RESUMO
Apicomplexan parasites replicate by several budding mechanisms with two well-characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two-hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two-hybrid. HAD2a has demonstrated enzyme-activity in vitro, localizes to the nascent daughter buds, and co-localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex.
Assuntos
Hidrolases/química , Monoéster Fosfórico Hidrolases/química , Proteínas de Protozoários/química , Toxoplasma/enzimologia , Sequência de Aminoácidos , Citocinese , Citoesqueleto/enzimologia , Genes Essenciais , Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2(K270E)-activated Gα relative to Gpa2(+) Gα. Deletion of sck1(+) or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2(+) or Gpa2(K270E), suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2(K270E) GDP-GTP exchange rate mutant, GTPase-defective Gpa2(R176H) weakly binds Sck1 in the two-hybrid screen and a deletion of sck1(+) in a Gpa2(R176H) strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting sck1(+) in a gpa2Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow S. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Glucose/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mutação , Ligação Proteica , Proteínas Quinases/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transdução de SinaisRESUMO
The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.
Assuntos
Citoesqueleto/metabolismo , Filamentos Intermediários/metabolismo , Multimerização Proteica , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Fusão Gênica Artificial , Citoesqueleto/ultraestrutura , Genes Reporter , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Toxoplasma/genéticaRESUMO
The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Galpha subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gbetagamma dimer to repress transcription of the glucose-regulated fbp1(+) gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2(+) chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Galpha function, thus extending our understanding of Galpha protein structure-function relationships.
Assuntos
Alelos , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Regulação Fúngica da Expressão Gênica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces , Sequência de Aminoácidos , AMP Cíclico/metabolismo , Glucose/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Heterotrimeric G proteins mediate signal transduction pathways to control development in fungal, plant, and animal cells. A recent study in the July issue of Molecular Cell identifies three proteins that, while not displaying sequence similarity to G protein subunits, appear to act as structural mimics of a Gbetagamma dimer to negatively regulate pseudohyphal growth in budding yeast.
Assuntos
Proteínas de Caenorhabditis elegans , Divisão Celular/fisiologia , Subunidades beta da Proteína de Ligação ao GTP , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Substâncias Macromoleculares , Estrutura Quaternária de Proteína/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3',5'-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Fosfodiesterase/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Genes Fúngicos , Genes Reporter , Camundongos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genéticaRESUMO
Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven "upstream" proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git1+ gene. Git1 is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git1+ demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated G alpha-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Git1 and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Glucose/farmacologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Alinhamento de Sequência , Esporos Fúngicos/fisiologia , Supressão GenéticaRESUMO
We previously constructed a collection of fission yeast strains that express various mammalian cyclic nucleotide phosphodiesterases (PDEs) and developed a cell-based high throughput screen (HTS) for small molecule PDE inhibitors. Here we describe a compound, BC54, that is a selective inhibitor of enzymes from the cAMP-specific PDE4 and PDE7 families. Consistent with the biological effect of other PDE4 and PDE7 inhibitors, BC54 displays potent anti-inflammatory properties and is superior to a combination of rolipram (a PDE4 inhibitor) and BRL50481 (a PDE7A inhibitor) for inducing apoptosis in chronic lymphocytic leukemia (CLL) cells. We further exploited PKA-regulated growth phenotypes in fission yeast to isolate two mutant alleles of the human PDE4B2 gene that encode enzymes possessing single amino acid changes that confer partial resistance to BC54. We confirm this resistance to both BC54 and rolipram via yeast-based assays and, for PDE4B2T407A, in vitro enzyme assays. Thus, we are able to use this system for both chemical screens to identify biologically-active PDE inhibitors and molecular genetic studies to characterize the interaction of these molecules with their target enzymes. Based on its potency, selectivity, and effectiveness in cell culture, BC54 should be a useful tool to study biological processes regulated by PDE4 and PDE7 enzymes.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Cicloexanos/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Rolipram/administração & dosagem , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genéticaRESUMO
Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs2+ were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git2+/cyr1+), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the git2(-) mutant alleles used in the suppressor selection and mutations in git1+, git3+, git5+, git7+, git10+, and git11+, which are all required for adenylate cyclase activation. Notably, these cgs2 mutant alleles fail to suppress mutations in gpa2+, which encodes the Galpha subunit of a heterotrimeric G protein required for adenylate cyclase activation, although the previously identified cgs2-2 allele does suppress loss of gpa2+. Further analysis of the cgs2-s1 allele reveals a synthetic interaction with the gpa2(R176H)-activated allele, with respect to derepression of fbp1-lacZ transcription in glucose-starved cells. In addition, direct measurements of cAMP levels show that cgs2-s1 cells maintain normal basal cAMP levels, but are severely defective in feedback regulation upon glucose detection. These results suggest that PDE activity in S. pombe may be coordinately regulated with adenylate cyclase activity as part of the feedback regulation mechanism to limit the cAMP response to glucose detection.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/genética , Retroalimentação Fisiológica/fisiologia , Glucose/metabolismo , Schizosaccharomyces/genética , Transdução de Sinais/fisiologia , Supressão Genética/genética , 3',5'-AMP Cíclico Fosfodiesterases/genética , Mapeamento Cromossômico , Primers do DNA , Retroalimentação Fisiológica/genética , Biblioteca Gênica , Mutação/genética , Radioimunoensaio , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genéticaRESUMO
We have used a high throughput small molecule screen, using a fission yeast-based assay, to identify novel phosphodiesterase 7 (PDE7) inhibitors. One of the most effective hit compounds was BC12, a barbituric acid-based molecule that exhibits unusually potent immunosuppressive and immunomodulatory actions on T lymphocyte function, including inhibition of T cell proliferation and IL-2 cytokine production. BC12 treatment confers a >95% inhibition of IL-2 secretion in phytohaemagglutinin (PHA) plus phorbol-12-myristate-13-acetate (PMA) stimulated Jurkat T cells. The effect of BC12 on IL-2 secretion is not due to decreased cell viability; rather, BC12 blocks up-regulation of IL-2 transcription in activated T cells. BC12 also inhibits IL-2 secretion in human peripheral T lymphocytes stimulated in response to CD3/CD28 co-ligation or the combination of PMA and ionomycin, as well as the proliferation of primary murine T cells stimulated with PMA and ionomycin. A BC12 analog that lacks PDE7 inhibitory activity (BC12-4) displays similar biological activity, suggesting that BC12 does not act via PDE7 inhibition. To investigate the mechanism of inhibition of IL-2 production by BC12, we performed microarray analyses using unstimulated and stimulated Jurkat T cells in the presence or absence of BC12 or BC12-4. Our studies show these compounds affect the transcriptional response to stimulation and act via one or more shared targets to produce both anti-inflammatory and pro-stress effects. These results demonstrate potent immunomodulatory activity for BC12 and BC12-4 in T lymphocytes and suggest a potential clinical use as an immunotherapeutic to treat T lymphocyte-mediated diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Barbitúricos/farmacologia , Inibidores Enzimáticos/farmacologia , Linfócitos T/efeitos dos fármacos , Barbitúricos/química , Proliferação de Células/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Humanos , Imunomodulação , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Análise em Microsséries , Linfócitos T/fisiologiaRESUMO
The basal complex in Toxoplasma functions as the contractile ring in the cell division process. Basal complex contraction tapers the daughter cytoskeleton toward the basal end and is required for daughter segregation. We have previously shown that the protein MORN1 is essential for basal complex assembly and likely acts as a scaffolding protein. To further our understanding of the basal complex, we combined subcellular fractionation with an affinity purification of the MORN1 complex and identified its protein composition. We identified two new components of the basal complex, one of which uniquely associated with the basal complex in mature parasites, the first of its kind. In addition, we identified several other novel cytoskeleton proteins with different spatiotemporal dynamics throughout cell division. Since many of these proteins are unique to Apicomplexa this study significantly contributes to the annotation of their unique cytoskeleton. Furthermore, we show that G-actin binding protein TgCAP is localized at the apical cap region in intracellular parasites, but quickly redistributes to a cytoplasmic localization pattern upon egress. © 2012 Wiley Periodicals, Inc.
