Essential Role of CgErg6p in Maintaining Oxidative Stress Tolerance and Iron Homeostasis in Candida glabrata.

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, CgYap1p, CgMsn4p and CgYap5p, 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.

Catechin potentiates the antifungal effect of miconazole in Candida glabrata.

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.

Erg6p is essential for antifungal drug resistance, plasma membrane properties and cell wall integrity in Candida glabrata.

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 UPC2 gene in Kluyveromyces lactis stress adaptation.

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.

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.

Differences in the arrangement of the Pdr5p multidrug transporter binding pocket of Saccharomyces cerevisiae and Kluyveromyces lactis.

Multidrug transporters are often responsible for failure of medical treatment, since they expel a variety of structurally and functionally unrelated drugs out of the cell. We found that the fluorescent probe diS-C3(3) is a substrate of not only Pdr5p of Saccharomyces cerevisiae (ScPdr5p) but also of its less-explored Kluyveromyces lactis homologue (KlPdr5p). This enabled us to compare the ability of azoles to competitively inhibit the Pdr5p-mediated probe efflux in the two species. In K. lactis, these azoles completely inhibit probe transport by KlPdr5p and also compete with each other for transport. This indicates that the probe and the azoles are bound by the same site(s) of the KlPdr5p binding pocket. On the other hand, the azoles' capacity to inhibit the probe transport by ScPdr5p is limited, as a result of their partial cotransport with the probe. While the azoles bind to only one or two separate binding sites, the probe is able to bind to all three of them. Moreover, the bulky ScPdr5p substrate enniatin B, which effectively inhibits both probe and azole transport by the pump, has negligible effect on KlPdr5p. Our data point to a tighter arrangement of the KlPdr5p binding pocket compared to that of ScPdr5p.

Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species.

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p 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 KlPdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.

Sterol Analysis in Kluyveromyces lactis.

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.

ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors.

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.

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.

The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

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.

Stress response and expression of fluconazole resistance associated genes in the pathogenic yeast Candida glabrata deleted in the CgPDR16 gene.

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.

Deletion of the PDR16 gene influences the plasma membrane properties of the yeast Kluyveromyces 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.

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.

Mutation of the CgPDR16 gene attenuates azole tolerance and biofilm production in pathogenic Candida glabrata.

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.

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.

Antibacterial activity of CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) generating reactive oxygen species.

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.