RESUMO
Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.
Assuntos
Ascomicetos/genética , Ascomicetos/virologia , Proteínas Fúngicas/genética , Alelos , Epistasia Genética , Proteínas Fúngicas/metabolismo , Loci Gênicos , Genótipo , Técnicas de Genotipagem , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Polimorfismo GenéticoRESUMO
Dicer gene dcl2, required for the RNA silencing antiviral defense response in the chestnut blight fungus Cryphonectria parasitica, is inducible upon mycovirus infection and promotes viral RNA recombination. We now report that the antiviral defense response requires only one of the four C. parasitica Argonaute-like protein genes, agl2. The agl2 gene is required for the virus-induced increase in dcl2 transcript accumulation. Agl2 and dcl2 transcripts accumulated to much higher levels in response to hairpin RNA production or infection by a mutant CHV1-EP713 hypovirus lacking the suppressor of RNA silencing p29 than to wild-type CHV1-EP713. Similar results were obtained for an agl2-promoter/EGFP-reporter construct, indicating that p29-mediated repression of agl2 transcript accumulation is promoter-dependent. Significantly, the agl2 deletion mutant exhibited stable maintenance of non-viral sequences in recombinant hypovirus RNA virus vectors and the absence of hypovirus-defective interfering (DI) RNA production. These results establish a key role for an Argonaute gene in the induction of an RNA silencing antiviral defense response and the promotion of viral RNA recombination. They also provide evidence for a mechanism by which a virus-encoded RNA silencing suppressor represses the transcriptional induction of an RNA silencing component.
Assuntos
Ascomicetos/genética , Ascomicetos/virologia , Genes Fúngicos , RNA Viral/genética , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , Inativação Gênica , Dados de Sequência Molecular , Mutação , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas , Recombinação Genética , Ribonucleases/genéticaRESUMO
We report characterization of the gene encoding putative transcription factor PRO1, identified in transcriptional profiling studies as being downregulated in the chestnut blight fungus Cryphonectria parasitica in response to infection by virulence-attenuating hypoviruses. Sequence analysis confirmed that pro1 encodes a Zn(II)(2)Cys(6) binuclear cluster DNA binding protein with significant sequence similarity to the pro1 gene product that controls fruiting body development in Sordaria macrospora. Targeted disruption of the C. parasitica pro1 gene resulted in two phenotypic changes that also accompany hypovirus infection, a significant reduction in asexual sporulation that could be reversed by exposure to high light intensity, and loss of female fertility. The pro1 disruption mutant, however, retained full virulence. Although hypovirus CHV1-EP713 infection was established in the pro1 disruption mutant, infected colonies continually produced virus-free sectors, suggesting that PRO1 is required for stable maintenance of hypovirus infection. These results complement the recent characterization of the hypovirus-responsive homologue of the Saccharomyces cerevisiae Ste12 C(2)H(2) zinc finger transcription factor gene, cpst12, which was shown to be required for C. parasitica female fertility and virulence.
Assuntos
Ascomicetos/enzimologia , Ascomicetos/virologia , Proteínas Fúngicas/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Doenças das Plantas/microbiologia , Vírus de RNA/fisiologia , Reprodução Assexuada , Aesculus/microbiologia , Sequência de Aminoácidos , Ascomicetos/patogenicidade , Ascomicetos/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Carboxila)/química , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Alinhamento de Sequência , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologia , Esporos Fúngicos/virologia , VirulênciaRESUMO
Intoxication of mammalian cells by Bacillus anthracis requires the coordinate activity of three distinct bacterial proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF). Among these proteins, PA has become the major focus of work on monoclonal antibodies and vaccines designed to treat or prevent anthrax infection since neither EF nor LF is capable of inducing cellular toxicity in its absence. Here, we present the development of a sensitive, precise, and biologically relevant bioassay platform capable of quantifying antibody-mediated PA neutralization. This bioassay is based on the ability of PA to bind and shuttle EF, a bacterial adenylate cyclase, into mammalian cells leading to an increase in cAMP that can be quantified using a sensitive chemiluminescent ELISA. The results of this study indicate that the cAMP-induction assay possesses the necessary performance characteristics for use as both a potency-indicating release assay in a quality control setting and as a surrogate pharmacodynamic marker for ensuring the continued bioactivity of therapeutic antibodies against PA during clinical trials.
Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Bioensaio/métodos , AMP Cíclico/metabolismo , Venenos de Víboras/metabolismo , Animais , Bacillus anthracis/imunologia , Células CHO , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Venenos de Víboras/imunologiaRESUMO
BACKGROUND: In animal models, treatment with 5H3, a fully human protective antigen-directed monoclonal antibody (PA-MAb), improved survival when administered close to the time of Bacillus anthracis lethal toxin (LeTx) bolus or live bacterial challenge. However, treatment with PA-MAb would be most valuable clinically if it were beneficial even when administered after the onset of shock and lethality due to LeTx. METHODS: We investigated the effects of PA-MAb versus placebo administered in rats (n=324) at the time of or 3, 6, 9, or 12 h after the initiation of a 24-h LeTx infusion. RESULTS: In rats receiving placebo, mean arterial blood pressure (MBP) and heart rate (HR) were decreased in nonsurvivors, compared with those in survivors, at 6 h and then worsened further, with lethality first evident at 8 h (median, 16 h; range, 8-152 h). At each treatment time, survival rates were greater for PA-MAb than for placebo, although improvement was decreased at later treatment times (P=.001, for the effect of time). Compared with placebo, PA-MAb significantly increased MBP during the 12 h after the initiation of treatment, but the increase was greatest for treatment at 3 h; similarly, PA-MAb significantly increased HR at all treatment times. CONCLUSION: In this rat model, improvements in outcome due to PA-MAb were significant when it was administered up to 6 h (and approached significance when administered up to 12 h) after initial exposure to LeTx. Clinically, PA-MAb may be beneficial even when administered after the onset of shock and lethality due to LeTx.
Assuntos
Antraz/terapia , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Hemodinâmica/efeitos dos fármacos , Sepse/terapia , Animais , Antraz/mortalidade , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/imunologia , Pressão Sanguínea , Frequência Cardíaca , Infusões Intravenosas , Ratos , Ratos Sprague-Dawley , Sepse/mortalidade , Fatores de TempoRESUMO
Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.
Assuntos
Adenilil Ciclases/biossíntese , Adenilil Ciclases/química , Bacillus anthracis/metabolismo , Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Adenilil Ciclases/genética , Adenilil Ciclases/isolamento & purificação , Adenilil Ciclases/farmacologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias , Bacillus anthracis/genética , Toxinas Bacterianas , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Escherichia coli/genética , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Transformação BacterianaRESUMO
Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. Current vaccines against anthrax use PA as their primary component since it confers protective immunity. In this work, we expressed soluble, recombinant PA in relatively high amounts in the periplasm of E. coli from shake flasks and bioreactors. The PA protein was purified using Q-Sepharose-HP and hydroxyapatite chromatography, and routinely found to be 96-98% pure. Yields of purified PA varied depending on the method of production; however, medium cell density fermentations resulted in approximately 370 mg/L of highly pure biologically active PA protein. These results exhibit the ability to generate gram quantities of PA from E. coli.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Escherichia coli/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Fermentação , Dados de Sequência Molecular , Periplasma/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.
Assuntos
Anticorpos Monoclonais/biossíntese , Linfócitos B/metabolismo , Proteínas de Membrana , Receptores do Fator de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Receptor do Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Injeções Intravenosas , Leucócitos Mononucleares/efeitos dos fármacos , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Macaca fascicularis , Masculino , Camundongos , Mutagênese Sítio-Dirigida , Testes de Neutralização , Receptores do Fator de Necrose Tumoral/administração & dosagem , Receptores do Fator de Necrose Tumoral/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Proteína Transmembrana Ativadora e Interagente do CAMLRESUMO
It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V(lambda) and 13/35 V(kappa) light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V(H) CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC(50) values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.
Assuntos
Anticorpos , Proteínas de Membrana/imunologia , Fator de Necrose Tumoral alfa/imunologia , Sequência de Aminoácidos , Anticorpos/química , Anticorpos/classificação , Anticorpos/genética , Anticorpos/imunologia , Fator Ativador de Células B , Regiões Determinantes de Complementaridade , Humanos , Cadeias Pesadas de Imunoglobulinas , Cadeias Leves de Imunoglobulina , Dados de Sequência Molecular , Biblioteca de Peptídeos , Filogenia , Mutação PuntualRESUMO
The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.1 A resolution by X-ray crystallography. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein also reveals an L-shaped channel suitable for a peptidoglycan substrate. Analysis of the structural features of this enzyme provides clues to the mechanism of action of S. aureus FemA.
Assuntos
Proteínas de Bactérias/química , Estrutura Terciária de Proteína , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Peptidoglicano/biossíntese , Alinhamento de SequênciaRESUMO
Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S. aureus (strain ISP3) genomic DNA library. Analysis of the predicted polypeptide sequence from the pth gene showed that the protein shared complete conservation of the three residues thought to be involved in the active site of E. coli Pth. The gene was cloned into a pQE-60 expression vector and expressed in E. coli, and the resulting His-tagged Pth protein was purified to greater than 95% purity from the soluble portion of the E. coli lysate in a single chromatographic step. His-tagged Pth was shown to be biologically active by its ability to hydrolyze diacetyl-[(3)H]Lys-tRNA(Lys) in a time- and concentration-dependent manner. Optimum hydrolyzing activity of Pth occurred at a pH value of 7.0 and a MgCl(2) concentration of 5 mM. The K(m) of the diacetyl-[(3)H]-Lys-tRNA(Lys) substrate for S. aureus Pth was determined to be 2.8 microM. A far UV circular dichroism spectrum revealed that His-tagged S. aureus Pth appears to have a structured core predominated by beta-sheet.