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This article cites 56 articles, 23 of which can be accessed free
A synthetic peptide homolog corresponding to the C-terminal 30 amino acids of Clostridium perfringens type A enterotoxin (CPE) was conjugated to a thyroglobulin carrier and used to immunize mice. Conjugate-immunized mice produced... more
A synthetic peptide homolog corresponding to the C-terminal 30 amino acids of Clostridium perfringens type A enterotoxin (CPE) was conjugated to a thyroglobulin carrier and used to immunize mice. Conjugate-immunized mice produced antibodies which neutralized native CPE cytotoxicity, at least in part, by blocking enterotoxin binding. This peptide may be useful for the development of a vaccine to protect against CPE-mediated disease.
Research Interests: Microbiology, Biology, Medicine, Biological Sciences, Infection and immunity, and 15 moreMice, Enterotoxins, Female, Animals, Infection, Immunization, Peptide, Antibody, Toxin, Amino Acid Sequence, Clostridium perfringens, Thyroglobulin, Enterotoxin, Molecular Sequence Data, and Medical and Health Sciences
Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE). Removal of the N-terminal 25 amino acids of CPE increased the primary... more
Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE). Removal of the N-terminal 25 amino acids of CPE increased the primary cytotoxic effect of CPE but did not affect binding. CPE sequences required for at least four epitopes were also identified.
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Research Interests: Biochemistry, Microbiology, Biology, Medicine, Multidisciplinary, and 15 moreMutation, Atmosphere, Air, Siderophores, Research article, Iron, Paraquat, Superoxide Dismutase, PLoS one, Oxygen, Biosynthesis, Bacillus Anthracis, Oxidation-Reduction, Benzamides, and reverse transcriptase polymerase chain reaction
In order to cause disease, pathogenic bacteria require specialized means to sense the various microenvironments presented to them in the context of a host and then to regulate the systems required for establishment, persistence, growth,... more
In order to cause disease, pathogenic bacteria require specialized means to sense the various microenvironments presented to them in the context of a host and then to regulate the systems required for establishment, persistence, growth, and induction of the pathologies associated with infection. The example presented in this chapter focuses on one such system utilized by Bacillus anthracis during anthrax infections, iron acquisition. Recently, research focused on the acquisition of one such nutrient essential for successful anthrax infections, iron, has come to the fore. The chapter summarizes the various mechanisms used by B. anthracis for obtaining iron, reviews the relative impact of each of these mechanisms on a successful anthrax infection, and presents the transcriptome regulated by low concentrations of iron. Once inside the bacterial cytoplasm, iron is released from heme when the heme monooxygenase, IsdG, breaks down the molecule. Siderophores are high-affinity iron-chelatin...
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Research Interests: Microbiology and Mbio
Germination of spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances... more
Germination of spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of and compares them to those of two other well-studied organisms, and germination is unique, as does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing germination mechanisms have been made on several fronts, major gaps in our understanding of germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of germination and potential avenues for the development of therapeutics, and discusses t...
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Bacillus anthracis —a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal... more
Bacillus anthracis —a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cel...
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Research Interests: Genetics, Innate immunity, Multidisciplinary, Macromolecular X-Ray Crystallography, Protein Structure and Function, and 11 moreMice, Animals, Iron, Enzyme, Virulence factor, Protein Conformation, Structure activity Relationship, Bacillus Anthracis, Hydrogen-Ion Concentration, Benzamides, and Shikimic acid
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Research Interests: Biology, Biological Chemistry, Medicine, Macromolecular X-Ray Crystallography, Biocatalysis, and 13 morePolyamines, Biological Sciences, Siderophores, CHEMICAL SCIENCES, Substrate Specificity, Amino Acid Sequence, Bacillus Anthracis, Protein Binding, Biosynthetic Pathways, Benzamides, Molecular Structure, Molecular Sequence Data, and Medical and Health Sciences
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This article cites 12 articles, 3 of which can be accessed free at:
Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process... more
Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under irondepleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during latestage growth and for wild-type levels of sporulation in sporulation medium...
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Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a... more
Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for tauro...
