RESUMEN
Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1Δ and tco2Δ) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 (Mbp1- and Swi4-like protein 1). Expression analysis revealed that MBS1 was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this, C. neoformans with the deletion of MBS1 exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes of C. neoformans. Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence of C. neoformans. In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis.
Asunto(s)
Antifúngicos/farmacología , Criptococosis/microbiología , Cryptococcus neoformans/patogenicidad , Farmacorresistencia Fúngica , Flucitosina/farmacología , Proteínas Fúngicas/metabolismo , Estrés Oxidativo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Recuento de Colonia Microbiana , Secuencia Conservada , Criptococosis/inmunología , Cryptococcus neoformans/efectos de los fármacos , Cryptococcus neoformans/fisiología , Daño del ADN , Ergosterol/biosíntesis , Femenino , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Pleiotropía Genética , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones , Viabilidad Microbiana/efectos de los fármacos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Proteínas Quinasas/metabolismo , Factores de Transcripción/genética , Virulencia , Factores de Virulencia/biosíntesisRESUMEN
Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect- or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes.
Asunto(s)
Cryptococcus neoformans/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Termotolerancia/fisiología , Factores de Transcripción/metabolismo , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fosforilación , Transducción de Señal , Temperatura , Termotolerancia/genética , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and â¼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens.