RESUMEN
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.
Asunto(s)
Carbunco/inmunología , Apoptosis/inmunología , Bacillus anthracis/patogenicidad , Macrófagos/microbiología , Mitocondrias/microbiología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico/metabolismo , Animales , Carbunco/enzimología , Carbunco/microbiología , Bacillus anthracis/metabolismo , Línea Celular , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Expresión Génica , Macrófagos/enzimología , Ratones , Mitocondrias/enzimología , Óxido Nítrico Sintasa de Tipo I/genéticaRESUMEN
UNLABELLED: Vascular dysfunction and thrombosis have been described in association with anthrax infection in humans and animals but the mechanisms of these dysfunctions, as well as the components involved in thrombi formation are poorly understood. Immunofluorescent microscopy was used to define the composition of thrombi in the liver of mice challenged with the Bacillus anthracis Sterne spores. Lethal infection with the toxigenic Sterne strain, in contrast to the non-lethal, non-toxigenic delta-Sterne strain, demonstrated time-dependent increase in the number of vegetative bacteria inside the liver sinusoids and central vein. Massive appearance of thrombi typically occluding the lumen of the vessels coincided with the sudden death of infected animals. Bacterial chains in the thrombi were stained positive for syndecan-1 (SDC-1), fibronectin, and were surrounded by fibrin polymers, GPIIb-positive platelets, von Willebrand Factor (vWF), CD45-positive leukocytes, and massive amount of shed SDC-1. Experiments with human umbilical vein endothelial cells (HUVECs) demonstrated the active role of the host response to the secreted pathogenic factors of bacteria during the onset of the pro-thrombotic condition. The bacterial culture supernatants, as well as the isolated proteins (the pore-forming toxin anthrolysin O and phospholipase C) induced release of vWF, while anthrolysin O, sphingomyelinase and edema toxin induced release of thrombin from HUVECs and polymerization of fibrin in the presence of human plasma. CONCLUSION: Our findings suggest that activation of endothelium in response to infection can contribute to the formation of occlusive thrombi consisting of aggregated bacteria, vWF, shed SDC-1, fibrin, activated platelets, fibronectin and leukocytes.
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Carbunco/metabolismo , Bacillus anthracis/fisiología , Plaquetas/metabolismo , Fibrina/metabolismo , Hígado/microbiología , Sindecano-1/metabolismo , Trombosis/metabolismo , Factor de von Willebrand/metabolismo , Animales , Carbunco/inmunología , Carbunco/microbiología , Bacillus anthracis/genética , Coagulación Sanguínea , Fibronectinas/metabolismo , Humanos , Leucocitos/inmunología , Hígado/patología , Ratones , Ratones Endogámicos DBA , Trombosis/inmunología , Trombosis/microbiologíaRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.
Asunto(s)
Ataxina-3/fisiología , Enfermedad de Machado-Joseph/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Péptidos/metabolismo , Fosfoproteínas/fisiología , Transducción de Señal/fisiología , Animales , Apoptosis , Ataxina-3/genética , Línea Celular , Fibroblastos , Glucógeno Sintasa Quinasa 3/fisiología , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/fisiología , Análisis por Matrices de Proteínas , Proteínas Proto-Oncogénicas c-akt/fisiología , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
Anthrax is a zoonotic disease caused by Bacillus anthracis. The infection is associated with inflammation and sepsis, but little is known about the acute-phase response during disease and the nature of the bacterial factors causing it. In this study, we examined the levels of the acute-phase proteins (APPs) in comparative experiments using mice challenged with spores and a purified B. anthracis protease InhA as a possible factor mediating the response. A strong increase in the plasma levels of APPs such as haptoglobin and serum amyloid A was observed during infection. Protein and mRNA levels of plasminogen activator inhibitor (PAI)-1 in the liver were also increased concurrently with bacterial dissemination at 72 h post-infection. Similar effects were observed at 6 h post injection with InhA. Induction of hepatic transforming growth factor-beta1, a PAI-1 inducer, was also found in the liver of InhA-injected mice. PAI-1 elevation by InhA resulted in an increased level of urokinase-type plasminogen activator complex with PAI-1 and a decreased level of D-dimers indicating inhibition of blood fibrinolysis. These results reveal an acute liver response to anthrax infection and provide a plausible pathophysiological link between the host inflammatory response and the pro-thrombotic haemostatic imbalance in the course of disease through PAI-1 induction in the liver.
