Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans

Objectives: Functional characterization of the erg1 mutant of ergosterol biosynthesis of Candida albicans. Methods: We disrupted the ERG1 gene of C. albicans, which encodes squalene epoxidase (EC 1.14.99.7). Since the disruption of both alleles of ERG1 was lethal, the second allele of a heterozygous disruptant was placed under the control of a regulable promoter, MET3p, which is repressed by methionine and cysteine. Results: The reverse-phase HPLC analysis of sterol, extracted from the conditional mutant strain, showed a total lack of ergosterol and instead accumulation of squalene. This imbalance in sterol composition led to defects in growth and increased susceptibilities to drugs including fluconazole, ketoconazole, cycloheximide, nystatin, amphotericin B and terbinafine. Reduced drug efflux activity of the erg1 mutant was associated with poor surface localization of Cdr1p, suggesting that enhanced passive diffusion and reduced efflux mediated by the ABC (ATP binding cassette) transporter Cdr1p increases drug susceptibility. Additionally, conditional erg1 mutant strains were unable to form hyphae in various media. Conclusions: Taken together, our results demonstrate that the absence of ergosterol, which is one of the constituents of membrane microdomains (rafts), has a direct effect on drug susceptibility and morphogenesis of C. albicans

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSƒ9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein

Expression of CDR1, a multidrug resistance gene of Candida albicans: transcriptional activation by heat shock, drugs and human steroid hormones

We have examined the expression of CDR1 (Candida drug resistance gene) in different stress conditions. There was a significant but transient enhancement of CDR1 expression associated with elevated temperatures. Most noteworthy transcriptional activation was observed with miconazole and vinblastine. Interestingly, β-estradiol and progesterone were also able to enhance CDR1 expression. Elevated levels of CDR1 and CDR2 (a homologue of CDR1) mRNA were found in some azole-resistant clinical isolates of C. albicans. CaMDR1 (benomyl-resistant) expression, however, did not differ among all the resistant isolates. Our results confirm the existence of multiple mechanisms of azole resistance in C. albicans

A physiological role for gene loops in yeast

DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3′-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression—a phenomenon defined as “transcriptional memory”—and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast

Identification of polymorphic mutant alleles of CaMDR1, a major facilitator of Candida albicans which confers multidrug resistance, and its in vitro transcriptional activation

CaMDR1 (Candida albicans Multi Drug Resistance) encodes a major facilitator whose expression in Saccharomyces cerevisiae confers resistance to several unrelated drugs. We describe here the identification and molecular characterization of seven mutant alleles of CaMDR1 (CaMDR1-1 to 1-7). The complete sequencing of CaMDR1 alleles revealed several in-frame point mutations leading to a change in amino-acid residues where insertion/replacement of an aspartate residue in a serine-asparagine-aspartate-rich domain was most noteworthy. Interestingly, these alleles showed a distinct drug resistance profile. The expression of CaMDR1, or of its alleles, in C. albicans cells was enhanced by benomyl, methotrexate and several other unrelated drugs, and was more pronounced in at least one of the azole-resistant clinical isolates

SRE1 and SRE2 are two specific steroid-responsive modules of Candida drug resistance gene 1 (CDR1) promoter

CDR1 gene encoding an ATP-driven drug extrusion pump has been implicated in the development of azole-resistance in Candida albicans. Although the upregulation of CDR1 expression by various environmental factors has been documented, the molecular mechanism underlying such process is poorly understood. We have demonstrated earlier that the CDR1 promoter encompasses a large number of cis-regulatory elements, presumably mediating its response to various drugs. In this study we have identified a novel steroid responsive region (SRR) conferring β-oestradiol and progesterone inducibility on the CDR1 promoter. The SRR is located −696 to −521 bp upstream of the transcription start site; it is modular in nature and can confer steroid responsiveness to a heterologous promoter (ADH1) linked to a GFP reporter gene. In vitro DNase I protection analyses of SRR revealed two progesterone responsive sequences (−628 to −594 and −683 to −648) and one β-oestradiol responsive sequence (−628 to −577), which was further corroborated by the gel mobility shift assay. Deletion analyses within the SRR further delimited these steroid responsive sequences into two distinct elements, viz. SRE1 and SRE2. While SRE1 (−677 to −648) responds only to progesterone, SRE2 (−628 to −598) responded to both progesterone and β-oestradiol. Both SRE1 and SRE2 were specific for steroids, as they did not respond to other drugs, such as cycloheximide, miconazole and terbinafine. In silico comparison of the SRE½ with the promoter sequences of other MDR (CDR2 and PDR5) and non-MDR (HSP90) steroid-responsive genes revealed a similarity with respect to conservation of three 5 bp stretches (AAGAA, CCGAA and ATTGG). Taken together, we have identified a novel steroid responsive cis-regulatory sequence in the CDR1 promoter, which presumably can be instrumental in understanding the steroid response cascade in Candida albicans