RESUMO
Treatment of not only bacterial but also fungal infections is currently a growing concern. A major reason is the acquisition of multidrug resistance in both prokaryotic and human cells. The multidrug resistance phenotype is a cellular response to the presence of cytotoxic substances in the environment. The basic mechanism of multidrug resistance is overexpression of the membrane proteins involved in the extrusion of toxic substances outside the cell. The resistance mechanism based on the efflux of inhibitors as a result of the overproduction of transport proteins was also observed in some plant and animal pathogens and human tumour cells. The phenomenon of multidrug resistance associated with an excessive and long-term use of antifungals, in particular of azole derivatives, was also confirmed in the yeast Candida glabrata which is becoming a growing concern for health care professionals. Reduced susceptibility to azole derivatives in particular, a high potential for adapting to stressors, and multiple mechanisms of resistance to structurally and functionally unrelated antifungal drugs make the species C. glabrata a potential threat to hospital patients.
Assuntos
Candida glabrata/efeitos dos fármacos , Farmacorresistência Fúngica Múltipla/fisiologia , Antifúngicos/farmacologia , Azóis/farmacologia , Candida glabrata/fisiologia , Humanos , Infecções Oportunistas/microbiologiaRESUMO
The yeasts, being favorite eukaryotic microorganisms used in food industry and biotechnologies for production of biomass and various substances, are also used as model organisms in genetic manipulation, molecular and biological research. In this respect, Saccharomyces cerevisiae is the best-known species but current situation in medicine and industry requires the use of other species. Here we summarize the basic taxonomic, morphological, physiological, genetic, etc. information about the pathogenic yeast Candida glabrata that is evolutionarily very closely related to baker's yeast.
Assuntos
Candida glabrata , Candida glabrata/citologia , Candida glabrata/genética , Candida glabrata/patogenicidade , Candida glabrata/fisiologia , Candidíase/microbiologia , Farmacorresistência Fúngica Múltipla , HumanosRESUMO
We have studied the dependence on mitochondrial ATP of expression of MAL genes specifying maltose utilization in yeast. It was found that bongkrekic acid does not prevent the maltose induced synthesis of alpha-glucosidase in derepressed cells of the wild-type and corresponding respiratory-deficient mutant of Saacharomyces cerevisiae. The results suggest that expression of nuclear genes specifying alpha-glucosidase and maltose catabolism in yeast is apparently not dependent on the proper function of mitochondrial adenine nucleotide translocase and does not even require the presence of normal levels of ATP in mitochondria.
Assuntos
Trifosfato de Adenosina/metabolismo , Glucosidases/biossíntese , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/enzimologia , alfa-Glucosidases/biossíntese , Ácido Bongcréquico/farmacologia , Indução Enzimática , Maltose/farmacologiaRESUMO
The Saccharomyces cerevisiae mutant strain containing the op1 mutation affecting the function of a mitochondrial ATP/ADP translocator has been crossed to the pel1 and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL). Using tetrad analysis of diploids issued from corresponding crosses a synthetic lethal interaction has been observed between the op1 and pel1 mutations resulting in the lack of growth of a corresponding double mutant on minimal medium containing glucose. The op1 pel1 double mutant also displayed a decreased susceptibility to fluconazole and a compromised growth even in complex medium containing glucose. The viability of mutant cells was strongly reduced, corresponding to <30 % and 10 % of colony-forming units observed after growth in complex and minimal medium, respectively. A lower viability of the double mutant in minimal medium was accompanied by an increased formation of mitochondrial petite mutants (as determined by mtDNA rescue into diploid cells). The results indicate that in the simultaneous absence of mitochondrial anionic phospholipids (PG plus CL) and ATP/ADP exchange across the inner mitochondrial membrane the yeast mitochondrial functions are severely limited, leading to a strongly compromised cell multiplication. Since under similar conditions the op1 crd1 double mutant was able to grow on minimal medium this deleterious effect of anionic phospholipid deficiency could be at least partially substituted by PG accumulated in the cardiolipin deficient delta crd1 mutant cells.
Assuntos
CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Cruzamentos Genéticos , Meios de Cultura , Farmacorresistência Fúngica , Fluconazol/farmacologia , Glucose/metabolismo , Potenciais da Membrana/fisiologia , Mitocôndrias , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae, chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.
Assuntos
Alcaloides/farmacologia , Antibacterianos/farmacologia , Mutação/genética , Consumo de Oxigênio/genética , Fenantridinas/farmacologia , Saccharomyces cerevisiae/genética , Benzofenantridinas , DNA Mitocondrial/efeitos dos fármacos , Isoquinolinas , Testes de Sensibilidade Microbiana , Consumo de Oxigênio/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial fumarase has been identified through the in vitro biochemical assay of enzyme activity after visual selection due to an increased acidification ability of its colonies. Cells of the fumarase-deficient mutant fermenting glucose accumulated extracellular fumaric acid. This accumulation was observed only in growing cultures and required functional mitochondrial electron transport from succinate dehydrogenase to oxygen.
