Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Suppression of Sensitivity to Drugs and Antibiotics by High External Cation Concentrations in Fission Yeast

  • John P. Alao ,

    Contributed equally to this work with: John P. Alao, Andrea M. Weber

    john.p.alao@cmb.gu.se

    Affiliation Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30, Göteborg, Sweden

  • Andrea M. Weber ,

    Contributed equally to this work with: John P. Alao, Andrea M. Weber

    Affiliation Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30, Göteborg, Sweden

  • Aidin Shabro,

    Affiliation Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30, Göteborg, Sweden

  • Per Sunnerhagen

    Affiliation Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE-405 30, Göteborg, Sweden

Suppression of Sensitivity to Drugs and Antibiotics by High External Cation Concentrations in Fission Yeast

  • John P. Alao, 
  • Andrea M. Weber, 
  • Aidin Shabro, 
  • Per Sunnerhagen
PLOS
x

Abstract

Background

Potassium ion homeostasis plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics and chemotherapeutic agents in Schizosaccharomyces pombe and other yeasts. However, the precise relationship between drug resistance in S. pombe and external potassium concentrations (particularly in its natural habitats) remains unclear. S. pombe can tolerate a wide range of external potassium concentrations which in turn affect plasma membrane polarization. We thus hypothesized that high external potassium concentrations suppress the sensitivity of this yeast to various drugs.

Methods

We have investigated the effect of external KCl concentrations on the sensitivity of S. pombe cells to a wide range of antibiotics, antimicrobial agents and chemotherapeutic drugs. We employed survival assays, immunoblotting and microscopy for these studies.

Results

We demonstrate that KCl, and to a lesser extent NaCl and RbCl can suppress the sensitivity of S. pombe to a wide range of antibiotics. Ammonium chloride and potassium hydrogen sulphate also suppressed drug sensitivity. This effect appears to depend in part on changes to membrane polarization and membrane transport proteins. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated.

Conclusions

High concentrations of external potassium and other cations suppress sensitivity to a wide range of drugs in S. pombe. Potassium-rich environments may thus provide S. pombe a competitive advantage in nature. Modulating potassium ion homeostasis may sensitize pathogenic fungi to antifungal agents.

Background

Understanding the complex relationship between K+ homeostasis and multidrug/cation sensitivity has important implications for a wide range of fields including microbial ecology, evolution, comparative genomics, fermentation and brewing, food spoilage, the treatment of infectious diseases and cancer therapy [18]. The fission yeast Schizosaccharomyces pombe utilizes an extensive repertoire of transporters and signaling pathways to regulate K+ homeostasis [9]. Proper maintenance of K+ homeostasis in turn plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics such as hygromycin B, and chemotherapeutic agents in S. pombe [8]. In particular, expression of the Trk1 and Trk2 K+ transporters and the Hal4 kinase have been shown to be key regulators of K+ import in S. pombe and other yeasts [9, 10]. hal4 or trk1Δ trk2Δ double mutants fail to adequately import K+, resulting in membrane hyperpolarization and multidrug sensitivity [8]. Addition of excess KCl to the media partially restored the resistance of these mutants to cations and multiple drugs. These genes have thus been proposed to facilitate resistance to multiple drugs in S. pombe [8]. Conversely, yeast mutants unable to effectively regulate H+ or K+ ion efflux have depolarized cell membranes and increased resistance to cations and hygromycin B [1113]. Similarly, membrane depolarization by addition of excess KCl or NaCl has been shown to suppress the sensitivity of wild type Saccharomyces cerevisiae to hygromycin B. Together, these studies suggest that plasma membrane potential influences sensitivity to cations and cationic drugs in S. pombe and other yeasts. Yet, the precise relationship between drug resistance in S. pombe and external KCl concentrations (particularly in its natural habitats) remains unclear. Furthermore, the relative effects of external KCl and NaCl concentrations on drug sensitivity in S. pombe remain poorly characterized.

Despite its extensive characterization under laboratory conditions, little is known about how K+ homeostasis influences the survival of S. pombe in its natural environment. S. pombe has frequently been isolated from a restricted range of fermenting plant products rich in potassium [9, 14]. Under natural conditions, S. pombe must compete for resources with other micro-organisms including lactic acid bacteria (LAB), non-LAB bacteria, other fungi and yeasts [6, 7]. LAB produce lactic acid as a byproduct of fermentation. They also produce bacteriocins (peptide antimicrobials) and other bacteriostatic molecules [57, 15]. These substances are believed to confer competitive advantages to LAB in their natural environment [6]. Additionally S. pombe must also be able to resist the potential effects of yeast killer toxins [16, 17], acetic acid [7] and antibiotics produced by non-LAB strains (e.g. Streptomyces spp.) [7, 18]. Studies on the microbial dynamics of fermenting millet and wine, both rich in potassium, suggest that S. pombe is particularly well adapted to its natural environment(s) [6]. As such, it must be able to withstand not only high external potassium levels and low pH but also a diverse range of antimicrobial substances. Wild type (wt) S. pombe strains are however relatively sensitive to several antibiotic and chemotherapeutic substances under standard laboratory conditions [8]. In both yeast and bacteria, such sensitivity is closely linked to the proper regulation of the plasma membrane potential [4, 8, 13, 19]. Cation homeostasis and osmoregulation have also been linked to drug resistance in S. cerevisiae, Candida albicans (C. albicans) and Aspergillus spp. [11, 2022]. The development of resistance to antifungal therapeutics continues to limit their clinical efficacy [21, 23, 24]. Understanding the link between ion homeostasis, osmoregulation and drug resistance may thus lead to the development of new treatment strategies. In a previous study [25] we noted that increasing the concentration of external K+ in media greatly suppressed the sensitivity of S. pombe to the antibiotics bleomycin and phleomycin. Since S. pombe can tolerate a wide range of external K+ concentrations which in turn affect plasma membrane polarization [8, 9], we hypothesized that potassium-rich natural environments may confer a competitive advantage to this and other yeast species.

In the present study, we have investigated the effect of external K+ concentrations on the sensitivity of S. pombe to a wide range of antibiotics, other antimicrobial agents and chemotherapeutic agents. In addition, we have also compared and contrasted the relative effects of KCl, its analogue RbCl, and NaCl on drug sensitivity in this yeast. We demonstrate that KCl and to a lesser extent NaCl and RbCl can suppress the sensitivity of S. pombe to a wide range of antibiotics. We also demonstrate that high external ammonium concentrations similarly suppress drug sensitivity in S. pombe. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated. Together, our findings suggest that low pH, high KCl, environments may provide a unique environmental niche for S. pombe in nature.

Materials and Methods

Drugs

All drugs except caspofungin were from Sigma Aldrich (Sigma Aldrich AB, Stockholm, Sweden). Caspofungin was from Santa Cruz Biotechnology (Heidelberg, Germany) All drugs were dissolved in either dH2O, dimethyl sulphoxide (DMSO) or ethanol and stored at -20°C.

Growth of S. pombe strains

S. pombe was cultured in YES medium [26] at 30°C unless otherwise indicated. For potassium free media (HGA media), we used a solution of 3% glucose and 0.05% NH4Cl in dH2O. pH was adjusted to 6.3 with 5% ammonia solution. Strains are listed in Table 1.

Drug sensitivity assays

Log phase cultures were resuspended in fresh media containing the desired drug with or without potassium chloride. After the required incubation period, the cultures were equilibrated for cell number, serially diluted and spotted unto YES agar plates. The plates were incubated at 30°C for 2–3 days. Alternatively, log phase cultures were serially diluted and spotted unto YES agar plates containing the desired alkali salt or drug concentrations and incubated at 30°C for 2–3 days.

