Very Early Blood Diffusion of the Active Lethal and Edema Factors of Bacillus anthracis After Intranasal Infection

Abstract Background Lethal and edema toxins are critical virulence factors of Bacillus anthracis. Few data are available on their presence in the early stage of intranasal infection. Methods To investigate the diffusion of edema factor (EF) and lethal factor (LF), we use sensitive quantitative methods to measure their enzymatic activities in mice intranasally challenged with a wild-type B anthracis strain or with an isogenic mutant deficient for the protective antigen. Results One hour after mouse challenge, although only 7% of mice presented bacteremia, LF and EF were detected in the blood of 100% and 42% of mice, respectively. Protective antigen facilitated the diffusion of LF and EF into the blood compartment. Toxins played a significant role in the systemic dissemination of B anthracis in the blood, spleen, and liver. A mouse model of intoxination further confirmed that LT and ET could diffuse rapidly in the circulation, independently of bacteria. Conclusions In this inhalational model, toxins have disseminated rapidly in the blood, playing a significant and novel role in the early systemic diffusion of bacteria, demonstrating that they may represent a very early target for the diagnosis and the treatment of anthrax.

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Expression of either Lethal Toxin or Edema Toxin by Bacillus anthracis Is Sufficient for Virulence in a Rabbit Model of Inhalational Anthrax

Infection and Immunity ◽

10.1128/iai.06340-11 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2414-2425 ◽

Cited By ~ 21

Author(s):

Julie A. Lovchik ◽

Melissa Drysdale ◽

Theresa M. Koehler ◽

Julie A. Hutt ◽

C. Rick Lyons

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Rabbit Model ◽

Lethal Toxin ◽

Content Type ◽

Edema Factor ◽

Systemic Dissemination ◽

Edema Toxin ◽

Ames Strain

ABSTRACTThe development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics againstBacillus anthracis. We investigated the roles ofB. anthraciscapsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability ofB. anthracisto traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA− mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA− mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.

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Toxin-Deficient Mutants of Bacillus anthracis Are Lethal in a Murine Model for Pulmonary Anthrax

Infection and Immunity ◽

10.1128/iai.00719-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6067-6074 ◽

Cited By ~ 61

Author(s):

Sara Heninger ◽

Melissa Drysdale ◽

Julie Lovchik ◽

Julie Hutt ◽

Mary F. Lipscomb ◽

...

Keyword(s):

Bacillus Anthracis ◽

Murine Model ◽

Parent Strain ◽

Protective Antigen ◽

Histopathological Examination ◽

Lethal Factor ◽

Lethal Dose ◽

Etiologic Agent ◽

Time To Death ◽

Edema Factor

ABSTRACT Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD50) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD50s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.

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Functional Analysis of Bacillus anthracis Protective Antigen by Using Neutralizing Monoclonal Antibodies

Infection and Immunity ◽

10.1128/iai.72.11.6313-6317.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6313-6317 ◽

Cited By ~ 71

Author(s):

Fabien Brossier ◽

Martine Lévy ◽

Annie Landier ◽

Pierre Lafaye ◽

Michèle Mock

Keyword(s):

Functional Analysis ◽

Monoclonal Antibodies ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Cell Receptor ◽

Lethal Challenge ◽

Edema Factor

ABSTRACT Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

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Toxicity of Anthrax Toxin Is Influenced by Receptor Expression

Clinical and Vaccine Immunology ◽

10.1128/cvi.00103-08 ◽

2008 ◽

Vol 15 (9) ◽

pp. 1330-1336 ◽

Cited By ~ 7

Author(s):

Sarah C. Taft ◽

Alison A. Weiss

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Chinese Hamster ◽

Receptor Expression ◽

Anthrax Toxin ◽

Cellular Receptor ◽

Wild Type ◽

Human Form ◽

Edema Factor ◽

Surface Pores

ABSTRACT Anthrax toxin protective antigen (PA) binds to its cellular receptor, and seven subunits self-associate to form a heptameric ring that mediates the cytoplasmic entry of lethal factor or edema factor. The influence of receptor type on susceptibility to anthrax toxin components was examined using Chinese hamster ovary (CHO) cells expressing the human form of one of two PA receptors: TEM8 or CMG2. Unexpectedly, PA alone, previously believed to only mediate entry of lethal factor or edema factor, was found to be toxic to CHO-TEM8 cells; cells treated with PA alone displayed reduced cell growth and decreased metabolic activity. PA-treated cells swelled and became permeable to membrane-excluded dye, suggesting that PA formed cell surface pores on CHO-TEM8 cells. While CHO-CMG2 cells were not killed by wild-type PA, they were susceptible to the PA variant, F427A. Receptor expression also conferred differences in susceptibility to edema factor.

