UPC2 gene deletion modifies sterol homeostasis and susceptibility to metabolic inhibitors in Kluyveromyces lactis.

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.

The Absence of PDR16 Gene Restricts the Overexpression of CaSNQ2 Gene in the Presence of Fluconazole in Candida albicans.

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.

Stb5p is involved in Kluyveromyces lactis response to 4-nitroquinoline-N-oxide stress.

In yeast, the STB5 gene encodes a transcriptional factor belonging to binuclear cluster class (Zn2Cys6) 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.

Erg6 gene is essential for stress adaptation in Kluyveromyces 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.