RESUMO
Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.
Assuntos
Candida albicans , Glicoproteínas de Membrana/toxicidade , Pichia , Vasculite/induzido quimicamente , Anafilaxia/induzido quimicamente , Anafilaxia/patologia , Animais , Parede Celular , Vasos Coronários/patologia , Masculino , Camundongos Endogâmicos , Miocárdio/patologia , Vasculite/patologiaRESUMO
The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.
Assuntos
Bacillus thuringiensis/química , Técnicas Bacteriológicas/métodos , Agentes de Controle Biológico/isolamento & purificação , Microbiologia Industrial/métodos , Glicoproteínas de Membrana/isolamento & purificação , Rhipicephalus/efeitos dos fármacos , Animais , Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/efeitos dos fármacos , Bacillus thuringiensis/crescimento & desenvolvimento , Bacillus thuringiensis/metabolismo , Agentes de Controle Biológico/toxicidade , Biomassa , Reatores Biológicos , Bovinos , Meios de Cultura/farmacologia , Feminino , Fermentação , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/toxicidade , Oviposição/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Coelhos , Esporos BacterianosRESUMO
Familial Danish Dementia (FDD), an early-onset non-amyloid-ß (Aß) cerebral amyloidosis, is neuropathologically characterized by widespread cerebral amyloid angiopathy, parenchymal amyloid and preamyloid deposits, as well as neurofibrillary degeneration indistinguishable to that seen in Alzheimer's disease (AD). The main amyloid subunit composing FDD lesions, a 34-amino acid de-novo generated peptide ADan, is the direct result of a genetic defect at the 3'-end of the BRI2 gene and the physiologic action of furin-like proteolytic processing at the C-terminal region of the ADan precursor protein. We aimed to study the impact of the FDD mutation, the additional formation of the pyroglutamate (pE) posttranslational modification as well as the relevance of C-terminal truncations -all major components of the heterogeneous FDD deposits- on the structural and neurotoxic properties of the molecule. Our data indicates that whereas the mutation generated a ß-sheet-rich hydrophobic ADan subunit of high oligomerization/fibrillization propensity and the pE modification further enhanced these properties, C-terminal truncations had the opposite effect mostly abolishing these features. The potentiation of pro-amyloidogenic properties correlated with the initiation of neuronal cell death mechanisms involving oxidative stress, perturbation of mitochondrial membrane potential, release of mitochondrial cytochrome c, and downstream activation of caspase-mediated apoptotic pathways. The amyloid-induced toxicity was inhibited by targeting specific components of these detrimental cellular pathways, using reactive oxygen scavengers and monoclonal antibodies recognizing the pathological amyloid subunit. Taken together, the data indicate that the FDD mutation and the pE posttranslational modification are both primary elements driving intact ADan into an amyloidogenic/neurotoxic pathway while truncations at the C-terminus eliminate the pro-amyloidogenic characteristics of the molecule, likely reflecting effect of physiologic clearance mechanisms.
Assuntos
Catarata/metabolismo , Morte Celular/fisiologia , Ataxia Cerebelar/metabolismo , Surdez/metabolismo , Demência/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Estresse Oxidativo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Caspase 3/metabolismo , Catarata/genética , Morte Celular/genética , Linhagem Celular Tumoral , Ataxia Cerebelar/genética , Surdez/genética , Demência/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/toxicidade , Camundongos , Mitocôndrias/genética , Estresse Oxidativo/genética , Conformação Proteica , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid ß-protein (Aß) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aß are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability.
Assuntos
Angiopatia Amiloide Cerebral Familiar/genética , Cistina/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutação , Neurotoxinas/química , Neurotoxinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Amiloide , Neuropatias Amiloides Familiares , Animais , Angiopatia Amiloide Cerebral Familiar/metabolismo , Angiopatia Amiloide Cerebral Familiar/fisiopatologia , Cistina/genética , Cistina/metabolismo , Humanos , Potenciação de Longa Duração , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.