Immunoblotting

Monoclonal antibodies directed against HA were from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal antibodies directed against phospho-(Thr180/Tyr182) p38 were from Cell Signaling Technology (Bionordika (Sweden) AB, Stockholm, Sweden). Monoclonal antibodies directed against α-tubulin were from Sigma-Aldrich (Sigma Aldrich AB). For immunoblotting, protein extracts were prepared as previously described [25] with addition of 1 × PhosStop phosphatase inhibitor cocktail (Roche Diagnostics Scandinavia AB, Bromma, Sweden). Proteins were separated by SDS-PAGE. Epitope-tagged proteins were detected with the appropriate monoclonal antibodies.

Microscopy

Cells were harvested, fixed in 70% ethanol and stored at 4°C until analyzed. Images were obtained with a Zeiss AxioCam on a Zeiss Axioplan 2 microscope with a 100 × objective using a 4,6-diamidino-2-phenylindole (DAPI) filter set. Fixed cells were mounted in VECTASHIELD mounting medium and visualized using differential interference contrast (DIC) or a DAPI filter set. For studies with doxorubicin, cells were pelleted and directly examined by fluorescence microscopy.

Results

Potassium chloride suppresses drug sensitivity in S. pombe

In order determine the effect of external K+ concentrations on drug sensitivity, S. pombe cells were exposed to the antibiotic DNA damaging agents bleomycin (5 μg/ ml), doxorubicin (400 nM) or phleomycin (10 μg/ ml) in the presence of increasing KCl concentrations. KCl effectively suppressed the sensitivity of wt S. pombe cells to bleomycin at concentrations as low as 60 mM (Fig. 1A). At these concentrations, KCl also completely suppressed the sensitivity of wt S. pombe cells to doxorubicin and phleomycin (Fig. 1B and 1C). We noted, however, that 60 mM KCl was insufficient to suppress the sensitivity of the checkpoint-deficient rad3Δ and rad24Δ mutants to doxorubicin and phleomycin (Fig. 1B, C, and S1A, F Fig.). Microscopic examination of wt cells exposed to phleomycin suggested that low doses of KCl do not prevent DNA damage induced by this agent (Fig. 1D). Activation of the Rad3- regulated DNA response pathway results in S. pombe induces Chk1 phosphorylation observable as a band shift on Western blots [27]. In agreement, the complete abolition of phleomycin-induced Chk1 phosphorylation was observed only at concentrations of 0.3 to 0.6 M KCl (Fig. 1E). Exposure to 0.6 M KCl in the presence of the aminoglycoside antibiotics G418 and hygromycin similarly abolished sensitivity of wt S. pombe to these agents (Fig. 1F). At 0.6 M, KCl also suppressed the sensitivity of wt S. pombe cells to cisplatin, anisomycin, and antimycin A (Fig. 1G, L, and M). In contrast, KCl did not abolish sensitivity to camptothecin, hydroxyurea (HU), tunicamycin, staurosporin, leptomycin B (LMB) or actinomycin D (Table 2, Fig. 1H-K, N, and O). Furthermore, KCl did not inhibit the effects of latrunculin B and methylbenzimidazol-2yl carbamate (MBC) on cytokinesis and mitosis respectively (S1G Fig.). Co- exposure to 0.6 M KCl also suppressed sensitivity to 50 mM LiCl (Fig. 1P). The suppressive effect of KCl on phleomycin sensitivity to was not due to inactivation of the drug. In fact, pre-incubation in KCl of the drug alone greatly enhanced the cytotoxic effect of phleomycin in wt and rad24Δ mutants (S1E Fig.).

thumbnail
Fig 1. KCl suppresses drug sensitivity in S. pombe

A. Wild type (wt) S. pombe cells were cultured in the presence of the 5 μg/ ml bleomycin alone or with indicated concentrations of KCl in the media for 24 h at 30°C. Equal cell numbers were serially diluted and plated on YES agar. Plates were incubated at 30°C for 2–3 days. B. Wt and rad3Δ mutants were exposed to 40 μg/ ml doxorubicin alone or with the indicated concentrations of KCl in the media for 24 h at 30°C and treated as in A. C. Wt and rad3Δ cells were treated as in B, except that the cells were exposed to 10 μg/ ml phleomycin. D. Wt S. pombe cells were incubated with 10 μg/ ml phleomycin alone or with the indicated concentrations of KCl in the media. Cells were fixed in 70% ethanol and examined by microscopy. E. A strain expressing HA- tagged Chk1 was incubated with 10 μg/ ml phleomycin in the presence of the indicated KCl concentrations. Total lysates were resolved by SDS- PAGE and probed with antibodies directed against HA. Tubulin was used to monitor equal gel loading. F. Wt cells were exposed to 100 μg/ ml of G418 or hygromycin for 24 h with or without 0.6 M KCl and then treated as in A. G- P. Wt cells were exposed to the indicated drugs with or without 0.6 M KCl for 24 h and treated as in A.

https://doi.org/10.1371/journal.pone.0119297.g001

Cations suppress drug sensitivity in S. pombe

To further understand the effect(s) of KCl on drug sensitivity, we investigated the degree to which KCl suppresses sensitivity to phleomycin, G418 and hygromycin. In the presence of 0.6 M KCl, however, doses as high as 400 μg/ ml phleomycin, 1000 μg/ ml G418, and 500 μg/ ml hygromycin did not affect the survival of wild type S. pombe cells (Fig. 2A-C). At a concentration of 0.6 M therefore, KCl increased resistance to phleomycin, G418 and hygromycin by a factor of 400, 200, and 50 times respectively (Table 3). The minimum inhibitory concentrations (MICs) in our assays were 1 μg/ ml, 5 μg/ ml and 10 μg/ ml for phleomycin, G418 and hygromycin respectively (S1B,C Fig.). Calcium chloride (0.1 M) also failed to suppress drug sensitivity in S. pombe (Fig. 2D). Previous studies have similarly reported that sensitivity to bleomycin (structurally similar to phleomycin) in particular, is strongly influenced by internal K+ levels [8]. The ability of KCl to suppress sensitivity to these drugs did not result from the induction of osmotic stress, since equiosmotic concentrations of sorbitol did not have this effect (Fig. 2E, F and S2H Fig.). In contrast, sodium chloride (NaCl), ammonium chloride (NH4Cl), rubidium chloride (RbCl) and potassium hydrogen phosphate (K2HPO4) all suppressed drug sensitivity (Fig. 2G-I, K, S2A, B Fig.). The ability of NH4Cl to suppress drug sensitivity was not limited to phleomycin since it also suppressed sensitivity to doxorubicin (Fig. 2J). The observation that K2HPO4 suppressed drug sensitivity in S. pombe ruled out a role for chloride (Cl-) ions in this activity (Fig. 2H). Similarly, the ability of NH4Cl to suppress drug sensitivity suggested that this activity is not restricted to metal ions (Fig. 2I-K, S2B Fig.). Under these conditions, KCl also facilitated the survival of S. pombe in the presence of 100 μg/ ml G418 and hygromycin (Fig. 2L). The ability of KCl, NaCl and RbCl to suppress drug sensitivity was not strictly identical. We noted that 0.6 M KCl suppressed the sensitivity of S. pombe to 1 mM but not 2 mM cadmium chloride (CdCl2) (S2C Fig.). In contrast, co-culture in the presence of 0.5 M NaCl suppressed the sensitivity to 2 mM CdCl2 (S2D Fig.). S. pombe cells grew poorly in liquid YES media in the presence of 0.6 M NaCl. KCl (0.6 M) did not suppress sensitivity to 0.4% acetic acid (S2F Fig.). The ability of KCl to suppress hygromycin and phleomycin sensitivity was also not affected by the presence of 0.1%- 0.3% acetic or lactic acid (S2G Fig.). Furthermore, acidification of the external pH with lactic acid (pH 6.4–3.1) had little or no effect on viability (S2G Fig.).

thumbnail
Fig 2. Suppression of drug sensitivity in S. pombe by alkali metal ions.

