RESUMO
Anthrax is a major zoonotic disease of wildlife, and in places like West Africa, it can be caused by Bacillus anthracis in arid nonsylvatic savannahs, and by B. cereus biovar anthracis (Bcbva) in sylvatic rainforests. Bcbva-caused anthrax has been implicated in as much as 38% of mortality in rainforest ecosystems, where insects can enhance the transmission of anthrax-causing bacteria. While anthrax is well-characterized in mammals, its transmission by insects points to an unidentified anthrax-resistance mechanism in its vectors. In mammals, a secreted anthrax toxin component, 83 kDa Protective Antigen (PA83), binds to cell-surface receptors and is cleaved by furin into an evolutionary-conserved PA20 and a pore-forming PA63 subunits. We show that PA20 increases the resistance of Drosophila flies and Culex mosquitoes to bacterial challenges, without directly affecting the bacterial growth. We further show that the PA83 loop known to be cleaved by furin to release PA20 from PA63 is, in part, responsible for the PA20-mediated protection. We found that PA20 binds directly to the Toll activating peptidoglycan-recognition protein-SA (PGRP-SA) and that the Toll/NF-κB pathway is necessary for the PA20-mediated protection of infected flies. This effect of PA20 on innate immunity may also exist in mammals: we show that PA20 binds to human PGRP-SA ortholog. Moreover, the constitutive activity of Imd/NF-κB pathway in MAPKK Dsor1 mutant flies is sufficient to confer the protection from bacterial infections in a manner that is independent of PA20 treatment. Lastly, Clostridium septicum alpha toxin protects flies from anthrax-causing bacteria, showing that other pathogens may help insects resist anthrax. The mechanism of anthrax resistance in insects has direct implications on insect-mediated anthrax transmission for wildlife management, and with potential for applications, such as reducing the sensitivity of pollinating insects to bacterial pathogens.
Assuntos
Vacinas contra Antraz/administração & dosagem , Antraz/tratamento farmacológico , Antígenos de Bactérias/administração & dosagem , Bacillus anthracis/efeitos dos fármacos , Toxinas Bacterianas/administração & dosagem , Drosophila melanogaster/crescimento & desenvolvimento , Mosquitos Vetores/microbiologia , Substâncias Protetoras/administração & dosagem , Animais , Antraz/microbiologia , Culex , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Feminino , MasculinoRESUMO
BACKGROUND: Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified. RESULTS: In this study, we demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, we identified the closest genetic GR homolog as D. melanogaster Estrogen Related Receptor (ERR) gene. We discovered that Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as we show that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene. CONCLUSIONS: This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/microbiologia , Hidrocortisona/análogos & derivados , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/patogenicidade , Animais , Simulação por Computador , Cortisona/efeitos adversos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ergosterol/efeitos adversos , Estrogênios/efeitos adversos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata , Mutação , Saccharomyces cerevisiae/metabolismoRESUMO
Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQ in mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD60). In rabbits challenged with 200 LD50 of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQ in humans from over 2â¯000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.
Assuntos
Antraz , Bacillus anthracis , Amodiaquina , Animais , Antraz/tratamento farmacológico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Levofloxacino , Camundongos , Coelhos , Revisões Sistemáticas como AssuntoRESUMO
Of the numerous infectious diseases afflicting humans, anthrax disease, caused by Bacillus anthracis, poses a major threat in its virulence and lack of effective treatment. The currently lacking standards of care, as well as the lengthy drug approval process, demonstrate the pressing demand for treatment for B. anthracis infections. The present study screened 1586 clinically approved drugs in an attempt to identify repurposable compounds against B. cereus, a relative strain that shares many physical and genetic characteristics with B. anthracis. Our study yielded five drugs that successfully inhibited B. cereus growth: dichlorophen, oxiconazole, suloctidil, bithionol, and hexestrol. These drugs exhibited varying levels of efficacy in broad-spectrum experiments against several Gram-positive and Gram-negative bacterial strains, with hexestrol showing the greatest inhibition across all tested strains. Through tests for the efficacy of each drug on B. cereus, bithionol was the single most potent compound on both solid and liquid media and exhibited even greater eradication of B. cereus in combination with suloctidil on solid agar. This multifaceted in vitro study of approved drugs demonstrates the potential to repurpose these drugs as treatments for anthrax disease in a time-efficient manner to address a global health need.
