium marker-8

Plasmid pXO1 encodes the tripartite anthrax toxin, which is the major virulence factor of Bacillus anthracis. In spite of the important role of pXO1 in anthrax pathogenesis, very little is known about its replication and maintenance in B. anthracis. We cloned a 5-kb region of the pXO1 plasmid into an Escherichia coli vector and showed that this plasmid can replicate when introduced into B. anthracis. Mutational analysis showed that open reading frame 45 (repX) of pXO1 was required for the replication of the miniplasmid in B. anthracis. Interestingly, repX showed limited homology to bacterial FtsZ proteins that are involved in cell division. A mutation in the predicted GTP binding domain of RepX abolished its replication activity. Genes almost identical to repX are contained on several megaplasmids in members of the Bacillus cereus group, including a B. cereus strain that causes an anthrax-like disease. Our results identify a novel group of FtsZ-related initiator proteins that are required for the replication of virulence plasmids in B. anthracis and possibly in related organisms. Such replication proteins may provide novel drug targets for the elimination of plasmids encoding the anthrax toxin and other virulence factors.

We report here that a bacterial toxin, anthrax lethal toxin (LeTx), at very low concentrations represses glucocorticoid receptor (GR) transactivation in a transient transfection system and the activity of an endogenous GR-regulated gene in both a cellular system and an animal model. This repression is noncompetitive and does not affect ligand binding or DNA binding, suggesting that anthrax lethal toxin (LeTx) probably exerts its effects through a cofactor(s) involved in the interaction between GR and the basal transcription machinery. LeTx-nuclear receptor repression is selective, repressing GR, progesterone receptor B (PR-B), and estrogen receptor [alpha] (ER[alpha]), but not the mineralocorticoid receptor (MR) or ER[beta]. GR repression was also caused by selected p38 mitogen-activated protein (MAP) kinase inhibitors, suggesting that the LeTx action may result in part from its known inactivation of MAP kinases. Simultaneous loss of GR and other nuclear receptor activities could render an animal more susceptible to lethal or toxic effects of anthrax infection by removing the normally protective antiinflammatory effects of these hormones, similar to the increased mortality seen in animals exposed to both GR antagonists and infectious agents or bacterial products. These finding have implications for development of new treatments and prevention of the toxic effects of anthrax.

The three proteins that comprise anthrax toxin, edema factor (EF), lethal factor (LF), and protective antigen (PA), assemble at the mammalian cell surface into toxic complexes. After binding to its receptor, PA is proteolytically activated, yielding a carboxyl-terminal 63-kDa fragment (P[A.sub.63]) that coordinates assembly of the complexes, promotes their endocytosis, and translocates EF and LF to the cytosol. [PA.sub.63] spontaneously oligomerizes to form symmetric ring-shaped heptamers that are capable of binding three molecules of EF and/or LF as competing ligands. To determine whether binding of these ligands depends on oligomerization of P[A.sub.63], we prepared two oligomerization-deficient forms of this protein, each mutated on a different P[A.sub.63]-P[A.sub.63] contact face. In solution or when bound to receptors on Chinese hamster ovary K1 cells, neither mutant alone bound ligand, but a mixture of them did. After the two mutants were proteolytically activated and mixed with ligand in solution, a ternary complex was isolated containing one molecule of each protein. Thus EF and LF bind stably only to P[A.sub.63] dimers or higher order oligomers. These findings are relevant to the kinetics and pathways of assembly of anthrax toxin complexes.

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained Bacillus anthracis-specific sequences. Double PCR using 'nested primers' increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of B. anthracis toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the vrrA gene variable region on the B. anthracis chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single B. anthracis strain.

PCR analysis of 198 Bacillus anthracis isolates indicated a variable region of DNA sequence differing in length among the isolates. Five polymorphisms were characterized by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat region is found within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not alter the reading frame of the encoded protein and only modified the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6.

ANTHRAX This special news package looks behind the scenes at one of the labs helping with the anthrax investigation; a second story looks at what doctors and researchers have learned [...]

The studies have shown that the defect in cytotoxicity is because of a defect in translocation and when bound to the host cellular receptor, an inability to undergo low pH-induced pore formation. The studies have helped in providing a model in which hydrogen bonds to the histidine ring are strengthened by receptor binding and the combination of both fluorination and receptor binding is enough for blocking low pH-induced pore formation.

Several studies are conducted to explain the direct interaction taking place between anthrax toxin receptor 1 and the actin cytoskeleton. The analyzed peptide is shown to exhibit an actin building activity, hence leading to the ordering of actin filaments into arrays.

The fluorescence spectroscopy technique is employed to determine the ligand specificity and binding affinity of the siderophore-mediated iron acquisition systems of the substrate-binding proteins (SBPs) in Bacillus cereus. A complete identification of the receptors for anthrax virulence-associated petrobactin is also presented.

The influence of host environment on the regulation of the expression of the protective antigen in anthrax with respect to 3' untranslated region sequence of the protective antigen forming hairpin structure. Results confirm that both the coding and the noncoding 3' untranslated region sequences form stable hairpin structures.

The single nucleotide polymorphisms (SNPs) are the most widespread type of sequence variation in genomes type and are emerging as highly valuable genealogical and phylogenetic markers is stated. As they are usually identified using a limited subset of the sequences from a population, they represent a highly biased sample of genetic variation.