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Proteínas de Fase Aguda/metabolismo , Carbunco/fisiopatología , Bacillus anthracis/enzimología , Bacillus anthracis/patogenicidad , Metaloproteasas/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Regulación hacia Arriba , Animales , Carbunco/inmunología , Carbunco/microbiología , Bacillus anthracis/fisiología , Proteínas Bacterianas/metabolismo , Femenino , Haptoglobinas/metabolismo , Humanos , Inflamación/inmunología , Inflamación/microbiología , Hígado/metabolismo , Ratones , Ratones Endogámicos DBA , Esporas Bacterianas/patogenicidad , Trombosis/inmunología , Trombosis/microbiologíaRESUMEN
Bacillus anthracis infection is associated with severe hemostatic disturbances but their roles and contribution to fatality remain incompletely characterized. We undertook analyses of circulating antithrombin levels during the course of infection using a comparison of lethal and nonlethal murine anthrax models. Plasma samples were obtained from DBA/2 mice challenged intraperitoneally with the spores of either toxigenic B. anthracis Sterne strain or nontoxigenic, avirulent delta Sterne strain. We found that plasma antithrombin levels were rapidly depleted in Sterne spore-challenged mice, concomitant with elevation of neutrophil elastase (NE) and massive syndecan shedding from the liver into circulation. The shed syndecan bound with antithrombin accelerated NE-mediated antithrombin proteolysis. The liver response to infection demonstrated strain-specific compensatory increases of antithrombin and syndecan gene transcription. Both bacterial strains induced changes in blood coagulation parameters consistent with the onset of disseminated intravascular coagulation. We propose that antithrombin depletion proceeding through activation of neutrophils and massive shedding of heparin-like syndecan from the liver into circulation contribute to anthrax coagulopathy.
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Carbunco , Antitrombinas/metabolismo , Bacillus anthracis/patogenicidad , Coagulación Intravascular Diseminada/fisiopatología , Elastasa de Leucocito/sangre , Sindecanos/metabolismo , Animales , Carbunco/complicaciones , Carbunco/microbiología , Carbunco/mortalidad , Bacillus anthracis/fisiología , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/etiología , Femenino , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos DBA , Activación Neutrófila , Esporas Bacterianas/fisiología , Sindecano-1/metabolismo , Sindecano-4/metabolismo , VirulenciaRESUMEN
We recently reported that the open-mesh (0.7 µ) polyacrylamide microparticles (MPs) with internally-coupled Cibacron affinity dye demonstrate protective effect in mice challenged into footpads with high doses (200 LD50) of anthrax (Sterne) spores. A single injection of MPs before spore challenge reduces inflammatory response, delays onset of mortality and promotes survival. In this study, we show that the effect of MPs was substantially increased at the lower spore dose (7 LD50). The inflammation of footpads was reduced to the background level, and 60% of animals survived for 16 days while all untreated infected animals died within 6 days with strong inflammation. The effects of MPs were promoted when the MPs were loaded with a combination of neutrophil-attracting chemokines IL-8 and MIP-1α which delayed the onset of mortality in comparison with untreated mice for additional 8 days. The MPs were not inherently cytotoxic against the bacteria or cultured murine Raw 264.7 cells, but stimulated these cells to release G-CSF, MCP-1, MIP-1α, and TNF-α. Consistent with this finding the injection of MPs induced neutrophil influx into footpads, stimulated production of TNF-α associated with migration of pERK1/2-positive cells with the Langerhans phenotype from epidermis to regional lymph nodes. Our data support the mechanism of protection in which the immune defense induced by MPs along with the exogenous chemokines counterbalances the suppressive effect caused by anthrax infection.