Assuntos
Aedes/efeitos dos fármacos , Bacillaceae/química , Culex/efeitos dos fármacos , Glicoproteínas de Membrana/toxicidade , Sequência de Aminoácidos , Animais , Quitina/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/farmacologia , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Esporos Bacterianos/químicaRESUMO
BACKGROUND: Familial British and Familial Danish dementias (FBD and FDD, respectively) are associated with mutations in the BRI2 gene. Processing of the mutated BRI2 protein leads to the accumulation in the brain of the 34-mer amyloid Bri (ABri) and amyloid Dan (ADan) peptides, accompanied by neurofibrillary tangles. Recently, transgenic mice successfully reproduced different aspects of FDD, while modeling of FBD in vivo has been more difficult. In this work we have modeled FBD and FDD in Drosophila and tested the hypothesis that ABri and ADan are differentially neurotoxic. RESULTS: By using site-directed insertion, we generated transgenic lines carrying ABri, ADan, Bri2-23 (the normal product of wild-type BRI2 processing) and amyloid-ß (Aß) 1-42 as a well-characterized neurotoxic peptide, alone or with a His-tag. Therefore, we avoided random insertion effects and were able to compare levels of accumulation accurately. Peptides were expressed with the GAL4-Upstream Activating Sequence (UAS) system using specific drivers. Despite low levels of expression, toxicity in the eye was characterized by mild disorganization of ommatidia and amyloid peptides accumulation. The highest toxicity was seen for ADan, followed by Aß42 and ABri. Pan-neuronal expression in the CNS revealed an age-dependent toxicity of amyloid peptides as determined by the ability of flies to climb in a geotaxis paradigm when compared to Bri2-23. This effect was stronger for ADan, detected at 7 days post-eclosion, and followed by ABri and Aß42, whose toxicity became evident after 15 and 21 days, respectively. Histological analysis showed mild vacuolization and thioflavine-S-negative deposits of amyloid peptides. In contrast, the over-expression of amyloid peptides in the specific subset of lateral neurons that control circadian locomotor activity showed no toxicity. CONCLUSIONS: Our results support the differential neurotoxicity of ADan and ABri in the Drosophila eye and CNS at low expression levels. Such differences may be partially attributed to rates of aggregation and accumulation. In the CNS, both peptides appear to be more neurotoxic than wild-type Aß42. These Drosophila models will allow a systematic and unambiguous comparison of differences and similarities in the mechanisms of toxicity of diverse amyloid peptides associated with dementia.
Assuntos
Catarata , Ataxia Cerebelar , Angiopatia Amiloide Cerebral Familiar , Surdez , Demência , Modelos Animais de Doenças , Glicoproteínas de Membrana/toxicidade , Proteínas Adaptadoras de Transdução de Sinal , Neuropatias Amiloides Familiares , Animais , Animais Geneticamente Modificados , Catarata/genética , Ataxia Cerebelar/genética , Angiopatia Amiloide Cerebral Familiar/genética , Surdez/genética , Demência/genética , Drosophila melanogaster , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismoRESUMO
AIMS: CAWS, Candida albicans water-soluble fraction, is an extracellular mannoprotein produced by C. albicans NBRC1385. It is a ligand of dectin-2, the C-type lectin receptor for innate immunity, and has strong potency for induction of vasculitis in DBA/2 mice. The structure of this mannoprotein is known to be modulated by the culture conditions. To clarify the structure required for vasculitis, CAWSs were prepared in the two culture conditions with or without pH control, and biological properties were compared. METHODS: CAWSs prepared by the standard protocol and pH controlled at 7.0 were designated as CAWS and CAWS727, respectively. The antigenicity was detected by the anti-Candida mannan IgG. These chemical structures were assessed by nuclear magnetic resonance analysis and the lectin array system. The in vitro activity of CAWSs was tested by tumor necrosis factor-α (TNF-α) induction using bone marrow-derived dendritic cells and spleen cell cultures. RESULTS: The antigenicity of CAWS727 was similar to CAWS but the nuclear magnetic resonance analysis showed a higher ratio of ß-mannosyl linkages were detected in CAWS727. The lectin array showed relative affinities of CAWS727 to α-mannosyl specific lectins were weaker than those of CAWS. CAWS induced severe vasculitis in DBA/2 mice while CAWS727 did not. CAWS significantly induced TNF-α but CAWS727 did slightly. In addition, CAWS-induced TNF-α production was inhibited by mixing with CAWS727 in a concentration dependent manner. CONCLUSION: The α-mannosyl linkages of Candida mannan is a key molecule for the immunotoxicity. CAWS727, which conatins ß-mannosyl linkages, competitively bound to lectin receptors, and resulted in reductions in the inflammatory response.
Assuntos
Arterite/imunologia , Células da Medula Óssea/imunologia , Candida albicans/química , Misturas Complexas/toxicidade , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Glicoproteínas de Membrana/toxicidade , Baço/imunologia , Animais , Arterite/induzido quimicamente , Arterite/patologia , Células da Medula Óssea/patologia , Candida albicans/imunologia , Misturas Complexas/química , Misturas Complexas/imunologia , Células Dendríticas/patologia , Relação Dose-Resposta Imunológica , Masculino , Manose/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Baço/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.