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Nutrient-dependent germination of Bacillus anthracis spores is stimulated when receptors located in the inner membrane detect combinations of amino acid and purine nucleoside germinants. B. anthracis pro-duces five distinct germinant... more
Nutrient-dependent germination of Bacillus anthracis spores is stimulated when receptors located in the inner membrane detect combinations of amino acid and purine nucleoside germinants. B. anthracis pro-duces five distinct germinant receptors, GerH, GerK, GerL, GerS and GerX. Otherwise isogenic mutant strains expressing only one of these receptors were created and tested for germination and virulence. The GerH receptor was necessary and sufficient for wild-type levels of germination with inosine-containing germinants in the absence of other receptors. GerK and GerL were sufficient for germination in 50 mM L-alanine. When mutants were inoculated intratrache-ally, any receptor, except for GerX, was sufficient to allow for a fully virulent infection. In contrast, when inoculated subcutaneously only the GerH receptor was able to facilitate a fully virulent infection. These results suggest that route of infection determines ger-minant receptor requirements. A mutant lacking all five ger...
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is a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host. is the leading cause of nosocomial infectious diarrhea worldwide. infection (CDI) arises after a... more
is a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host. is the leading cause of nosocomial infectious diarrhea worldwide. infection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step in pathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest that germination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores ar...
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Research Interests: Molecular Microbiology, Western blotting, Biological Sciences, Bacterial Toxins, Animals, and 12 moreAnthrax, Amino Acid Sequence, Structure activity Relationship, Protective Antigen, Lethal Factor, Cytoplasm, Diphtheria Toxin, Molecular Sequence Data, binding sites, CHO cells, Cricetinae, and Medical and Health Sciences
Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). We have recently determined the crystal structure of the 735-residue PA in its monomeric and heptameric forms (Petosa et... more
Anthrax lethal toxin comprises two proteins: protective antigen (PA; MW 83 kDa) and lethal factor (LF; MW 87 kDa). We have recently determined the crystal structure of the 735-residue PA in its monomeric and heptameric forms (Petosa et al. 1997). It bears no resemblance to other bacterial toxins of known three-dimensional structure, and defines a new structural class which includes homologous toxins from other Gram-positive bacteria. We have proposed a model of membrane insertion in which the water-soluble heptamer undergoes a substantial pH-induced conformational change involving the creation of a 14-stranded beta-barrel. Recent work by Collier's group (Benson et al. 1998) lends strong support to our model of membrane insertion. 'Lethal factor' is the catalytic component of anthrax lethal toxin. It binds to the surface of the cell-bound PA heptamer and, following endocytosis and acidification of the endosome, translocates to the cytosol. We have made substantial progress towards an atomic resolution crystal structure of LF. Progress towards a structure of the 7:7 translocation complex between the PA heptamer and LF will also be discussed.
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Over the past two decades, Clostridium difficile infections have been increasing in both number and severity throughout the world. As with other spore forming bacteria, germination is a vital step in the life cycle of this pathogen.... more
Over the past two decades, Clostridium difficile infections have been increasing in both number and severity throughout the world. As with other spore forming bacteria, germination is a vital step in the life cycle of this pathogen. Studies have examined differences in sporulation and toxin production among a number of C. difficile clinical isolates; however, few have examined differences in germination and the relationship between this phenotype and disease severity. Here, over 100 C. difficile isolates from the University of Michigan Health System were examined for overall germination in response to various combinations of known germinants (taurocholate) and co-germinants (glycine and histidine). Significant variation was observed among isolates under all conditions tested. Isolates representing ribotype 014-020, which was the most frequently isolated ribotype at our hospital, exhibited increased germination in the presence of taurocholate and glycine when compared to isolates rep...
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The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is... more
The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block...
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... Although anthrax lethal toxin (LT) may contribute significantly to septicemia and death of the host ... Anthrax is primarily a disease of herbivorous animals caused by Bacillus anthracis, a gram ... Title not supplied (PMID:17145935).... more
... Although anthrax lethal toxin (LT) may contribute significantly to septicemia and death of the host ... Anthrax is primarily a disease of herbivorous animals caused by Bacillus anthracis, a gram ... Title not supplied (PMID:17145935). Inhalational anthrax is a complex human disease that ...
