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The rising number of invasive fungal infections caused by drug-resistant Candida strains is one of the greatest challenges for the development of novel antifungal strategies. The scarcity of available antifungals has drawn attention to the potential of natural products as antifungals and in combinational therapies. One of these is catechins-polyphenolic compounds-flavanols, found in a variety of plants. In this work, we evaluated the changes in the susceptibility of Candida glabrata strain characterized at the laboratory level and clinical isolates using the combination of catechin and antifungal azoles. Catechin alone had no antifungal activity within the concentration range tested. Its use in combination with miconazole resulted in complete inhibition of growth in the sensitive C. glabrata isolate and a significant growth reduction in the azole resistant C. glabrata clinical isolate. Simultaneous use of catechin and miconazole leads to increased intracellular ROS generation. The enhanced susceptibility of C. glabrata clinical isolates to miconazole by catechin was accompanied with the intracellular accumulation of ROS and changes in the plasma membrane permeability, as measured using fluorescence anisotropy, affecting the function of plasma membrane proteins., (© 2023. The Author(s).)

The human pathogenic fungus Candida glabrata is the second leading cause of candidemia, a life-threatening invasive mycosis. Clinical outcomes are complicated by reduced susceptibility of C. glabrata to azoles together with its ability to evolve stable resistance to both azoles and echinocandins following drug exposure. Compared to other Candida spp., C. glabrata displays robust oxidative stress resistance. In this study, we investigated the impact of CgERG6 gene deletion on the oxidative stress response in C. glabrata. CgERG6 gene encodes sterol-24-C-methyltransferase, which is involved in the final steps of ergosterol biosynthesis. Our previous results showed that the Cgerg6Δ mutant has a lower ergosterol content in its membranes. Here, we show that the Cgerg6Δ mutant displays increased susceptibility to oxidative stress inducing agents, such as menadione, hydrogen peroxide and diamide, accompanied with increased intracellular ROS production. The Cgerg6Δ mutant is not able to tolerate higher concentrations of iron in the growth media. We observed increased expression of transcription factors, Cg Yap1p, Cg Msn4p and Cg Yap5p, together with increased expression of catalase encoding the CgCTA1 gene and vacuolar iron transporter CgCCC1 in the Cgerg6Δ mutant cells. However, it seems that the CgERG6 gene deletion does not influence the function of mitochondria.

ERG6 gene encodes C-24 methyltransferase, one of the specific enzymes that differ in mammalian and yeast sterol biosynthesis. To explore the function of CgErg6p in the yeast pathogen Candida glabrata, we have constructed the Cgerg6Δ deletion mutant. We found that C. glabrata cells lacking CgErg6p exhibit reduced susceptibility to both antifungal azoles and polyenes. The reduced content of ergosterol in the Cgerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthetic pathway. The absence of CgErg6p leads to plasma membrane hyperpolarization and decrease in its fluidity compared to the parental C. glabrata strain. The absence of sterols containing C-24 alkyls influenced the susceptibility of Cgerg6Δ mutant cells to alkali metal cations and several other metabolic inhibitors. Our results thus show that sterols lacking C-24 alkyls are not sufficient substitutes for maintaining yeast plasma membrane function. The absence of CgErg6p influences also the cell wall integrity and calcineurin signaling in C. glabrata., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

