scholarly journals Certhrax Is an Antivirulence Factor for the Anthrax-Like OrganismBacillus cereusStrain G9241

2018 ◽  
Vol 86 (6) ◽  
Author(s):  
Yuliya I. Seldina ◽  
Courtney D. Petro ◽  
Stephanie L. Servetas ◽  
James M. Vergis ◽  
Christy L. Ventura ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Toxin Gene ◽  
Healthy Human ◽  
Content Type ◽  
Toxin Binding ◽  
Edema Factor ◽  
Capsule Locus

ABSTRACTBacillus cereusG9241 caused a life-threatening anthrax-like lung infection in a previously healthy human. This strain harbors two large virulence plasmids, pBCXO1 and pBC210, that are absent from typicalB. cereusisolates. The pBCXO1 plasmid is nearly identical to pXO1 fromBacillus anthracisand carries genes (pagA1,lef, andcya) for anthrax toxin components (protective antigen [called PA1 in G9241], lethal factor [LF], and edema factor [EF], respectively). The plasmid also has an intact hyaluronic acid capsule locus. The pBC210 plasmid has a tetrasaccharide capsule locus, a gene for a PA1 homolog called PA2 (pagA2), and a gene (cer) for Certhrax, an ADP-ribosyltransferase toxin that inactivates vinculin. LF, EF, and Certhrax require PA for entry into cells. In this study, we asked what role PA1, PA2, LF, and Certhrax play in the pathogenicity of G9241. To answer this, we generated isogenic deletion mutations in the targeted toxin gene components and then assessed the strains for virulence in highly G9241-susceptible (A/J) and moderately G9241-sensitive (C57BL/6) mice. We found that full virulence of G9241 required PA1 and LF, while PA2 contributed minimally to pathogenesis of G9241 but could not functionally replace PA1 as a toxin-binding subunitin vivo. Surprisingly, we discovered that Certhrax attenuated the virulence of G9241; i.e., a ΔcerΔlefmutant strain was more virulent than a Δlefmutant strain following subcutaneous inoculation of A/J mice. Moreover, the enzymatic activity of Certhrax contributed to this phenotype. We concluded that Certhrax acts as an antivirulence factor in the anthrax-like organismB. cereusG9241.

scholarly journals Mouse Monoclonal Antibodies to Anthrax Edema Factor Protect against Infection

2011 ◽  
Vol 79 (11) ◽  
pp. 4609-4616 ◽  
Author(s):  
Clinton E. Leysath ◽  
Kuang-Hua Chen ◽  
Mahtab Moayeri ◽  
Devorah Crown ◽  
Rasem Fattah ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Essential Element ◽  
Content Type ◽  
Time To Death ◽  
Edema Factor ◽  
Edema Toxin

ABSTRACTBacillus anthracisis the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic CBdomain. MAb 7F10 shows potent neutralization of edema toxin activityin vitroandin vivo; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.

scholarly journals Small-Molecule Inhibitors of Lethal Factor Protease Activity Protect against Anthrax Infection

2013 ◽  
Vol 57 (9) ◽  
pp. 4139-4145 ◽  
Author(s):  
Mahtab Moayeri ◽  
Devorah Crown ◽  
Guan-Sheng Jiao ◽  
Seongjin Kim ◽  
Alan Johnson ◽  
...  
Keyword(s):  
Small Molecule ◽  
Neutralizing Antibodies ◽  
Small Molecule Inhibitors ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Similar Efficacy ◽  
Content Type ◽  
Edema Factor ◽  
Anthrax Infection

ABSTRACTBacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated theirin vivoefficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

scholarly journals Differential Contribution of Bacillus anthracis Toxins to Pathogenicity in Two Animal Models

2012 ◽  
Vol 80 (8) ◽  
pp. 2623-2631 ◽  
Author(s):  
Haim Levy ◽  
Shay Weiss ◽  
Zeev Altboum ◽  
Josef Schlomovitz ◽  
Itai Glinert ◽  
...  
Keyword(s):  
Animal Models ◽  
Guinea Pigs ◽  
Protective Antigen ◽  
Comprehensive Evaluation ◽  
Lethal Factor ◽  
Rabbit Model ◽  
Content Type ◽  
Edema Factor ◽  
Genes Encoding ◽  
Guinea Pig Model

ABSTRACTThe virulence ofBacillus anthracis, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of thepag,lef, andcyagenes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing apagdeletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion ofpagor even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism ofB. anthracisin the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation ofB. anthracisvirulence in general and in particular for the design of relevant next-generation anthrax vaccines.

