RESUMO
Toxins play a major role in the pathogenesis of Bacillus anthracis by subverting the host defenses. However, besides toxins, B. anthracis expresses effector proteins, whose role in pathogenesis are yet to be investigated. Here we present that suppressor-of-variegation, enhancer-of-zeste, trithorax protein from B. anthracis (BaSET) methylates human histone H1, resulting in repression of NF-κB functions. Notably, BaSET is secreted and undergoes nuclear translocation to enhance H1 methylation in B. anthracis-infected macrophages. Compared with wild type Sterne, delayed growth kinetics and altered septum formation were observed in the BaSET knock-out (BaΔSET) bacilli. Uncontrolled BaSET expression during complementation of the BaSET gene in BaΔSET partially restored growth during stationary phase but resulted in substantially shorter bacilli throughout the growth cycle. Importantly, in contrast to Sterne, the BaΔSET B. anthracis is avirulent in a lethal murine bacteremia model of infection. Collectively, BaSET is required for repression of host transcription as well as proper B. anthracis growth, making it a potentially unique virulence determinant.
Assuntos
Antraz/enzimologia , Bacillus anthracis , Proteínas de Bactérias/biossíntese , Epigênese Genética , Macrófagos/metabolismo , NF-kappa B/metabolismo , Proteínas Metiltransferases/biossíntese , Transcrição Gênica , Fatores de Virulência/biossíntese , Animais , Antraz/genética , Antraz/patologia , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Células HeLa , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , NF-kappa B/genética , Proteínas Metiltransferases/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. METHODS: We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K(i) and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. RESULTS: We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. CONCLUSIONS: These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.
Assuntos
Bacillus anthracis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Antagonistas do Ácido Fólico/química , Simulação de Acoplamento Molecular , Tetra-Hidrofolato Desidrogenase/química , Antraz/tratamento farmacológico , Antraz/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Antagonistas do Ácido Fólico/uso terapêutico , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
BACKGROUND: Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. MATERIAL AND METHODS: Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. RESULTS: Serum ADA activity was signiï¬cantly higher in patients with cutaneous anthrax than in the controls (p<0.001). A positive correlation was observed between ADA activity and lymphocyte counts (r=0.589, p=0.021) in the patient group. CONCLUSIONS: This study suggests that serum ADA could be used as a biochemical marker in cutaneous anthrax.
Assuntos
Adenosina Desaminase/sangue , Antraz/sangue , Antraz/enzimologia , Dermatopatias Bacterianas/sangue , Dermatopatias Bacterianas/enzimologia , Adulto , Estudos de Casos e Controles , Demografia , Feminino , Humanos , MasculinoRESUMO
Bacillus anthracis, a causative agent of anthrax, is able to germinate and survive within macrophages. A recent study suggested that B. anthracis-derived nitric oxide (bNO) is a key aspect of bacterial defense that protects bacterial DNA from oxidative burst in the macrophages. However, the virulent effect of bNO in host cells has not been investigated. Here, we report that bNO contributes macrophage killing by S-nitrosylation of bioenergetic-relating proteins within mitochondria. Toxigenic Sterne induces expression of the bnos gene and produces bNO during early stage of infection. Nitroso-proteomic analysis coupled with a biotin-switch technique demonstrated that toxigenic infection induces protein S-nitrosylation in B. anthracis-susceptible RAW264.7. For each target enzyme tested (complex I, complex III and complex IV), infection by B. anthracis Sterne caused enzyme inhibition. Nω-nitro-L-arginine methyl ester, a NO synthase inhibitor, reduced S-nitrosylation and partially restored cell viability evaluated by intracellular ATP levels in macrophages. Our data suggest that bNO leads to energy depletion driven by impaired mitochondrial bioenergetic machinery that ultimately contributes to macrophage death. This novel mechanism of anthrax pathogenesis may offer specific approach to the development of therapeutics.
Assuntos
Antraz/imunologia , Apoptose/imunologia , Bacillus anthracis/patogenicidade , Macrófagos/microbiologia , Mitocôndrias/microbiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Animais , Antraz/enzimologia , Antraz/microbiologia , Bacillus anthracis/metabolismo , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Macrófagos/enzimologia , Camundongos , Mitocôndrias/enzimologia , Óxido Nítrico Sintase Tipo I/genéticaRESUMO
Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.