KlUpc2p, a transcription factor belonging to the fungal binuclear cluster family, is an important regulator of ergosterol biosynthesis and azole drug resistance in Kluyveromyces lactis. In this work, we show that the absence of KlUpc2p generates Rag -  phenotype and modulates the K. lactis susceptibility to oxidants and calcofuor white. The KlUPC2 deletion leads to increased expression of KlMGA2 gene, encoding an important regulator of hypoxic and lipid biosynthetic genes in K. lactis and also KlHOG1 gene. The absence of KlUpc2p does not lead to statistically significant changes in glycerol, corroborating the expression of KlGPD1 gene, encoding NAD + -dependent glycerol-3-phosphate dehydrogenase, that is similar in both the deletion mutant and the parental wild-type strain. Increased sensitivity of Klupc2 mutant cells to brefeldin A accompanied with significant increase in KlARF2 gene expression point to the involvement of KlUpc2p in intracellular signaling. Our observations highlight the connections between ergosterol and fatty acid metabolism to modulate membrane properties and point to the possible involvement of KlUpc2p in K. lactis oxidative stress response., (© 2022. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Kluyveromyces lactis Upc2p is an ortholog of Upc2p/Ecm22p transcription factors involved in regulation of sterol import and sterol homeostasis in Saccharomyces cerevisiae. In this work, we investigated the role of Upc2p in K. lactis. The absence of KlUpc2p significantly reduced the tolerance of mutant cells to antifungal azoles and Li + cations. Reduced expression of genes from the late ergosterol pathway results in a decreased ergosterol content and altered plasma membrane-associated functions in Klupc2 mutant cells-the plasma membrane is hyperpolarized, and its fluidity is reduced. KlUpc2p contributes to transcriptional upregulation of KlENA1, KlPMA1 and KlYAP1 under azole stress. Our study demonstrates that KlUpc2p is involved in the regulation of ergosterol homeostasis in K. lactis. The analysis of KlPMA1 and KlPDR12 transcripts in wild-type and Klupc2Δ mutant strains showed that KlUpc2p acts as an activator or as a repressor depending upon its target., (© 2020 John Wiley & Sons, Ltd.)

In yeast, the PDR16 gene encodes one of the PITP proteins involved in lipid metabolism and is regarded as a factor involved in clinical azole resistance of fungal pathogens. In this study, we prepared Candida albicans CaPDR16/pdr16Δ and Capdr16Δ/Δ heterozygous and homozygous mutant strains and assessed their responses to different stresses. The CaPDR16 deletion strains exhibited increased susceptibility to antifungal azoles and acetic acid. The addition of Tween80 restored the growth of Capdr16 mutants in the presence of azoles. However, the PDR16 gene deletion has not remarkable influence on sterol profile or membrane properties (membrane potential, anisotropy) of Capdr16Δ and Capdr16Δ/Δ mutant cells. Changes in halotolerance of C. albicans pdr16 deletion mutants were not observed. Fluconazole treatment leads to increased expression of ERG genes both in the wild-type and Capdr16Δ and Capdr16Δ/Δ mutant cells, and the amount of ergosterol and its precursors remain comparable in all three strains tested. Fluconazole treatment induced the expression of ATP-binding cassette transporter gene CaSNQ2 and MFS transporter gene CaTPO3 in the wild-type strain but not in the Capdr16Δ and Capdr16Δ/Δ mutants. The expression of CaSNQ2 gene markedly increased also in cells treated with hydrogen peroxide irrespective of the presence of CaPdr16p. CaPDR16 gene thus belongs to genes whose presence is required for full induction of CaSNQ2 and CaTPO3 genes in the presence of fluconazole in C. albicans.

In yeast, the STB5 gene encodes a transcriptional factor belonging to binuclear cluster class (Zn 2 Cys 6 ) of transcriptional regulators specific to ascomycetes. In this study, we prepared the Kluyveromyces lactis stb5Δ strain and assessed its responses to different stresses. We showed that KlSTB5 gene is able to complement the deficiencies of Saccharomyces cerevisiae stb5Δ mutant. The results of phenotypic analysis suggested that KlSTB5 gene deletion did not sensitize K. lactis cells to oxidative stress inducing compounds but led to Klstb5Δ resistance to 4-nitroquinoline-N-oxide and hygromycin B. Expression analysis indicated that the loss of KlSTB5 gene function induced the transcription of drug efflux pump encoding genes that might contribute to increased 4-nitroquinoline-N-oxide and hygromycin B tolerance. Our results show that KlStb5p functions as negative regulator of some ABC transporter genes in K. lactis.

We investigated the effect of Kluyveromyces lactis ERG6 gene deletion on plasma membrane function and showed increased susceptibility of mutant cells to salt stress, cationic drugs and weak organic acids. Contrary to Saccharomyces cerevisiae, Klerg6 mutant cells exhibited increased tolerance to tunicamycin. The content of cell wall polysacharides did not significantly vary between wild-type and mutant cells. Although the expression of the NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGPD1) in the Klerg6 mutant cells was only half of that in the parental strain, it was induced in the presence of calcofluor white. Also, cells exposed to this drug accumulated glycerol. The absence of KlErg6p led to plasma membrane hyperpolarization but had no statistically significant influence on the plasma membrane fluidity. We propose that the phenotype of Klerg6 mutant cells to a large extent was a result of the reduced activity of specific plasma membrane proteins that require proper lipid composition for full activity.

Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter Kl Pdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. Kl Pdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool ( Kolaczkowski et al. , 1996 ). An active Kl Pdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)

The ERG6 gene encodes an S-adenosylmethionine dependent sterol C-24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation in both K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to the Sec14 family. Decreased activity of KlPdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The overexpression of efflux pumps is an important mechanism leading to the development of multidrug resistance phenomenon. The transcription factor KlPdr1p, belonging to the Zn 2 Cys 6 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.

Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

In yeasts, the PDR16 gene encodes a phosphatidylinositol transfer protein which belongs to the Sec14 homologue (SFH) family and localizes to lipid droplets, microsomes and at the cell periphery. The loss of its function alters the lipid droplet metabolism and plasma membrane properties, and renders yeast cells more sensitive to azole antimycotics. In this study, the entire chromosomal CgPDR16 ORF was replaced by the ScURA3 gene both in azole sensitive and azole resistant strains of Candida glabrata bearing a gain-of-function mutation in the CgPDR1 gene, and their responses to different stresses were assessed. The CgPDR16 deletion was found to sensitize the mutant strains to azole antifungals without changes in their osmo- and halotolerance. Fluconazole treated pdr16Δ mutant strains displayed a reduced expression of several genes involved in azole tolerance. The gain-of-function CgPDR1 allele as well as the cycloheximide and hydrogen peroxide treatments of cells enhanced the expression of the CgPDR16 gene. The results indicate that CgPDR16 belongs to genes whose expression is induced by chemical and oxidative stresses. The loss of its function can attenuate the expression of drug efflux pump encoding genes that might also contribute to the decreased azole tolerance in pdr16Δ mutant cells., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The plasma membrane is the first line of cell defense against changes in external environment, thus its integrity and functionality are of utmost importance. The plasma membrane properties depend on both its protein and lipid composition. The PDR16 gene is involved in the control of Kluyveromyces lactis susceptibility to drugs and alkali metal cations. It encodes the homologue of the major K. lactis phosphatidylinositol transfer protein Sec14p. Sec14p participates in protein secretion, regulation of lipid synthesis, and turnover in vivo. We report here that the plasma membrane of the Klpdr16Δ mutant is hyperpolarized and its fluidity is lower than that of the parental strain. In addition, protoplasts prepared from the Klpdr16Δ cells display decreased stability when subjected to hypo-osmotic conditions. These changes in membrane properties lead to an accumulation of radiolabeled fluconazole and lithium cations inside mutant cells. Our results point to the fact that the PDR16 gene of K. lactis (KlPDR16) influences the plasma membrane properties in K. lactis that lead to subsequent changes in susceptibility to a broad range of xenobiotics.

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., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The PDR16 gene encodes the homologue of Sec14p, participating in protein secretion, regulation of lipid synthesis and turnover in vivo and acting as a phosphatidylinositol transfer protein in vitro. This gene is also involved in the regulation of multidrug resistance in Saccharomyces cerevisiae and pathogenic yeasts. Here we report the results of functional analysis of the CgPDR16 gene, whose mutation has been previously shown to enhance fluconazole sensitivity in Candida glabrata mutant cells. We have cloned the CgPDR16 gene, which was able to complement the pdr16Δ mutation in both C. glabrata and S. cerevisiae. Along with fluconazole, the pdr16Δ mutation resulted in increased susceptibility of mutant cells to several azole antifungals without changes in sensitivity to polyene antibiotics, cycloheximide, NQO, 5-fluorocytosine and oxidants inducing the intracellular formation of reactive oxygen species. The susceptibility of the pdr16Δ mutant strain to itraconazole and 5-fluorocytosine was enhanced by CTBT [7-chlorotetrazolo(5,1-c)benzo(1,2,4)triazine] inducing oxidative stress. The pdr16Δ mutation increased the accumulation of rhodamine 6G in mutant cells, decreased the level of itraconazole resistance caused by gain-of-function mutations in the CgPDR1 gene, and reduced cell surface hydrophobicity and biofilm production. These results point to the pleiotropic phenotype of the pdr16Δ mutant and support the role of the CgPDR16 gene in the control of drug susceptibility and virulence in the pathogenic C. glabrata., (Copyright © 2013 John Wiley & Sons, Ltd.)

