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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.

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.

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 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.