RESUMEN
Phagocytosis of Candida albicans by either primary bone marrow-derived mouse macrophages or RAW 264.7 cells upregulated transcription of PRA1, which encodes a cell wall/membrane-associated antigen previously described as a fibrinogen binding protein. However, a pra1 null mutant was still able to bind fibrinogen, showing that Pra1p is not uniquely required for fibrinogen binding. As well, Pra1 tagged with green fluorescent protein did not colocalize with AlexaFluor 546-labeled human fibrinogen, and while PRA1 expression was inhibited when Candida was grown in fetal bovine serum-containing medium, Candida binding to fibrinogen was activated by these conditions. Therefore, it appears that Pra1p can play at most a minor role in fibrinogen binding to C. albicans. PRA1 gene expression is induced in vitro by alkaline pH, and therefore its activation in phagosomes suggested that phagosome maturation was suppressed by the presence of Candida cells. LysoTracker red-labeled organelles failed to fuse with phagosomes containing live Candida, while phagosomes containing dead Candida underwent a normal phagosome-to-phagolysosome maturation. Immunofluorescence staining with the early/recycling endosomal marker transferrin receptor (CD71) suggested that live Candida may escape macrophage destruction through the inhibition of phagolysosomal maturation.
Asunto(s)
Candida albicans/inmunología , Fibrinógeno/metabolismo , Proteínas Fúngicas/metabolismo , Macrófagos/microbiología , Animales , Línea Celular , Células Cultivadas , Proteínas Fúngicas/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Ratones , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Fagosomas/microbiología , Unión ProteicaRESUMEN
Prostaglandins are hormone-like chemicals involved in the inflammatory response and in defense against pathogens. We investigated the effect of extracellular prostaglandin E2 (PGE2) on the human fungal pathogen Candida albicans. Transcriptional analysis of C. albicans treated with PGE2 indicated differential expression of genes involved in alternative carbon source catabolism, and showed repression of genes encoding components of both the translational machinery and the homolog of Saccharomyces cerevisiae Mac1 regulon needed for iron uptake. Prostaglandin-mediated repression of the Mac1 regulon required the Tup1p transcriptional regulator, and did not occur in hyphal cells. Analysis of the promoter of the Mac1 regulon component FRE7 revealed a well-conserved palindromic Mac1p binding site that was critical for expression of a downstream reporter. To identify elements other than Tup1 that are involved in regulation of the Mac1 regulon, we screened a C. albicans transcription factor mutant library with this FRE7 promoter fused to a lacZ reporter. Oaf1p, Stp4p, Azf1p, and Cas1p mutants showed moderate enhancement of reporter expression. Azf1p and Cas1p were shown to be transducers of the PGE2 dependent signaling pathway.
Asunto(s)
Candida albicans/efectos de los fármacos , Dinoprostona/farmacología , Transcripción Genética , Candida albicans/genética , Candida albicans/fisiología , Dinoprostona/metabolismo , Regulación Fúngica de la Expresión Génica , Análisis por MicromatricesRESUMEN
We used Drosophila melanogaster macrophage-like Schneider 2 (S2) cells as a model to study cell-mediated innate immunity against infection by the opportunistic fungal pathogen Candida albicans. Transcriptional profiling of S2 cells coincubated with C. albicans cells revealed up-regulation of several genes. One of the most highly up-regulated genes during this interaction is the D. melanogaster translational regulator 4E-BP encoded by the Thor gene. Analysis of Drosophila 4E-BP(null) mutant survival upon infection with C. albicans showed that 4E-BP plays an important role in host defense, suggesting a role for translational control in the D. melanogaster response to C. albicans infection.
Asunto(s)
Candida albicans/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/microbiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Animales , Candidiasis , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Determinación de Punto Final , Regulación de la Expresión Génica , Genes de Insecto , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación/genética , Factores de Iniciación de Péptidos/genética , Fagocitosis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Supervivencia , Transcripción GenéticaRESUMEN
The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the "TEDS-rule" site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5(S366D) were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.