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ABSTRACT
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Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model). The isolation procedure involved,... more
Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model). The isolation procedure involved, after collagenase dispersion of the liver, separation of the hepatocytes into small- and large-cell fractions by centrifugal elutriation, and subsequent selection of cells deficient in asialoglycoprotein receptor(s) by plating onto asialofetuin (ASF)-coated plates. The number of cell surface binding sites for the asialoglycoprotein receptor was measured with both asialoorosomucoid and ASF as ligands. There was a 50% reduction of binding sites for both ligands in the original cell suspensions obtained from preneoplastic livers. The reduction in receptor binding sites was most pronounced in the large cell fraction (less than or equal to 30% of control value) after separating the original cell suspension by elutriation into small and large cell fractions...
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... 39 Anthrax Spore Germination Nathan Fisher , Katherine A. Carr , Jonathan D. Giebel , and Philip C. Hanna INTRODUCTION ... Page 8. 46 Chapter 3 Anthrax Spore Germination ecules, germinant entry may involve some manner of selectivity... more
... 39 Anthrax Spore Germination Nathan Fisher , Katherine A. Carr , Jonathan D. Giebel , and Philip C. Hanna INTRODUCTION ... Page 8. 46 Chapter 3 Anthrax Spore Germination ecules, germinant entry may involve some manner of selectivity through outer protective barriers. ...
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It was with great interest that I read the report Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor by Nicholas S. Duesbery et al. (1 May, p. 734) and Evelyn Strauss's excellent accompanying Research... more
It was with great interest that I read the report Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor by Nicholas S. Duesbery et al. (1 May, p. 734) and Evelyn Strauss's excellent accompanying Research News article New clue to how anthrax kills (1 May, p. 676). I ...
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Research Interests: Genetics, Macrophages, Multidisciplinary, Antibiotic Resistance, Recombinant DNA Technology, and 13 moreVirulence, Cell line, Mice, Animals, Salmonella Typhimurium, Fatty Acid Oxidation, Enzyme, Molecular cloning, Fatty Acid, Base Sequence, Pathogenic Bacteria, Molecular Sequence Data, and Genetic factors
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Prime-boost vaccination using plasmid DNA and replication-defective adenovirus vectors has emerged as a highly effective strategy for vaccinating against viral pathogens. However, its ability to provide protection against bacterial... more
Prime-boost vaccination using plasmid DNA and replication-defective adenovirus vectors has emerged as a highly effective strategy for vaccinating against viral pathogens. However, its ability to provide protection against bacterial disease has never been assessed. Here we evaluate prime-boost vaccination approaches for immunizing against anthrax. We show that mice primed with DNA and boosted with an adenovirus vector, both expressing domain four of Bacillus anthracis protective antigen (PA), have higher antibody and toxin-neutralizing titers than mice immunized with either single modality alone. DNA-primed/adenovirus-boosted mice also had significantly higher antibody and toxin-neutralizing titers than mice immunized with Anthrax Vaccine Adsorbed. High levels of antigen-specific interferon-gamma-secreting cells were present in vaccinated mice indicating that a cell-mediated immune response had also been stimulated. Both DNA-primed/adenovirus-boosted and adenovirus-primed/adenovirus-boosted mice were fully protected from Sterne strain spore challenge. We also show that a single injection with an adenovirus vector-expressing domain four of PA can provide partial protection from spore challenge 2 weeks after immunization and full protection 3 weeks after immunization. These results demonstrate that adenovirus-based prime-boost vaccination can provide rapid protection from anthrax and that this approach may be an effective strategy for immunizing against bacterial as well as viral pathogens.
