The ERG6 gene is crucial for the biosynthesis of ergosterol, a key component of yeast cell membranes. Our study examines the impact of ERG6 gene deletion on the membrane composition and physicochemical properties of the pathogenic yeast Candida glabrata. Specifically, we investigated changes in selected sterol content, phospholipid composition, transmembrane potential, and PDR16 gene activity. Sterol levels were measured using high-performance liquid chromatography, the phospholipid profile was analysed via thin-layer chromatography, transmembrane potential was assessed with fluorescence spectroscopy, and gene expression levels were determined by quantitative PCR. Our findings revealed a depletion of ergosterol, increased zymosterol and eburicol content, an increased phosphatidylcholine and a reduced phosphatidylethanolamine content in the Δerg6 strain compared to the wt. Additionally, the Δerg6 strain exhibited membrane hyperpolarization without changes in PDR16 expression. Furthermore, the Δerg6 strain showed increased sensitivity to the antifungals myriocin and aureobasidine A. These results suggest that ERG6 gene deletion leads to significant alterations in membrane composition and may activates an alternative ergosterol synthesis pathway in the C. glabrata Δerg6 deletion mutant.
- Keywords
- Candida glabrata, ERG6, Eburicol, Ergosterol, Phospholipids, Transmembrane potential,
- MeSH
- Antifungal Agents pharmacology MeSH
- Cell Membrane * metabolism chemistry drug effects MeSH
- Candida glabrata * genetics metabolism drug effects cytology MeSH
- Gene Deletion * MeSH
- Ergosterol metabolism biosynthesis MeSH
- Phospholipids metabolism MeSH
- Fungal Proteins * genetics metabolism MeSH
- Membrane Potentials drug effects MeSH
- Gene Expression Regulation, Fungal MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Antifungal Agents MeSH
- Ergosterol MeSH
- Phospholipids MeSH
- Fungal Proteins * MeSH
The non-conventional yeast Kluyveromyces marxianus has recently emerged as a promising candidate for many food, environment, and biotechnology applications. This yeast is thermotolerant and has robust growth under many adverse conditions. Here, we show that its ability to grow under potassium-limiting conditions is much better than that of Saccharomyces cerevisiae, suggesting a very efficient and high-affinity potassium uptake system(s) in this species. The K. marxianus genome contains two genes for putative potassium transporters: KmHAK1 and KmTRK1. To characterize the products of the two genes, we constructed single and double knock-out mutants in K. marxianus and also expressed both genes in an S. cerevisiae mutant, that lacks potassium importers. Our results in K. marxianus and S. cerevisiae revealed that both genes encode efficient high-affinity potassium transporters, contributing to potassium homeostasis and maintaining plasma-membrane potential and cytosolic pH. In K. marxianus, the presence of HAK1 supports growth at low K+ much better than that of TRK1, probably because the substrate affinity of KmHak1 is about 10-fold higher than that of KmTrk1, and its expression is induced ~80-fold upon potassium starvation. KmHak1 is crucial for salt stress survival in both K. marxianus and S. cerevisiae. In co-expression experiments with ScTrk1 and ScTrk2, its robustness contributes to an increased tolerance of S. cerevisiae cells to sodium and lithium salt stress.
- Keywords
- Kluyveromyces marxianu, K+–H+ symporter, affinity, potassium, transporter, uniporter,
- MeSH
- Potassium * metabolism MeSH
- Fungal Proteins genetics metabolism MeSH
- Gene Knockout Techniques MeSH
- Kluyveromyces * genetics metabolism growth & development MeSH
- Hydrogen-Ion Concentration MeSH
- Membrane Potentials MeSH
- Cation Transport Proteins * genetics metabolism MeSH
- Gene Expression Regulation, Fungal MeSH
- Saccharomyces cerevisiae * genetics metabolism growth & development MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Potassium * MeSH
- Fungal Proteins MeSH
- Cation Transport Proteins * MeSH
Yeasts need a high intracellular concentration of potassium to grow. The main K+ uptake system in Saccharomyces cerevisiae is the Trk1 transporter, a complex protein with four MPM helical membrane motifs. Trk1 has been shown to exist in low- or high-affinity modes, which reflect the availability of potassium in the environment. However, when and how the affinity changes, and whether the potassium availability is the only signal for the affinity switch, remains unknown. Here, we characterize the Trk1 kinetic parameters under various conditions and find that Trk1's KT and Vmax change gradually. This gliding adjustment is rapid and precisely reflects the changes in the intracellular potassium content and membrane potential. A detailed characterization of the specific mutations in the P-helices of the MPM segments reveals that the presence of proline in the P-helix of the second and third MPM domain (F820P and L949P) does not affect the function of Trk1 in general, but rather specifically prevents the transporter's transition to a high-affinity state. The analogous mutations in the two remaining MPM domains (L81P and L1115P) result in a mislocalized and inactive protein, highlighting the importance of the first and fourth P-helices in proper Trk1 folding and activity at the plasma membrane.
