RESUMEN
The activity of albaconazole (UR-9825; J. Uriach & Cía. S.A., Barcelona, Spain) was compared to that of fluconazole against 12 isolates of Cryptococcus neoformans in vitro and against 1 isolate in vivo in a rabbit model of cryptococcal meningitis. Albaconazole was 100-fold more potent in vitro than fluconazole on a per-weight basis and was fungicidal at potentially relevant concentrations for two isolates. MICs ranged from
Asunto(s)
Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Cryptococcus neoformans/efectos de los fármacos , Meningitis Criptocócica/tratamiento farmacológico , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Triazoles/farmacología , Triazoles/uso terapéutico , Animales , Recuento de Colonia Microbiana , Fluconazol/uso terapéutico , Meningitis Criptocócica/microbiología , Pruebas de Sensibilidad Microbiana , Conejos , Análisis de SupervivenciaRESUMEN
The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.
Asunto(s)
Cryptococcus neoformans/patogenicidad , Manosa-6-Fosfato Isomerasa/fisiología , Secuencia de Aminoácidos , Animales , Criptococosis/microbiología , Cryptococcus neoformans/enzimología , Modelos Animales de Enfermedad , Genes Fúngicos , Humanos , Masculino , Manosa-6-Fosfato Isomerasa/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutagénesis , Conejos , Homología de Secuencia de Aminoácido , VirulenciaRESUMEN
Cryptococcus neoformans is a leading cause of life-threatening fungal infection in immunocompromised patients. Inositol-phosphoryl ceramide synthase 1 (Ipc1) is a fungus-specific enzyme, encoded by the essential IPC1 gene, that catalyzes the formation of complex sphingolipids and may also regulate the levels of phytoceramide and diacylglycerol. Here, we investigated the functions of this essential gene by modulating its expression in C. neoformans using a galactose-inducible promoter. Down-regulation of IPC1 significantly lowers the expression of certain virulence traits such as melanin pigmentation and, remarkably, impairs pathogenicity of C. neoformans in an established rabbit model. Interestingly, we found that IPC1 down-regulation significantly decreases the intracellular growth of C. neoformans in the J774.16 murine macrophage-like cells. Finally, we studied the effect of IPC1 expression under different stress conditions and found that down-regulation of IPC1 confers a defect on in vitro growth at low pH. Because this environment is similar to that in the phagolysosome of J774.16 macrophage-like cells, our findings indicate that down-regulation of IPC1 confers a growth defect in vivo through a pH-dependent mechanism. In conclusion, our study is the first to define a novel and crucial function of Ipc1 in fungal pathogenesis.