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) is an antifungal and chemosensitizing agent that induces oxidative stress in yeast and filamentous fungi and enhances the cytotoxic activity of 5-fluorocytosine and azole antimycotics. This study reports the effect of CTBT on bacterial cells. CTBT inhibited the growth of both Gram-positive and Gram-negative bacterial species. The action of CTBT was bactericidal. In Escherichia coli, CTBT induced an increased formation of reactive oxygen species (ROS), as determined with a ROS specific probe 2',7'-dichlorodihydrofluorescein diacetate. In zone inhibition assays, bacterial cells were more sensitive to CTBT compared with paraquat, menadione and hydrogen peroxide. The deletion of oxidative stress related genes resulted in increased susceptibility of E. coli mutant strains to CTBT treatment. Exogenous antioxidants such as ascorbic acid, cysteine and glutathione exhibited a protective effect against the growth inhibition induced by CTBT. CTBT may be a useful tool in the studies of ROS generation, oxidant sensing and oxidative stress response in different bacterial species., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2013 John Wiley & Sons, Ltd.)

7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) is an antifungal agent that induces oxidative stress and enhances the activity of other antifungals with different modes of action. A genome-wide screening of Saccharomyces cerevisiae genomic library in the high-copy-number plasmid revealed three genes, YAP1, PDE2, and STB3, which increased the CTBT tolerance of the parental strain. The YAP1 gene is known to activate many genes in response to oxidants. The PDE2 and STB3 genes encode the high-affinity cAMP phosphodiesterase and the transcription factor recognizing the ribosomal RNA processing element in promoter sequences, respectively. The protective effects of their overexpression against CTBT toxicity was observed in the absence of certain proteins involved in stress responses, cell wall integrity signaling, and chromatin remodeling. The enhanced CTBT tolerance of the YAP1, PDE2, and STB3 transformants was a consequence of their high antioxidant enzyme activities at the beginning of CTBT treatment in comparison with that of the parental strain, for that they inactivated the CTBT-induced reactive oxygen species. These results point to the complex interplay among the oxidant sensing, cAMP-protein kinase A signaling, and transcription reprogramming of yeast cells, leading to their better adaptation to the stress imposed by CTBT., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Multidrug resistance (MDR), a ubiquitous phenomenon conserved from bacteria to humans, causes serious problems in the treatment of human cancers and infections of bacterial and fungal origin. The development of MDR in yeast is frequently associated with gain-of-function mutations in the Zn(2)Cys(6) transcription factors activating the expression of several plasma membrane exporters. In the aerobic yeast Kluyveromyces lactis the Zn(2)Cys(6) transcription factor KlPdr1p is involved in the control of multidrug resistance. The aim of the present study was to identify the changes in K. lactis proteome of the Klpdr1Δ deletion mutant compared with the wild-type expressing the KlPDR1 gene from a multicopy plasmid. A total of 15 differentially expressed proteins, out of 20 spots with different intensities detected, were identified. In the Klpdr1Δ deletion mutant, the increase in the abundance of proteins involved in carbohydrate metabolism (mainly glycolysis/gluconeogenesis) was observed. Most of the proteins overexpressed in the wild type strain containing the KlPDR1 gene on multicopy plasmid were involved in the stress defence and redox homeostasis. The results indicate a close connection between MDR and oxidative stress response associated with the post-translational mechanisms regulating the levels of active forms of proteins involved in K. lactis MDR., (Copyright © 2012 Elsevier B.V. All rights reserved.)

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, and C. glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2',7'-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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.

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.