Asunto(s)
Candida albicans/crecimiento & desarrollo , Miosina Tipo I/fisiología , Actinas/fisiología , Animales , Candida albicans/citología , Candida albicans/metabolismo , Pared Celular/química , Quitina/metabolismo , Medios de Cultivo/química , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Hifa/citología , Hifa/crecimiento & desarrollo , Mutación , Canales de Potasio/agonistas , Canales de Potasio/metabolismoRESUMEN
Phagocytic cells such as neutrophils and macrophages are potential components of the immune defense that protects mammals against Candida albicans infection. We have tested the interaction between the mouse macrophage cell line RAW 264.7 and a variety of mutant strains of C. albicans. We used an end point dilution assay to monitor the killing of C. albicans at low multiplicities of infection (MOIs). Several mutants that show reduced virulence in mouse systemic-infection models show reduced colony formation in the presence of macrophage cells. To permit analysis of the macrophage-Candida interaction at higher MOIs, we introduced a luciferase reporter gene into wild-type and mutant Candida cells and used loss of the luminescence signal to quantify proliferation. This assay gave results similar to those for the end point dilution assay. Activation of the macrophages with mouse gamma interferon did not enhance anti-Candida activity. Continued coculture of the Candida and macrophage cells eventually led to death of the macrophages, but for the RAW 264.7 cell line this was not due to apoptotic pathways involving caspase-8 or -9 activation. In general Candida cells defective in the formation of hyphae were both less virulent in animal models and more sensitive to macrophage engulfment and growth inhibition. However the nonvirulent, hypha-defective cla4 mutant line was considerably more resistant to macrophage-mediated inhibition than the wild-type strain. Thus although mutants sensitive to engulfment are typically less virulent in systemic-infection models, sensitivity to phagocytic macrophage cells is not the unique determinant of C. albicans virulence.
Asunto(s)
Candida albicans/inmunología , Interferón gamma/farmacología , Macrófagos/inmunología , Animales , Apoptosis , Candida albicans/clasificación , Candida albicans/crecimiento & desarrollo , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Línea Celular , Genotipo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , FagocitosisAsunto(s)
Candida albicans/enzimología , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Proteínas de Saccharomyces cerevisiae , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Candida albicans/genética , Dominio Catalítico , Secuencia Conservada , Relación Dosis-Respuesta a Droga , Humanos , Datos de Secuencia Molecular , Fosforilación , Proteína Fosfatasa 2 , Proteína Fosfatasa 2C , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygous cacdc35 Delta cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.
Asunto(s)
Adenilil Ciclasas/metabolismo , Candida albicans/enzimología , Transducción de Señal , Adenilil Ciclasas/genética , Adenilil Ciclasas/inmunología , Adenilil Ciclasas/aislamiento & purificación , Animales , Secuencia de Bases , Candida albicans/crecimiento & desarrollo , Candida albicans/inmunología , Candida albicans/patogenicidad , Candidiasis/microbiología , Línea Celular , Deleción Cromosómica , Cromosomas Fúngicos , ADN de Hongos , Femenino , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , VirulenciaRESUMEN
Candida albicans has a number of transcriptional regulatory circuits that control aspects of cell type and cell morphogenesis. Recent work has uncovered a cryptic mating-type locus, and a variety of transcription factors that are important in regulation of the transition from yeast growth to hyphal growth. In some cases, the signalling pathways regulating these transcription factors are becoming defined. Analysis of phenotypic switching implicates internal factors, as well as external signals, in control of cellular morphogenesis.
Asunto(s)
Candida albicans/fisiología , Transcripción Genética , Candida albicans/citología , Candida albicans/patogenicidad , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fenotipo , Transducción de Señal , Factores de Transcripción/fisiología , VirulenciaRESUMEN
Cpp1p is a putative mitogen-activated protein (MAP) kinase phosphatase that suppresses Candida albicans hyphal formation at 25 degrees C through its probable substrate, the Cek1p filamentation MAP kinase. Here we report that expression of the serum-induced genes SAP4-6 and HYR1 increased several fold in hyphal forms of a cpp1/cpp1 null mutant, while the rate and extent of hyphal development up to 5 h were normal. Therefore, we provide evidence that Cpp1p represses hyphal gene expression by acting through a Cek1p-independent mechanism. SAP4-6 and HYR1 transcripts were undetectable in a null mutant of another key regulator of filamentation, Efg1p; thus, Efg1p and Cpp1p oppose each other during the expression of these genes in hyphal forms.
Asunto(s)
Ácido Aspártico Endopeptidasas/genética , Candida albicans/enzimología , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción , Candida albicans/genética , ARN Mensajero/análisisRESUMEN
In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.
Asunto(s)
Candida albicans/genética , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Carioferinas , Receptores Citoplasmáticos y Nucleares , Secuencia de Aminoácidos , Southern Blotting , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Genes Fúngicos/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteína Exportina 1RESUMEN
Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.
