Antibody titers of PEG-PLA block copolymer nanosphere containing chimeric recombinant protein of protective antigen and lethal factor of Bacillus anthracis

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

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Injection of Staphylococcus aureus EDIN by the Bacillus anthracis Protective Antigen Machinery Induces Vascular Permeability

Infection and Immunity ◽

10.1128/iai.00186-09 ◽

2009 ◽

Vol 77 (9) ◽

pp. 3596-3601 ◽

Cited By ~ 27

Author(s):

Monica Rolando ◽

Patrick Munro ◽

Caroline Stefani ◽

Patrick Auberger ◽

Gilles Flatau ◽

...

Keyword(s):

Bacillus Anthracis ◽

Vascular Permeability ◽

Protective Antigen ◽

Lethal Factor ◽

Vascular Leakage ◽

Host Cells ◽

Mitogen Activated Protein Kinase ◽

Systemic Injection ◽

Pulmonary Endothelium ◽

Endothelium Permeability

ABSTRACT Systemic injection of Bacillus anthracis lethal toxin (LT) produces vascular leakage and animal death. Recent studies suggest that LT triggers direct endothelial cell cytotoxicity that is responsible for the vascular leakage. LT is composed of heptamers of protective antigen (PA), which drives the endocytosis and translocation into host cells of the lethal factor (LF), a mitogen-activated protein kinase kinase protease. Here we investigated the consequences of injection of an endothelium-permeabilizing factor using LT as a “molecular syringe.” To this end, we generated the chimeric factor LE, corresponding to the PA-binding domain of LF (LF1-254) fused to EDIN exoenzyme. EDIN ADP ribosylates RhoA, leading to actin cable disruption and formation of transcellular tunnels in endothelial cells. We report that systemic injection of LET (LE plus PA) triggers a PA-dependent increase in the pulmonary endothelium permeability. We also report that native LT induces a progressive loss of endothelium barrier function. We established that there is a direct correlation between the extent of endothelium permeability induced by LT and the cytotoxic activity of LT. This suggests new ways to design therapeutic drugs against anthrax directed toward vascular permeability.

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Comparative Secretome Analyses of Three Bacillus anthracis Strains with Variant Plasmid Contents

Infection and Immunity ◽

10.1128/iai.73.6.3646-3658.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3646-3658 ◽

Cited By ~ 39

Author(s):

Janine M. Lamonica ◽

MaryAnn Wagner ◽

Michel Eschenbrenner ◽

Leanne E. Williams ◽

Tabbi L. Miller ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Isocitrate Lyase ◽

Lethal Factor ◽

Protein A ◽

Surface Protein ◽

Computer Assisted ◽

Two Dimensional Gel Electrophoresis ◽

Peptide Mass ◽

Proteomic Approach

ABSTRACT Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1+ pXO2+) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1+ pXO2−) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1− pXO2−). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1+. This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.

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Detection and Fate of Bacillus anthracis (Sterne) Vegetative Cells and Spores Added to Bulk Tank Milk†

Journal of Food Protection ◽

10.4315/0362-028x-66.12.2349 ◽

2003 ◽

Vol 66 (12) ◽

pp. 2349-2354 ◽

Cited By ~ 29

Author(s):

MICHAEL L. PERDUE ◽

JEFF KARNS ◽

JIM HIGGINS ◽

JO ANN VAN KESSEL

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Raw Milk ◽

Molecular Data ◽

Molecular Techniques ◽

Nucleotide Sequence Analysis ◽

Vegetative Cells ◽

Bulk Tank Milk ◽

Bulk Tank

A preparation of Bacillus anthracis (Sterne strain) spores was used to evaluate commercially available reagents and portable equipment for detecting anthrax contamination by using real-time PCR and was used to assess the fate of spores added directly to bulk tank milk. The Ruggedized Advanced Pathogen Identification Device (RAPID) was employed to detect spores in raw milk down to a concentration of 2,500 spores per ml. Commercially available primers and probes developed to detect either the protective antigen gene or the lethal factor gene both provided easily read positive signals with the RAPID following extraction from milk with a commercially available DNA extraction kit. Nucleotide sequence analysis of the vrrA gene with the use of DNA extracted from spiked milk provided molecular data that readily identified the spores as B. anthracis with a 100% BLAST match to the Sterne and Ames strains and easily distinguished them from B. cereus. Physical-fate and thermal-stability studies demonstrated that spores and vegetative cells have a strong affinity for the cream fraction of whole milk. A single treatment at standard pasteurization temperatures, while 100% lethal to vegetative cells, had no effect on spore viability even 14 days after the treatment. Twenty-four hours after the first treatment, a second treatment at 72°C for 15 s reduced the viability of the population by ca. 99% but still did not kill all of the spores. From these studies, we conclude that standard pasteurization techniques for milk would have little effect on the viability of B. anthracis spores and that raw or pasteurized milk poses no obstacles to the rapid detection of the spores by molecular techniques.

