Tesi di dottorato di ricerca
MOLECULAR MECHANISMS OF DRUG RESISTANCE IN CANDIDA ALBICANS: ROLE OF TRANSCRIPTION FACTORS MCM1 AND ADA2
Settore scientifico disciplinare
Corso di studi
MICROBIOLOGIA E GENETICA
tutor Senesi, Sonia
Data inizio appello
Candida albicans is an opportunistic fungal pathogen responsible for localized as well as disseminated infections. C. albicans is the most commonly isolated specie from blood cultures, accounting for over 60% of all Candida isolates. The fungistatic drug fluconazole is the most widely used to treat fungal infections, thanks to its favorable bio-availability and to its low toxicity. The frequent and widespread use of this antifungal has led to the outcome of drug resistant clinical isolates. Various drug resistance mechanisms are known that render C. albicans resistant to fluconazole, among those, the overexpression of efflux pumps, which extrude the drug out of the fungal cell, and the overexpression of the fluconazole target, the Erg11 enzyme. Overexpression of drug resistance genes is often associated to mutations in trans-acting transcription factors. The present study aimed at better understanding the role of known transcription factors involved in fluconazole resistance in C. albicans. In particular the role of TFs Mcm1 and Ada2 in resistance gene promoter activation has been evaluated. The transcription factors Mrr1 and Cap1 mediate MDR1 upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutive MDR1 overexpression. The essential MADS box transcription factor Mcm1 also binds to the MDR1 promoter, but its role in inducible or constitutive MDR1 upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, the importance of Mcm1 for MDR1 expression was investigated. The results obtained indicated that Mcm1 was dispensable for MDR1 upregulation by H2O2, but required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to cause MDR1 overexpression. These results demonstrate a differential requirement of the co-regulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 could induce MDR1 expression independently of Mcm1, whereas Mrr1 required either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump.<br>Other transcription factors that mediate drug resistance gene regulation are Tac1, which regulates CDR1 and CDR2 expression, and Upc2, regulating ERG11 gene expression. Also in this case, gain-of-function mutations, which render these transcription factors hyperactive, result in constitutive overexpression of the target genes.<br>Ada2 is part of the SAGA/ADA complex and has been shown to be recruited to 200 promoters upstream of genes involved in different stress-response functions and metabolic processes. As for Mcm1, the importance of Ada2 for MDR1 expression was investigated, as well as for CDR2 and ERG11 expression. Ada2 was found to be dispensable for MDR1 upregulation by H2O2, but required for MDR1 activation by hyperactive Cap1. When activated by benomyl or a gain-of-function mutation, Mrr1 induced MDR1 expression even better in the absence of Ada2. CDR2 expression by hyperactive Tac1 was facilitated in the presence of Ada2 and an opposite behaviour was observed when CDR2 expression was stimulated by the presence of fluphenazine in the medium. Finally, further experiments are required to better understand the role of Ada2 in the expression of ERG11.<br>Overall, these findings provide a more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.