scholarly journals Micropatterned Macrophage Analysis Reveals Global Cytoskeleton Constraints Induced by Bacillus anthracis Edema Toxin

2015 ◽  
Vol 83 (8) ◽  
pp. 3114-3125 ◽  
Author(s):  
Yannick Trescos ◽  
Emilie Tessier ◽  
Clémence Rougeaux ◽  
Pierre L. Goossens ◽  
Jean-Nicolas Tournier
Keyword(s):  
Bacillus Anthracis ◽  
Immune Cells ◽  
Adherens Junction ◽  
Peritoneal Macrophages ◽  
Cell Physiology ◽  
Actin Reorganization ◽  
Content Type ◽  
Cellular Physiology ◽  
Edema Toxin ◽  
Polar Organization

Bacillus anthracissecretes the edema toxin (ET) that disrupts the cellular physiology of endothelial and immune cells, ultimately affecting the adherens junction integrity of blood vessels that in turn leads to edema. The effects of ET on the cytoskeleton, which is critical in cell physiology, have not been described thus far on macrophages. In this study, we have developed different adhesive micropatterned surfaces (L and crossbow) to control the shape of bone marrow-derived macrophages (BMDMs) and primary peritoneal macrophages. We found that macrophage F-actin cytoskeleton adopts a specific polar organization slightly different from classical human HeLa cells on the micropatterns. Moreover, ET induced a major quantitative reorganization of F-actin within 16 h with a collapse at the nonadhesive side of BMDMs along the nucleus. There was an increase in size and deformation into a kidney-like shape, followed by a decrease in size that correlates with a global cellular collapse. The collapse of F-actin was correlated with a release of focal adhesion on the patterns and decreased cell size. Finally, the cell nucleus was affected by actin reorganization. By using this technology, we could describe many previously unknown macrophage cellular dysfunctions induced by ET. This novel tool could be used to analyze more broadly the effects of toxins and other virulence factors that target the cytoskeleton.

scholarly journals Bacillus anthracis Edema Toxin Activates Nuclear Glycogen Synthase Kinase 3β

2008 ◽  
Vol 76 (11) ◽  
pp. 4895-4904 ◽  
Author(s):  
Jason L. Larabee ◽  
Kevin DeGiusti ◽  
James L. Regens ◽  
Jimmy D. Ballard
Keyword(s):  
Cell Cycle ◽  
Bacillus Anthracis ◽  
Signaling Pathways ◽  
Glycogen Synthase ◽  
Cellular Systems ◽  
Glycogen Synthase Kinase ◽  
Cell Physiology ◽  
Cell Fractionation ◽  
Edema Toxin ◽  
Gsk 3Β

ABSTRACT Bacillus anthracis edema toxin (ET) generates high levels of cyclic AMP and impacts a complex network of signaling pathways in targeted cells. In the current study, we sought to identify kinase signaling pathways modulated by ET to better understand how this toxin alters cell physiology. Using a panel of small-molecule inhibitors of mammalian kinases, we found that inhibitors of glycogen synthase kinase 3 beta (GSK-3β) protected cells from ET-induced changes in the cell cycle. GSK-3β inhibitors prevented declines in cellular levels of cyclin D1 and c-Jun following treatment of macrophages with ET. Strikingly, cell fractionation experiments and confocal immunofluorescence microscopy revealed that ET activates a compartmentalized pool of GSK-3β residing in the nuclei, but not in the cytoplasm, of macrophages. To investigate the outcome of this event, we examined the cellular location and activation state of β-catenin, a critical substrate of GSK-3β, and found that the protein was inactivated within the nucleus following intoxication with ET. To determine if ET could overcome the effects of stimuli that inactivate GSK-3β, we examined the impact of the toxin on the Wnt signaling pathway. The results of these experiments revealed that by targeting GSK-3β residing in the nucleus, ET circumvents the upstream cytoplasmic inactivation of GSK-3β, which occurs following exposure to Wnt-3A. These findings suggest ET arrests the cell cycle by a mechanism involving activation of GSK-3β residing in the nucleus, and by using this novel mechanism of intoxication, ET avoids cellular systems that would otherwise reverse the effects of the toxin.

