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1.
J Biotechnol ; 147(2): 136-43, 2010 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-20356564

RESUMO

Strains of Saccharomyces cerevisiae capable of lysis upon conditional down-regulation of cell-wall biogenesis genes (SRB1 and PKC1) have been reported. Here, we show that they lyse and release recombinant protein not only under laboratory conditions, but (more importantly) under conditions found in the human stomach and duodenum. These findings provide proof that, in principle, such conditional lysis strains could be used as an integral part of a system for the oral delivery of therapeutic proteins. However, the current mechanism of conditional lysis is based on the use of the MET3 promoter which requires addition of methionine and cysteine for down-regulation of SRB1 and PKC1. This requirement makes it difficult to apply in vivo. We reasoned that promoters, suitable for in vivo down-regulation of lysis-inducing genes, could be identified amongst yeast genes whose transcript abundance is reduced under conditions found in the human gut. A microarray experiment identified a number of candidate genes with significantly reduced transcript levels under simulated human gut conditions. The greatest effects were seen with ANB1, TIR1, and MF(ALPHA)2), and we propose that their promoters have the potential to be used in vivo to achieve yeast lysis in the gut.


Assuntos
Parede Celular/química , Duodeno/química , Veículos Farmacêuticos/química , Saccharomyces cerevisiae/química , Estômago/química , Proliferação de Células , Parede Celular/genética , Parede Celular/metabolismo , Cisteína/metabolismo , Duodeno/metabolismo , Mucosa Gástrica/metabolismo , Perfilação da Expressão Gênica , Genes Fúngicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Metionina/metabolismo , Mutação , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regiões Promotoras Genéticas , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Mol Microbiol ; 67(1): 47-62, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18078440

RESUMO

Candida albicans hypha formation which has been stimulated via the Ras1-cAMP-Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl-cAMP. The CAI4-Ras1(G13V) strain, which carries a dominant-active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4-Ras1(G13V) strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP-repressed genes and cells repressed for hypha formation. Because several cAMP-controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1-Cdc35 pathway, thus leading to an alteration of C. albicans morphology.


Assuntos
Candida albicans/fisiologia , AMP Cíclico/metabolismo , Dodecanol/farmacologia , Farneseno Álcool/farmacologia , Proteínas Fúngicas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Candida albicans/citologia , Candida albicans/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico , Temperatura Alta , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , RNA Fúngico/efeitos dos fármacos , RNA Fúngico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo
3.
Microbiology (Reading) ; 149(Pt 10): 2961-2976, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14523128

RESUMO

The cAMP-dependent pathway, which regulates yeast-to-hypha morphogenesis in Candida albicans, is controlled by changes in cAMP levels determined by the processes of synthesis and hydrolysis. Both low- and high-affinity cAMP phosphodiesterases are encoded in the C. albicans genome. CaPDE2, encoding the high-affinity cAMP phosphodiesterase, has been cloned and shown to be toxic in Saccharomyces cerevisiae upon overexpression under pGAL1, but functional under the moderate pMET3. Deletion of CaPDE2 causes elevated cAMP levels and responsiveness to exogenous cAMP, higher sensitivity to heat shock, severe growth defects at 42 degrees C and highly reduced levels of EFG1 transcription. In vitro in hypha-inducing liquid medium CaPDE2, deletion prohibits normal hyphal, but not pseudohyphal growth. On solid medium capde2 mutants form aberrant hyphae, with fewer branches and almost no lateral buds, which are deficient in hypha-to-yeast reversion. The phenotypic defects of capde2 mutants show that the cAMP-dependent pathway plays specific roles in hyphal and pseudohyphal development, its regulatory role however, being greater in liquid than on solid medium in vitro. The increased expression of CaPDE2 after serum addition correlates well with a drop in cAMP levels following the initial rise in response to the hyphal inducer. These results suggest that Capde2p mediates a desensitization mechanism by lowering basal cAMP levels in response to environmental stimuli in C. albicans.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Candida albicans/enzimologia , Proteínas Fúngicas , Hifas/crescimento & desenvolvimento , 3',5'-AMP Cíclico Fosfodiesterases/genética , Candida albicans/crescimento & desenvolvimento , AMP Cíclico/farmacologia , Proteínas de Ligação a DNA/genética , Saccharomyces cerevisiae/enzimologia , Soro/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica
4.
Microbiology (Reading) ; 146 ( Pt 9): 2133-2146, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10974101