RESUMEN
BACKGROUND: It has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice. RESULTS: The isolated anthrax hemolytic proteins AnlB (sphingomyelinase) and AnlO (cholesterol-binding pore-forming factor), as well as ClnA (B. cereus homolog of B. anthracis phosphatidyl choline-preferring phospholipase C) cause accelerated shedding of Synd1 and E-cadherin from epithelial cells and compromise epithelial barrier integrity within a few hours. In comparison with hemolysins in a similar range of concentrations, anthrax lethal toxin (LT) also accelerates shedding albeit at slower rate. Individual components of LT, lethal factor and protective antigen are inactive with regard to shedding. Inhibition experiments favor a hypothesis that activities of tested bacterial shedding inducers converge on the stimulation of cytoplasmic tyrosine kinases of the Syk family, ultimately leading to activation of cellular sheddase. Both LT and AnlO modulate ERK1/2 and p38 MAPK signaling pathways, while JNK pathway seems to be irrelevant to accelerated shedding. Accelerated shedding of Synd1 also takes place in DBA/2 mice challenged with Bacillus anthracis (Sterne) spores. Elevated levels of shed ectodomain are readily detectable in circulation after 24 h. CONCLUSION: The concerted acceleration of shedding by several virulence factors could represent a new pathogenic mechanism contributing to disruption of epithelial or endothelial integrity, hemorrhage, edema and abnormal cell signaling during anthrax infection.
Asunto(s)
Bacillus anthracis/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Sindecano-1/metabolismo , Factores de Virulencia/metabolismo , Animales , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Cadherinas/metabolismo , Línea Celular , Proteínas Hemolisinas/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Ratones , Ratones Endogámicos DBA , Esfingomielina Fosfodiesterasa/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Bacillus cereus, a food poisoning bacterium closely related to Bacillus anthracis, secretes a multitude of virulence factors including enterotoxins, hemolysins, and phospholipases. However, the majority of the in vitro experiments evaluating the cytotoxic potential of B. cereus were carried out in the conditions of aeration, and the impact of the oxygen limitation in conditions encountered by the microbe in natural environment such as gastrointestinal tract remains poorly understood. This research reports comparative analysis of ATCC strains 11778 (BC1) and 14579 (BC2) in aerobic and microaerobic (static) cultures with regard to their toxicity for human lung epithelial cells. We showed that BC1 increased its toxicity upon oxygen limitation while BC2 was highly cytotoxic in both growth conditions. The combined effect of the pore-forming, cholesterol-dependent hemolysin, cereolysin O (CLO), and metabolic product(s) such as succinate produced in microaerobic conditions provided substantial contribution to the toxicity of BC1 but not BC2 which relied mainly on other toxins. This mechanism is shared between CB1 and B. anthracis. It involves the permeabilization of the cell membrane which facilitates transport of toxic bacterial metabolites into the cell. The toxicity of BC1 was potentiated in the presence of bovine serum albumin which appeared to serve as reservoir for bacteria-derived nitric oxide participating in the downstream production of reactive oxidizing species with the properties of peroxynitrite. In agreement with this the BC1 cultures demonstrated the increased oxidation of the indicator dye Amplex Red catalyzed by peroxidase as well as the increased toxicity in the presence of externally added ascorbic acid.
RESUMEN
In this study the hydrogel microparticles (MPs) were used to enhance migration of neutrophils in order to improve outcome of anthrax infection in a mouse model. Two MP formulations were tested. In the first one the polyacrylamide gel MPs were chemically coupled with Cibacron Blue (CB) affinity bait. In the second one the bait molecules within the MPs were additionally loaded with neutrophil-attracting chemokines (CKs), human CXCL8 and mouse CCL3. A non-covalent interaction of the bait with the CKs provided their gradual release after administration of the MPs to the host. Mice were challenged into footpads with Bacillus anthracis Sterne spores and given a dose of MPs a few hours before and/or after the spores. Pre-treatment with a single dose of CK-releasing MPs without any additional intervention was able to induce influx of neutrophils to the site of spore inoculation and regional lymph nodes correlating with reduced bacterial burden and decreased inflammatory response in footpads. On average, in two independent experiments, up to 53% of mice survived over 13 days. All control spore-challenged but MP-untreated mice died. The CB-coupled particles were also found to improve survival likely due to the capacity to stimulate release of endogenous CKs, but were less potent at decreasing the inflammatory host response than the CK-releasing MPs. The CK post-treatment did not improve survival compared to the untreated mice which died within 4 to 6 days with a strong inflammation of footpads, indicating quick dissemination of spores though the lymphatics after challenge. This is the first report on the enhanced innate host resistance to anthrax in response to CKs delivered and/or endogenously induced by the MPs.