Assuntos
Antraz/tratamento farmacológico , Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Simulação por Computador , Engenharia de Proteínas , gama-Glutamiltransferase/química , Sequência de Aminoácidos , Animais , Antraz/enzimologia , Antraz/genética , Bacillus anthracis/genética , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/uso terapêutico , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismo , gama-Glutamiltransferase/uso terapêuticoRESUMO
Bacillus anthracis is a gram-positive spore-forming bacterium that causes anthrax. With the increased threat of anthrax in biowarfare, there is an urgent need to characterize new antimicrobial targets from B. anthracis. One such target is dihydrodipicolinate synthase (DHDPS), which catalyzes the committed step in the pathway yielding meso-diaminopimelate and lysine. In this study, we employed CD spectroscopy to demonstrate that the thermostability of DHDPS from B. anthracis (Ba-DHDPS) is significantly enhanced in the presence of the substrate, pyruvate. Analytical ultracentrifugation studies show that the tetramer-dimer dissociation constant of the enzyme is 3-fold tighter in the presence of pyruvate compared with the apo form. To examine the significance of this substrate-mediated stabilization phenomenon, a dimeric mutant of Ba-DHDPS (L170E/G191E) was generated and shown to have markedly reduced activity compared with the wild-type tetramer. This demonstrates that the substrate, pyruvate, stabilizes the active form of the enzyme. We next determined the high resolution (2.15 A) crystal structure of Ba-DHDPS in complex with pyruvate (3HIJ) and compared this to the apo structure (1XL9). Structural analyses show that there is a significant (91 A(2)) increase in buried surface area at the tetramerization interface of the pyruvate-bound structure. This study describes a new mechanism for stabilization of the active oligomeric form of an antibiotic target from B. anthracis and reveals an "Achilles heel" that can be exploited in structure-based drug design.
Assuntos
Antraz/enzimologia , Bacillus anthracis/enzimologia , Proteínas de Bactérias/química , Hidroliases/química , Ácido Pirúvico/química , Substituição de Aminoácidos , Antraz/tratamento farmacológico , Antraz/genética , Antibacterianos/química , Antibacterianos/uso terapêutico , Bacillus anthracis/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Armas Biológicas , Ácido Diaminopimélico/química , Ácido Diaminopimélico/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Hidroliases/antagonistas & inibidores , Hidroliases/genética , Hidroliases/metabolismo , Lisina/química , Lisina/genética , Lisina/metabolismo , Mutação de Sentido Incorreto , Estrutura Quaternária de Proteína/fisiologia , Ácido Pirúvico/metabolismoRESUMO
Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0 +/- 0.02, 1.0 +/- 0.04, 2.1 +/- 0.12, and 2.0 +/- 0.08 microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05 +/- 0.002, 0.153 +/- 0.004, 1.30 +/- 0.10, and 1.29 +/- 0.40 microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52 kcal/mol and formed a single hydrogen bond with a distance of 1.973 A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.
Assuntos
Aldeído-Cetona Transferases/antagonistas & inibidores , Aldeído-Cetona Transferases/química , Antibacterianos/química , Bacillus anthracis/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Pirimidinas/química , Aldeído-Cetona Transferases/genética , Aldeído-Cetona Transferases/metabolismo , Antraz/tratamento farmacológico , Antraz/enzimologia , Antibacterianos/uso terapêutico , Domínio Catalítico , Inibidores Enzimáticos/uso terapêutico , Ligação de Hidrogênio , Ligação Proteica , Pirimidinas/uso terapêutico , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L(-/-) mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.
Assuntos
Antraz/enzimologia , Bacillus anthracis , Endorribonucleases/biossíntese , Infecções por Escherichia coli/enzimologia , Escherichia coli , Interferon Tipo I/biossíntese , Animais , Antraz/genética , Antraz/imunologia , Bacillus anthracis/imunologia , Catepsina E/biossíntese , Catepsina E/genética , Catepsina E/imunologia , Endorribonucleases/genética , Endorribonucleases/imunologia , Endossomos/enzimologia , Endossomos/genética , Endossomos/imunologia , Escherichia coli/imunologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Camundongos , Camundongos Knockout , Estabilidade de RNA/genética , Estabilidade de RNA/imunologiaRESUMO
Anthrax lethal toxin (LT) was previously shown to enhance transcriptional activity of NF-kappaB in tumor necrosis factor-alpha-activated primary human endothelial cells. Here we show that this LT-mediated increase in NF-kappaB activation is associated with the enhanced degradation of the inhibitory proteins IkappaBalpha and IkappaBbeta but not IkappaBepsilon. Moreover, this was accompanied by enhanced activation of the IkappaB kinase complex (IKK), which is responsible for targeting IkappaB proteins for degradation. Importantly, LT enhancement of IkappaBalpha degradation was completely blocked by a selective IKKbeta inhibitor, whereas IkappaBbeta degradation was attenuated, suggesting a mechanistic link. Consistent with the above data, LT-cotreated cells show elevated phosphorylation of two IKK substrates, IkappaBalpha and p65, both of which were blocked by incubation with the IKKbeta inhibitor. Consistent with NF-kappaB activation, LT increased transcription of the NF-kappaB regulated gene CD40. Conversely, LT inhibited transcription of another NF-kappaB-regulated gene, CCL2. This inhibition was linked to the LT-mediated suppression of another CCL2-regulating transcription factor, AP-1 (activator protein-1). These data suggest that LT-mediated enhancement of NF-kappaB is IKK-dependent, but importantly, the net effect of LT on the transcription of proinflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax.
Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Antígenos CD40/biossíntese , Quimiocina CCL2/biossíntese , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Antraz/enzimologia , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Humanos , Proteínas I-kappa B/metabolismo , Inflamação/enzimologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Furin is a protease that is ubiquitous in mammalian metabolism. One of the innovations that make sudden acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) more infectious than its ancestor viruses is the addition of a furin cleavage site. Conditions associated with elevated furin levels, including diabetes, obesity, and hypertension, overlap greatly with vulnerability to the severe form of coronavirus disease 2019 (COVID-19). We suggest that diet and lifestyle modifications that reduce the associated comorbidities may prevent the development of severe COVID-19 by, in part, lowering circulating furin levels. Likewise, natural and pharmaceutical inhibitors of furin may be candidate prophylactic interventions or, if used early in the COVID-19, may prevent the development of critical symptoms.
Assuntos
Antraz/sangue , COVID-19/sangue , Diabetes Mellitus/sangue , Furina/sangue , Hipertensão/sangue , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Antraz/enzimologia , COVID-19/enzimologia , Diabetes Mellitus/enzimologia , Humanos , Hipertensão/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologiaRESUMO
Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85ß (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.
Assuntos
Antraz/enzimologia , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/toxicidade , Bacillus anthracis/enzimologia , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Peptídeo Hidrolases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Motivos de Aminoácidos , Animais , Antraz/genética , Antraz/microbiologia , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genéticaRESUMO
Bacillus anthracis, the etiological agent of anthrax, is a spore-forming gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs), sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A-dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deadly pathogen.
Assuntos
Antraz/prevenção & controle , Antígenos de Bactérias/toxicidade , Bacillus anthracis/enzimologia , Toxinas Bacterianas/toxicidade , Fosfolipases A2 do Grupo II/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Animais , Antraz/enzimologia , Antraz/imunologia , Lavagem Broncoalveolar , Células Cultivadas , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Fosfolipases A2 do Grupo II/imunologia , Cobaias , Interações Hospedeiro-Patógeno , Imunidade Inata , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/microbiologia , Masculino , RNA Mensageiro/metabolismoRESUMO
Inhalation of anthrax spores rapidly develops into a deadly bacteraemia and toxaemia. Anthrax toxins include the lethal factor (LF), a mitogen-activated protein kinase (MAPK)-kinase-specific metalloprotease, which acts in the cell cytosol and plays a major part in anthrax pathogenesis. Recently, screening methods have led to the discovery of LF inhibitors that are membrane permeable. This will pave the way for design of novel anthrax therapeutics that are capable of inhibiting the metalloprotease activity of LF in vivo.
Assuntos
Antraz/enzimologia , Toxinas Bacterianas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Metaloproteases/antagonistas & inibidores , Peptídeos/farmacologia , Antígenos de Bactérias , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be downregulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.
Assuntos
Antraz/microbiologia , Bacillus anthracis/fisiologia , Macrófagos/enzimologia , Macrófagos/microbiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Animais , Antraz/enzimologia , Antraz/metabolismo , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Humanos , Macrófagos/química , Macrófagos/metabolismo , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , Mapeamento de Peptídeos , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esporos Bacterianos/fisiologiaRESUMO
Lethal factor (LF), along with its receptor-binding partner protective antigen (PA), forms lethal toxin (LT), a critical virulence factor for Bacillus anthracis. LF is a Zn(2+) protease that cleaves specific mitogen activated protein kinase kinases (MAPKKs), inactivating signal transduction intermediates required for normal immune function. Initial research emphasized the role of LT in attenuating pro-inflammatory responses by macrophages, the primary targets of infection. More recent studies have revealed that LT affects a broad range of immune cells. In addition to direct effects on macrophages and neutrophils, LT suppresses the costimulatory functions of dendritic cells, thereby impeding essential cross-talk between innate and adaptive immune responses. Moreover, LT acts directly on T and B lymphocytes, blocking antigen receptor-dependent proliferation, cytokine production and Ig production. In this manner, LT mounts a broad-based attack on host immunity, thus providing B. anthracis with multiple mechanisms for avoiding protective host responses.
