RESUMEN
Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BDelta, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors.
Asunto(s)
Aspergilosis/microbiología , Aspergillus fumigatus/genética , Regulación Fúngica de la Expresión Génica , Genes Esenciales , Genes Fúngicos , Regiones Promotoras Genéticas , Animales , Antifúngicos/uso terapéutico , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/patogenicidad , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Masculino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Nitrato Reductasas/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fenotipo , ARN Mensajero/análisis , Recombinación Genética , Esterol 14-Desmetilasa , Transcripción Genética , Virulencia/genética , Virulencia/fisiologíaRESUMEN
BACKGROUND: Neutrophils represent the first line of defence against aggressions. The programmed death of neutrophils is delayed by pro-inflammatory stimuli to ensure a proper resolution of the inflammation in time and place. The pro-inflammatory stimuli include granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently, we have demonstrated that although neutrophils have an identical spontaneous apoptosis in elderly subjects compared to that in young subjects, the GM-CSF-induced delayed apoptosis is markedly diminished. The present study investigates whether an alteration of the GM-CSF stimulation of MAPKs play a role in the diminished rescue from apoptosis of PMN of elderly subjects. METHODS: Neutrophils were separated from healthy young and elderly donors satisfying the SENIEUR protocol. Neutrophils were stimulated with GM-CSF and inhibitors of the MAPKinase pathway. Apoptosis commitment, phosphorylation of signaling molecules, caspase-3 activities as well as expression of pro- and anti-apoptotic molecules were performed in this study. Data were analyzed using Student's two-tailed t-test for independent means. Significance was set for p
Asunto(s)
Envejecimiento/fisiología , Senescencia Celular/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Proteínas de Neoplasias/genética , Neutrófilos/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/genética , Envejecimiento/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Senescencia Celular/efectos de los fármacos , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Proteína X Asociada a bcl-2RESUMEN
Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387-391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.