Assuntos
Fumarato Hidratase/genética , Fumaratos/metabolismo , Mutação , Saccharomyces cerevisiae/enzimologia , Anaerobiose , Antifúngicos/farmacologia , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados/farmacologia , Fumarato Hidratase/antagonistas & inibidores , Fumarato Hidratase/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metacrilatos , Consumo de Oxigênio , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , EstrobilurinasRESUMO
Cells of the pel1 mutant of Saccharomyces cerevisiae were found to contain an extremely low content of cardiolipin, a decreased level of phosphatidylcholine and an increased level of phosphatidylinositol. Disruption of the PEL1 gene in cells containing a null mutation in the CHO1 gene was lethal. Despite its putative functional homology with CHO1, the overexpression of the PEL1 gene in the cho1 null mutant did not restore the wild-type properties of the transformed cells and failed to stimulate the incorporation of L-[3-3H]serine into total lipids of the intact yeast cells.
Assuntos
CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Cardiolipinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Antígenos/genética , Cardiolipinas/genética , Genes Fúngicos/fisiologia , Metabolismo dos Lipídeos , Mutação/fisiologia , Fenótipo , Fosfatidilserinas/fisiologiaRESUMO
Pure wine yeast cultures are increasingly used in winemaking to perform controlled fermentations and produce wine of reproducible quality. For the genetic manipulation of natural wine yeast strains dominant selective markers are obviously useful. Here we demonstrate the successful use of the mutated PDR3 gene as a dominant molecular marker for the selection of transformants of prototrophic wine yeast Saccharomyces cerevisiae. The selected transformants displayed a multidrug resistance phenotype that was resistant to strobilurin derivatives and azoles used to control pathogenic fungi in agriculture and medicine, respectively. Random amplification of DNA sequences and electrophoretic karyotyping of the host and transformed strains after microvinification experiments resulted in the same gel electrophoresis patterns. The chemical and sensory analysis of experimental wines proved that the used transformants preserved all their useful winemaking properties indicating that the pdr3-9 allele does not deteriorate the technological properties of the transformed wine yeast strain.
Assuntos
Proteínas de Ligação a DNA/genética , Farmacorresistência Fúngica Múltipla/genética , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Genes Dominantes , Marcadores Genéticos , Mutação , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Transformação Genética , VinhoRESUMO
Yersinia enterocolitica was detected in 160 of 2760 examined samples that were prepared from dental cavity of 920 pigs. From the isolates 49 strains were characterized with respect to their genetic and phenotypic markers of virulence. All strains were belonging to serogroup 0:3, biovar 4. Forty-one isolates (84%) harboured the virulence plasmid detected by DNA colony hybridization. Of the other assays of virulence--autoagglutination, binding of crystal violet, pyrazinamidase activity, calcium dependence and Congo red binding--the latter two exhibited the best correlation with the detected presence of plasmid DNA.
Assuntos
Boca/microbiologia , Suínos/microbiologia , Yersinia enterocolitica/classificação , Animais , Técnicas de Tipagem Bacteriana/veterinária , Plasmídeos/genética , Eslováquia , Virulência/genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/isolamento & purificaçãoRESUMO
Two structurally related food additives, 3-(5-nitro-2-furyl)acrylic acid (5-NFAA) and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) showed a marked mutagenic effect on Salmonella typhimurium strains TA100 and TA98 and Escherichia coli WP2 uvrA+ and WP2 uvrA. On the molar basis 5-NFAA was about two orders of magnitude less effective than AF-2. In Salmonella typhimurium TA100 anaerobic conditions stimulated the mutagenic effect of 5-NFAA which was more pronounced in nitrofuran-reductase deficient strain of Salmonella typhimurium TA100 FR50. 5-NFAA increased the number of isoleucine revertants and induced mitotic recombination at tryptophan, threonine and adenine loci of the diploid strains Saccharomyces cerevisiae a/b and Saccharomyces cerevisiae SBTD. Activity of 5-NFAA was lower than that of AF-2. The test on sex-linked recessive lethal mutations in Drosophila melanogaster indicated that only 5-NFAA is mutagenic, increasing the mutation frequency about 10-fold above the control. Results with AF-2 fell within the control range.