Assuntos
Bacillus anthracis/patogenicidade , Fagossomos/metabolismo , Fagossomos/microbiologia , Animais , Antraz/microbiologia , Antraz/patologia , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/toxicidade , Antioxidantes/metabolismo , Bacillus anthracis/crescimento & desenvolvimento , Bacillus anthracis/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Modelos Animais de Doenças , Humanos , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/toxicidade , Fagócitos/metabolismo , Fagócitos/microbiologia , Fagócitos/patologia , Fosfolipases/genética , Fosfolipases/isolamento & purificação , Fosfolipases/toxicidade , Esporos Bacterianos/citologia , Esporos Bacterianos/patogenicidadeRESUMO
Filarial nematodes are parasites that have the ability to persist in their hosts for extended periods of time due to the employment of various mechanisms to divert or down-regulate the host's immune responses. One of these mechanisms is the production of immunomodulatory excretory-secretory (ES) products. This review will discuss the properties of one such product, ES-62, which over the years, has been shown to interact with and modulate the activities of a variety of cells of the immune system including B and T lymphocytes, dendritic cells, macrophages and mast cells. Overall, ES-62 diverts the immune system towards an anti-inflammatory phenotype and consistent with this it has been shown to have therapeutic potential in models of inflammatory disease associated with autoimmunity and allergy.
Assuntos
Dipetalonema/metabolismo , Proteínas de Helminto/metabolismo , Proteínas de Helminto/farmacologia , Imunomodulação/efeitos dos fármacos , Animais , Dipetalonema/imunologia , Proteínas de Helminto/imunologia , Proteínas de Helminto/toxicidade , Humanos , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Inflamação/induzido quimicamente , Inflamação/etiologia , Inflamação/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Glicoproteínas de Membrana/toxicidade , Modelos Biológicos , Fosforilcolina/imunologiaRESUMO
Neogenin is a receptor for netrins and proteins of the repulsive guidance molecule (RGM) family. It regulates several key developmental processes within the nervous system. The binding of RGMa to neogenin induces the inhibition of neurite outgrowth and the collapse of the growth cone of neurons. Here, we report that a disintegrin and metalloprotease (ADAM) transmembrane protein regulates the sensitivity of neurons to RGMa, by inducing the shedding of the ectodomain of neogenin. The extracellular domain of neogenin is directly associated with and cleaved off by the tumor necrosis factor-α converting enzyme (TACE), also called ADAM17. TACE is endogenously expressed in embryonic cortical neurons and regulates the cleavage of neogenin, and the inhibition of TACE in turn enhances RGMa-induced inhibition of neurite outgrowth and collapse of the growth cone. Conversely, exogenous expression of TACE abolishes the effect of RGMa. Therefore, TACE may play a role in modulating the RGM-induced repulsive behavior of neurons by regulating the expression of neogenin on the cell surface.
Assuntos
Proteínas ADAM/fisiologia , Córtex Cerebral/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/toxicidade , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/toxicidade , Neurônios/efeitos dos fármacos , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Células CHO , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Cricetinae , Cricetulus , Proteínas Ligadas por GPI , Células HEK293 , Humanos , Proteínas de Membrana/química , Neurônios/metabolismo , Neurônios/fisiologia , Ratos , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Bacillus anthracis/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Células Epiteliais/efeitos dos fármacos , Fermentação , Glicoproteínas de Membrana/toxicidade , Aerobiose , Bacillus anthracis/genética , Bacillus anthracis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Meios de Cultivo Condicionados/química , Células Epiteliais/metabolismo , Deleção de Genes , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Pulmão/citologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/toxicidade , Proteínas Recombinantes/toxicidade , Ácido Succínico/toxicidade , Fatores de Virulência/metabolismo , Fatores de Virulência/farmacologia , Fatores de Virulência/toxicidadeRESUMO
Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human alpha-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human alpha-defensins HNP-4 and HD-6 and human beta-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Colesterol/farmacologia , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Hemólise/efeitos dos fármacos , Glicoproteínas de Membrana/toxicidade , Estreptolisinas/toxicidade , alfa-Defensinas/farmacologia , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Coelhos , Soro/fisiologia , alfa-Defensinas/química , alfa-Defensinas/metabolismoRESUMO
Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.
Assuntos
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Venenos de Serpentes/genética , Venenos de Serpentes/isolamento & purificação , Sequência de Aminoácidos , Animais , Masculino , Glicoproteínas de Membrana/toxicidade , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Ratos , Ratos Wistar , Venenos de Serpentes/toxicidadeRESUMO
The surface layer protein encoding genes from five mosquito-pathogenic Bacillus sphaericus isolates were amplified and sequenced. Negative staining of the S-layer protein extracted from the cell wall of wild-type B. sphaericus C3-41 was prepared. It showed a flat-sheet crystal lattice structure. Two genes encoding the entire and N-terminally truncated S-layer protein (slpC and DeltaslpC respectively), were ligated into plasmid pET28a and expressed in Escherichia coli. SDS-PAGE revealed that about 130 KD and 110 KD proteins could be expressed in the cytoplasm of recombinant E. coli BL21(pET28a/slpC) and E. coli BL21(pET28a/DeltaslpC) respectively. Furthermore, an intracellular sheet-like or fingerprint-shape structure was investigated in two recombinant strains, which expressed SlpC and DeltaSlpC protein respectively, by ultrathin microscopy study, but bioassay results suggested that the S-layer protein of wild B. sphaericus C3-41 and recombinant E. coli BL21 (pET28a/slpC) have no direct toxicity against mosquito larvae. These results should provide information for further understanding of the function of S-layer protein of pathogenic B. sphaericus.
Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Regulação Bacteriana da Expressão Gênica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/toxicidade , Filogenia , Animais , Bacillus/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Parede Celular/química , Clonagem Molecular , Cristalização , Culicidae/efeitos dos fármacos , Culicidae/crescimento & desenvolvimento , DNA Bacteriano/genética , Larva/efeitos dos fármacos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/toxicidade , Análise de Sequência de DNARESUMO
Anthrolysin O (ALO) is a toxin produced by Bacillus anthracis, the causative agent of anthrax. It is a member of the cholesterol-dependent cytolysin (CDC) group of toxins, many of which are potential vaccine candidates that protect against their producing organisms. Pore formation by ALO was studied by transmission electron microscopy and pores were found to be consistent with those formed by other members of this toxin family. We constructed and characterised a novel genetic toxoid of anthrolysin O, Delta6mALO, which was able to bind to cells but was incapable of pore-formation or haemolysis. The capacity of the haemolytic and non-haemolytic forms of ALO to protect against challenge with the toxin or B. anthracis was determined. Immunisation with both active and non-haemolytic forms of ALO elicited protection against lethal i.v. challenge with ALO but neither was protective against B. anthracis in a murine i.p. challenge model. Immunisation with another CDC, pneumolysin, did not confer cross-protection against challenge with ALO. Histopathological investigation following lethal i.v. challenge with ALO revealed acute pathology in the lungs with occlusion of alveolar vessels by fibrin deposits.
Assuntos
Vacinas contra Antraz/imunologia , Antraz/prevenção & controle , Antígenos de Bactérias/toxicidade , Bacillus anthracis/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/toxicidade , Intoxicação/prevenção & controle , Toxoides/imunologia , Animais , Antraz/imunologia , Vacinas contra Antraz/genética , Vacinas contra Antraz/toxicidade , Antitoxinas/sangue , Proteínas de Bactérias/genética , Eritrócitos/efeitos dos fármacos , Eritrócitos/ultraestrutura , Feminino , Hemólise , Humanos , Imunoglobulina G/sangue , Pulmão/patologia , Glicoproteínas de Membrana/genética , Camundongos , Microscopia Eletrônica de Transmissão , Intoxicação/imunologia , Análise de Sobrevida , Toxoides/genética , Toxoides/toxicidadeRESUMO
Cellular immunity plays a major role in the control of human cytomegalovirus (HCMV) infection. CD4(+) T lymphocytes have been shown to contribute to this function but their precise role is a matter of debate. Although CD4(+) T cells have been shown to kill target cells through the perforin/granzyme pathway, whether HCMV-specific CD4(+) T cells are capable of killing HCMV-infected targets has not yet been documented. In the present paper, we have taken advantage of well established cellular reagents to address this issue. Human CD4(+) T-cell clones specific for the major immediate-early protein IE1 were shown to perform perforin-based cytotoxicity against peptide-pulsed targets. However, when tested on infected anitgen presenting cell targets, cytotoxicity was not detectable, although gamma interferon (IFN-gamma) production was significant. Furthermore, cytotoxicity against peptide-pulsed targets was inhibited by HCMV infection, whereas IFN-gamma production was not modified, suggesting that antigen processing was not altered. Remarkably, degranulation of CD4(+) T cells in the presence of infected targets was significant. Together, our data suggest that impaired cytotoxicity is not due to failure to recognize infected targets but rather to a mechanism specifically related to cytotoxicity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Citomegalovirus/imunologia , Proteínas Imediatamente Precoces/imunologia , Proteínas Virais/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Clonais , Humanos , Glicoproteínas de Membrana/toxicidade , Perforina , Proteínas Citotóxicas Formadoras de Poros/toxicidadeRESUMO