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

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz497 ◽

2019 ◽

Vol 221 (4) ◽

pp. 660-667 ◽

Cited By ~ 3

Author(s):

Clémence Rougeaux ◽

François Becher ◽

Pierre L Goossens ◽

Jean-Nicolas Tournier

Keyword(s):

Bacillus Anthracis ◽

Quantitative Methods ◽

Protective Antigen ◽

Early Stage ◽

Lethal Factor ◽

Wild Type ◽

Intranasal Infection ◽

Edema Factor ◽

Systemic Dissemination ◽

Few Data

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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Glycerol Monolaurate Inhibits Virulence Factor Production in Bacillus anthracis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.4.1302-1305.2005 ◽

2005 ◽

Vol 49 (4) ◽

pp. 1302-1305 ◽

Cited By ~ 24

Author(s):

Sara M. Vetter ◽

Patrick M. Schlievert

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Polyacrylamide Gel Electrophoresis ◽

Lethal Factor ◽

Sodium Dodecyl ◽

The United States ◽

Human Populations ◽

Transcriptional Level ◽

Mrna Levels ◽

Glycerol Monolaurate

ABSTRACT Anthrax, caused by Bacillus anthracis, has been brought to the public's attention because of the 2001 bioterrorism attacks. However, anthrax is a disease that poses agricultural threats in the United States as well as human populations in Europe, China, Africa, and Australia. Glycerol monolaurate (GML) is a compound that has been shown to inhibit exotoxin production by Staphylococcus aureus and other gram-positive bacteria. Here, we study the effects of GML on growth and toxin production in B. anthracis. The Sterne strain of B. anthracis was grown to post-exponential phase with 0-, 10-, 15-, or 20-μg/ml concentrations of GML and then assayed quantitatively for protective antigen (PA) and lethal factor (LF). After 8 h, GML at concentrations greater than 20 μg/ml was bacteriostatic to growth of the organism. However, a 10-μg/ml concentration of GML was not growth inhibitory, but amounts of PA and LF made were greatly reduced. This effect was not global for all proteins when total secreted protein from culture fluids was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Through quantitative reverse transcription-PCR assays, this toxin-inhibitory effect was shown to occur at the transcriptional level, since amounts of mRNA for pagA (PA), lef (LF), and cya (edema factor) were reduced. Surprisingly, mRNA levels of atxA, a regulator of exotoxin gene expression, rose in the presence of GML. These data will be useful in developing therapeutic tools to treat anthrax disease, whether in animals or humans. These results also suggest that mechanisms of virulence regulation exist independent of atxA.

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The Humoral Immune Response to Various Domains of Protective Antigen of Bacillus anthracis in Cutaneous Anthrax Cases in India

Defence Science Journal ◽

10.14429/dsj.66.10805 ◽

2016 ◽

Vol 66 (6) ◽

pp. 645 ◽

Cited By ~ 3

Author(s):

Anshul Varshney ◽

Nidhi Puranik ◽

M. Kumar ◽

A.K. Goel

Keyword(s):

Immune Response ◽

Bacillus Anthracis ◽

Humoral Immune Response ◽

Vaccine Development ◽

Protective Antigen ◽

Lethal Factor ◽

Serum Samples ◽

Public Health Importance ◽

Humoral Immune ◽

Cutaneous Anthrax

Anthrax, caused by Bacillus anthracis is known to occur globally since antiquity. Besides being an important biothreat agent, it is an important public health importance pathogen also in countries like India. B. anthracis secretes three distinct toxins, namely protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the central moiety of the anthrax toxin complex and therefore has been a molecule of choice for vaccine development. PA has four different domains with different functions. In this study, the major domains of PA were cloned and expressed in bacterial system. The purified recombinant proteins were used to determine the humoral immune response by ELISA using 43 human cutaneous anthrax serum samples. The maximum immunoreactivity was observed with the whole PA protein followed by domain 2, 4 and 1. The study corroborated that in addition to full PA, individual domain 2 and 4 can also be good target for vaccine development as well as for serodiagnostic assays for cutaneous anthrax

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Involvement of Residues 147VYYEIGK153 in Binding of Lethal Factor to Protective Antigen of Bacillus anthracis,

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.2001.4477 ◽

2001 ◽

Vol 281 (5) ◽

pp. 1358-1359

Author(s):

Pankaj Gupta ◽

Aparna Singh ◽

Vibha Chauhan ◽

Rakesh Bhatnagar

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor

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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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