RESUMO

Complementation studies and allele replacement in Saccharomyces cerevisiae revealed that PSA1/VIG9, an essential gene that encodes GDP-mannose pyrophosphorylase, is the wild-type SRB1 gene. Cloning and sequencing of the srb1-1 allele showed that it determines a single amino acid change from glycine to aspartic acid at residue 276 (srb1(D276)). Genetic evidence is presented showing that at least one further mutation is required for the sorbitol dependence of srb1(D276). A previously reported complementing gene, which this study has now identified as PDE2, is a multi-copy suppressor of sorbitol dependence and is not, as was previously suggested, the SRB1 gene. srb and pde2 mutants share a number of phenotypes, including lysis upon hypotonic shock and enhanced transformability. These data are consistent with the idea that the Ras/cAMP pathway might modulate cell-wall construction.


Assuntos
Parede Celular/metabolismo , AMP Cíclico/metabolismo , Genes Fúngicos , Nucleotidiltransferases/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sorbitol/metabolismo , Proteínas ras/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Supressores , Dados de Sequência Molecular , Mutação , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Pressão Osmótica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação Genética
5.
Microbiology (Reading) ; 145 ( Pt 2): 309-316, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10075413

RESUMO

The cell wall integrity determinants PKC1 and SRB1/PSA1/VIG9 of Saccharomyces cerevisiae were expressed under the control of the tightly regulated promoter pMET3. Substitution of the cell-cycle-regulated SRB1/PSA1 native promoter with pMET3 led to faster cell growth, larger cell volumes, and a twofold reduction of the steady-state SRB1/PSA1 mRNA level. In addition, the new pattern of expression of SRB1/PSA1 resulted in a dominant flocculation phenotype at all phases of batch growth. By contrast, expression of PKC1 from pMET3 increased the flocculation capacity of cells only at stationary phase. Methionine-mediated repression of either PSA1/SRB1 or PKC1 resulted in enhanced cell clumping. Cells in which both these genes had been replaced with their respective pMET3-regulated cassettes were highly flocculent under both expression and repression conditions. These results suggest that greater exposure of flocculin on the cell surface, caused by either cell wall distortion (through depletion of Pkc1p) or aberrant regulation of mannosylation (through constitutive production of Srb1p), results in an increased flocculation ability.


Assuntos
Parede Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteína Quinase C , Saccharomyces cerevisiae/fisiologia , Parede Celular/metabolismo , Regulação para Baixo , Repressão Enzimática , Floculação , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Metionina/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transcrição Gênica
6.
Microbiology (Reading) ; 144 ( Pt 9): 2417-2426, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9782489

RESUMO

Two genomic fragments have been isolated from Candida albicans which strongly hybridize to SRB1/PSA1/VIG9, an essential gene which encodes GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae. A common 2.5 kb Xbal-Pstl fragment has been identified, which Southern analysis suggests is most likely unique in the C. albicans genome. The fragment contains an ORF, which is 82% identical and 90% homologous to the Srb1p/Psa1p/Vig9p from S. cerevisiae, contains one additional amino acid at position 254 and is able to functionally complement the major phenotypic characteristics of S. cerevisiae srb1 null and conditional mutations. The authors therefore conclude that they have cloned and sequenced from C. albicans the bona fide homologue of SRB1/PSA1/VIG9, named hereafter CaSRB1. Northern analysis data indicate that the gene is expressed in C. albicans under conditions of growth in the yeast and hyphal form and suggest that its expression might be regulated.


Assuntos
Candida albicans/enzimologia , Candida albicans/genética , Genes Fúngicos , Nucleotidiltransferases/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Candida albicans/crescimento & desenvolvimento , Clonagem Molecular , Primers do DNA/genética , Escherichia coli/genética , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Fenótipo , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
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