Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species

<p>Abstract</p> <p>Background</p> <p>The virulence of <it>Candida </it>species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various <it>Candida </it>species has not been studied so far.</p> <p>Results</p> <p>The tolerance/sensitivity of four pathogenic <it>Candida </it>species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na⁺/H⁺antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, <it>C. dubliniensis </it>and <it>C. parapsilosis </it>respectively, were identified, cloned and functionally expressed in the plasma membranes of <it>Saccharomyces cerevisiae </it>cells lacking their own cation exporters. Both <it>Cp</it>Cnh1 and <it>Cd</it>Cnh1 antiporters had broad substrate specificity and transported Na⁺, K⁺, Li⁺, and Rb⁺. Their activity in <it>S. cerevisiae </it>cells differed; <it>Cp</it>Cnh1p provided cells with a much higher salt tolerance than the <it>Cd</it>Cnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters.</p> <p>Conclusion</p> <p>We have cloned two genes encoding putative Na⁺/H⁺antiporters in <it>C. parapsilosis </it>and <it>C. dubliniensis</it>, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na⁺/H⁺antiporters is one of the factors determining the tolerance of pathogenic <it>Candida </it>species to high external concentrations of alkali metal cations.</p

Alternative Glycerol Balance Strategies among Saccharomyces Species in Response to Winemaking Stress

Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.BO was supported by CAPES the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Brazilian Ministry of Education). This work has been supported by grants AGL2012-39937-C02-01 and AGL2015-67504-C3-1-R from the Spanish Government, FEDER, and Generalitat Valenciana PROMETEOII/2014/042 to AQ, GA CR 15-03708S from the Czech National Foundation to HS, and by the European Commission FP7: Marie Curie Initial Training Network CORNUCOPIA no. 264717 to AQ and HS.Peer reviewe

Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics

AbstractPotassium uptake defective Saccharomyces cerevisiae strains (Δtrk1,2 and Δtrk1,2 Δtok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi-copy plasmid and chromosomally expressed GFP-mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4μM) compared to Δtrk1,2 Δtok1 cells (EC50 2.6μM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350-fold more sensitive to CsCl and 1500-fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology

HIV-1 Vpu Protein Mediates the Transport of Potassium in Saccharomyces cerevisiae

Human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that belongs to the viroporin family. Viroporins interact with cell membranes, triggering membrane permeabilization and promoting release of viral particles. In vitro electrophysiological methods have revealed changes in membrane ion currents when Vpu is present; however, in vivo the molecular mechanism of Vpu at the plasma membrane is still uncertain. We used the yeast Saccharomyces cerevisiae as a genetic model system to analyze how Vpu ion channel impacts cellular homeostasis. Inducible expression of Vpu impaired cell growth, suggesting that this viral protein is toxic to yeast cultures. This toxicity decreased with extracellular acidic pH. Also, Vpu toxicity diminished as the extracellular K(+) concentration was increased. However, expression of the Vpu protein suppresses the growth defect of K(+) uptake-deficient yeast (Δtrk1,2). The phenotype rescue of these highly hyperpolarized cells was almost total when they were grown in medium supplemented with high concentrations of KCl (100 mM) at pH 7.0 but was significantly reduced when the extracellular K(+) concentration or pH was decreased. These results indicate that Vpu has the ability to modify K(+) transport in both yeast strains. Here, we show also that Vpu confers tolerance to the aminoglycoside antibiotic hygromycin B in Δtrk1,2 yeast. Our results suggest that Vpu interferes with cell growth of wild-type yeast but improves proliferation of the hyperpolarized trk1,2 mutant by inducing plasma membrane depolarization. Furthermore, evaluation of the ion channel activity of the Vpu protein in Δtrk1,2 yeast could aid in the development of a high-throughput screening assay for molecules that target the retroviral protein.This study was supported by Grants PI PI05/00013 and PI08/0912 from Fondo de Investigación Sanitaria. L.H. and N.M. were holders of Predoctoral Fellowships from Instituto de Salud Carlos III.S

The Type VI secretion system deploys anti-fungal effectors against microbial competitors

This work was supported by the Wellcome Trust (Senior Research Fellowship in Basic Biomedical Science to S.J.C., 104556; 097377, J.Q.; 101873 & 200208, N.A.R.G.), the MRC (MR/K000111X/1, S.J .C; MC_UU_12016/5, M.T.), and the BBSRC (BB/K016393/1 & BB/P020119/1, J.Q.). We thank Maximilian Fritsch, Mario López Martín and Birte Hollmann for help with strain construction; Gary Eitzen for construction of pGED1; Donna MacCallum for the gift of Candida glabrata ATCC2001; Joachim Morschhäuser for the gift of pNIM1; Gillian Milne (Microscopy and Histology facility, University of Aberdeen) for assistance with TEM; and Peter Taylor, Michael Porter, Laura Monlezun and Colin Rickman for advice and technical assistance.Peer reviewedPostprin

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

AbstractThe family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume

Reakcje genotypow pszenicy ozimej na porazenie przez Mycosphaerella graminicola

Reaction of six winter wheat culti vars and lines (Vlasta, Šárka, Charger, 00ST022, SG-U8044C a SG-U2113B) to Mycosphaerella graminicola isolate BR-331 and UH-05 on leaf segments of the detached second seedling leaf of cultivars placed on water agar with bezimidazole in clear plastic box were tested. The isolate BR-331 produced high occurrence of the disease in the cultivar Šárka, middle occurrence (the percentage covered by lesions bearing pycnidia) in the cultivar Vlasta and SG-U8044C and low occurrence in the line 00ST0022. The isolate UH-105 produced high occurrence in the cultivar 00ST022 and middle occurrence in the cultivar Vlasta. The cultivar Charger was resistant. The cultivar Šárka was attacked at least. Results show on different virulence of M. graminicola isolates to wheat cultivars.Badano reakcje 6. odmian i linii pszenicy ozimej (Vlasta, Šárka, Charger, 00ST022, SG-US8044C, i SG-U2113B) na porażenie przez izolaty RB - 331 i UH - 105 grzyba Mycosphaerella graminicola. Doświadczenia przeprowadzono na fragmentach drugich liści siewek pszenicy umieszczonych w czystych, plastikowych pudelkach z wodnym agarem zawierającym benzimidazol. Stopień porażenia izolatem BR - 331 był najwyższy na liściach odmiany Šárka, średni na odmianie Vlasta i linii SG - U8044C oraz niski na roślinach linii 00ST022. W przypadku izolatu UH -105 najsilniejsze objawy odnotowano na liściach linii OOST022, średnie porażenie stwierdzono na roślinach Vlasta. Na liściach odmiany Charger brak było objawów choroby. Wyniki doświadczenia wskazują na zróżnicowaną wirulencję izolatów Mycosphaerella graminicola względem testowanych genotypów pszenicy