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. 2011 May;55(5):2061-6.
doi: 10.1128/AAC.01467-10. Epub 2011 Feb 22.

Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1

Selene Mogavero  1 , Arianna Tavanti, Sonia Senesi, P David Rogers, Joachim Morschhäuser
Affiliations

Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1

Selene Mogavero et al. Antimicrob Agents Chemother. 2011 May.

Abstract

Overexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeast Candida albicans. 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, we investigated the importance of Mcm1 for MDR1 expression. We found that Mcm1 was dispensable for MDR1 upregulation by H2O2 but was required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and in the 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 for the coregulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induce MDR1 expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.

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Figures

Fig. 1.
Fig. 1.
Depletion of Mcm1 from reporter strains by treatment with doxycycline. Strains can42MPG2A and -B were grown in the absence (−) or presence (+) of doxycycline (Dox) and treated with benomyl or H2O2 as described in Materials and Methods. Whole-cell protein extracts were prepared and analyzed by Western immunoblotting with an anti-Myc antibody. The position of Myc-tagged Mcm1 is indicated; the lower band is a nonspecific cross-reacting protein. The positions of molecular mass markers are given on the right of the blot. Shown are the results for strain can42MPG2A; the same results were obtained with can42MPG2B.
Fig. 2.
Fig. 2.
Activation of the MDR1 promoter by H2O2 and benomyl in the wild-type strain, SC5314; the conditional mcm1 mutant, MRcan42; and the control strain, MRcan43. Parental strains and transformants carrying a PMDR1-GFP reporter fusion were grown in the absence (−) or presence (+) of doxycycline (Dox) and treated with H2O2 or benomyl (Ben) as described in Materials and Methods. The mean fluorescence of the cells was determined by flow cytometry. The results obtained with two independently generated reporter strains (SCMPG2A and -B, can42MPG2A and -B, or can43MPG2A and -B) are shown in each case. The background fluorescence of the parental strains, which do not contain the GFP gene, is indicated by the black part of each bar.
Fig. 3.
Fig. 3.
Effect of Mcm1 depletion on MDR1 overexpression mediated by hyperactive forms of Cap1 and Mrr1. The wild-type strain, SC5314; the conditional mcm1 mutant, MRcan42; the control strain, MRcan43; and independent derivatives (A and B) of MRcan42 and MRcan43 that were rendered homozygous for the CAP1ΔC333 or the MRR1P683S allele were grown for 4 h in the absence (light-gray bars) or presence (dark-gray bars) of doxycycline as described in Materials and Methods. MDR1 mRNA levels were determined by real-time RT-PCR and are presented as relative expression levels compared to those of the reference strain, SC5314, in the absence of doxycycline, which were set to 1. The graph shows the means and standard deviations of two independent experiments, with duplicate measurements performed with each strain. *, MDR1 expression levels in SC5314 and the parental strains MRcan42 and MRcan43 were too low to be visible in the graph.
Fig. 4.
Fig. 4.
Model of the roles of the transcription factors Cap1, Mrr1, and Mcm1 in MDR1 upregulation by inducing chemicals or gain-of-function mutations in Cap1 and Mrr1. The thinner bent arrows indicate reduced MDR1 promoter activity in the absence of the missing transcription factor. (A) H2O2 activates Cap1, resulting in accumulation of the transcription factor in the nucleus, where it can induce MDR1 expression, together with Mrr1, in an Mcm1-independent fashion. (B) Benomyl activates Mrr1 in an unknown way and also, at least partially, Cap1. When Cap1 is available at the MDR1 promoter, Mrr1 and Cap1 can induce MDR1 expression independently of Mcm1 (a); in the absence of Cap1, Mrr1 requires Mcm1 to induce MDR1 expression (b). (C) A hyperactive form of Cap1 (labeled Cap1*) can induce the MDR1 promoter in the absence of inducing stimuli and independently of Mcm1 and Mrr1 (a), but full induction requires the presence of Mrr1 (b). (D) A hyperactive Mrr1 containing a gain-of-function mutation (labeled Mrr1*) can induce the MDR1 promoter independently of Cap1 (which is localized in the cytoplasm in the absence of inducing stimuli) but requires the coregulator Mcm1.
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