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

2004 ◽  
Vol 72 (11) ◽  
pp. 6313-6317 ◽  
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.

scholarly journals Inhibition of Anthrax Protective Antigen Outside and Inside the Cell

2006 ◽  
Vol 51 (1) ◽  
pp. 245-251 ◽  
Author(s):  
Marina V. Backer ◽  
Vimal Patel ◽  
Brian T. Jehning ◽  
Kevin P. Claffey ◽  
Vladimir A. Karginov ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Fluorescent Microscopy ◽  
Lethal Factor ◽  
Fluorescent Dye ◽  
Cell Uptake ◽  
Raw 264.7 Cells ◽  
Receptor Mediated Endocytosis ◽  
Edema Factor

ABSTRACT In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.

scholarly journals Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice

2016 ◽  
Vol 23 (3) ◽  
pp. 213-218 ◽  
Author(s):  
Mahtab Moayeri ◽  
Jacqueline M. Tremblay ◽  
Michelle Debatis ◽  
Igor P. Dmitriev ◽  
Elena A. Kashentseva ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Inbred Mouse ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Serum Levels ◽  
Mouse Strains ◽  
Common Component ◽  
Content Type ◽  
Neutralizing Agent

ABSTRACTBacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated theirin vivoefficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.

scholarly journals Combinations of Monoclonal Antibodies to Anthrax Toxin Manifest New Properties in Neutralization Assays

2013 ◽  
Vol 81 (6) ◽  
pp. 1880-1888 ◽  
Author(s):  
Mary Ann Pohl ◽  
Johanna Rivera ◽  
Antonio Nakouzi ◽  
Siu-Kei Chow ◽  
Arturo Casadevall
Keyword(s):  
Monoclonal Antibodies ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Lethal Factor ◽  
Current Treatment ◽  
Emergent Properties ◽  
Content Type ◽  
Effective Form

ABSTRACTMonoclonal antibodies (MAbs) are potential therapeutic agents againstBacillus anthracistoxins, since there is no current treatment to counteract the detrimental effects of toxemia. In hopes of isolating new protective MAbs to the toxin component lethal factor (LF), we used a strain of mice (C57BL/6) that had not been used in previous studies, generating MAbs to LF. Six LF-binding MAbs were obtained, representing 3 IgG isotypes and one IgM. One MAb (20C1) provided protection from lethal toxin (LeTx) in anin vitromouse macrophage system but did not provide significant protectionin vivo. However, the combination of two MAbs to LF (17F1 and 20C1) provided synergistic increases in protection bothin vitroandin vivo. In addition, when these MAbs were mixed with MAbs to protective antigen (PA) previously generated in our laboratory, these MAb combinations produced synergistic toxin neutralizationin vitro. But when 17F1 was combined with another MAb to LF, 19C9, the combination resulted in enhanced lethal toxicity. While no single MAb to LF provided significant toxin neutralization, LF-immunized mice were completely protected from infection withB. anthracisstrain Sterne, which suggested that a polyclonal response is required for effective toxin neutralization. In total, these studies show that while a single MAb against LeTx may not be effective, combinations of multiple MAbs may provide the most effective form of passive immunotherapy, with the caveat that these may demonstrate emergent properties with regard to protective efficacy.

scholarly journals In vivo dynamics of active edema and lethal factors during anthrax

2016 ◽  
Vol 6 (1) ◽  
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.

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

2012 ◽  
Vol 80 (7) ◽  
pp. 2414-2425 ◽  
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.

scholarly journals Anthrax Toxin Translocation Complex Reveals insight into the Lethal Factor Unfolding and Refolding Mechanism

2020 ◽  
Author(s):  
Alexandra J Machen ◽  
Mark T Fisher ◽  
Bret D Freudenthal
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Pore Channel ◽  
Edema Factor ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Partially Unfolded ◽  
Α Helix

AbstractTranslocation is essential to the anthrax toxin mechanism. Protective antigen (PA), the translocon component of this AB toxin, forms an oligomeric pore with three key clamp sites that aid in the efficient entry of lethal factor (LF) or edema factor (EF), the enzymatic components of the toxin, into the cell. LF and EF translocate through the PA pore (PApore) with the pH gradient between the endosome and the cytosol facilitating rapid translocation in vivo. Structural details of the translocation process have remained elusive despite their biological importance. To overcome the technical challenges of studying translocation intermediates, we developed a novel method to immobilize, transition, and stabilize anthrax toxin to mimic important physiological steps in the intoxication process. Here, we report a cryoEM snapshot of PApore translocating the N-terminal domain of LF (LFN). The resulting 3.3 Å structure of the complex shows density of partially unfolded LFN near the canonical PApore binding site as well as in the α clamp, the Φ clamp, and the charge clamp. We also observe density consistent with an α helix emerging from the 100 Å β barrel channel suggesting LF secondary structural elements begin to refold in the pore channel. We conclude the anthrax toxin β barrel aids in efficient folding of its enzymatic payload prior to channel exit. Our hypothesized refolding mechanism has broader implications for pore length of other protein translocating toxins.Significance StatementToxins like the anthrax toxin aid bacteria in establishing an infection, evading the immune system, and proliferating inside a host. The anthrax toxin, a proteinaceous AB toxin secreted by Bacillus anthracis, consists of lethal factor and protective antigen. In this work, we explore the molecular details of lethal factor translocation through protective antigen pore necessary for cellular entry. Our cryo electron microscopy results provide evidence of lethal factor secondary structure refolding prior to protective antigen pore exit. Similar to the ribosome exit tunnel, the toxin pore channel likely contributes to native folding of lethal factor. We predict other AB toxins with extended pores also initiate substrate refolding inside the translocon for effective intoxication during bacterial infection, evasion, and proliferation.

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