Candida albicans water soluble fraction (CAWS) is a water-soluble extracellular mannoprotein-beta-glucan complex obtained from the culture supernatant of Candida albicans, which grows in a chemically defined medium. CAWS induced toxic reactions, such as acute anaphylactoid reaction, by intravenous administration and coronary arteritis by intraperitoneal administration. To clarify the structure responsible for these toxic reactions, C. albicans was cultured in pH- and temperature-controlled conditions and prepared with CAWS with or without the beta-1,2-linked mannosyl segment (BM). The structure of CAWS was assessed by immunochemical and spectroscopic methodologies, and we found that CAWS prepared under the natural culture conditions contained only small amounts of BM and CAWS prepared at neutral conditions at 27 degrees C contained a significantly higher percentage of BM. Both the acute lethal toxicity and coronary arteritis induction was significantly more severe in the absence of BM. Activation of a complement pathway, the lectin pathway, by CAWS was significantly stronger in the absence of BM. These facts strongly suggest that BM linkages in CAWS negatively modulate acute and chronic toxicity of CAWS, and may be strongly related to the lectin pathway of the complement activation.
Assuntos
Anafilaxia/induzido quimicamente , Candida albicans/patogenicidade , Manose/farmacologia , Glicoproteínas de Membrana/toxicidade , Vasculite/induzido quimicamente , beta-Glucanas/toxicidade , Animais , Ativação do Complemento , Espectroscopia de Ressonância Magnética , Masculino , Lectina de Ligação a Manose/farmacologia , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos ICRRESUMO
BACKGROUND: Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC) family, Anthrolysin O (ALO). RESULTS: In the present studies we show that recombinant ALO (rALO) or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs), monocytes, monocyte-derived macrophages (MDMs), lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH) release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+), UT231 (ALO-), and a complemented strain expressing ALO, UT231 (pUTE544), and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. CONCLUSION: The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.
Assuntos
Bacillus anthracis/patogenicidade , Proteínas de Bactérias/toxicidade , Colesterol/metabolismo , Citotoxinas/toxicidade , Macrófagos/efeitos dos fármacos , Glicoproteínas de Membrana/toxicidade , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Bacillus anthracis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Citotoxinas/genética , Citotoxinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , Proteínas Recombinantes/toxicidade , Azul TripanoRESUMO
Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells.
Assuntos
Toxinas Bacterianas/toxicidade , Mariposas/microbiologia , Photorhabdus/patogenicidade , Fatores de Virulência/toxicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Citoesqueleto/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Escherichia coli/genética , Escherichia coli/metabolismo , Ordem dos Genes , Hemócitos/efeitos dos fármacos , Larva/microbiologia , Substâncias Macromoleculares , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/toxicidade , Microscopia Eletrônica de Transmissão , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/toxicidade , Serratia/genética , Fatores de Virulência/genéticaRESUMO
Candida albicans water soluble fraction (CAWS), water soluble fraction of Candida albicans mainly composed of mannoprotein-beta-glucan complex, has various biological effects, such as anaphylactoid shock and coronary arteritis. These toxicological effects fit CAWS as one of PAMPs, pathogen-associated molecular patterns. Acute anaphylactoid reaction is known to be induced by lipopolysaccharide from Escherichia coli O9 (O9 LPS), which possesses the mannose homopolysaccharide as the O-antigen region. In the present study, we compared immunotoxicological and immunochemical similarity between CAWS and O9 LPS. CAWS strongly reacted with Candida serum factors, and the reactivity was found to be partially competed with O9 LPS. CAWS induced lethal toxicity was inhibited by pretreatment of mice with i.v. injection of CAWS. The lethality was found to be inhibited by i.v. injection of O9 LPS. Vice versa, O9 LPS induced acute lethal toxicity was also inhibited by pretreatment of mice with CAWS. These results suggested that CAWS, fungal PAMPs, and O9 LPS from Gram-negative bacteria share, at least in part, immunochemical and immunotoxicological activities.