In Saccharomyces cerevisiae, the simultaneous resistance to various cytotoxic compounds known as multidrug resistance (MDR) is caused by overexpression of membrane efflux pumps under the control of two main transcriptional activators Pdr1p and Pdr3p. In this work we describe the results of functional analysis of a single Kluyveromyces lactis homolog of the PDR1 gene, which encodes a zinc finger Zn(2)Cys(6)-containing transcription factor. The KlPDR1 deletion generated a strain hypersusceptible to oligomycin, antimycin A and azole antifungals. Overexpression of KlPDR1 from a multicopy plasmid in the Klpdr1Delta mutant strain increased the tolerance of transformants to all the drugs tested (oligomycin, antimycin A and azole antifungals). The plasmid-borne KlPDR1 gene was able to complement drug hypersensitivity of the S. cerevisiae pdr1Deltapdr3Delta mutant strain. The KlPDR1 was found to be necessary for upregulation of the ATP-binding cassette transporter encoded by the KlPDR5 gene and rhodamine 6G efflux out of the cells. The KlPDR5 and some other K. lactis pleiotropic drug resistance (PDR) orthologues were found to contain putative PDR-responsive elements in their promoters. These results demonstrate that KlPdr1p is involved in K. lactis MDR and is required for cell's tolerance to various cytotoxic compounds.

The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs ("phylogenetic footprints"). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.

The b-Zip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the KNQ1 gene, encoding a multidrug transporter of the major facilitator superfamily in Kluyveromyces lactis and containing two potential Yap1p response elements in its promoter, is a putative transcriptional target of KlYap1p, the structural and functional homologue of ScYap1p. In this work, we provide evidence that KlYAP1 controls the expression of the KNQ1 gene. Using a P(KNQ1)-gusA fusion construct we showed that the expression of KNQ1 is induced upon cell treatment with the oxidizing agents H2O2 and menadione and that this induction is mediated by KlYap1p. These results were confirmed by Northern-blot analysis showing that the expression of KNQ1 is responsive to hydrogen peroxide and dependent on the presence of KlYap1p. The role of KlYAP1 in the control of KNQ1 expression was further demonstrated by EMSA experiments and drug resistance assays. These results clearly demonstrate the involvement of the KlYap1p transcription factor in the control of KNQ1 gene expression.

Several transport systems play an important role in conferring multiple drug resistance, presumably due to their catalysis of the energy-dependent extrusion of a large number of structurally and functionally unrelated compounds out of the cells. In the present work, the gene named KNQ1 (encoding Kluyveromyces lactis membrane permease) was cloned by functional complementation of the cycloheximide-hypersensitivity phenotype of the Saccharomyces cerevisiae mutant strain lacking a functional PDR5 gene. The isolated gene exhibited 48.9% identity with the S. cerevisiae ATR1 gene conferring resistance to aminotriazole and 4-nitroquinoline- N-oxide and encoded a protein of 553 amino acids. When present in multicopy, it efficiently complemented the phenotype associated with the Delta pdr5 or Delta pdr1Delta pdr3 mutations in S. cerevisiae. Overexpression of the KNQ1 gene in K. lactis wild-type strains led to resistance against several cytotoxic compounds, like 4-nitroquinoline- N-oxide, 3-aminotriazole, bifonazole and ketoconazole. The gene was assigned to K. lactis chromosome III and its expression was found to be responsive to oxidative stress induced by hydrogen peroxide. Based on the phenotype of homologous and heterologous transformants, we propose that the gene encodes a membrane-associated component of the machinery responsible for decreasing the concentration of several toxic compounds in the cytoplasm of yeast cells.

By virtue of heterologous functional complementation of the Saccharomyces cerevisiae Delta pdr5 mutant strain, using a Kluyveromyces lactis genomic library, three different K. lactis chromosomal inserts were obtained. Transformation of the S. cerevisiae Delta pdr1 Delta pdr3 mutant strain, hypersensitive to drugs, with isolated plasmids resulted in resistance to cycloheximide and fluconazole. Transformation of K. lactis host strains, using the cloned chromosomal fragments, led to an increased level of resistance to some mitochondrial inhibitors and azole antifungals. The nucleotide sequence of the cloned inserts revealed that two of them contain the drug efflux transporter gene Kl-PDR5 and the third contains a DNA segment homologous to chromosome VII of S. cerevisiae. Along with three novel ORFs, encoding two proteins of unknown molecular function and one putative hexose transporter, this segment also contained the Kl-RPL28 gene, found to be responsible for the cycloheximide resistance of heterologous transformants. This gene codes for the large subunit ribosomal protein (149 amino acids) that shares 89.9% identity with its S. cerevisiae counterpart. The coding region of Kl-RPL28 was found to be interrupted with one intron near the 5' end. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the accession number AF493565.