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Killed but Metabolically Active Bacillus anthracis Vaccines Induce Broad and Protective Immunity against Anthrax

Infection and Immunity ◽

10.1128/iai.00530-08 ◽

2009 ◽

Vol 77 (4) ◽

pp. 1649-1663 ◽

Cited By ~ 24

Author(s):

Justin Skoble ◽

John W. Beaber ◽

Yi Gao ◽

Julie A. Lovchik ◽

Laurie E. Sower ◽

...

Keyword(s):

Bacillus Anthracis ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Protective Antigen ◽

Excision Repair ◽

Uv Light ◽

Lethal Factor ◽

Point Mutations ◽

Anthrax Vaccine ◽

Edema Factor

ABSTRACTBacillus anthracisis the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizingB. anthracisthat is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting thespoIIEanduvrABgenes, renderingB. anthracisextremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into thelefandcyagenes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMAB. anthracisvaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMAB. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMAB. anthracisfully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMAB. anthraciswere partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

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Murine Innate Immune Response to Virulent Toxigenic and Nontoxigenic Bacillus anthracis Strains

Infection and Immunity ◽

10.1128/iai.01712-06 ◽

2007 ◽

Vol 75 (4) ◽

pp. 1757-1764 ◽

Cited By ~ 12

Author(s):

Melissa Drysdale ◽

Gwyneth Olson ◽

Theresa M. Koehler ◽

Mary F. Lipscomb ◽

C. Rick Lyons

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Systemic Infection ◽

Neutrophil Recruitment ◽

Cell Recruitment ◽

Edema Factor ◽

Edema Toxin ◽

Neutrophil Depletion ◽

Intravenous Inoculation

ABSTRACT Effective treatment of anthrax is hampered by our limited understanding of the pathophysiology of Bacillus anthracis infection. We used a genetically complete (pXO1+ pXO2+) virulent B. anthracis strain and four isogenic toxin-null mutants to determine the effects of the anthrax edema toxin (ET; edema factor [EF] plus protective antigen [PA]) and lethal toxin (LT; lethal factor [LF] plus PA) on the host innate response during systemic infection. Using the spleen as an indicator for host response, we found that intravenous inoculation of LT-deficient mutants into C57BL/6 mice significantly increased production of several cytokines over that observed after infection with the parent strain or an EF-deficient mutant. Bacteria producing one or both of the toxins were capable of inducing significant apoptosis of cells present in spleens, whereas apoptosis was greatly reduced in mice infected with nontoxigenic mutants. Mice infected with toxin-producing strains also showed increased splenic neutrophil recruitment compared to mice infected with nontoxigenic strains and neutrophil depletion prior to infection with toxin-producing strains, leading to decreased levels of apoptosis. Together, these studies indicate that anthrax LT suppresses cytokine secretion during infection, but both EF and LF play roles in inducing neutrophil recruitment and enhancing apoptosis. Interestingly, in the absence of LF the effect of EF-induced cell recruitment is further enhanced, perhaps because LF so effectively suppresses the secretion of chemokines.

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Direct imaging of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry

10.1101/2021.01.22.427304 ◽

2021 ◽

Author(s):

Carly Merritt ◽

Elizabeth M. Chun ◽

Rasem J. Fattah ◽

Mahtab Moayeri ◽

Dennis Paliga ◽

...

Keyword(s):

Bacillus Anthracis ◽

Fluorescent Protein ◽

Protective Antigen ◽

Lethal Factor ◽

Chimeric Protein ◽

Anthrax Toxin ◽

Specific Cell ◽

Anthrax Lethal Toxin ◽

Surface Receptors ◽

Edema Factor

SUMMARYThe virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals is not fully elucidated. Herein, we describe a novel assay to image anthrax toxin intoxication in live animals, and we use the assay to visualize TEM-8- and CMG-2-dependent intoxication. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy. By using this assay, we show that CMG-2 is critical for intoxication in the liver and heart, whereas TEM-8 is required for full intoxication in the kidney and spleen. Other tissues examined were largely unaffected by single deficiences in either receptor, suggesting extensive overlap in TEM-8 and CMG-2 expression. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for identifying on- and off-targets for reengineered toxin variants in the clinical development of cancer treatments.BackgroundAssays for imaging of anthrax toxin intoxication in animals are not available.ResultsAnthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals.ConclusionShared and distinct functions of toxin receptors in cellular entry were uncovered. Significance. A simple and versatile assay for anthrax toxin intoxication is described.

