Potassium Uptake Mediated by Trk1 Is Crucial for Candida glabrata Growth and Fitness

Abstract

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.

Potassium uptake systems of Candida krusei

“…To confirm the predicted potassium uptake function of both putative C. krusei Trk transporters, we cloned the corresponding ORFs into two S. cerevisiae multicopy vectors behind the NHA1 promoter and tagged their 3′ termini with the GFP sequence in one of the two plasmid series (Table ). The weak and constitutive promoter of the NHA1 gene, which encodes a potassium and sodium efflux system (the Nha1 antiporter), has been already repeatedly successfully used for the expression of heterologous Trk transporters in S. cerevisiae (e.g., TRK1 genes from Z. rouxii or C. glabrata ; Llopis‐Torregrosa, Husekova, & Sychrova, ; Stribny et al, ; Zimmermannova, Salazar, Sychrova, & Ramos, ).…”

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii

“…We first amplified CaSAT1 from YEp352‐SAT1, resulting in several specific PCR fragments (data not shown). Llopis‐Torregrosa et al () suggested that regulatory elements upstream and downstream sat1 gene were not necessary to confer ClonNAT resistance. Thus, the chimeric primer pairs were designed to include ACT1 exon 1, the subsequent intron, and the full‐length sat1 ORF (Table and Figure a).…”

“…In absence of a Z. rouxii ‐tailored CRISPR‐Cas9 system, Cre‐ loxP system is preferable over hisG and lacZ systems as marker recycling method as it may be applied in prototrophic strains and avoids a counterselection such as growth on media supplemented with 5‐fluoroorotic acid (5‐FOA) for URA3 excision (Steensma & Ter Linde, ). To construct a Z. rouxii plasmid containing cre gene and ClonNAT R as dominant marker, we cloned cre gene under the control of the inducible pScGAL1 promoter (pScGAL1 + cre) into pCg2XpH‐N (Llopis‐Torregrosa et al, ) by HR in S. cerevisiae (Figure a). First, the pScGAL1 + cre construct was PCR‐amplified from pZCRE donor plasmid (Pribylova, De Montigny, et al, ) with the chimeric primers CRE‐pGRB‐SacI‐XbaI‐F1 and CRE‐pGRB‐XhoI‐XbaI‐R1 (Figure b).…”

“…(a) pHfluorins to pScGAL1 + cre swap cassette strategy used to build the centromeric vector pGRCRE containing and ClonNAT R selectable marker. Schematic representations of pCg2XpH‐N (Llopis‐Torregrosa et al, ) and the novel pGRCRE plasmid were drawn. C. albicans ACT1 exon 1 and intron in‐frame fused with sat1 gene are depicted as brawn block ad black line, respectively, while 43‐bp‐long pCg2XpH‐N target sequences as light grey blocks (homologous recombination, HR).…”

“…To generate pGRCRE, the fragment containing the cre gene behind the S. cerevisiae GAL1 promoter (pScGAL1; abbreviated as pScGAL1 + cre ) was PCR‐amplified from plasmid pZCRE (Pribylova, De Montigny, et al, ) with primers CRE‐pGRB‐SacI‐XbaI‐F1 and CRE‐pGRB‐XhoI‐XbaI‐R1 (Table ). Before cloning, two copies of ScPGK1 promoter‐pHluorin module were excised from pCg2XpH‐N (Llopis‐Torregrosa et al, ) with SacI and XhoI endonucleases. The linearized vector was gel‐purified and cloned with the PCR insert pScGAL1 + cre in S. cerevisiae Y13925, creating pGRCRE.…”