A- C. Wt S. pombe cells were incubated with various concentrations of phleomycin (phleo), G418 and hygromycin (hygro) ± 0.6 M KCl for 24 h at 30°C. Equal cell numbers were serially diluted and plated on YES agar. Plates were incubated at 30°C for 2–3 days. D. Wt S. pombe cells were incubated with 10 μg/ ml phleomycin ± 0.1 M CaCl2 for 4 h at 30°C and treated as in A. E. Wt S. pombe cells were exposed to 10 μg/ ml phleomycin ± 0.6 M KCl or 1.2 M sorbitol for 6 h, fixed in 70% ethanol, stained with DAPI and examined by microscopy. F. Wt S. pombe cells were treated as in E. Equal cell numbers were serially diluted and plated on YES agar. Plates were incubated at 30°C for 2–3 days. G. Wt S. pombe cells were incubated with 10 μg/ ml phleomycin or in the presence of the indicated NaCl concentrations for 24 h at 30°C and treated as in A. H. Wt cells were incubated with 10 μg/ ml phleomycin ± 0.25 M K2HPO for 4 h at 30°C and treated as in A. I. Wt cells were incubated with 10 μg/ ml phleomycin ± 0.25 M NH4Cl for 24 h at 30°C and treated as in A. H. I. S. pombe cells were incubated with 0.6 M RbCl ± 0.6 M KCl for 24 h at 30°C. J. S. pombe cells were incubated with 40 μg/ ml doxorubicin ± 1 M NH4Cl for 24 h at 30°C. K. Cells were treated as in I, fixed in 70% ethanol, stained with DAPI and examined by microscopy. L. Log phase wt cultures were serially diluted and plated on YES agar containing 100 μg/ ml hygromycin ± 0.6 M KCl for 72 h at 30°C.

https://doi.org/10.1371/journal.pone.0119297.g002

thumbnail
Table 3. Relative effect of external KCl on drug sensitivity in S. pombe.

https://doi.org/10.1371/journal.pone.0119297.t003

KCl suppresses drug sensitivity independently of Sty1, Hal4, and Trk1/2

The MAPK Sty1 plays a central role in mediating resistance to environmental stresses in S. pombe [28]. Sty1 has also been shown to regulate the Hal4 kinase, which together with the Trk1 and Trk2 transporters regulates K+ uptake in S. pombe [8, 29]. Sty1 was not required for KCl- mediated suppression of phleomycin sensitivity in S. pombe (Fig. 3A and 3B). In contrast to wt cells however, sty1Δ mutants displayed an elongated phenotype when exposed to phleomycin in the presence of 0.15–0.6 M KCl (Figs. 2E and 3A). Furthermore, lower concentrations of KCl (0.15b –0.3 M) were more effective at suppressing phleomycin sensitivity than higher concentrations (0.6 M) in this mutant (Fig. 3B). Immunoblotting demonstrated only minimal activation of Sty1 at concentrations of KCl (0.04–0.3 M) sufficient to suppress sensitivity to phleomycin (Fig. 3C). In addition, sty1Δ mutants grew worse in the presence of phleomycin and 0.15–0.6 M KCl than in the presence of 0.6 M KCl alone (Fig. 3B). Exposure to phleomycin alone did not induce Sty1 activation (Fig. 3E). In our study, the sensitivity of sty1Δ mutants to G418 was not greater than observed for wt cells (S3A Fig.). Together, our observations suggest that Sty1 is not required for KCl- mediated suppression of phleomycin per se. Sty1 does seem to enhance the survival of S. pombe cells however, when exposed to the combined stresses of phleomycin and KCl exposure (Fig. 3B) [28].

thumbnail
Fig 3. Sty1 is not required for the suppressive effect of KCl on drug sensitivity.

A. sty1Δ mutants were incubated with 10 μg/ ml phleomycin ± the indicated concentration of KCl for 4 h. Cells were fixed in 70% ethanol, stained with DAPI and examined by microscopy. B. Cell were treated as in A for 4 h, serially diluted on YES plates and incubated for 2–3 days at 30°C. As an extra control, the mutant was also exposed to 0.6 M KCl alone. C. Wt S. pombe cells were incubated with the indicated concentrations of KCl for 10 min at 30°C. Total lysates were resolved by SDS- PAGE and probed with antibodies directed against phos. p38. Tubulin was used to monitor equal gel loading. D. Wt, hal4Δ and trk1Δ trk2Δ cells were exposed to 10 μg/ ml phleomycin ± the indicated concentrations of KCl for 4 h at 30°C and treated as in B. E. Wt S. pombe cells were incubated with 10 μg/ ml phleomycin ± 0.6 M KCl. Total lysates were treated as in C. F. Wt, hal4Δ and trk1Δ trk2Δ cells were exposed to increasing doses of G418 for 4 h and treated as in B. G. hal4Δ and trk1Δ trk2Δ cells were exposed to 10 μg/ ml phleomycin ± 0.6 M KCl or o.6 M KCl alone and treated as in B.

https://doi.org/10.1371/journal.pone.0119297.g003

Trk1 and Trk2, together with Hal4, regulate K+ uptake in S. pombe [8, 30]. Mutants lacking trk1+ and trk2+, or hal4+, display membrane hyperpolarization and sensitivity to cations and cationic drugs [8]. As previously reported [8], high external K+ concentrations suppressed the sensitivity of hal4Δ mutants to cationic drugs. In our study, 0.3 M KCl suppressed sensitivity to 10 μg/ml phleomycin to the same degree in wt and hal4Δ mutants (Fig. 3D). Interestingly, higher concentrations (0.6 M) of KCl appeared to be less effective at suppressing the sensitivity of hal4Δ mutants to phleomycin (Fig. 3D). Co-exposure to KCl also suppressed the sensitivity of trk1Δ trk2Δ double mutants to phleomycin, albeit less efficiently than in wt and hal4Δ mutants (Fig. 3D). When cultured in the presence of 0.6 M KCl, we noted little or no effect on the viability of hal4Δ and trk1Δ trk2Δ mutants (Fig. 3G). The reduced viability observed following exposure to KCl and phleomycin may be due to the inability of these mutants to import sufficient levels of K+. We also observed that while hal4Δ mutants were more sensitive to G418 than wt cells, trk1Δ trk2Δ mutants were more resistant than hal4Δ mutants to this agent (Fig. 3F). We also observed that Hal4 was not required for KCl-mediated suppression of LiCl sensitivity (S3E Fig.). Factors other than membrane polarity are thus likely to influence sensitivity to particular drugs in S. pombe in the presence of medium to high KCl concentrations. The Na+ (Li+) / H+ antiporter Sod2 was not required for KCl-mediated suppression of LiCl and phleomycin sensitivity in S. pombe (S3B,E Fig.).

