Association of ADH 1 and DDR 48 expression with azole resistance in Candida albicans

Background: Candida albicans is a frequent opportunistic pathogenic fungus that causes mucosal and systemic infections. The alcohol dehydrogenase 1 (ADH1) and DDR48 genes were found to be upregulated in fluconazole resistant Candida albicans. Therefore, understanding the function of drug resistance of genes will help in the development of new antifungal agents that can reverse drug resistance. This study aimed to investigate the role of ADH1 and DDR48 genes in development of fluconazole resistant C. albicans. Subjects and Methods: This study involved 19 fluconazole susceptible and 6 fluconazole resistant C. albicans isolates from clinical specimens. The MICs of fluconazole were determined by the E-test. Quantitative expressions of Candida Drug Resistance (ADH1, CDR1, DDR48 and FLU1) genes were assessed by real time PCR. Results: There was a statistically significant higher expression levels of CDR1, FLU1, ADH1 and DDR48 in resistant and susceptible dose dependent isolates than in susceptible isolates (P = 0.009, 0.008, 0.01, 0.014 respectively). Strong positive correlations were observed between the expression levels of each of ADH1and DDR4 with azole resistance genes CDR1 and FLU1 [(rs) = 0.945, 0.815, respectively; P < 0.001; Spearman’s (rs) = 0.852 and 0.76, respectively; P < 0.001]. Conclusion: This is the first study that showed positive correlation between DDR48 and azole resistance genes. It has also indicated that ADH1 and DDR48 are associated with the resistance mechanisms of C. albicans to fluconazole. Identification of new drugs that target the The InTernaTIonal arabIc Journal of anTImIcrobIal agenTs Issn 2174-9094 2014 Vol. 4 No. 3:2 doi: 10.3823/754 This article is available from: www.iajaa. org / www.medbrary. com 2 Introduction Candida albicans is a human fungal pathogen that causes serious infections in immunocompromised population [1]. In hospitalized patients, this organism can disseminate hematogenously and infect virtually all organs [2]. Fluconazole and other azoles enter C. albicans cells by facilitated diffusion [3]. These antifungal drugs inhibit cytochrome P450 enzymes: lanosterol demethylase, a key enzyme in ergosterol biosynthesis [4], and C-22 sterol desaturase [5]. C. albicans is becoming increasingly resistant to azole antifungal agents, particularly fluconazole, a problem of growing importance due to the widespread use of a limited number of antifungal agents, particularly azoles [6, 7]. Various mechanisms can lead to the acquired resistance of Candida spp. to azole drugs, and most involve the induction of the efflux pumps encoded by CDR (Candida Drug Resistance) or MDR (Multiple Drug Resistance) genes, or the over-expression or acquisition of point mutations in the gene encoding the target enzyme lanosterol demethylase [8]. Over-expression of drug-efflux pumps Cdr1, Cdr2 and Mdr1 has been linked to fluconazole resistance [9]. Multidrug efflux transporters of the ABC (ATP-binding cassette) superfamily and of the Major Facilitator Superfamily (MFS) play a key role in the low level of accumulation of azoles in the yeast cell [10]. The ABC transporter superfamily, including Cdr1 and Cdr2, are membrane proteins that have two membrane-spanning domains and two nucleotidebinding domains that utilize ATP to drive substrates across the membrane [11]. CDR1 and CDR2 genes have been shown to be upregulated in resistant strains, leading to an enhanced efflux of the drug [10] and hence leading to resistance to multiple azoles [12, 13]. Cross-resistance between fluconazole and other azoles has been reported [14-16], the mechanistic basis for this cross-resistance most often involves the upregulation of genes encoding the CDR pumps that act as ATP-binding cassette efflux transporters [13, 17, 18]. On the other hand, MFS drug pumps, including Mdr1 and Flu1, have no nucleotide-binding domain but instead use the proton motive force of the membrane as an energy source [11]. Both MDR1 and FLU1 genes were over-expressed specifically in fluconazole-resistant C. albicans isolates. Disruption of FLU1 gene in C. albicans mutants with deletions in multidrug efflux transporter genes, including CDR1, CDR2 and MDR1, resulted in enhanced susceptibility to several azole derivatives, indicating the contribution of FLU1 in azole resistance [19]. The Adh1p (alcohol dehydrogenase protein) which is encoded by the ADH1 gene, plays an important role in intracellular energy metabolism [20], as the most critical mechanism governing drug resistance proteins encoded by these genes will help in eradication of fluconazole resistant C. albicans in infection.