RESUMO
The rising prevalence of multidrug-resistant hospital-acquired infections has increased the need for new antibacterial agents. In this study, a library of 1586 FDA-approved drugs was screened against A. calcoaceticus, a representative of the Acinetobacter calcoaceticus-baumannii complex. Three compounds were found to have previously undiscovered antibacterial properties against A. calcoaceticus: antifungal Miconazole, anthelminthic Dichlorophen, and Bithionol. These three drugs were tested against a wide range of Gram-positive and Gram-negative bacteria and confirmed to have broad-spectrum antibacterial properties. Combinations of these three drugs were also tested against the same bacteria, and two novel combination therapies with synergistic effects were discovered. In the future, antibacterial properties of these three drugs and two combination therapies will be evaluated against pathogenic bacteria using an animal model.
RESUMO
Inflammasomes activate caspase-1 in response to molecular signals from pathogens and other dangerous stimuli as a part of the innate immune response. A previous study discovered a small-molecule, 4-fluoro- N'-[1-(2-pyridinyl)ethylidene]benzohydrazide, which we named DN1, that reduces the cytotoxicity of anthrax lethal toxin (LT). We determined that DN1 protected cells irrespectively of LT concentration and reduced the pathogenicity of an additional bacterial exotoxin and several viruses. Using the LT cytotoxicity pathway, we show that DN1 does not prevent LT internalization and catalytic activity or caspase-1 activation. Moreover, DN1 does not affect the proteolytic activity of host cathepsin B, which facilitates the cytoplasmic entry of toxins. PubChem Bioactivities lists two G protein-coupled receptors (GPCR), type-1 angiotensin II receptor and apelin receptor, as targets of DN1. The inhibition of phosphatidylinositol 3-kinase, phospholipase C, and protein kinase B, which are downstream of GPCR signaling, synergized with DN1 in protecting cells from LT. We hypothesize that DN1-mediated antagonism of GPCRs modulates signal transduction pathways to induce a cellular state that reduces LT-induced pyroptosis downstream of caspase-1 activation. DN1 also reduced the susceptibility of Drosophila melanogaster to toxin-associated bacterial infections. Future experiments will aim to further characterize how DN1 modulates signal transduction pathways to inhibit pyroptotic cell death in LT-sensitive macrophages. DN1 represents a novel chemical probe to investigate host cellular mechanisms that mediate cell death in response to pathogenic agents.
Assuntos
Antraz/fisiopatologia , Antibacterianos/farmacologia , Antígenos de Bactérias/toxicidade , Bacillus anthracis/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Morte Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Antraz/tratamento farmacológico , Antraz/metabolismo , Antraz/microbiologia , Antibacterianos/química , Bacillus anthracis/genética , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Caspase 1/genética , Caspase 1/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Drosophila melanogaster , Feminino , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Exploiting the host endocytic trafficking pathway is a common mechanism by which bacterial exotoxins gain entry to exert virulent effects upon the host cells. A previous study identified a small-molecule, 1-(2,6-dimethyl-1-piperidinyl)-3-[(2-isopropyl-5-methylcyclohexyl)oxy]-2-propanol, that blocks the process of anthrax lethal toxin (LT) cytotoxicity. Here, we report the characterization of the bioactivity of this compound, which we named RC1. We found that RC1 protected host cells independently of LT concentration and also blocked intoxication by other bacterial exotoxins, suggesting that the target of the compound is a host factor. Using the anthrax LT intoxication pathway as a reference, we show that while anthrax toxin is able to bind to cells and establish an endosomal pore in the presence of the drug, the toxin is unable to translocate into the cytosol. We demonstrate that RC1 does not inhibit the toxin directly but rather reduces the enzymatic activity of host cathepsin B that mediates the escape of toxins into the cytoplasm from late endosomes. We demonstrate that the pathogenicity of Human cytomegalovirus and Herpes simplex virus 1, which relies on cathepsin B protease activity, is reduced by RC1. This study reveals the potential of RC1 as a broad-spectrum host-oriented therapy against several aggressive and deadly pathogens.