KCl inhibits drug import in S. pombe

Mutants lacking hal4+ have previously been shown to import higher levels of doxorubicin than wt cells [8]. In our study, co-exposure to KCl (0.6 M) and doxorubicin blocked the import of the drug. In marked contrast however, culture in the presence of KCl following exposure to doxorubicin did not result in the efflux of the drug (Fig. 4A). Previous studies have demonstrated the increased uptake of cationic compounds by S. pombe cells with hyperpolarized membranes [8]. Microscopic analyses clearly demonstrated that co-exposure to KCl inhibited the uptake of doxorubicin compared to cells exposed to the latter alone (Fig. 4A panels 2 and 3). Additional analyses demonstrated that the ability of KCl to prevent the import of doxorubicin into the cell was concentration-dependent (Fig. 4E). Furthermore, the ability of KCl to inhibit doxorubicin import correlated with its ability to suppress sensitivity to the drug in hal4Δ mutants (Fig. 4B). In contrast, exposure to 1.2 M sorbitol, did not prevent doxorubicin uptake (S2H Fig.). Mutants lacking the phosphatase Pzh1 are unable to effectively export K+ ions from interior of the cell. High internal K+ levels result in sensitivity to this ion, but resistance to Na+ [12]. Following exposure to phleomycin, pzh1Δ mutants appeared similar to wt cells (Fig. 4C). Survival assays demonstrated however, that pzh1Δ mutants were slightly more resistant to G418 than wt cells (Fig. 4D). Furthermore, pzh1Δ mutants were significantly more resistant to phleomycin than wt cells (Fig. 4D). Following long term exposure to phleomycin however, co-exposure to KCl suppressed sensitivity in a manner similar to that observed in wt cells (S3C,D Fig.). The relative KCl concentration-dependent sensitivity of pzh1Δ mutants to phleomycin indicated that their internal K+ ion levels are insufficient to block the activity of the drug. Our finding that pzh1Δ mutants were relatively more resistant to phleomycin than G418 provided further evidence for the exquisite sensitivity of bleomycin and phleomycin to internal K+ concentrations.

thumbnail
Fig 4. KCl blocks doxorubicin uptake in S. pombe.

A. hal4Δ mutants were exposed to 40 μg/ ml doxorubicin alone and together with 0.6 M KCl for 4 h or with doxorubicin for 2 h followed by coexposure to doxorubicin and KCl for another 2 h and examined by microscopy. B. Wt and hal4Δ mutants were treated as in A for 4 h, serially diluted and plated unto YES agar. Plates were incubated at 30°C for 2–3 days. C. Wt and pzh1Δ mutants were exposed to 10 μg/ ml phleomycin ± 0.6 M KCl for 4h, fixed in 70% ethanol and examined by microscopy. D. Wt and pzh1Δ mutants were exposed to 10 μg/ ml G418 or 5 μg/ ml phleomycin for 4 h and treated as in B. E. Wt cells were treated with 40 μg/ ml doxorubicin alone or together with 0.06M or 0.6 M KCl for 2 h.

https://doi.org/10.1371/journal.pone.0119297.g004

The findings above provided additional evidence for KCl-induced membrane depolarization in modulating the sensitivity of S. pombe to cationic drugs [8]. To further test this hypothesis, we investigated the effect of KCl on sensitivity to sodium orthovanadate (Na3VO4). The negatively charged VO43- acts as an inhibitor of protein tyrosine and alkaline phosphatases. As predicted, KCl significantly enhanced the toxicity of Na3VO4 (Fig. 5A). In addition, KCl had no effect on sensitivity to 1% potassium metabisulfite (K2S2O5) or 1% sodium metabisulfite (Na2S2O5) in S. pombe (Fig. 5B). To further investigate the role for potassium and sodium in suppressing drug sensitivity, we investigated their activity in HGA medium (3% glucose and 0.05% NH4Cl in dH2O. pH was adjusted to 6.3 with 5% ammonia solution). Taken together, these experiments demonstrated that high external concentrations of KCl and NaCl are sufficient to suppress drug sensitivity in S. pombe (Fig. 5C).

thumbnail
Fig 5. KCl enhances sensitivity to Na3VO4 in S. pombe.

A. Wt cells were incubated with 7.5 mM of Na3VO4 ± 0.6 M KCl for 24 h, serially diluted and plated unto YES agar. Plates were incubated at 30°C for 2–3 days. B. Wt cells were incubated with 1% Na2S2O5 or 1% Na2S2O5 ± 0.6 M KCl and treated as in A. C. Wt cells were incubated in HGA- medium (see Materials and methods) with phleomycin ± KCl or NaCl and treated as in A.

https://doi.org/10.1371/journal.pone.0119297.g005

Effect of KCl on fungicide and antiporter sensitivity

We next investigated the effect of high external KCl concentrations on drugs that disrupt fungal cell membranes or cation homeostasis (ionophores). Clotrimazole disrupt fungal cell membranes by inhibiting ergosterol synthesis, resulting in the leakage of ions and small molecules from the cell [31, 32]. KCl did not protect S. pombe from the lethal effects of 15 μg/ ml clotrimazole at either 0.06 or 0.6 M (Fig. 6A). Clotrimazole is an organic compound and this might account for the inability of KCl to inhibit its activity. In contrast to clotrimazole, polyene antifungal drugs such as amphotericin B and nystatin bind directly to ergosterol, leading the formation of pores in the membrane and the leakage of ions and small molecules from the cell [33, 34]. In addition, amphotericin B may also inhibit the Na+/ K+ pump contributing to cell death [35]. Interestingly, KCl exerted differential effects on the sensitivity of S. pombe to these drugs. Co-exposure to 0.06 M KCl inhibited the lethal effects of 1μg/ ml amphotericin B (Fig. 6B). In stark contrast, this suppression of sensitivity was not observed when wt S. pombe cells were co-exposed to amphotericin B and 0.6 M KCl (Fig. 6B). Co exposure to 3 μg/ ml nystatin and 0.06 M KCl did not affect sensitivity to this drug, while 0.6 M KCl enhanced sensitivity under similar conditions (Fig. 6C). Further analyses indicated that KCl similarly enhances sensitivity to amphotericin B at concentrations between 0.3 M and 0.6 M (S4A Fig.). Together, these findings demonstrated that KCl at concentrations of 0.15 M and above enhances sensitivity to the polyene antifungals amphotericin B and nystatin. It remains unclear if this KCl- induced increase in sensitivity is due to the non-ionic nature of these drugs. Caspofungin is an echinocandin antifungal drug that inhibits cell wall synthesis by inhibiting the enzyme (1→3)-β-D-glucan synthase [36]. In our studies, co-exposure to 0.6 M KCl completely inhibited sensitivity to 1 μg/ ml caspofungin (Fig. 6D). In contrast to its effect on the sensitivity of S. pombe to phleomycin (Fig. 2D, E), sorbitol (1.2 M) similarly abolished the lethal effects of caspofungin (S4B Fig.). Caspofungin is water soluble, suggesting that KCl may counter its activity by influencing membrane polarity. We next investigated the effect of KCl on sensitivity to the ionophore antibiotics nigericin and valinomycin [37, 38]. Nigericin is completely insoluble in water and functions as an antiporter for K+ and other ions, inducing cell death in part by causing acidification of the cytoplasm and ion leakage from the cell [38]. KCl (0.6 M) abolished sensitivity to 10 μg/ ml nigericin (Fig. 6E). Co-exposure to NH4Cl similarly suppressed sensitivity to nigericin. In contrast, 0.6 M KCl enhanced sensitivity to 100 μg/ ml valinomycin (Fig. 6F). Interestingly, exposure to 100 μg/ ml valinomycin alone, did not affect viability in S. pombe. Furthermore, co-exposure to NH4Cl did not induce sensitivity to this drug. Like nigericin, valinomycin is insoluble in water and induces K+ leakage from the cell. It thus remains unclear why KCl differentially affects the sensitivity of S. pombe to nigericin and valinomycin.

thumbnail
Fig 6. Effect of KCl on S. pombe sensitivity to fungicides.