Assuntos
Citoesqueleto/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Citoesqueleto/genética , Proteômica/métodos , Proteínas de Protozoários/genética , Toxoplasma/genéticaRESUMO
The Schizosaccharomyces pombe fbp1 gene is transcriptionally repressed by protein kinase A (PKA) that is activated by extracellular glucose via a cAMP-signaling pathway. We previously used an fbp1-ura4 reporter that places uracil biosynthesis under the control of the glucose-sensing pathway to identify mutations in genes of the cAMP pathway. More recently, this reporter has been used in high throughput screens for small molecule inhibitors of heterologously-expressed cyclic nucleotide phosphodiesterases (PDEs) that hydrolyse cAMP to 5' AMP. Here we show that strains lacking the adenylyl cyclase gene respond to either exogenous cAMP or cGMP to activate PKA, thus regulating fbp1-ura4 expression and other PKA-regulated processes such as conjugation and the nuclear export of an Rst2-GFP fusion protein. Expression of cGMP-specific PDEs or ones that hydrolyse both cAMP and cGMP increases the amount of exogenous cGMP required to activate PKA in order to repress fbp1-ura4 expression, creating conditions that allow detection of inhibitors of these PDEs. As proof of this concept, we screened a collection of compounds previously identified as inhibitors of cAMP-specific PDE4 or PDE7 enzymes for their ability to inhibit the mammalian cGMP-specific PDE5A enzyme. We identified compound BC76, which inhibits PDE5A in an in vitro enzyme assay with an IC(50) of 232nM. Further yeast-based assays show that BC76 inhibits PDE1, PDE4, PDE5, PDE8, PDE10 and PDE11, thus demonstrating the utility of this system for detecting and characterising inhibitors of either cAMP- or cGMP-metabolising PDEs.
Assuntos
Benzimidazóis/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Inibidores da Fosfodiesterase 5/farmacologia , Pirróis/farmacologia , Proteínas Recombinantes/biossíntese , Schizosaccharomyces/genética , Animais , Bovinos , Humanos , CamundongosRESUMO
Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor alpha from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Fosfodiesterase/análise , Inibidores de Fosfodiesterase/farmacologia , Schizosaccharomyces/metabolismo , Anti-Inflamatórios/farmacologia , Domínio Catalítico , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/química , Ensaios Enzimáticos , Humanos , Hidrólise/efeitos dos fármacos , Concentração Inibidora 50 , Lipopolissacarídeos/farmacologia , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/classificação , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
G protein-mediated signaling is implicated in yeast and fungal cAMP pathways. By two-hybrid screens and pull-down experiments, we show that the fission yeast Gpa2 Galpha binds an N-terminal domain of adenylate cyclase, comprising a moderately conserved sequence within a region otherwise poorly related to other fungal adenylate cyclases. Overexpressing this domain in yeast perturbs cAMP signaling, which is restored by Gpa2 coexpression. Mutations affecting this domain, over 1,100 residues from the catalytic domain, alter glucose-triggered cAMP signaling. This is evidence for direct activation of adenylate cyclase by a fungal G protein and suggests a distinct activation mechanism from that of mammals.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Glucose/fisiologia , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia , Adenilil Ciclases/genética , Substituição de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Ativação Enzimática , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Genótipo , Cinética , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Galpha protein GNA-1 is a direct positive regulator of adenylyl cyclase. deltagna-1 strains are female-sterile, and deltagna-1 strains have, reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a deltagna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. deltagna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not deltagna-1 cr-1, defects, which is consistent with previous results demonstrating that deltagna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the deltagna-1 mutation are sterile; however, unlike cr-1 and deltagna-1 strains, the deltagna-1 cr-1 mutant does not produce protoperithecia. The deltagna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < deltaga-1 < cr-1 = deltagna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Neurospora crassa/metabolismo , Adenilil Ciclases/genética , Sequência de Bases , Contagem de Colônia Microbiana , DNA Fúngico/genética , Proteínas Fúngicas/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Mutação , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Fenótipo , TemperaturaRESUMO
