RESUMEN
The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae. We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2(C505T) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC- and 5FC/FLC-resistant fcy1Delta strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.
Asunto(s)
Antifúngicos/farmacología , Candidiasis/microbiología , Codón sin Sentido/genética , Fluconazol/farmacología , Flucitosina/farmacología , Mutación Missense/genética , Northern Blotting , Southern Blotting , Candida/efectos de los fármacos , Candida/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la PolimerasaRESUMEN
We report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1.
Asunto(s)
Candida/efectos de los fármacos , Candida/genética , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN , Proteínas Fúngicas/genética , Humanos , Proteínas de Transporte de Membrana/genética , Metiltransferasas/genética , Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Fungal histidine kinase receptors (HKRs) sense and transduce many extracellular signals. We investigated the role of HKRs in morphogenetic transition, osmotolerance, oxidative stress response, and mating ability in the opportunistic yeast Candida lusitaniae. We isolated three genes, SLN1, NIK1, and CHK1, potentially encoding HKRs of classes VI, III, and X, respectively. These genes were disrupted by a transformation system based upon the "URA3 blaster" strategy. Functional analysis of disruptants was undertaken, except for the sln1 nik1 double mutant and the sln1 nik1 chk1 triple mutant, which are not viable in C. lusitaniae. The sln1 mutant revealed a high sensitivity to oxidative stress, whereas both the nik1 and chk1 mutants exhibited a more moderate sensitivity to peroxide. We also showed that the NIK1 gene was implicated in phenylpyrrole and dicarboximide compound susceptibility while HKRs seem not to be involved in resistance toward antifungals of clinical relevance. Concerning mating ability, all disruptants were still able to reproduce sexually in vitro in unilateral or bilateral crosses. The most important result of this study was that the sln1 mutant displayed a global defect of pseudohyphal differentiation, especially in high-osmolarity and oxidative-stress conditions. Thus, the SLN1 gene could be crucial for the C. lusitaniae yeast-to-pseudohypha morphogenetic transition. This implication is strengthened by a high level of SLN1 mRNAs revealed by semiquantitative reverse transcription-PCR when the yeast develops pseudohyphae. Our findings highlight a differential contribution of the three HKRs in osmotic and oxidant adaptation during the morphological transition in C. lusitaniae.
Asunto(s)
Adaptación Fisiológica , Candida/crecimiento & desarrollo , Candida/metabolismo , Proteínas Fúngicas/metabolismo , Hifa/crecimiento & desarrollo , Receptores de Superficie Celular/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Alelos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Antifúngicos/farmacología , Candida/efectos de los fármacos , Candida/genética , Clonación Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes del Tipo Sexual de los Hongos , Prueba de Complementación Genética , Hidantoínas/farmacología , Hifa/citología , Hifa/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Mutación/genética , Estrés Oxidativo/efectos de los fármacos , Pirroles/farmacología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genéticaRESUMEN
Inactivation of the FCY2 (cytosine permease), FCY1 (cytosine deaminase), and FUR1 (uracil phosphoribosyltransferase) genes in Candida lusitaniae produced two patterns of resistance to flucytosine. Mutant fur1 demonstrated resistance to 5-fluorouracil, whereas mutants fcy1 and fcy2 demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.
Asunto(s)
Candida/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Flucitosina/farmacología , Fluorouracilo/farmacología , Alelos , Antifúngicos/farmacología , Candida/enzimología , Candida/genética , Clonación Molecular , Citosina Desaminasa/genética , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Pentosiltransferasa/genética , Plásmidos/genética , Análisis de Secuencia de ADNRESUMEN
In a previous work, we described the possible relationship between a defect of purine-cytosine permease and the acquisition of a cross-resistance to the antifungal combination flucytosine (5FC) and fluconazole (FLC) in Candida lusitaniae (T. Noël, F. François, P. Paumard, C. Chastin, D. Brethes, and J. Villard, Antimicrob. Agents Chemother. 47:1275-1284, 2003). Using degenerate PCR and chromosome walking, we cloned two FCY2-like genes in C. lusitaniae. Northern blot analysis revealed that only one gene was expressed; it was named FCY2. The other one behaved as a pseudogene and was named FCY21. In order to better characterize the possible role of FCY2 in cross-resistance to 5FC-FLC, disruption experiments with auxotrophic strain 6936 ura3(D95V) FCY2 with an integrative vector carrying the URA3 gene and a partial sequence of the C. lusitaniae FCY2 gene were undertaken. Southern blot analysis revealed that homologous recombination events occurred in all transformants analyzed at rates of 50% at resident locus FCY2 and 50% at resident locus URA3, resulting in the genotypes ura3 fcy2::URA3 and ura3::URA3 FCY2, respectively. It was then demonstrated that only transformants harboring a disrupted fcy2 gene were resistant to 5FC, susceptible to FLC, and resistant to the 5FC-FLC combination. Finally, complementation experiments with a functional FCY2 gene restored 5FC and FLC susceptibilities to the wild-type levels. The results of this study provide molecular evidence that inactivation of the sole FCY2 gene promotes cross-resistance to the antifungal association 5FC-FLC in C. lusitaniae.