Assuntos
Antraz/imunologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Leucócitos Mononucleares/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Metaloproteases/imunologia , Animais , Antraz/enzimologia , Antraz/metabolismo , Antígenos de Bactérias/metabolismo , Bacillus anthracis/enzimologia , Toxinas Bacterianas/metabolismo , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Metaloproteases/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismoRESUMO
The MEK family of protein kinases plays key roles in regulating cellular responses to mitogens as well as environmental stress. Inappropriate activation of these kinases contributes to tumorigenesis. In contrast, anthrax lethal factor, the principal virulence factor of anthrax toxin, has been demonstrated to selectively inactivate MEKs. In this article we will discuss recent advances in our understanding of molecular aspects of the pathogenesis of anthrax, emphasizing the potential role of MEK signalling in this disease, and outline novel strategies to use anthrax lethal toxin in the treatment of cancer.
Assuntos
Antraz/patologia , Antígenos de Bactérias , Toxinas Bacterianas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Antraz/enzimologia , Bacillus anthracis , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Humanos , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , MAP Quinase Quinase 3 , MAP Quinase Quinase 5 , MAP Quinase Quinase 6 , MAP Quinase Quinase 7 , Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismoRESUMO
The immunization of guinea pigs with trivaccine and monovaccines against plaque, tularemia and anthrax induces a decrease in the activity of acidic phosphatase in lymphocytes, as well as a decrease in the number of lymphocytes containing this enzyme. A decrease in the activity of alkaline phosphatase and peroxidase had been found to occur in neutrophil leukocytes. Besides, neutrophil leukocytes have shown an increase in the activity of acidic phosphatase and nonspecific esterases. The study based on the evaluation of the activity of the above-mentioned enzymes in lymphocytes and neutrophils has not revealed the predominant influence exercised by any of the antigens, different in their nature and used separately or in the form of a combined preparation, on immunogenesis.
Assuntos
Antraz/enzimologia , Leucócitos/enzimologia , Peste/enzimologia , Tularemia/enzimologia , Animais , Antraz/imunologia , Bacillus anthracis/imunologia , Vacinas Bacterianas/administração & dosagem , Francisella tularensis/imunologia , Cobaias , Histocitoquímica , Imunidade , Leucócitos/imunologia , Peste/imunologia , Vacina contra a Peste/administração & dosagem , Fatores de Tempo , Tularemia/imunologiaRESUMO
Interaction of the cells of Bacillus anthracis strain CH-7 with benzylpenicillin was studied. The cells of strain CH-7 were shown to contain the penicillinase gene in the repressed state. Spontaneous derepression of the gene at a rate of 10(-8) resulting in the synthesis of penicillinase was observed. Penicillinase was synthesized constitutionally and its synthesis did not depend on the presence of benzylpenicillin in the cultivation medium. The therapeutic effect of benzylpenicillin in the treatment of the experimental infection induced by the B. anthracis strain producing penicillinase was estimated. The efficacy was shown to depend on the time of the beginning of the antibiotic therapy. When the clinical signs of the infection were evident in the animals contaminated with the penicillinase-producing strain of B. anthracis, their treatment with the mean daily doses of benzylpenicillin failed.
Assuntos
Antraz/tratamento farmacológico , Bacillus anthracis/efeitos dos fármacos , Penicilina G/farmacologia , Animais , Antraz/enzimologia , Antraz/genética , Bacillus anthracis/genética , Testes de Sensibilidade Microbiana , Penicilinase/biossíntese , Penicilinase/genética , CoelhosRESUMO
Gastrointestinal (GI) anthrax results from the ingestion of Bacillus anthracis. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated B. anthracis Sterne strain (pXO1+ pXO2-) spores that resulted in rapid animal death. B. anthracis Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only B. anthracis, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.
Assuntos
Antraz/imunologia , Antraz/microbiologia , Bacillus anthracis/imunologia , Colo/imunologia , Disbiose/imunologia , Disbiose/microbiologia , Gastroenteropatias/imunologia , Gastroenteropatias/microbiologia , Tolerância Imunológica , Animais , Antraz/enzimologia , Antraz/patologia , Colo/microbiologia , Colo/patologia , Disbiose/patologia , Epitélio/imunologia , Epitélio/microbiologia , Epitélio/patologia , Gastroenteropatias/enzimologia , Gastroenteropatias/patologia , Imunidade Inata , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Linfócitos T/imunologiaRESUMO
Anthrax is caused by the gram-positive bacterium Bacillus anthracis. The pathogenesis of this disease is dependent on the presence of two binary toxins, edema toxin (EdTx) and lethal toxin (LeTx). LeTx, the major virulence factor contributing to anthrax, contains the effector moiety lethal factor (LF), a zinc-dependent metalloprotease specific for targeting mitogen-activated protein kinase kinases. This review will focus on the protease-specific activity and function of LF, and will include a discussion on the implications and consequences of this activity, both in terms of anthrax disease, and how this activity can be exploited to gain insight into other pathologic conditions.