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Current knowledge of CPE action is briefly summarized in Figure 1. After specific binding to a protein receptor(s), the entire CPE molecule rapidly inserts into membranes forming a complex of 150,000 Mr. Almost simultaneously with... more
Current knowledge of CPE action is briefly summarized in Figure 1. After specific binding to a protein receptor(s), the entire CPE molecule rapidly inserts into membranes forming a complex of 150,000 Mr. Almost simultaneously with insertion, there is a sudden change in ion fluxes. The molecular events behind the induction of ion flux changes remain undefined, but might involve either direct formation of membrane pores by CPE or activation of pre-existing membrane pores. As intracellular ion levels change, cellular metabolism is affected and processes such as macromolecular syntheses are inhibited. One of the ion flux effects resulting from CPE treatment involves increased Ca2+ influx; as more Ca2+ enters the cell, morphologic damage and permeability alterations for larger molecules occur. It remains to be determined if both morphologic damage and larger permeability alterations are necessarily linked but, for example, it could be envisioned that CPE-induced Ca2+ influx causes a cytoskeletal collapse leading to altered membrane permeability. The cytoskeleton has been shown to be sensitive to intracellular Ca2+ levels and is important in normal membrane structure/function relationships. As the cumulative effects of CPE inhibit cellular metabolism, cell death occurs. The precise irreversible CPE lethal action still must be identified. As CPE-treated intestinal epithelial cells die in vivo, histopathologic damage appears. This damage results in loss of normal intestinal function causing secretion of fluids and electrolytes. This effect is clinically manifested as diarrhea. The strongly cytotoxic action of CPE clearly distinguished the action enterotoxin from STa or CT.(ABSTRACT TRUNCATED AT 250 WORDS)
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Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and... more
Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 μM and 19 μM against SbnE and 180 μM and 200 μM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus , B. anthracis , E. coli , and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents.
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Bacillus anthracis spores are the etiologic agent of anthrax. Nutrient germinant receptors (nGRs) packaged within the inner membrane of the spore sense the presence of specific stimuli in the environment and trigger the process of... more
Bacillus anthracis spores are the etiologic agent of anthrax. Nutrient germinant receptors (nGRs) packaged within the inner membrane of the spore sense the presence of specific stimuli in the environment and trigger the process of germination, quickly returning the bacterium to the metabolically active, vegetative bacillus. This ability to sense the host environment and initiate germination is a required step in the infectious cycle. The nGRs are comprised of three subunits: the A-, B-, and C-type proteins. To date there are limited structural data for the A- and B-type nGR subunits. Here the transmembrane topologies of the B. anthracis GerH A , GerH B , and GerH C proteins are presented. C-terminal green fluorescent protein (GFP) fusions to various lengths of the GerH proteins were overexpressed in vegetative bacteria, and the subcellular locations of these GFP fusion sites were analyzed by flow cytometry and protease sensitivity. GFP fusion to full-length GerH C confirmed that the...
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After entry of infectious anthrax spores into the body, host-specific signals induce spore germination, outgrowth of vegetative bacilli and the expression of lethal toxin and other virulence factors. Anthrax lethal toxin (LeTx) is a... more
After entry of infectious anthrax spores into the body, host-specific signals induce spore germination, outgrowth of vegetative bacilli and the expression of lethal toxin and other virulence factors. Anthrax lethal toxin (LeTx) is a virulence factor responsible for the major pathologies seen during systemic anthrax infections. Injection of sterile LeTx into test animals mimics the shock and sudden death seen during active bacterial infections. Once large levels of LeTx are produced within the body, destruction of bacteria by administration of antibiotics is usually unsuccessful. The LeTx is believed to be secreted into the bloodstream where it circulates freely throughout the body and binds and enters host cells. Once in the cytoplasm, the lethal factor acts as a zinc-metalloprotease disrupting normal homoeostatic functions. Macrophages are a uniquely sensitive cell type that seem to be vital global mediators of toxin-induced pathologies. Removal of macrophages from mice renders them insensitive to LeTx challenge. Low levels of lethal toxin induce macrophage production, in vitro, of the shock-inducing cytokines TNF and Il-1beta. Higher levels of LeTx cause over-production of reactive oxygen intermediates, bursting of macrophages and release of mediators of shock. We believe that agents capable of blocking key steps of the lethal toxin cascade may prove useful in combating anthrax pathologies.
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... All Rights Reserved. Murine Macrophage Transcriptional Responses to Bacillus anthracis Infection and Intoxication Nicholas H. Bergman,l' 2 Karla D. Passalacqua, 2 Renee Gaspard,3 Lynne M. Shetron-Rama, 2 John Quackenbush,3... more
... All Rights Reserved. Murine Macrophage Transcriptional Responses to Bacillus anthracis Infection and Intoxication Nicholas H. Bergman,l' 2 Karla D. Passalacqua, 2 Renee Gaspard,3 Lynne M. Shetron-Rama, 2 John Quackenbush,3 and Philip C. Hanna2 . ...