- Keywords
- Saccharomyces cerevisiae, cation homeostasis, membrane potential, potassium uptake,
- Publication type
- Journal Article MeSH
There are only a few antifungal drugs used systemically in treatment, and invasive fungal infections that are resistant to these drugs are an emerging problem in health care. In this study, we performed a high-copy-number genomic DNA (gDNA) library screening to find and characterize genes that reduce susceptibility to amphotericin B, caspofungin, and voriconazole in Saccharomyces cerevisiae We identified the PDR16 and PMP3 genes for amphotericin B, the RMD9 and SWH1 genes for caspofungin, and the MRS3 and TRI1 genes for voriconazole. The deletion mutants for PDR16 and PMP3 were drug susceptible, but the other mutants had no apparent susceptibility. Quantitative-PCR analyses suggested that the corresponding drugs upregulated expression of the PDR16, PMP3, SWH1, and MRS3 genes. To further characterize these genes, we also profiled the global expression patterns of the cells after treatment with the antifungals and determined the genes and paths that were up- or downregulated. We also cloned Candida albicans homologs of the PDR16, PMP3, MRS3, and TRI1 genes and expressed them in S. cerevisiae Heterologous expression of Candida homologs also provided reduced drug susceptibility to the budding yeast cells. Our analyses suggest the involvement of new genes in antifungal drug resistance.
- Keywords
- amphotericin B, antifungal agents, caspofungin, drug resistance, genomics, multidrug resistance, voriconazole,
- MeSH
- Amphotericin B pharmacology MeSH
- Antifungal Agents pharmacology MeSH
- Candida albicans drug effects genetics metabolism MeSH
- Drug Resistance, Fungal genetics MeSH
- Caspofungin pharmacology MeSH
- Microbial Sensitivity Tests MeSH
- Saccharomyces cerevisiae Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae drug effects genetics metabolism MeSH
- Saccharomycetales drug effects genetics metabolism MeSH
- Voriconazole pharmacology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Amphotericin B MeSH
- Antifungal Agents MeSH
- Caspofungin MeSH
- Saccharomyces cerevisiae Proteins MeSH
- Voriconazole MeSH
Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.
- Keywords
- Candida glabrata, Membrane potential, Potassium transport, Saccharomyces cerevisiae, Salt tolerance, Trk1,
- MeSH
- Cell Membrane metabolism MeSH
- Candida glabrata genetics metabolism MeSH
- Potassium metabolism MeSH
- Fungal Proteins genetics metabolism MeSH
- Homeostasis MeSH
- Hydrogen-Ion Concentration MeSH
- Cation Transport Proteins genetics metabolism MeSH
- Gene Expression Regulation, Fungal MeSH
- Sodium metabolism MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Potassium MeSH
- Fungal Proteins MeSH
- Cation Transport Proteins MeSH
- Sodium MeSH
Saccharomyces species, which are mostly used in the food and beverage industries, are known to differ in their fermentation efficiency and tolerance of adverse fermentation conditions. However, the basis of their difference has not been fully elucidated, although their genomes have been sequenced and analyzed. Five strains of four Saccharomyces species (S. cerevisiae, S. kudriavzevii, S. bayanus, and S. paradoxus), when grown in parallel in laboratory conditions, exhibit very similar basic physiological parameters such as membrane potential, intracellular pH, and the degree to which they are able to quickly activate their Pma1 H+-ATPase upon glucose addition. On the other hand, they differ in their ability to proliferate in media with a very low concentration of potassium, in their osmotolerance and tolerance to toxic cations and cationic drugs in a growth-medium specific manner, and in their capacity to survive anhydrobiosis. Overall, S. cerevisiae (T73 more than FL100) and S. paradoxus are the most robust, and S. kudriavzevii the most sensitive species. Our results suggest that the difference in stress survival is based on their ability to quickly accommodate their cell size and metabolism to changing environmental conditions and to adjust their portfolio of available detoxifying transporters.
- Keywords
- Intracellular pH, Membrane potential, Saccharomyces, Stress tolerance,
- MeSH
- Fermentation MeSH
- Fungal Proteins genetics metabolism MeSH
- Stress, Physiological MeSH
- Glucose metabolism MeSH
- Proton-Translocating ATPases genetics metabolism MeSH
- Saccharomyces classification genetics growth & development physiology MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
- Names of Substances
- Fungal Proteins MeSH
- Glucose MeSH
- Proton-Translocating ATPases MeSH
The maintenance of potassium homeostasis is crucial for all types of cells, including Candida glabrata. Three types of plasma-membrane systems mediating potassium influx with different transport mechanisms have been described in yeasts: the Trk1 uniporter, the Hak cation-proton symporter and the Acu ATPase. The C. glabrata genome contains only one gene encoding putative system for potassium uptake, the Trk1 uniporter. Therefore, its importance in maintaining adequate levels of intracellular potassium appears to be critical for C. glabrata cells. In this study, we first confirmed the potassium-uptake activity of the identified gene's product by heterologous expression in a suitable S. cerevisiae mutant, further we generated a corresponding deletion mutant in C. glabrata and analysed its phenotype in detail. The obtained results show a pleiotropic effect on the cell physiology when CgTRK1 is deleted, affecting not only the ability of trk1Δ to grow at low potassium concentrations, but also the tolerance to toxic alkali-metal cations and cationic drugs, as well as the membrane potential and intracellular pH. Taken together, our results find the sole potassium uptake system in C. glabrata cells to be a promising target in the search for its specific inhibitors and in developing new antifungal drugs.
- MeSH
- Cell Membrane metabolism MeSH
- Candida glabrata metabolism physiology MeSH
- Potassium metabolism MeSH
- Homeostasis physiology MeSH
- Ion Transport physiology MeSH
- Cations metabolism MeSH
- Membrane Potentials physiology MeSH
- Cation Transport Proteins metabolism MeSH
- Gene Expression Regulation, Fungal physiology MeSH
- Saccharomyces cerevisiae metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Potassium MeSH
- Cations MeSH
- Cation Transport Proteins MeSH
- Trk1 protein, Candida albicans MeSH Browser
