Screening And Functional Characterization Of Novel Genes Involved In Azole Resistance Of Filamentous Fungi

Azoles are widely used for controlling fungal infections in clinics and agriculture. However, fungi can become resistant to azoles by multiple mechanisms, which lead to high rates of infection and low efficacy of drugs. Increased knowledge on mechanisms of antifungal drug resistance could allow rational drug use and identification of new classes of drugs. The regulatory mechanisms of antifungal resistance have been intensively studied in yeast, such as Candida albicans, while, in filamentous fungi, reports in this field are very few. Several transcription factors, which regulate responses to azoles, have been found in yeast. However, in filamentous fungi, most of these proteins do not have homologues or their homologues are not involved in antifungal drug resistance, which suggests that filamentous fungi may have different transcription factors which mediate transcriptional response to azoles. In addition, many genes involved in ergosterol-synthesis pathway are upregulated under azoles stress, but most of their roles in azole resistance were unkown in filamentous fungi. Therefore, it is necessary to elucidate the mechanisms of antifungal drug resistance in filamentous fungi.Using Neurospora crassa as the model organism, this study aimed to discover new genes involved in azoles resistance through analyzing its transcriptional responses to azoles and performing azoles sensitivity test to gene knockout strains. Meanwhile, homologous genes of new genes found in Neurospora crassa were deleted in pathogenic fungi Fusarium verticillioides and azoles sensitivity of their knockout mutants were tested to make sure its roles in different filamentous fungi. The main results of this study are as follows:1) Two gene knockout strains showed hypersensitivity to ketoconazole and itraconazole. The corresponding genes encode a light inducible protein CCG-8and a hypothetical protein, respectively. The hypothetical protein was named ads-4(antifungal drug sensitive-4) in this study, ccg-8knockout mutant showed wild-type phenotype to osmotic and oxidative stress, while ads-4knockout mutant showed hypersensitivity to oxidative stress and it also produced less conidia than wild type. Homologues of CCG-8and ADS-4broadly present in filamentous fungi. Deletion of either CCG-8or ADS-4in F.verticillioides led to hypersensitivity to azoles, suggesting that ccg-8and ads-4are involved in azoles resistance in different filamentous fungi.2) ERG is a key gene of ergosterol-synthesis pathway. Deletion of N. crassa ERG5increased sensitivity to azoles and deleted its homologous gene in F. verticillioides also led to sensitivity, which suggested that ERG5could be a new target in developing new drugs.