The vaccine efficacy of the gene sequence encoding the signal peptide of the antigen known as antigen 2 or proline-rich antigen (Ag2/PRA), an immunodominant antigen present in the cell wall of the fungal pathogen Coccidioides immitis, was investigated in a murine model of coccidioidomycosis. Expression plasmids for Ag2/PRA(1-18) DNA (signal sequence), Ag2/PRA(19-194) DNA (lacking the signal sequence), and Ag2/PRA(1-194) DNA (full length) were inserted in the pVR1012 vector, and the constructs were used to vaccinate the highly susceptible BALB/c mouse strain. Immunization with the signal gene sequence significantly reduced the fungal burden in the lungs and spleens of mice 12 days after intraperitoneal challenge with a lethal dose of 2,500 C. immitis arthroconidia, to a level comparable to the protection induced in mice immunized with the full-length Ag2/PRA(1-194) DNA. The Ag2/PRA(19-194) gene protected mice but to a significantly lower level than the signal sequence or the full-length Ag2 gene. The immunizing capacity of Ag2/PRA(1-18) was not attributable to a nonspecific immunostimulatory effect of DNA, as evidenced by the fact that mice immunized with a frameshift mutation of Ag2/PRA(1-18) were not protected against challenge. Furthermore, a synthetic peptide corresponding to the translated sequence of Ag2/PRA(1-18) DNA protected mice, albeit at a lower level than the Ag2/PRA(1-18) DNA vaccine. The protection induced with the signal gene vaccine correlated with the production of gamma interferon when splenocytes from Ag2/PRA(1-18)-immunized mice were stimulated with recombinant full-length Ag2 and was not associated with the production of anti-Coccidioides immunoglobulin G antibody. This is the first study to establish that a signal peptide sequence alone, administered as a gene vaccine or synthetic peptide, can induce protective immunity against a microbial pathogen.
Assuntos
Antígenos de Fungos/genética , Coccidioidomicose/prevenção & controle , Vacinas Fúngicas/imunologia , Glicoproteínas/genética , Epitopos Imunodominantes/genética , Sinais Direcionadores de Proteínas/fisiologia , Vacinas de DNA/imunologia , Animais , Anticorpos Antifúngicos/sangue , Anticorpos Antifúngicos/imunologia , Antígenos de Fungos/imunologia , Coccidioides/genética , Coccidioides/imunologia , Coccidioidomicose/imunologia , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas , Vacinas Fúngicas/genética , Expressão Gênica , Glicoproteínas/imunologia , Epitopos Imunodominantes/imunologia , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , PlasmídeosRESUMO
Molecular genetic analyses in Schizosaccharomyces pombe are greatly enhanced by our ability to delete chromosomal genes via homologous recombination and to introduce genes expressed from autonomous plasmids. In this paper, we describe a novel approach to generating marked deletion cassettes that bypasses the need for the long, PAGE-purified oligonucleotides required in the currently used PCR-based deletion approach. We also describe additional uses of this two-step PCR method for constructing chromosomal insertion cassettes. Finally, we describe how gap repair in S. pombe can facilitate plasmid constructions in a manner that circumvents the reliance on compatible restriction sites in the DNA molecules that are being joined. Several applications of this gap repair plasmid construction strategy are discussed.
Assuntos
Deleção de Genes , Plasmídeos/genética , Schizosaccharomyces/genética , Mutagênese Insercional/métodosRESUMO
Coccidioides immitis is a fungal pathogen of humans and is classified as a Select Agent. We have identified a new potential vaccine candidate for this pathogen using cDNA expression library immunization (ELI). A C. immitis spherule-phase cDNA library containing 800-1000 genes was divided into 10 pools and each was tested for its protective capacity in BALB/c mice against intraperitoneal challenge with 2500 arthroconidia of this dimorphic fungus. The most protective pool, designated Pool 7, was fractionated into five sublibraries, each containing 60 genes, and of these, only Pool 7-3 induced a significant level of protection in mice. Fractionation of Pool 7-3 into six sublibraries, each with 10 genes, yielded a protective fraction, designated Pool 7-3-5. Subsequent fraction of the latter pool into 10 sublibraries, each with one clone, yielded a clone designated 7-3-5-5 that was highly protective. Clone 7-3-5-5 was sequenced and found to contain a 672bp ORF encoding a 224 amino acid protein having a 19 amino acid signal sequence on the N-terminus and a 15 amino acid C-terminal GPI anchor site. The 7-3-5-5 clone, designated ELI-Antigen 1 (ELI-Ag1), showed partial homology with a hypothetical protein from Neurospora crassa. This is the first study to identify a protective antigen from a fungus using ELI, and it is also the first report in which sequential fractionation of an expression library successfully identified a single protective gene.