Asunto(s)
Bacillus anthracis/fisiología , Quimiocina CCL3/administración & dosificación , Interleucina-8/administración & dosificación , Esporas Bacterianas , Animales , Portadores de Fármacos , Femenino , Ratones , Ratones Endogámicos DBARESUMEN
BACKGROUND: Inhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of intoxication in experimental animals is drastically different from that found during the infectious process. In order to close a gap between our understanding of anthrax molecular pathology and the most prominent clinical features of the infectious process we undertook bioinformatic and experimental analyses of potential proteolytic virulence factors of B. anthracis distinct from lethal toxin. METHODS: Secreted proteins (other than lethal and edema toxins) produced by B. anthracis were tested for tissue-damaging activity and toxicity in mice. Chemical protease inhibitors and rabbit immune sera raised against B. anthracis proteases were used to treat mice challenged with B. anthracis (Sterne) spores. RESULTS: B. anthracis strain delta Ames (pXO1-, pXO2-) producing no lethal and edema toxins secrets a number of metalloprotease virulence factors upon cultivation under aerobic conditions, including those with hemorrhagic, caseinolytic and collagenolytic activities, belonging to M4 and M9 thermolysin and bacterial collagenase families, respectively. These factors are directly toxic to DBA/2 mice upon intratracheal administration at 0.5 mg/kg and higher doses. Chemical protease inhibitors (phosphoramidon and 1, 10-phenanthroline), as well as immune sera against M4 and M9 proteases of B. anthracis, were used to treat mice challenged with B. anthracis (Sterne) spores. These substances demonstrate a substantial protective efficacy in combination with ciprofloxacin therapy initiated as late as 48 h post spore challenge, compared to the antibiotic alone. CONCLUSION: Secreted proteolytic enzymes are important pathogenic factors of B. anthrasis, which can be considered as effective therapeutic targets in the development of anthrax treatment and prophylactic approaches complementing anti-lethal toxin therapy.
Asunto(s)
Carbunco/tratamiento farmacológico , Bacillus anthracis/enzimología , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/uso terapéutico , Animales , Antibacterianos/uso terapéutico , Bacillus anthracis/efectos de los fármacos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Ciprofloxacina/uso terapéutico , Quimioterapia Combinada , Femenino , Glicopéptidos/uso terapéutico , Inmunización Pasiva , Ratones , Ratones Endogámicos DBA , Fenantrolinas/uso terapéutico , Inhibidores de Proteasas/farmacología , Factores de Virulencia/metabolismoRESUMEN
Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissection. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and the protective capacity of the host.
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Carbunco/metabolismo , Carbunco/microbiología , Bacillus anthracis/fisiología , Ganglios Linfáticos/metabolismo , Análisis por Matrices de Proteínas , Animales , Carbunco/mortalidad , Biomarcadores , Modelos Animales de Enfermedad , Inmunohistoquímica , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Ratones , Fosfoproteínas/metabolismo , Proteoma , Proteómica/métodos , Transducción de Señal , Esporas BacterianasRESUMEN
Chemokines (CKs) secreted by the host cells into surrounding tissue establish concentration gradients directing the migration of leukocytes. We propose an in vivo CK gradient remodeling approach based on sustained release of CKs by the crosslinked poly(N-isopropylacrylamide) hydrogel open meshwork nano-particles (NPs) containing internal crosslinked dye affinity baits for a reversible CK binding and release. The sustained release is based on a new principle of affinity off-rate tuning. The NPs with Cibacron Blue F3G-A and Reactive Blue-4 baits demonstrated a low-micromolar affinity binding to IL-8, MIP-2, and MCP-1 with a half-life of several hours at 37°C. The capacity of NPs loaded with IL-8 and MIP-1α to increase neutrophil recruitment to lymph nodes (LNs) was tested in mice after footpad injection. Fluorescently-labeled NPs used as tracers indicated the delivery into the sub-capsular compartment of draining LNs. The animals administered the CK-loaded NPs demonstrated a widening of the sub-capsular space and a strong lymph node influx of leukocytes, while mice injected with control NPs without CKs or bolus doses of soluble CKs alone showed only a marginal neutrophil response. This technology provides a new means therapeutically direct or restore immune cell traffic, and can also be employed for simultaneous therapy delivery.
RESUMEN
Nitric oxide (NO) is a key physiological regulator in eukaryotic and prokaryotic organisms. It can cause a variety of biological effects by reacting with its targets or/and indirectly inducing oxidative stress. NO can also be produced by bacteria including the pathogenic Bacillus anthracis; however, its role in the infectious process only begins to emerge. NO incapacitates macrophages by S-nitrosylating the intracellular proteins and protects B. anthracis from oxidative stress. It is also implicated in the formation of toxic peroxynitrite. In this study we further assessed the effects of B. anthracis NO produced by the NO synthase (bNOS) on bacterial metabolism and host cells in experiments with the bNOS knockout Sterne strain. The mutation abrogated accumulation of nitrite and nitrate as tracer products of NO in the culture medium and markedly attenuated growth in both aerobic and microaerobic conditions. The regulatory role of NO was also suggested by the abnormally high rate of nitrate denitrification by the mutant in the presence of oxygen. Anaerobic regulation mediated by NO was reflected in reduced fermentation of glucose by the mutant correlating with the reduced toxicity of bacteria toward host cells in culture. The toxic effect of NO required permeabilization of the target cells as well as the activity of fermentation-derived metabolite in the conditions of reduced pH. The host cells demonstrated increased phosphorylation of major survivor protein kinase AKT correlating with reduced toxicity of the mutant in comparison with Sterne. Our global proteomic analysis of lymph from the lymph nodes of infected mice harboring bacteria revealed numerous changes in the pattern and levels of proteins associated with the activity of bNOS influencing key cell physiological processes relevant to energy metabolism, growth, signal transduction, stress response, septic shock, and homeostasis. This is the first in vivo observation of the bacterial NO effect on the lymphatic system.
RESUMEN
This study aimed to characterize a soluble proteome of popliteal lymph nodes during lymphadenitis induced by intradermal injection of Bacillus anthracis Sterne spores in mice using tandem LC-MS/MS and reverse-phase protein microarray with antibodies specific to epitopes of phosphorylated proteins. More than 380 proteins were detected in the normal intra-nodal lymph, while the infectious process resulted in the profound changes in the protein abundances and appearance of 297 unique proteins. These proteins belong to an array of processes reflecting response to wounding, inflammation and perturbations of hemostasis, innate immune response, coagulation and fibrinolysis, regulation of body fluid levels and vascular disturbance among others. Comparison of lymph and serum revealed 83 common proteins. Also, using 71 antibodies specific to total and phosphorylated forms of proteins we carried initial characterization of circulating lymph phosphoproteome which brought additional information regarding signaling pathways operating in the lymphatics. The results demonstrate that the proteome of intra-nodal lymph serves as a sensitive sentinel of the processes occurring within the lymph nodes during infection. The acute innate response of the lymph nodes to anthrax is accompanied by cellular damage and inflammation with a large number of up- and down-regulated proteins many of which are distinct from those detected in serum. MS data are available via ProteomeXchange with identifier PXD001342.
Asunto(s)
Carbunco/metabolismo , Bacillus anthracis , Regulación de la Expresión Génica , Ganglios Linfáticos/metabolismo , Proteoma/biosíntesis , Enfermedades Cutáneas Bacterianas/metabolismo , Animales , Carbunco/patología , Ganglios Linfáticos/patología , Ratones , Enfermedades Cutáneas Bacterianas/patologíaRESUMEN
Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO) synthase (baNOS) plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages. In this study we for the first time present evidence that bacteria-derived NO participates in the generation of highly reactive oxidizing species which could be abolished by the NOS inhibitor L - NAME, free thiols, and superoxide dismutase but not catalase. The formation of toxicants is likely a result of the simultaneous formation of NO and superoxide leading to a labile peroxynitrite and its stable decomposition product, nitrogen dioxide. The toxicity of bacteria could be potentiated in the presence of bovine serum albumin. This effect is consistent with the property of serum albumin to serves as a trap of a volatile NO accelerating its reactions. Our data suggest that during infection in the hypoxic environment of pre-mortal host the accumulated NO is expected to have a broad toxic impact on host cell functions.
