PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans

Abstract

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates ofCandida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 39 untranslated region (39 UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 39 UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 39 UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was~35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-a, were recovered from AS (MTL-a/MTL-a), while a single type of PAP1 allele (PAP1-a) was recovered from AR isolates (MTL-a/MTL-a). Among the heterozygous deletions of PAP1-a (Dpap1-a/PAP1-a) and PAP1-a (PAP1-a/Dpap1-a), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

The ABCs of Candida albicans Multidrug Transporter Cdr1

“…In another study, it was found that poly(A) polymerase homozygosity and hyperadenylation are responsible for the increased stability of CDR1 mRNA (71). However, the half-life of Cdr1p did not change between AS and AR isolates, thus ruling out any possible role for Cdr1 protein stability in drug resistance (70).…”

Genomic Plasticity of the Human Fungal Pathogen Candida albicans

“…For example, LOH appears to play an important role in acquired azole resistance in C. albicans: homozygosis of the hyperactive alleles of ERG11, TAC1, or MRR1 (which encode the target of azole drugs and transcription factors that upregulate drug efflux pumps) is associated with increased azole resistance (39,50,204). Homozygosis of the PAP1 allele within the MTL locus also confers different levels of resistance to drugs (88,121). Similarly, resistance to the antifungal 5-flucytosine (5-FC) was associated with homozygosis of a hyperactive FUR1 allele (45).…”

Antifungal activity of linalool in cases of Candida spp. isolated from individuals with oral candidiasis

“…to antifungal drugs is related to their prolonged use. Among the mechanisms that contribute to this phenomenon are the overexpression or mutation of genes, such as ERG11, CDR1, CDR2 and MDR1 that encode efflux pumps (Sanglard et al, 2003;Basso Junior et al, 2010;Manoharlal et al, 2010), as well as the ERG-3 gene encoding the enzyme ∆ 5.6 sterol dessaturase, important in the synthesis of ergosterol (Sanglard et al, 2003) and alterations in the lipid composition of the fungal plasma membrane, which hinders the influx of the drug into the cell (Loffler et al, 2000). It should be pointed out that these mechanisms can occur simultaneously, contributing to increase the phenomenon of resistance.…”