Asunto(s)
Actinas/fisiología , Proteínas del Citoesqueleto , Proteínas Motoras Moleculares/fisiología , Miosina Tipo I , Miosinas/fisiología , Proteínas de Saccharomyces cerevisiae , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Actinas/metabolismo , Secuencia de Aminoácidos , Movimiento Celular/fisiología , Proteínas Fúngicas/metabolismo , Ligandos , Proteínas de Microfilamentos/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Morfogénesis/fisiología , Cadenas Pesadas de Miosina/metabolismo , Miosinas/metabolismo , Unión Proteica , Proteínas/metabolismo , Saccharomyces cerevisiae , Técnicas del Sistema de Dos Híbridos , Proteína del Síndrome de Wiskott-AldrichRESUMEN
The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins. We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)). Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated 'transplants', in which the C-terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes de Fusión/fisiología , Saccharomyces cerevisiae/fisiología , Humanos , Feromonas/fisiologíaRESUMEN
The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Portadoras , Proteínas Fúngicas/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , Factores de Transcripción , Alelos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Dominio Catalítico , Ciclo Celular/genética , Proteínas Fúngicas/genética , Glicerol/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Proteínas de la Membrana , Ósmosis , Feromonas/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Proteínas Fúngicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Datos de Secuencia Molecular , Mutagénesis , Mapeo Peptídico , Conformación Proteica , Saccharomyces cerevisiae/genéticaRESUMEN
Ste20p from Saccharomyces cerevisiae is a member of the Ste20/p21-activated protein kinase family of protein kinases. The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis. This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence. Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity. The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes. Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication. In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Compartimento Celular , Ciclo Celular , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1 delta/cek1 delta null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 microliter suspension containing 1 x 10(5), 1 x 10(6) and 1 x 10(7) blastopores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1 delta/cek1 delta null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Candida albicans/enzimología , Candida albicans/genética , Candidiasis/etiología , Mastitis/etiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Animales , Candida albicans/patogenicidad , Candidiasis/microbiología , Candidiasis/patología , Modelos Animales de Enfermedad , Femenino , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos , Prueba de Complementación Genética , Masculino , Mastitis/microbiología , Mastitis/patología , Ratones , Ratones Endogámicos BALB C , Embarazo , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Candida albicans/crecimiento & desarrollo , Proteínas Fúngicas/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Candida albicans/citología , Candida albicans/patogenicidad , Candidiasis/mortalidad , Diferenciación Celular , Proteínas Fúngicas/genética , Dosificación de Gen , Ratones , Modelos Biológicos , Mutación , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Supresión Genética , Factores de Transcripción/metabolismo , Virulencia/genéticaRESUMEN
Serine/threonine protein kinases of the Ste20/PAK family have been implicated in the signalling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades. In the yeast Saccharomyces cerevisiae, Ste20 is involved in transmitting the mating-pheromone signal from the betagamma-subunits (encoded by the STE4 and STE18 genes, respectively) of a heterotrimeric G protein to a downstream MAP kinase cascade. We have identified a binding site for the G-protein beta-subunit (Gbeta) in the non-catalytic carboxy-terminal regions of Ste20 and its mammalian homologues, the p21-activated protein kinases (PAKs). Association of Gbeta with this site in Ste20 was regulated by binding of pheromone to the receptor. Mutations in Gbeta and Ste20 that prevented this association blocked activation of the MAP kinase cascade. Considering the high degree of structural and functional conservation of Ste20/PAK family members and G-protein subunits, our results provide a possible model for a role of these kinases in Gbetagamma-mediated signal transduction in organisms ranging from yeast to mammals.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Animales , Proteínas Fúngicas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM , Ratones , Feromonas/metabolismo , Unión Proteica , Ratas , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Candida albicans strains with a deletion of the mitogen-activated protein kinase tyrosine phosphatase gene (CPP1) are derepressed in the yeast-to-hyphal transition on solid surfaces in vitro at ambient temperatures and this gene is therefore required for repression of the yeast-to-hyphal switch. The pathology caused by a CPP1 null mutant strain was compared with that of the null mutant into which the wild-type CPP1 gene was introduced by homologous recombination and with the wild-type parent strain in a murine mycotic mastitis model. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 1 x 10(5), 1 x 10(6) and 1 x 10(7) cell-forming units before euthanasia. Infected and non-infected control glands were evaluated histopathologically. The null mutant strains showed less severe pathology than the two control strains. The Cpplp tyrosine phosphatase may thus be considered a virulence determinant during localized infection in C. albicans.
Asunto(s)
Candida albicans/patogenicidad , Mastitis/microbiología , Proteínas Tirosina Fosfatasas/genética , Animales , Proteínas Bacterianas/genética , Candida albicans/genética , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Virulencia/genéticaRESUMEN
Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.