Insight into the Kluyveromyces lactis Pdr1p regulon.

The overexpression of efflux pumps is an important mechanism leading to the development of multidrug resistance phenomenon. The transcription factor KlPdr1p, belonging to the Zn2Cys6 family, is a central regulator of efflux pump expression in Kluyveromyces lactis. To better understand how KlPDR1-mediated drug resistance is achieved in K. lactis, we used DNA microarrays to identify genes whose expression was affected by deletion or overexpression of the KlPDR1 gene. Eighty-nine targets of the KlPDR1 were identified. From those the transcription of 16 genes was induced in the transformant overexpressing KlPDR1* and simultaneously repressed in the Klpdr1Δ deletion mutant. Almost all of these genes contain putative binding motifs for the AP-1-like transcription factors in their promoters. Furthermore, we studied the possible interplay between KlPdr1p and KlYap1p transcription factors. Our results show that KlYap1p does not significantly contribute to the regulation of KlPDR1 gene expression in the presence of azoles. However, KlPDR1 expression markedly increased in the presence of hydrogen peroxide and hinged upon the presence of KlYap1p. Our results show that although both KlPdr1p and KlYap1p transcription factors are involved in the control of K. lactis multidrug resistance, further studies will be needed to determine their interplay.

Isolation and functional analysis of the KlPDR16 gene.

The fight against multidrug-resistant pathogens requires an understanding of the underlying cellular mechanisms. In this work, we isolate and characterize one of the multidrug resistance determinants in Kluyveromyces lactis, the KlPDR16 gene. We show that KlPdr16p (345 aa), which belongs to the KlPdr1p regulon, is a functional homologue of the Saccharomyces cerevisiae Pdr16p. Deletion of KlPDR16 resulted in hypersensitivity of K. lactis cells to antifungal azoles, oligomycin, rhodamine 6G, 4-nitroquinoline-N-oxide and alkali metal cations. The Klpdr16∆ mutation led to a decreased content of ergosterol in whole-cell extract. In spite of the hypersensitivity of Klpdr16∆ mutant cells to rhodamine 6G and oligomycin, the transcript level of the KlPDR5 gene and the rhodamine 6G efflux in the mutant was the same as in the parental strain. Increased accumulation of rhodamine 6G in Klpdr16∆ cells indicates that KlPDR16 limits the rate of passive drug diffusion across the membrane, without affecting the glucose-induced drug export. The results obtained show that KlPDR16, similar to its orthologues in other yeast species, influences the passive drug diffusion into the yeast cell.

Gain-of-function mutation in the KlPDR1 gene encoding multidrug resistance regulator in Kluyveromyces lactis.

KlPdr1p is a single Kluyveromyces lactis homologue of Saccharomyces cerevisiae ScPdr1p/ScPdr3p, the main transcriptional regulators of genes involved in S. cerevisiae multidrug resistance. KlPDR1 deletion leads to a sharp increase in K. lactis drug susceptibility. The presence of putative PDRE and YRE regulatory elements in the KlPDR1 gene promoter suggests an autoregulation of its transcription as well as its control by KlYap1p, the transcription factor involved in oxidative stress response. In this study, one plasmid-borne Klpdr1-1 allele that led to amino acid substitution (L273P) in the KlPdr1p was isolated. Overexpression of the Klpdr1-1 allele from a multicopy plasmid in the K. lactis wild-type and Klpdr1Δ mutant strain increased the tolerance of transformants to oligomycin. The plasmid-borne Klpdr1-1 allele increased the activation of the ScPDR5 promoter and complemented the drug hypersensitivity of the S. cerevisiae pdr1Δ pdr3Δ mutant strain. The results indicate that L273P amino acid substitution is the result of a gain-of-function mutation in the KlPDR1 gene that confers KlPdr1p hyperactivity, as revealed by a high expression of the ABC transporter gene KlPDR5, leading to multidrug resistance and rhodamine 6G efflux out of the cells.

Autoactivated KlPDR1 gene in the control of multidrug resistance in Kluyveromyces lactis.

The KlPDR1 gene encodes a zinc finger transcription factor that has recently been shown to be involved in the control of multidrug resistance of Kluyveromyces lactis . In this work, we provide evidence that the K. lactis KlPDR1 gene is under positive autoregulation by KlPdr1p, which plays a role in the activation of the main multidrug resistance transporter gene KlPDR5. Electrophoretic mobility shift assays, as well as the use of gusA reporter constructs, enabled us to identify the 5'-tataTCCGGGTAactt-3' sequence motif in the KlPDR1 promoter (in the position -326 to -319 bp) as the PDRE (pleiotropic drug responsive element) for the binding of KlPdr1p. The drug sensitivity of Klpdr1Δ mutant cells was complemented by introducing the plasmid-born KlPDR1 gene. The KlPdr1p activated the expression of the P(KlPDR1)-gusA fusion gene, and the expression of the KlPDR1 gene was induced by fluconazole. The PDRE was also found in the promoter of KlPDR5, a gene encoding the ATP-dependent efflux pump responsible for the drug resistance phenomenon in K. lactis.

Interplay among regulators of multidrug resistance in Kluyveromyces lactis.

The KlYAP1 and KlPDR1 genes encode two main transcriptional regulators involved in the control of multidrug resistance in Kluyveromyces lactis. Deletion of KlPDR1 or KlYAP1 genes in K. lactis generated strain hypersusceptible to diamide, benomyl, fluconazole and oligomycin. Overexpression of genes KlPDR1 or KlYAP1 from a multicopy plasmid in the Klpdr1Δ mutant strain increased the tolerance of transformants to all the drugs tested. YRE response elements were found in the promoter of the KlPDR1 gene. Gel retardation assays confirmed the binding of KlYap1p to the YREs in the KlPDR1 gene promoter indicating that KlYap1p can control the KlPDR1 gene expression.