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Autogenous Regulation of the Bacillus anthracis pag Operon

Journal of Bacteriology ◽

10.1128/jb.181.15.4485-4492.1999 ◽

1999 ◽

Vol 181 (15) ◽

pp. 4485-4492 ◽

Cited By ~ 45

Author(s):

Alex R. Hoffmaster ◽

Theresa M. Koehler

Keyword(s):

Steady State ◽

Bacillus Anthracis ◽

Target Cell ◽

Protective Antigen ◽

Lethal Factor ◽

Repeat Sequence ◽

Virulence Plasmid ◽

Multicopy Plasmid ◽

Transcription Terminator ◽

Edema Factor

ABSTRACT Protective antigen (PA) is an important component of the edema and lethal toxins produced by Bacillus anthracis. PA is essential for binding the toxins to the target cell receptor and for facilitating translocation of the enzymatic toxin components, edema factor and lethal factor, across the target cell membrane. The structural gene for PA, pagA (previously known aspag), is located on the 182-kb virulence plasmid pXO1 at a locus distinct from the edema factor and lethal factor genes. Here we show that a 300-bp gene located downstream of pagA is cotranscribed with pagA and represses expression of the operon. We have designated this gene pagR (for protective antigen repressor). Two pagA mRNA transcripts were detected in cells producing PA: a short, 2.7-kb transcript corresponding to thepagA gene, and a longer, 4.2-kb transcript representing a bicistronic message derived from pagA and pagR. The 3′ end of the short transcript mapped adjacent to an inverted repeat sequence, suggesting that the sequence can act as a transcription terminator. Attenuation of termination at this site results in transcription of pagR. A pagR mutant exhibited increased steady-state levels of pagA mRNA, indicating that pagR negatively controls expression of the operon. Autogenous control of the operon may involve atxA, a trans-acting positive regulator of pagA. The steady-state level of atxA mRNA was also increased in thepagR mutant. The mutant phenotype was complemented by addition of pagR in trans on a multicopy plasmid.

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In vivo dynamics of active edema and lethal factors during anthrax

Scientific Reports ◽

10.1038/srep23346 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 15

Author(s):

Clémence Rougeaux ◽

François Becher ◽

Eric Ezan ◽

Jean-Nicolas Tournier ◽

Pierre L. Goossens

Keyword(s):

Quantitative Methods ◽

Protective Antigen ◽

Lethal Factor ◽

Infection Process ◽

Long Distance ◽

Infectious Process ◽

Edema Factor ◽

Lethal Factors ◽

First Time

Abstract Lethal and edema toxins are critical virulence factors of Bacillus anthracis. However, little is known about their in vivo dynamics of production during anthrax. In this study, we unraveled for the first time the in vivo kinetics of production of the toxin components EF (edema factor) and LF (lethal factor) during cutaneous infection with a wild-type toxinogenic encapsulated strain in immuno-competent mice. We stratified the asynchronous infection process into defined stages through bioluminescence imaging (BLI), while exploiting sensitive quantitative methods by measuring the enzymatic activity of LF and EF. LF was produced in high amounts, while EF amounts steadily increased during the infectious process. This led to high LF/EF ratios throughout the infection, with variations between 50 to a few thousands. In the bloodstream, the early detection of active LF and EF despite the absence of bacteria suggests that they may exert long distance effects. Infection with a strain deficient in the protective antigen toxin component enabled to address its role in the diffusion of LF and EF within the host. Our data provide a picture of the in vivo complexity of the infectious process.

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Discriminating Virulence Mechanisms among Bacillus anthracis Strains by Using a Murine Subcutaneous Infection Model

Infection and Immunity ◽

10.1128/iai.00647-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 429-435 ◽

Cited By ~ 27

Author(s):

Hitendra S. Chand ◽

Melissa Drysdale ◽

Julie Lovchik ◽

Theresa M. Koehler ◽

Mary F. Lipscomb ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Lethal Dose ◽

Infection Model ◽

Virulence Plasmid ◽

Edema Factor ◽

Lethal Infection ◽

Toxin Protein ◽

Ames Strain

ABSTRACT Bacillus anthracis strains harboring virulence plasmid pXO1 that encodes the toxin protein protective antigen (PA), lethal factor, and edema factor and virulence plasmid pXO2 that encodes capsule biosynthetic enzymes exhibit different levels of virulence in certain animal models. In the murine model of pulmonary infection, B. anthracis virulence was capsule dependent but toxin independent. We examined the role of toxins in subcutaneous (s.c.) infections using two different genetically complete (pXO1+ pXO2+) strains of B. anthracis, strains Ames and UT500. Similar to findings for the pulmonary model, toxin was not required for infection by the Ames strain, because the 50% lethal dose (LD50) of a PA-deficient (PA−) Ames mutant was identical to that of the parent Ames strain. However, PA was required for efficient s.c. infection by the UT500 strain, because the s.c. LD50 of a UT500 PA− mutant was 10,000-fold higher than the LD50 of the parent UT500 strain. This difference between the Ames strain and the UT500 strain could not be attributed to differences in spore coat properties or the rate of germination, because s.c. inoculation with the capsulated bacillus forms also required toxin synthesis by the UT500 strain to cause lethal infection. The toxin-dependent phenotype of the UT500 strain was host phagocyte dependent, because eliminating Gr-1+ phagocytes restored virulence to the UT500 PA− mutant. These experiments demonstrate that the dominant virulence factors used to establish infection by B. anthracis depend on the route of inoculation and the bacterial strain.

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