Asunto(s)
Antifúngicos/farmacología , Candida/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Flucitosina/farmacología , Proteínas de Transporte de Nucleobases/genética , Recombinación Genética , Secuencia de Aminoácidos , Candida/genética , Paseo de Cromosoma , Clonación Molecular , Citosina , ADN de Hongos/análisis , ADN de Hongos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Proteínas de Transporte de Nucleobases/metabolismo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
We report on five clinical isolates routinely identified as Candida lusitaniae that the ID 32C system was unable to discriminate from the closely related species Candida pulcherrima. When additional tests did not allow accurate identification, the less usual mating type test identified all of them as Clavispora lusitaniae. Mating type testing appears to be a valuable tool for assessing the true incidence of this emerging non-albicans Candida species.
Asunto(s)
Candida/clasificación , Candida/fisiología , Técnicas de Tipificación Micológica , FenotipoRESUMEN
OBJECTIVES: The aim of the present study was to expand the MIC database for Candida lusitaniae in order to further determine its antifungal susceptibility pattern. METHODS: The activities of amphotericin B, fluconazole, itraconazole, voriconazole and flucytosine were determined in vitro against 80 clinical isolates of C. lusitaniae. A set of 59 clinical isolates of Candida albicans and of 51 isolates of Candida glabrata was included to compare the susceptibilities to amphotericin B. The MICs were determined by Etest with RPMI 1640 agar, and with both this medium and antibiotic medium 3 (AM3) agar for testing of amphotericin B. RESULTS: All isolates were highly susceptible to fluconazole. The susceptibility to itraconazole was good; only 4% of isolates had dose-dependent susceptibility (MICs 0.25-0.5 mg/L). Voriconazole was very active in vitro (100% of isolates were inhibited at < or =0.094 mg/L). Flucytosine MICs ranged widely (0.004->32 mg/L). The set included 19% of flucytosine-resistant isolates. For amphotericin B, 100% of isolates were inhibited at < or =0.75 mg/L (MIC(50) 0.047 mg/L; MIC(90) 0.19 mg/L) and at < or =4 mg/L (MIC(50) 0.25 mg/L; MIC(90) 0.75 mg/L) on RPMI and on AM3, respectively. A single isolate was categorized as resistant to amphotericin B (MIC 0.75 and 4 mg/L on RPMI and on AM3, respectively). Amphotericin B thus appeared very active in vitro against C. lusitaniae. Whatever the test medium, the level of susceptibility of C. lusitaniae to amphotericin B did not differ much from those of C. albicans and C. glabrata. CONCLUSION: C. lusitaniae appears to be susceptible to amphotericin B, azole antifungal agents, and, to a lesser extent, flucytosine.
Asunto(s)
Antifúngicos/farmacología , Candida/efectos de los fármacos , Anfotericina B/farmacología , Candidiasis/microbiología , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Candida lusitaniae is an emerging opportunistic pathogen which exhibits an unusual antifungal susceptibility pattern. We describe a case of fatal renal infection due to C. lusitaniae in a very low birth weight neonate who was treated with short courses of fluconazole given alternately with amphotericin B. A colony morphology switching was detected on the standard primary culture medium by changes in colony size. Switching was shown to affect deeply the susceptibility to amphotericin B. Afterwards, the switched phenotype developed a cross resistance to fluconazole and itraconazole. Several issues raised by this case are discussed in the light of an extensive review of the literature. Our observations point out the importance of both the detection of colony morphology switching and the close monitoring of antifungal susceptibility in the management of infections due to C. lusitaniae. A judicious therapeutic strategy should prevent the acquisition of multidrug resistance during antifungal therapy.
Asunto(s)
Candida/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Farmacorresistencia Fúngica , Resistencia a Múltiples Medicamentos , Fungemia/tratamiento farmacológico , Recién Nacido de muy Bajo Peso , Antifúngicos/farmacología , Secuencia de Bases , Candida/clasificación , Candidiasis/diagnóstico , Recuento de Colonia Microbiana , Fungemia/diagnóstico , Humanos , Recién Nacido , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/microbiología , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Técnicas de Tipificación Micológica , Reacción en Cadena de la Polimerasa , ARN de Hongos/análisis , Factores de Riesgo , Sensibilidad y EspecificidadRESUMEN
An unusual interaction between flucytosine and fluconazole was observed when a collection of 60 Candida lusitaniae clinical isolates was screened for cross-resistance. Among eight isolates resistant to flucytosine (MIC >/= 128 micro g/ml) and susceptible to fluconazole (0.5 < MIC < 2 micro g/ml), four became flucytosine-fluconazole cross resistant when both antifungals were used simultaneously. Fluconazole resistance occurred only in the presence of high flucytosine concentrations, and the higher the fluconazole concentration used, the greater the flucytosine concentration necessary to trigger the cross-resistance. When the flucytosine- and fluconazole-resistant cells were grown in the presence of fluconazole alone, the cells reversed to fluconazole susceptibility. Genetic analyses of the progeny from crosses between resistant and sensitive isolates showed that resistance to flucytosine was derived from a recessive mutation in a single gene, whereas cross-resistance to fluconazole seemed to vary like a quantitative trait. We further demonstrated that the four clinical isolates were susceptible to 5-fluorouracil and that cytosine deaminase activity was unaffected. Kinetic transport studies with [(14)C]flucytosine showed that flucytosine resistance was due to a defect in the purine-cytosine permease. Our hypothesis was that extracellular flucytosine would subsequently behave as a competitive inhibitor of fluconazole uptake transport. Finally, in vitro selection of spontaneous and induced mutants indicated that such a cross-resistance mechanism could also affect other Candida species, including C. albicans, C. tropicalis, and C. glabrata. This is the first report of a putative fluconazole uptake transporter in Candida species and of a possible resistance mechanism associated with a deficiency in the uptake of this drug.