Assuntos
Acrilatos/toxicidade , Conservantes de Alimentos/toxicidade , Furilfuramida/toxicidade , Mutagênicos , Nitrofuranos/toxicidade , Animais , Aberrações Cromossômicas , Drosophila melanogaster , Escherichia coli/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Linfócitos/efeitos dos fármacos , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Salmonella typhimurium/efeitos dos fármacosRESUMO
For successful transformation of prototrophic industrial yeast strains dominant selectable markers are necessary. In the present study we show the applicability of a selection system based on the phenotype of multidrug resistance. The mutant pdr3-9 allele on centromeric or episomal vector, encoding a more efficient transcriptional activator with Y276H amino acid substitution, was used as a dominant selectable marker for selection of transformants. The pdr3-9 allele conferred resistance of transformed cells to cycloheximide, chloramphenicol, mucidin and oligomycin both in the absence and in the presence of a chromosomal copy of the PDR3 gene. Both multicopy YEp352/pdr3-9 and centromeric pFL38/pdr3-9 vectors bearing the mutant pdr3-9 allele have proved to be a valuable tool for a direct selection of transformants of industrial strains of Saccharomyces cerevisiae.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Fúngicos , Marcadores Genéticos , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transformação Genética , Antifúngicos/farmacologia , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Genes Dominantes , Testes de Sensibilidade Microbiana , Mutação , Plasmídeos/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Uracila/metabolismoRESUMO
N,N-Dimethyl-l-methyldodecylamine oxide inhibited the growth of bacteria, yeast and filamentous fungi; further it induced lysis of osmotically stabilized protoplasts of Saccharomyces cerevisiae and human erythrocytes. This effect is based evidently on a change in the organization and function of cell membranes.
Assuntos
Aminas/farmacologia , Bactérias/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Membrana Eritrocítica/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Fragilidade Osmótica/efeitos dos fármacos , Óxidos/farmacologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Viability of petite-negative yeast, such as Kluyveromyces lactis, is dependent on functional mitochondrial genome encoding essential components of both mitochondrial protein synthesizing system and oxidative phosphorylation. We have isolated several nuclear mutants impaired in mitochondrial functions that were unable to grow on non-fermentable carbon and energy sources. They were used for the isolation and molecular characterization of the three genes encoding apocytochrome c, apocytochrome c1 and the protein involved in the biogenesis of cytochrome oxidase. All cytochrome-deficient mutants were viable and did not survive the ethidium bromide mutagenesis. Petite-positive Saccharomyces cerevisiae requires intact mitochondrial genome when its phosphatidylglycerolphosphate synthase was inactivated due to mutation in the PEL1 gene. Using PEL-lacZ fusion genes it was demonstrated that Pel1p is a mitochondrial protein (expressed in response to myo-inositol and choline). The pel1 mutant was deficient in phosphatidylglycerol (PG) and cardiolipin (CL) and its rho-/rho0 mutants grew extremely slowly on complex medium with glucose. Under the same conditions the growth rate of the crd1 rho- double mutants was similar to that of its parent crd1 mutant deficient in cardiolipin synthase and accumulating PG. The results demonstrate that the petite negativity in yeast is not dependent on an intact respiratory chain or functional oxidative phosphorylation. The presence of the negatively charged PG or CL seems to be essential for the maintenance of specific mitochondrial functions required for the normal mitotic growth of yeast cells.
Assuntos
Genoma Fúngico , Kluyveromyces/crescimento & desenvolvimento , Kluyveromyces/genética , Mitocôndrias/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Citocromos/genética , Transporte de Elétrons , Proteínas Fúngicas/genética , Membranas Intracelulares/metabolismo , Kluyveromyces/metabolismo , Mitocôndrias/metabolismo , Mitose , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We have isolated a respiration-deficient nuclear mutant of the yeast Kluyveromyces lactis that exhibited diminished levels of all cytochromes and did not grow on glycerol and other nonfermentable carbon sources. The mutant named cyc1 was transformed with a K. lactis genomic library and the DNA fragment conferring its wild-type properties was isolated and sequenced. The sequence of the isolated gene showed extensive homology with other eukaryotic cytochrome-c genes. The highest level of homology, based on the deduced amino acid sequences, was observed between the gene products of K. lactis and Hansenula anomala.
Assuntos
Apoproteínas/deficiência , Grupo dos Citocromos c/deficiência , Proteínas Fúngicas/genética , Genes Fúngicos , Kluyveromyces/genética , Sequência de Aminoácidos , Animais , Apoproteínas/genética , Carbono/metabolismo , Meios de Cultura/metabolismo , Grupo dos Citocromos c/genética , Citocromos c , DNA Fúngico/genética , Fermentação , Biblioteca Gênica , Glicerol/metabolismo , Humanos , Kluyveromyces/enzimologia , Kluyveromyces/crescimento & desenvolvimento , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The effect of ten derivatives of carbonylcyanide phenylhydrazone on growth of bacteria, yeast and different species of filamentous fungi was investigated. In yeast and mitochondria isolated from rat liver the effect of these derivatives on the respiratory activity was also followed. The relative efficiency of the individual derivatives of carbonylcyanide phenylhydrazone was determined on the basis of the results obtained. It was shown that derivatives, in which the substituent on the benzene ring causes simultaneously an increase of acidity and lipophilicity of the derivative as compared with the non-substituted carbonylcyanide phenylhydrazone (4-trifluoromethoxy-, 3-chloro-, 4-chloro and 3,4-dichloro-derivatives) were most effective.
Assuntos
Bacillus cereus/crescimento & desenvolvimento , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Fungos/crescimento & desenvolvimento , Nitrilas/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/análogos & derivados , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio , Ratos , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
In yeast the resistance to kresoxim-methyl and azoxystrobin, like the resistance to strobilurin A (mucidin) is under the control of both mitochondrial cob gene and the PDR network of nuclear genes involved in multidrug resistance. The mucidin-resistant mucl (G137R) and muc2 (L275S) mutants of Saccharomyces cerevisiae containing point mutations in mtDNA were found to be cross-resistant to kresoxim-methyl and azoxystrobin. Cross-resistance to all three strobilurin fungicides was also observed in yeast transformants containing gain-of-function mutations in the nuclear PDR3 gene. On the other hand, nuclear mutants containing disrupted chromosomal copies of the PDR1 and PDR3 genes or the PDR5 gene alone were hypersensitive to kresoxim-methyl, azoxystrobin and strobilurin A. The frequencies of spontaneous mutants selected for resistance either to kresoxim-methyl, azoxystrobin or strobilurin A were similar and resulted from mutations both in mitochondrial and nuclear genes. The results indicate that resistance to strobilurin fungicides, differing in chemical structure and specific activity, can be caused by the same molecular mechanism involving changes in the structure of apocytochrome b and/or increased efflux of strobilurins from fungal cells.
Assuntos
Acrilatos/farmacologia , Farmacorresistência Fúngica Múltipla/genética , Ácidos Graxos Insaturados/farmacologia , Fenilacetatos/farmacologia , Pirimidinas/farmacologia , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/genética , Antifúngicos/farmacologia , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Genes Fúngicos , Metacrilatos , Testes de Sensibilidade Microbiana , Mitocôndrias/genética , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Estrobilurinas , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: Analysis of the prevalence and species representation of pathogenic yeasts in patients with vulvovaginal candidiasis. Determination of in vitro susceptibility of yeast isolates to clinically used antimycotic agents. DESIGN: A retrospective clinical study of patients with positive vaginal cultures for the presence of pathogenic yeast species. SETTING: I. gynekologicko-pôrodnícka klinika LF UK a FN, Zochova 7,811 03 Bratislava, Slovenská republika. METHODS: Identification of yeast pathogens on the chromogenic medium CHROMagar CANDIDA and with API-CANDIDA identification system. In vitro susceptibility assays of clinical yeast isolates to antifungal agents using the plate dilution method, NCCLS method and ATB-FUNGUS test system. RESULTS: The highest prevalence of vulvovaginal candidasis was found in women aged between 20-30 years. Candida albicans was the most commonly identified species of pathogenic yeasts (87.4%). Of the non-albicans species, C. glabrata (6.3%) was the most prevalent species. C. glabrata and C. krusei clinical isolates were found to be generally less susceptible to several antifungals in vitro as compared to C. albicans strains. A minimal number of resistant yeast isolates was observed for econazole, clotrimazole and nystatin. A relatively high number of resistant strains was observed for some other azole antifungals (miconazole, ketoconazole, itraconazole, fluconazole). CONCLUSION: A successful treatment of vaginal mycotic infections requires the results of the microbiological analyses. They will bring evidence to a physician of the presence and fate of the pathogen, of its sensitivity to antifungals, both of which are essential for the rational and successful therapy of Candida vaginitis.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candidíase Vulvovaginal/microbiologia , Farmacorresistência Fúngica , Adulto , Candida/isolamento & purificação , Feminino , Humanos , Testes de Sensibilidade Microbiana , Estudos RetrospectivosRESUMO
We determined the susceptibility to oxidative stress and assessed the four virulence factors of the 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. All the isolates were susceptible to hydrogen peroxide, diamide, and 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) inducing an increased formation of reactive oxygen species in fungal cells. The mean relative cell surface hydrophobicity (CSH) of isolates was 21.9, ranging from 1.92 to 56.96. All isolates showed biofilm formation. A high biofilm formation was observed among 60.5% of isolates. Positive correlations were observed between biofilm formation and moderate values of CSHs. The 76.3% and 84.2% of isolates displayed varying degrees of proteinase and phospholipase activity, respectively. These results demonstrate a differential distribution of factors contributing to virulence of C. glabrata clinical isolates and point to their significance in pathogenesis that would be targeted by novel antifungals.