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Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidad , Óxido Nítrico/metabolismo , Albúmina Sérica Bovina/metabolismo , Animales , Bovinos , Humanos , Hipoxia , Óxido Nítrico/toxicidad , Superóxidos/metabolismo , Superóxidos/toxicidadRESUMEN
Bacillus anthracis generates virulence factors such as lethal and edema toxins, capsule, and hemolytic proteins under conditions of reduced oxygenation. Here, we report on the acute cytotoxicity of culture supernatants (Sups) of six nonencapsulated B. anthracis strains grown till the stationary phase under static microaerobic conditions. Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible. Sups displayed a reduction of pH to 5.3-5.5, indicating the onset of acid anaerobic fermentation; however, low pH itself was not a major factor of toxicity. The pore-forming hemolysin, anthrolysin O (ALO), contributed to the toxicity in a concentration-dependent manner. Its effect was found to be synergistic with a metabolic product of B. anthracis, succinic acid. Cells exposed to Sups demonstrated cytoplasmic membrane blebbing, increased permeability, loss of ATP, mitochondrial membrane potential collapse, and arrest of cell respiration. The toxicity was reduced by inhibition of ALO by cholesterol, decomposition of reactive oxygen species, and inhibition of mitochondrial succinate dehydrogenase. Cell death appears to be caused by an acute primary membrane permeabilization by ALO, followed by a burst of reactive radicals from the mitochondria fuelled by the succinate, which is generated by bacteria in the hypoxic environment. This mechanism of metabolic toxicity is relevant to the late-stage conditions of hypoxia and acidosis found in anthrax patients and might operate at anatomical locations of the host deprived from oxygen supply.
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Bacillus anthracis/metabolismo , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/toxicidad , Células Epiteliales/efectos de los fármacos , Fermentación , Glicoproteínas de Membrana/toxicidad , Aerobiosis , Bacillus anthracis/genética , Bacillus anthracis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Medios de Cultivo Condicionados/química , Células Epiteliales/metabolismo , Eliminación de Gen , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Pulmón/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/toxicidad , Proteínas Recombinantes/toxicidad , Ácido Succínico/toxicidad , Factores de Virulencia/metabolismo , Factores de Virulencia/farmacología , Factores de Virulencia/toxicidadRESUMEN
AIM: To establish whether activation of adenosine type-3 receptors (A3Rs) and inhibition of interleukin-1ß-induced inflammation is beneficial in combination with antibiotic therapy to increase survival of mice challenged with anthrax spores. METHODS: DBA/2 mice were challenged with Bacillus anthracis spores of the toxigenic Sterne strain 43F2. Survival of animals was monitored for 15 d. Ciprofloxacin treatment (50 mg/kg, once daily, intraperitoneally) was initiated at day +1 simultaneously with the administration of inhibitors, and continued for 10 d. Two doses (2.5 mg/kg and 12.5 mg/kg) of acetyl-tyrosyl-valyl-alanyl-aspartyl-chloromethylketone (YVAD) and three doses (0.05, 0.15 and 0.3 mg/kg) of 1-[2-Chloro-6-[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-ß-D- ribofuranuronamide (Cl-IB-MECA) were tested. Animals received YVAD on days 1-4, and Cl-IB-MECA on days 1-10 once daily, subcutaneously. Human lung epithelial cells in culture were challenged with spores or edema toxin and the effects of IB-MECA on phosphorylation of AKT and generation of cAMP were tested. RESULTS: We showed that the outcome of antibiotic treatment in a murine anthrax model could be substantially improved by co-administration of the caspase-1/4 inhibitor YVAD and the A3R agonist Cl-IB-MECA. Combination treatment with these substances and ciprofloxacin resulted in up to 90% synergistic protection. All untreated mice died, and antibiotic alone protected only 30% of animals. We conclude that both substances target the aberrant host signaling that underpins anthrax mortality. CONCLUSION: Our findings suggest new possibilities for combination therapy of anthrax with antibiotics, A3R agonists and caspase-1 inhibitors.