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 Vaccine Precipitated Induces Edema Toxin-Neutralizing, Edema Factor-Specific Antibodies in Human Recipients

2017 ◽  
Vol 24 (11) ◽  
Author(s):  
Eric K. Dumas ◽  
Timothy Gross ◽  
Jason Larabee ◽  
Lance Pate ◽  
Hannah Cuthbertson ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Cell Function ◽  
Immune Cell ◽  
Protective Antigen ◽  
Serum Samples ◽  
Anthrax Vaccine ◽  
Content Type ◽  
Specific Antibodies ◽  
Edema Factor ◽  
Edema Toxin

ABSTRACT Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP (n = 33) versus anthrax vaccine adsorbed (AVA; n = 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 ± 58.6) of EF antibodies than AVA (4.2% and 7.8 ± 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines.

scholarly journals Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth

2016 ◽  
Vol 198 (6) ◽  
pp. 986-993 ◽  
Author(s):  
Ning Liu ◽  
Yingying Li ◽  
Chunyan Dong ◽  
Xiaohan Xu ◽  
Pan Wei ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Underlying Mechanism ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Ros Production ◽  
Ampk Activation ◽  
Intracellular Growth ◽  
Serine Threonine Kinase ◽  
Content Type ◽  
Amp Activated Protein Kinase

ABSTRACTAMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process ofBrucellainfection remains unknown. Our data demonstrate thatB. abortusinfection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKβ, and TAK1 are not required for the activation of AMPK byB. abortusinfection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found thatB. abortusinfection-induced IRE1-dependent activation of AMPK promotesB. abortusintracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production.IMPORTANCEPrevious studies showed thatB. abortusinfection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused byB. abortusinfection plays important role in NADPH oxidase-derived ROS production.

scholarly journals The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Bacillus anthracis Infection in BALB/c Mice and Cynomolgus Macaques

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Trudy H. Grossman ◽  
Michael S. Anderson ◽  
Lindsay Drabek ◽  
Melanie Gooldy ◽  
Henry S. Heine ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Day Treatment ◽  
Inhalation Exposure ◽  
Treated Group ◽  
Average Dose ◽  
Once Daily ◽  
Content Type ◽  
Cynomolgus Macaques ◽  
Observation Period

ABSTRACT The fluorocycline TP-271 was evaluated in mouse and nonhuman primate (NHP) models of inhalational anthrax. BALB/c mice were exposed by nose-only aerosol to Bacillus anthracis Ames spores at a level of 18 to 88 lethal doses sufficient to kill 50% of exposed individuals (LD50). When 21 days of once-daily dosing was initiated at 24 h postchallenge (the postexposure prophylaxis [PEP] study), the rates of survival for the groups treated with TP-271 at 3, 6, 12, and 18 mg/kg of body weight were 90%, 95%, 95%, and 84%, respectively. When 21 days of dosing was initiated at 48 h postchallenge (the treatment [Tx] study), the rates of survival for the groups treated with TP-271 at 6, 12, and 18 mg/kg TP-271 were 100%, 91%, and 81%, respectively. No deaths of TP-271-treated mice occurred during the 39-day posttreatment observation period. In the NHP model, cynomolgus macaques received an average dose of 197 LD50 of B. anthracis Ames spore equivalents using a head-only inhalation exposure chamber, and once-daily treatment of 1 mg/kg TP-271 lasting for 14 or 21 days was initiated within 3 h of detection of protective antigen (PA) in the blood. No (0/8) animals in the vehicle control-treated group survived, whereas all 8 infected macaques treated for 21 days and 4 of 6 macaques in the 14-day treatment group survived to the end of the study (56 days postchallenge). All survivors developed toxin-neutralizing and anti-PA IgG antibodies, indicating an immunologic response. On the basis of the results obtained with the mouse and NHP models, TP-271 shows promise as a countermeasure for the treatment of inhalational anthrax.

scholarly journals Immunomodulatory Effects of Cinobufagin on Murine Lymphocytes and Macrophages

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Yang Yu ◽  
Hui Wang ◽  
Xianhua Meng ◽  
Lu Hao ◽  
Yue Fu ◽  
...  
Keyword(s):  
Immune Cells ◽  
Combined Treatment ◽  
Peritoneal Macrophages ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Cycle Distribution ◽  
Bioactive Component ◽  
Fetal Bovine ◽  
Spleen Lymphocytes ◽  
Mtt Assays ◽  
Enhance Activity

Cinobufagin (CBG), a major bioactive component of the traditional Chinese medicine ChanSu, has been reported to have potent pharmacological activity. In this study, we aimed to elucidate the effects of CBG on the activity of immune cells in mice. Peritoneal macrophages and splenocytes from mice were prepared and cultured in RPMI1640 supplemented with 10% fetal bovine serum. Concanavalin (ConA), lipopolysaccharide (LPS), and CBG (0.0125, 0.05, 0.15, or 0.25 μg/mL) were added to the culture medium, and the phagocytic activity of macrophages was detected by MTT assays. Additionally, lymphocyte secretion of interleukin- (IL-)2 and IL-10 was detected by enzyme-linked immunosorbent assay, and the cell cycle distribution and cell surface markers were detected by flow cytometry. Our results demonstrated that CBG promoted lymphocyte proliferation; this effect was suppressed by combined treatment with ConA or LPS. Moreover, CBG also significantly improved the CD4+/CD8+ratio in spleen lymphocytes and increased the percentage of spleen lymphocytes in the S phase. Finally, we found that CBG enhanced the secretion of IL-2 and IL-10 and increased the phagocytosis ability of macrophages. In summary, CBG could enhance activity of immune cells.

scholarly journals Glutamate Racemase Mutants of Bacillus anthracis

2015 ◽  
Vol 197 (11) ◽  
pp. 1854-1861 ◽  
Author(s):  
So-Young Oh ◽  
Stefan G. Richter ◽  
Dominique M. Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Glutamic Acid ◽  
Bacillus Anthracis ◽  
Causative Agent ◽  
Bacterial Growth ◽  
Bacterial Infections ◽  
Content Type ◽  
Antibiotic Development ◽  
Drug Candidates ◽  
Glutamate Racemase ◽  
Cell Shapes

ABSTRACTd-Glutamate is an essential component of bacterial peptidoglycan and a building block of the poly-γ-d-glutamic acid (PDGA) capsule ofBacillus anthracis, the causative agent of anthrax. Earlier work suggested that two glutamate racemases, encoded byracE1andracE2, are each essential for growth ofB. anthracis, supplyingd-glutamic acid for the synthesis of peptidoglycan and PDGA capsule. Earlier work could not explain, however, why two enzymes that catalyze the same reaction may be needed for bacterial growth. Here, we report that deletion ofracE1orracE2did not prevent growth ofB. anthracisSterne (pXO1+pXO2−), the noncapsulating vaccine strain, or ofB. anthracisAmes (pXO1+pXO2+), a fully virulent, capsulating isolate. While mutants with deletions inracE1andracE2were not viable,racE2deletion delayed vegetative growth ofB. anthracisfollowing spore germination and caused aberrant cell shapes, phenotypes that were partially restored by exogenousd-glutamate. Deletion ofracE1orracE2fromB. anthracisAmes did not affect the production or stereochemical composition of the PDGA capsule. A model is presented wherebyB. anthracis, similar toBacillus subtilis, utilizes two functionally redundant racemase enzymes to synthesized-glutamic acid for peptidoglycan synthesis.IMPORTANCEGlutamate racemases, enzymes that convertl-glutamate tod-glutamate, are targeted for antibiotic development. Glutamate racemase inhibitors may be useful for the treatment of bacterial infections such as anthrax, where the causative agent,B. anthracis, requiresd-glutamate for the synthesis of peptidoglycan and poly-γ-d-glutamic acid (PDGA) capsule. Here we show thatB. anthracispossesses two glutamate racemase genes that can be deleted without abolishing either bacterial growth or PDGA synthesis. These data indicate that drug candidates must inhibit both glutamate racemases, RacE1 and RacE2, in order to blockB. anthracisgrowth and achieve therapeutic efficacy.

scholarly journals Leishmanicidal Activity of Isoselenocyanate Derivatives

2018 ◽  
Vol 63 (2) ◽  
pp. e00904-18 ◽  
Author(s):  
Celia Fernández-Rubio ◽  
Esther Larrea ◽  
José Peña Guerrero ◽  
Eduardo Sesma Herrero ◽  
Iñigo Gamboa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Amphotericin B ◽  
Cell Cycle Progression ◽  
Leishmania Major ◽  
Antitumor Agents ◽  
Peritoneal Macrophages ◽  
Reference Drug ◽  
Content Type ◽  
Leishmanicidal Activity

ABSTRACTConventional chemotherapy against leishmaniasis includes agents exhibiting considerable toxicity. In addition, reports of drug resistance are not uncommon. Thus, safe and effective therapies are urgently needed. Isoselenocyanate compounds have recently been identified with potential antitumor activity. It is well known that some antitumor agents demonstrate effects againstLeishmania. In this study, thein vitroleishmanicidal activities of several organo-selenium and organo-sulfur compounds were tested againstLeishmania majorandLeishmania amazonensisparasites, using promastigotes and intracellular amastigote forms. The cytotoxicity of these agents was measured in murine peritoneal macrophages and their selectivity indexes were calculated. One of the tested compounds, the isoselenocyanate derivative NISC-6, showed selectivity indexes 2- and 10-fold higher than those of the reference drug amphotericin B when evaluated inL. amazonensisandL. major, respectively. The American strain (L. amazonensis) was less sensitive to NISC-6 thanL. major, showing a trend similar to that observed previously for amphotericin B. In addition, we also observed that NISC-6 significantly reduced the number of amastigotes per infected macrophage. On the other hand, we showed that NISC-6 decreases expression levels ofLeishmaniagenes involved in the cell cycle, such astopoisomerase-2(TOP-2),PCNA, andMCM4, therefore contributing to its leishmanicidal activity. The effect of this compound on cell cycle progression was confirmed by flow cytometry. We observed a significant increase of cells in the G1phase and a dramatic reduction of cells in the S phase compared to untreated cells. Altogether, our data suggest that the isoselenocyanate NISC-6 may be a promising candidate for new drug development against leishmaniasis.

scholarly journals Bioterrorism: Lessons Learned Since the Anthrax Mailings

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Michael J. Imperiale ◽  
Arturo Casadevall
Keyword(s):  
United States ◽  
Bacillus Anthracis ◽  
Life Science ◽  
Science Research ◽  
The United States ◽  
September 11 ◽  
Lessons Learned ◽  
Content Type ◽  
Risks And Benefits ◽  
September 11 Attacks

ABSTRACT In the fall of 2001, Bacillus anthracis spores were spread through letters mailed in the United States. Twenty-two people are known to have been infected, and five of these individuals died. Together with the  September 11 attacks, this resulted in a reevaluation of the risks and benefits of life science research with the potential for misuse. In this editorial, we review some of the results of these discussions and their implications for the future.

scholarly journals Immunostimulatory Effects of Recombinant Erysipelothrix rhusiopathiae Expressing Porcine Interleukin-18 in Mice and Pigs

2012 ◽  
Vol 19 (9) ◽  
pp. 1393-1398 ◽  
Author(s):  
Yohsuke Ogawa ◽  
Yu Minagawa ◽  
Fang Shi ◽  
Masahiro Eguchi ◽  
Yoshihiro Muneta ◽  
...  
Keyword(s):  
Immune Responses ◽  
Peritoneal Macrophages ◽  
Erysipelothrix Rhusiopathiae ◽  
Interleukin 18 ◽  
Murine Splenocytes ◽  
Content Type ◽  
Immunostimulatory Effect ◽  
Ifn Γ ◽  
Acquired Immune Responses

ABSTRACTInterleukin-18 (IL-18), which was originally called gamma interferon (IFN-γ)-inducing factor, has been shown to play an important role in innate and acquired immune responses. In this study, attenuatedErysipelothrix rhusiopathiaestrains were engineered to produce porcine IL-18 (poIL-18) and evaluated for their potential immunostimulatory effect in animals. Recombinant poIL-18 was successfully expressed in the recombinantE. rhusiopathiaestrains YS-1/IL-18 and KO/IL-18. The culture supernatant of YS-1/IL-18 was confirmed to induce IFN-γ production in murine splenocytesin vitro, and this production was inhibited by incubation with anti-poIL-18 monoclonal antibodies. Furthermore, more IFN-γ production was induced upon stimulation of splenocytes with concanavalin A for splenocytes from mice that were intraperitoneally inoculated with YS-1/IL-18 than for splenocytes from control mice inoculated with the parent strain YS-1. Peritoneal macrophages from mice preinoculated with YS-1/IL-18 exhibited enhanced phagocytosis ofSalmonella entericasubsp.entericaserovar Typhimurium compared with peritoneal macrophages from control mice preinoculated with YS-1. We also confirmed the immunostimulatory effect on humoral immune responses against antigens ofE. rhusiopathiaeandMycoplasma hyopneumoniaein gnotobiotic pigs that were orally preinoculated with KO/IL-18. Thus, these results provide evidence thatE. rhusiopathiaeis a promising vector for the expression of host cytokines and suggest the potential utility ofE. rhusiopathiaevector-encoded cytokines in the activation of host innate and acquired immune responses.

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