A- C. Wt cells were exposed to 10 μg/ ml clomitrazole, 1 μg/ ml amphotericin B or 5 μg/ ml nystatin alone and together with the indicated concentrations of KCl for 24 h, serially diluted and plated unto YES agar. Plates were incubated at 30°C for 2–3 days. D- E. Wt cells were exposed to 1 μg/ ml caspofungin or 40 μg/ ml nigericin ± 0.6 M KCl and treated as in A. F. Wt cells were exposed to 20 μg/ ml nigericin ± 0.3 M KCl or 0.25 M NH4Cl for 24 h and treated as in A. G. Wt cells were exposed to 100 μg/ ml valinomycin ± 0.6 M KCl for 24 h and treated as in A. H. Wt cells were exposed to 80 μg/ ml valinomycin ± 0.6 M KCl or 0.25 M NH4Cl for 24 h and treated as in A.

https://doi.org/10.1371/journal.pone.0119297.g006

Discussion

In this study, we investigated the effect of high external K+ and other ion concentrations on drug sensitivity in S. pombe. In S. pombe and other yeasts, a close relationship exists between the regulation of K+ etc. homeostasis and the polarity of the cell membrane. Deletion of hal4+ or co-deletion of trk1+ and trk2+ results in membrane depolarization and sensitivity to cations [8]. Interestingly, mutants lacking hal4+ or both trk1+ and trk2+ display hypersensitivity to a range of antibiotics, chemotherapeutic agents and other drugs [8]. These compounds, despite differences in structure and chemical classes, are all cationic. It has been proposed that the membrane hyperpolarization induced by deleting hal4+ or trk1+ and trk2+ facilitates the import of these compounds into the cell. Elevating the external KCl concentration partially restored resistance to cations and drugs in hal4Δ and trk1Δ trk2Δ mutants [8]. Furthermore, external KCl and NaCl concentrations have been shown to suppress the sensitivity of S. cerevisiae to hygromycin [13]. The ability of elevated external KCl concentrations to suppress drug sensitivity in hal4Δ and trk1Δ trk2Δ mutants suggested to us that a similar effect might occur in wt S. pombe. We thus investigated the effect of elevating the external concentration of K+ and other ions on the sensitivity of S. pombe to various cations and drugs (Table 3 and S5 Fig.).

In general, elevating the external K+ concentration of the media to 0.3–0.6 M was sufficient to suppress the sensitivity of wt S. pombe to cationic drugs such as bleomycin, phleomycin, G418 and hygromycin. KCl was more effective at suppressing sensitivity to water soluble compounds. Nevertheless, KCl did not suppress sensitivity to hydroxyurea which is highly soluble in water. Conversely, co-exposure to KCl also suppressed sensitivity to anisomycin and antimycin which are nonpolar compounds. Thus, no clear relationship between that ability of KCl to suppress sensitivity and the solubility, polarity or class of drugs investigated in this study was identified (Fig. 1 and S5 Fig.). The ability of KCl to suppress drug sensitivity was dependent on the K+ ion and not hyperosmosis per se, as sorbitol did not suppress drug sensitivity in a similar manner. The ability of metal cations to suppress drug sensitivity in S. pombe was not restricted to K+ since alkali cations Na+ and Rb+ also suppressed drug sensitivity in S. pombe. Indeed, co-exposure to NH4Cl also suppressed drug sensitivity. The observation that K2HPO4 similarly suppressed drug sensitivity ruled out a role for Cl- ions. Furthermore, the activity of these cations on cell survival was not strictly identical since Na+ was more effective at suppressing sensitivity to cadmium than K+. Interestingly, K+ facilitated the growth of S. pombe on solid rich media in the presence of NaCl. In terms of relative survival and culture mass, K+ was the most effective suppressor of drug sensitivity. The degree to which KCl suppressed drug sensitivity in S. pombe also varied amongst bleomycin, phleomycin, G418 and hygromycin. A previous study suggested that the deletion of hal4+ or trk1+ and trk2+ enhanced sensitivity to bleomycin, to a greater degree than other drugs tested [8]. In our study, KCl was similarly most effective at inhibiting the sensitivity of wt S. pombe cells to bleomycin and phleomycin. KCl also suppressed sensitivity to hygromycin to a greater degree than to G418. Interestingly, trk1Δ trk2Δ mutants were resistant to G418 relative to wt and hal4Δ mutants. Hence, the degree to which KCl suppresses sensitivity is dependent on the particular drug in question (Table 2 and Fig. 3). Nevertheless, our findings clearly show that the exogenous elevation of external K+, Na+ or Rb+ ion concentrations suppresses the sensitivity of S. pombe of a large number of diverse drug classes.

Previous studies suggest that membrane polarization serves as a pleotropic drug resistance mechanism. Membrane potential in S. pombe is regulated by the antagonistic relationship between K+ import and proton export by Pma1 plasma membrane ATPase [8, 12, 13, 30]. The inability of hal4 or trk1 trk2 double mutants to effectively import K+ results in membrane hyperpolarization and increased sensitivity to metal cations and cationic drugs [8]. External addition of low KCl concentrations (50 mM) suppressed the sensitivity of these mutants to various cationic molecules [8]. Similarly, S. cerevisiae Pma1 mutants have hyperpolarized cell membranes and are resistant to hygromycin [13]. These findings suggest that membrane polarization influences sensitivity to cations. In our studies, external KCl concentrations of at least 150 mM were required to completely suppress drug sensitivity in wt S. pombe cells. Microscopic analyses using doxorubicin demonstrated that KCl prevents the import of the drug. Furthermore, pzh1Δ mutants are unable to effectively export K+ ions leading to membrane depolarization [12] and were partially resistant to G418 and phleomycin. We also demonstrated that Pzh1 and Sod2 respectively required for K+ and Na+ export were not required for the suppression of drug sensitivity by KCl. The C. albicans and S. cerevisiae homologues of S. pombe Pzh1, CaPpz1 and Ppz1 respectively, have similarly been linked to hygromycin B and spermine resistance [11, 39]. The partially conserved function of Pzh1 family proteins thus suggests a conserved functional role that influences drug resistance. Furthermore, Sty1 activity was required for tolerating exposure to KCl but not its effect on drug sensitivity. Our findings thus support the notion that membrane polarization confers pleiotropic drug resistance in S. pombe [8]. The differential sensitivity of bleomycin and phleomycin to external K+ concentrations compared to G418 and hygromycin suggests however, that membrane polarity alone cannot account for the suppressive effect of this ion on drug sensitivity. Mutants lacking the Trk1 and Trk2 K+ transporters must clearly still be able to import this ion. It has been proposed that amino acid permeases and glucose transporters may facilitate K+ import in the absence of Trk1 and Trk2 [40, 41]. Furthermore, the existence of a membrane potential- and voltage- sensitive ATPase alternative K+ importer in S. pombe has been proposed [42]. The L-carnitine transporter Agp2 in S. cerevisiae has been shown to mediate bleomycin uptake [43, 44]. Exposure to high KCl concentrations may alter the substrate specificity or uptake kinetics of these transporters. This may also account for the differential effect of KCl on sensitivity to bleomycin, hygromycin and G418 observed by us and others [8]. In S. cerevisiae, the drug:H+ transporters Qdr2 and Qdr3 facilitate resistance to bleomycin, cisplatin, spermine and other toxic compounds [45, 46]. In addition, Qdr2 and Qdr3 also play a role in regulating K+ concentrations in cells by facilitating the import of the ion [47]. These drug:H+ transporters may thus play a role in mediating the KCl- induced suppression of drug sensitivity. Our unexpected finding that co-deletion of trk1+ and trk2+ conferred resistance to G418 suggests that the Trk1 and Trk2 transporters may be involved in the uptake of this drug (Fig. 3).

We also examined the effect of high external KCl concentrations on the sensitivity of S. pombe to the ionophores nigericin and valinomycin. Nigericin is an H+/ K+ exchanger and induces cell death in part by causing the leakage of the latter ion from the cell [42, 48]. In our studies, high external KCl concentrations inhibited sensitivity to nigericin in S. pombe. Although previous reports demonstrated that high external K+ and Na+ concentrations suppress nigericin activity [48], we have now demonstrated that NH4Cl exerts a similar effect. Nigericin is anionic at physiological pH, suggesting that the protective effect of KCl and NH4Cl was not due to their effect on membrane potential. The precise mechanism whereby KCl and NH4Cl suppress sensitivity to nigericin remains unclear. One likely possibility is that they override the physiological effects of ion efflux induced by this ionophore [48]. In contrast to nigericin, valinomycin induces K+ influx in S. pombe [48] and did not affect viability in our assays. Strikingly, co-exposure to valinomycin and high KCl concentrations did result in a significant loss of viability. Co- exposure to NH4Cl and valinomycin affected viability to a far lesser degree. It is possible that in the presence of valinomycin, high external KCl concentrations result in the accumulation of toxic levels of K+ ions. Mutants lacking Pzh1 are unable to export K+ ions and are thus similarly sensitive to high KCl concentrations [12]. KCl also influenced the sensitivity of S. pombe to the polyene antibiotics amphotericin B and nystatin. At lower external KCl concentrations (0.06 M), sensitivity to amphotericin B was suppressed. In contrast, we did not observe this effect when the external KCl concentration was raised to 0.6 M (S4A Fig.). At this concentration, co-exposure to KCl also leads to a loss of viability in the presence of otherwise non-lethal concentrations of nystatin. The polyene antibiotics bind ergosterol in the fungal cell membrane, causing pore formation and ion leakage from the cell [21]. Low external KCl concentrations may compensate for the drug-induced loss of intracellular ions, while higher concentrations are toxic as a consequence of membrane disruption. KCl did not suppress sensitivity to clomitrazole, which disrupts the cell membrane by inhibiting ergosterol synthesis. A previous study in S. cerevisiae demonstrated that co-exposure 150 mM KCl, but not 300 mM sorbitol increased sensitivity to fluconazole [22]. Future studies will investigate the role of K+ ion homeostasis in modulating the sensitivity of S. pombe to azoles. It remains possible that lower concentrations of KCl or higher concentrations of sorbitol can suppress sensitivity to azoles. Both KCl and sorbitol activate the MAPK- regulated stress response pathway, which has been shown to mediate resistance to azoles in yeast (reviewed in [21]). Inhibition of the heat shock protein Hsp90, a molecular chaperone and downstream target of the MAPK pathway has been shown to enhance sensitivity to fluconazole in both S. cerevisiae and C. albicans [21, 49, 50]. KCl and sorbitol may thus influence sensitivity to azoles via activation of the MAPK- regulated stress response pathway. Additionally, low doses of KCl may counteract the disruptive effects of azoles on cation homeostasis [51]. Sensitivity to the echinocandin caspofungin, another disruptor of fungal cell membranes, was suppressed by KCl and sorbitol. Unlike the other antifungal drugs tested by us, caspofungin is water soluble, possibly explaining the protective effect of KCl. Clinically, the emergence of resistance to echinocandin antifungals has been linked to the MAPK- regulated stress response pathway which plays a role in maintaining cell wall integrity [21, 49]. Both KCl and sorbitol activate the MAPK- regulated stress response pathway in S. pombe. Hence, activation of these pathways by either compound may contribute towards suppressing sensitivity to caspofungin.

Our studies clearly indicate that external K+ and other ion concentrations modulate the sensitivity of S. pombe to a diverse array of ions and drugs. These findings raise a number of interesting questions. S. pombe is clearly suited to tolerate relatively high concentrations of K+ ions in the surrounding medium. The evolutionary significance of this ability remains unclear. S. pombe is frequently found in environments with relatively high potassium concentrations e.g grapes and millet [14]. Furthermore, potassium concentrations are can be particularly high in desiccating environments [52]. S. pombe likely competes for limited nutrients with antibiotic-producing microorganisms [53]. It is tempting to imagine that high potassium concentrations may confer a competitive advantage to S. pombe by facilitating resistance to antibiotics. Resistance to antifungal agents remains an important issue clinically. Furthermore, the use of polyene antibiotics is associated with significant toxicity. Our findings and those of others [8], suggest that drugs which interfere with ion homeostasis could be used to modulate the sensitivity of fungal pathogens to fungicides. Importantly, we demonstrate that it is the K+ and other cations and not osmotic stress that suppress sensitivity to various drugs. Nonetheless, activation of the MAPK- regulated stress response pathway by KCl and osmotic stress may suppress sensitivity to some antifungal agents.

Conclusions

We have demonstrated that high external concentrations of K+ and some other alkali ions significantly suppress the sensitivity of S. pombe to numerous antibiotic and cytotoxic compounds. The ability of KCl to suppress drug sensitivity was not limited to any particular drug class. Nevertheless, KCl was particularly effective at suppressing sensitivity to cationic compounds. Changes in membrane polarization are thus likely to underlie this effect. However, changes to the specificity and kinetics of membrane transporters may also be involved. Our findings suggest that potassium rich environments may allow S. pombe to compete more effectively with organisms that produce antimicrobial agents in its natural environment. Modulating potassium homeostasis in fungal pathogens may also provide a strategy to suppress their resistance to some antifungal agents.

Supporting Information

S1 Fig. Effect of KCl on drug sensitivity in S. pombe.

A. Wt and rad24Δ mutant strains were incubated in the presence of 10 μg/ ml phleomycin alone or with the indicated concentrations of KCl. Equal cell numbers were plated on YES agar and incubated for 2–3 days at 30°C. B- D. Minimum inhibitory concentrations (MICs) for phleomycin, G418 and hygomycin B in wt S. pombe cells was determined by incubating cultures for 24 h in the presence of the indicated drug concentrations. E. S. pombe cells were incubated with 10 μg/ ml phleomycin, or phleomycin (10 mg/ ml) incubated in an equal volume of 0.6 M KCl for 1 h and then diluted to 10 μg/ ml. The cells were exposed for 4 h and then plated in equal numbers on YES agar. F. rad24Δ mutants were exposed to 10 μg/ ml phleomycin alone and with the indicated concentrations of KCl for 4 h. The cells were fixed in 70% ethanol, stained with DAPI and examined by fluorescence microscopy. Arrows indicate cells with mis-segregated chromosomes. G. Wt cells were treated with 10 μM latrunculin B (LatB) or 50 μg/ ml MBC for 4 h and 7 h respectively, fixed in ethanol and treated as in G.

https://doi.org/10.1371/journal.pone.0119297.s001

(PPTX)

S2 Fig. Effect of external ions on drug and metal sensitivity in S. pombe.

A. Cells were exposed to 10 μg/ ml phleomycin ± the indicated concentrations of RbCl for 24 h. Equal numbers of cells were plated on YES agar. B. Cells were treated as in A with phleomycin ± the indicated concentrations of NH4Cl. C- D. Cells were treated as in A but with the indicated compounds. E. Relative growth of S. pombe cells in the presence of 10 μg/ ml phleomycin ± the indicated compounds for 24h. Data represent the means of 3 experiments ± S.E. F. S. pombe cells were exposed to 0.4% acetic acid for 24 h and treated as in A. G. Wild type cells were exposed to the indicated compounds for 24 h and treated as in A. H. Wild type S. pombe cells were incubated with 40 μg/ ml doxorubicin alone and with 0.6 M KCl or 1.2 M sorbitol for 2 h and examined by fluorescent microscopy.

https://doi.org/10.1371/journal.pone.0119297.s002

(PPTX)

S3 Fig. KCl suppresses drug sensitivity in S. pombe independently of Sty1, Sod2, Pzh1 and Hal4.

A. Wt and sty1Δ strains were incubated with the indicated concentrations of G418 for 24 h. Equal cell numbers were plated on YES agar and incubated at 30°C for 2–3 days. B. Wt and sod2Δ strains were incubated for 4 h in the presence of 20 μg/ ml phleomycin ± 0.6 M KCl and then treated as in A. C. pzh1Δ mutants were incubated for 4 h in the presence of 10 μg/ ml phleomycin ± 0.3 M KCl and then treated as in A. D. pzh1Δ mutants from C were fixed in 70% ethanol, stained with DAPI and examined by florescence microscopy. E. Wild type, hal4Δ and sod2Δ strains were incubated with 0.004 M LiCl for 4 h and then treated as in A.

https://doi.org/10.1371/journal.pone.0119297.s003

(PPTX)

S4 Fig. Effect of KCl on the sensitivity of S. pombe to cell wall disrupting agents.

A. S. pombe cells were exposed to 1.0 μg/ ml amphotericin B ± the indicated concentrations of KCl for 24 h. Equal cell numbers were plated on YES agar and incubated at 30°C for 2–3 days. B. Cells were exposed to 1.0 μg/ ml caspofungin alone or together with the indicated concentrations of sorbitol and treated as in A.

https://doi.org/10.1371/journal.pone.0119297.s004

(PPTX)

S5 Fig. Molecular structures of compounds investigated in this study.

Figures were obtained from the supplier web site or www.wikipedia.com.

https://doi.org/10.1371/journal.pone.0119297.s005

(PPTX)

Acknowledgments

We thank J. Ariño for the pzh1Δ and sod2Δ strains, and K. Shiozaki for the hal4Δ and trk1Δ trk2Δ strains.

Author Contributions

Conceived and designed the experiments: JPA PS. Performed the experiments: JPA AMW AS. Analyzed the data: JPA. Wrote the paper: JPA.

References

  1. 1. Aouida M, Tounekti O, Leduc A, Belhadj O, Mir L, Ramotar D. Isolation and characterization of Saccharomyces cerevisiae mutants with enhanced resistance to the anticancer drug bleomycin. Current genetics 2004, 45(5):265–272. pmid:15007625
  2. 2. Asher V, Sowter H, Shaw R, Bali A, Khan R. Eag and HERG potassium channels as novel therapeutic targets in cancer. World journal of surgical oncology 2010, 8:113. pmid:21190577
  3. 3. Bianchi L, Wible B, Arcangeli A, Taglialatela M, Morra F, Castaldo P, et al. herg encodes a K+ current highly conserved in tumors of different histogenesis: a selective advantage for cancer cells? Cancer research 1998, 58(4):815–822. pmid:9485040
  4. 4. Castaneda-Garcia A, Do TT, Blazquez J. The K+ uptake regulator TrkA controls membrane potential, pH homeostasis and multidrug susceptibility in Mycobacterium smegmatis. The Journal of antimicrobial chemotherapy 2011, 66(7):1489–1498. pmid:21613307
  5. 5. Lafon-Lafourcade S, Lonvaud-Funel A, Carre E. Lactic acid bacteria of wines: stimulation of growth and malolactic fermentation. Antonie van Leeuwenhoek 1983, 49(3):349–352. pmid:6354084
  6. 6. Navarro L, Zarazaga M, Saenz J, Ruiz-Larrea F, Torres C. Bacteriocin production by lactic acid bacteria isolated from Rioja red wines. Journal of applied microbiology 2000, 88(1):44–51. pmid:10735242
  7. 7. Nkanga EJ, Hagedorn C. Detection of antibiotic-producing Streptomyces inhabiting forest soils. Antimicrobial agents and chemotherapy 1978, 14(1):51–59. pmid:686709
  8. 8. Thornton G, Wilkinson CR, Toone WM, Jones N. A novel pathway determining multidrug sensitivity in Schizosaccharomyces pombe. Genes Cells 2005, 10(10):941–951. pmid:16164595
  9. 9. Calero F, Ramos J. K+ fluxes in Schizosaccharomyces pombe. FEMS yeast research 2003, 4(1):1–6.
  10. 10. Arino J, Ramos J, Sychrova H. Alkali metal cation transport and homeostasis in yeasts. Microbiol Mol Biol Rev 2010, 74(1):95–120. pmid:20197501
  11. 11. Adam C, Erdei E, Casado C, Kovacs L, Gonzalez A, Majoros L, et al. Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans. Microbiology (Reading, England) 2012, 158(Pt 5):1258–1267. pmid:22343349
  12. 12. Balcells L, Gomez N, Casamayor A, Clotet J, Arino J. Regulation of salt tolerance in fission yeast by a protein-phosphatase-Z-like Ser/Thr protein phosphatase. uropean journal of biochemistry / FEBS 1997, 250(2):476–483. pmid:9428701
  13. 13. Perlin DS, Brown CL, Haber JE. Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae. The Journal of biological chemistry 1988, 263(34):18118–18122. pmid:3056938
  14. 14. Brown WR, Liti G, Rosa C, James S, Roberts I, Robert V, et al. A Geographically Diverse Collection of Schizosaccharomyces pombe Isolates Shows Limited Phenotypic Variation but Extensive Karyotypic Diversity. G3 (Bethesda, Md 2011, 1(7):615–626.
  15. 15. Konings WN, Kok J, Kuipers OP, Poolman B. Lactic acid bacteria: the bugs of the new millennium. Current opinion in microbiology 2000, 3(3):276–282.
  16. 16. Kagan BL. Mode of action of yeast killer toxins: channel formation in lipid bilayer membranes. Nature 1983, 302(5910):709–711. pmid:6300695
  17. 17. Schmitt MJ, Breinig F. Yeast viral killer toxins: lethality and self-protection. Nature reviews 2006, 4(3):212–221.
  18. 18. Faparusi SI, Bassir O. Effect of Extracts of the Bark of Saccoglottis gabonensis on the Microflora of Palm Wine. Applied microbiology 1972, 24(6):853–856. pmid:16349950
  19. 19. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415(6870):389–395. pmid:11807545
  20. 20. Canovas D, de Lorenzo V. Osmotic stress limits arsenic hypertolerance in Aspergillus sp. P37. FEMS microbiology ecology 2007, 61(2):258–263. pmid:17578525
  21. 21. Shapiro RS, Robbins N, Cowen LE. Regulatory circuitry governing fungal development, drug resistance, and disease. Microbiol Mol Biol Rev 2011, 75(2):213–267. pmid:21646428
  22. 22. Stella CA, Burgos HI. Effect of potassium on Saccharomyces cerevisiae resistance to fluconazole. Antimicrobial agents and chemotherapy 2001, 45(5):1589–1590. pmid:11302836
  23. 23. Alcazar-Fuoli L, Mellado E. Current status of antifungal resistance and its impact on clinical practice. British journal of haematology 2014, 166(4):471–484. pmid:24749533
  24. 24. Miceli MH, Lee SA. Emerging moulds: epidemiological trends and antifungal resistance. Mycoses 2011, 54(6):e666–678. pmid:21672045
  25. 25. Alao JP, Huis In 't Veld PJ, Buhse F, Sunnerhagen P. Hyperosmosis enhances radiation and hydroxyurea resistance of Schizosaccharomyces pombe checkpoint mutants through the spindle checkpoint and delayed cytokinesis. Molecular microbiology 2010, 77(1):143–157. pmid:20444100
  26. 26. Forsburg SL. Growth and manipulation of S. pombe. Current protocols in molecular biology / edited by Ausubel Frederick M [et al] 2003, Chapter 13:Unit 13 16.
  27. 27. Walworth NC, Bernards R. rad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science 1996, 271(5247):353–356. pmid:8553071
  28. 28. Alao JP, Sunnerhagen P. Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress? Molecular microbiology 2008, 68(2):246–254. pmid:18366437
  29. 29. Wang LY, Shimada K, Morishita M, Shiozaki K. Response of fission yeast to toxic cations involves cooperative action of the stress-activated protein kinase Spc1/Sty1 and the Hal4 protein kinase. Molecular and cellular biology 2005, 25(10):3945–3955. pmid:15870269
  30. 30. Calero F, Gomez N, Arino J, Ramos J. Trk1 and Trk2 define the major K(+) transport system in fission yeast. Journal of bacteriology 2000, 182(2):394–399. pmid:10629185
  31. 31. Lupetti A, Danesi R, Campa M, Del Tacca M, Kelly S. Molecular basis of resistance to azole antifungals. Trends in molecular medicine 2002, 8(2):76–81. pmid:11815273
  32. 32. Vanden Bossche H, Marichal P, Gorrens J, Coene MC, Willemsens G, Bellens D, et al. Biochemical approaches to selective antifungal activity. Focus on azole antifungals. Mycoses 1989, 32 Suppl 1:35–52. pmid:2561184
  33. 33. Georgopapadakou NH, Walsh TJ. Antifungal agents: chemotherapeutic targets and immunologic strategies. Antimicrobial agents and chemotherapy 1996, 40(2):279–291. pmid:8834867
  34. 34. Sheehan DJ, Hitchcock CA, Sibley CM. Current and emerging azole antifungal agents. Clinical microbiology reviews 1999, 12(1):40–79. pmid:9880474
  35. 35. Vertut-Doi A, Hannaert P, Bolard J. The polyene antibiotic amphotericin B inhibits the Na+/K+ pump of human erythrocytes. Biochemical and biophysical research communications 1988, 157(2):692–697. pmid:2849435
  36. 36. Deresinski SC, Stevens DA. Caspofungin. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 2003, 36(11):1445–1457.
  37. 37. Ahmed S, Booth IR. The use of valinomycin, nigericin and trichlorocarbanilide in control of the protonmotive force in Escherichia coli cells. The Biochemical journal 1983, 212(1):105–112. pmid:6307285
  38. 38. Guffanti AA, Davidson LF, Mann TM, Krulwich TA. Nigericin-induced death of an acidophilic bacterium. Journal of general microbiology 1979, 114(1):201–206. pmid:42667
  39. 39. Yenush L, Mulet JM, Arino J, Serrano R. The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression. The EMBO journal 2002, 21(5):920–929. pmid:11867520
  40. 40. Madrid R, Gomez MJ, Ramos J, Rodriguez-Navarro A. Ectopic potassium uptake in trk1 trk2 mutants of Saccharomyces cerevisiae correlates with a highly hyperpolarized membrane potential. The Journal of biological chemistry 1998, 273(24):14838–14844. pmid:9614085
  41. 41. Wright MB, Ramos J, Gomez MJ, Moulder K, Scherrer M, Munson G, et al. Potassium transport by amino acid permeases in Saccharomyces cerevisiae. The Journal of biological chemistry 1997, 272(21):13647–13652. pmid:9153214
  42. 42. Villalobo A. Potassium transport coupled to ATP hydrolysis in reconstituted proteoliposomes of yeast plasma membrane ATPase. The Journal of biological chemistry 1982, 257(4):1824–1828. pmid:6120168
  43. 43. Aouida M, Leduc A, Wang H, Ramotar D. Characterization of a transport and detoxification pathway for the antitumour drug bleomycin in Saccharomyces cerevisiae. The Biochemical journal 2004, 384(Pt 1):47–58. pmid:15248838
  44. 44. Aouida M, Page N, Leduc A, Peter M, Ramotar D. A genome-wide screen in Saccharomyces cerevisiae reveals altered transport as a mechanism of resistance to the anticancer drug bleomycin. Cancer research 2004, 64(3):1102–1109. pmid:14871844
  45. 45. Teixeira MC, Cabrito TR, Hanif ZM, Vargas RC, Tenreiro S, Sa-Correia I. Yeast response and tolerance to polyamine toxicity involving the drug: H+ antiporter Qdr3 and the transcription factors Yap1 and Gcn4. Microbiology (Reading, England) 2011, 157(Pt 4):945–956.
  46. 46. Tenreiro S, Vargas RC, Teixeira MC, Magnani C, Sa-Correia I. The yeast multidrug transporter Qdr3 (Ybr043c): localization and role as a determinant of resistance to quinidine, barban, cisplatin, and bleomycin. Biochemical and biophysical research communications 2005, 327(3):952–959. pmid:15649438
  47. 47. Vargas RC, Garcia-Salcedo R, Tenreiro S, Teixeira MC, Fernandes AR, Ramos J, et al. Saccharomyces cerevisiae multidrug resistance transporter Qdr2 is implicated in potassium uptake, providing a physiological advantage to quinidine-stressed cells. Eukaryotic cell 2007, 6(2):134–142. pmid:17189489
  48. 48. Graven SN, Estrada OS, Lardy HA. Alkali metal cation release and respiratory inhibition induced by nigericin in rat liver mitochondria. Proceedings of the National Academy of Sciences of the United States of America 1966, 56(2):654–658. pmid:5229984
  49. 49. Cowen LE, Steinbach WJ. Stress, drugs, and evolution: the role of cellular signaling in fungal drug resistance. Eukaryotic cell 2008, 7(5):747–764. pmid:18375617
  50. 50. Yang XX, Maurer KC, Molanus M, Mager WH, Siderius M, van der Vies SM. The molecular chaperone Hsp90 is required for high osmotic stress response in Saccharomyces cerevisiae. FEMS yeast research 2006, 6(2):195–204. pmid:16487343
  51. 51. Zhang YQ, Gamarra S, Garcia-Effron G, Park S, Perlin DS, Rao R. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS pathogens 2010, 6(6):e1000939. pmid:20532216
  52. 52. Hohmann S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 2002, 66(2):300–372. pmid:12040128
  53. 53. Gullberg E, Cao S, Berg OG, Ilback C, Sandegren L, Hughes D, et al. Selection of resistant bacteria at very low antibiotic concentrations. PLoS pathogens 2011, 7(7):e1002158. pmid:21811410