RESUMEN
Hemorrhagic meningitis is a fatal complication of anthrax, but its pathogenesis remains poorly understood. The present study examined the role of B. anthracis-secreted metalloprotease InhA on monolayer integrity and permeability of human brain microvasculature endothelial cells (HBMECs) which constitute the blood-brain barrier (BBB). Treatment of HBMECs with purified InhA resulted in a time-dependent decrease in trans-endothelial electrical resistance (TEER) accompanied by zonula occluden-1 (ZO-1) degradation. An InhA-expressing B. subtilis exhibited increased permeability of HBMECs, which did not occur with the isogenic inhA deletion mutant (ΔinhA) of B. anthracis, compared with the corresponding wild-type strain. Mice intravenously administered with purified InhA or nanoparticles-conjugated to InhA demonstrated a time-dependent Evans Blue dye extravasation, leptomeningeal thickening, leukocyte infiltration, and brain parenchymal distribution of InhA indicating BBB leakage and cerebral hemorrhage. Mice challenged with vegetative bacteria of the ΔinhA strain of B. anthracis exhibited a significant decrease in leptomeningeal thickening compared to the wildtype strain. Cumulatively, these findings indicate that InhA contributes to BBB disruption associated with anthrax meningitis through proteolytic attack on the endothelial tight junctional protein zonula occluden (ZO)-1.
Asunto(s)
Bacillus anthracis/enzimología , Proteínas Bacterianas/metabolismo , Barrera Hematoencefálica/microbiología , Barrera Hematoencefálica/patología , Hemorragia Cerebral/microbiología , Hemorragia Cerebral/patología , Metaloproteasas/metabolismo , Animales , Carbunco/microbiología , Carbunco/patología , Western Blotting , Citoplasma/metabolismo , Impedancia Eléctrica , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Células Endoteliales/patología , Femenino , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Nanosferas , Permeabilidad , Fosfoproteínas/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
The causative agent of anthrax, Bacillus anthracis, is capable of circumventing the humoral and innate immune defense of the host and modulating the blood chemistry in circulation to initiate a productive infection. It has been shown that the pathogen employs a number of strategies against immune cells using secreted pathogenic factors such as toxins. However, interference of B. anthracis with the innate immune system through specific interaction of the spore surface with host proteins such as the complement system has heretofore attracted little attention. In order to assess the mechanisms by which B. anthracis evades the defense system, we employed a proteomic analysis to identify human serum proteins interacting with B. anthracis spores, and found that plasminogen (PLG) is a major surface-bound protein. PLG efficiently bound to spores in a lysine- and exosporium-dependent manner. We identified α-enolase and elongation factor tu as PLG receptors. PLG-bound spores were capable of exhibiting anti-opsonic properties by cleaving C3b molecules in vitro and in rabbit bronchoalveolar lavage fluid, resulting in a decrease in macrophage phagocytosis. Our findings represent a step forward in understanding the mechanisms involved in the evasion of innate immunity by B. anthracis through recruitment of PLG resulting in the enhancement of anti-complement and anti-opsonization properties of the pathogen.
Asunto(s)
Bacillus anthracis/inmunología , Complemento C3b/inmunología , Fibrinolisina/metabolismo , Inmunidad Innata/inmunología , Plasminógeno/metabolismo , Animales , Bacillus anthracis/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/inmunología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Electroforesis en Gel Bidimensional , Humanos , Inmunidad Innata/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Proteínas Opsoninas/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Unión Proteica/efectos de los fármacos , Conejos , Proteínas Recombinantes/metabolismo , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK) and downstream transcriptional factors [STAT1 (Y701), ATF2 (T69/71), MSK1 (S360) and CREB (S133)]. NF-κB phosphorylation was also increased. Activation of p53 (S15, S46) correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473), along with phosphorylation of FOX 01/03 (T24/31) which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication.