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Suppression of sensitivity to drugs and antibiotics by high external cation concentrations in fission yeast.

Alao JP, Weber AM, Shabro A, Sunnerhagen P - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

No MeSH data available.


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.
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pone.0119297.g003: 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.

Mentions: 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].


Suppression of sensitivity to drugs and antibiotics by high external cation concentrations in fission yeast.

Alao JP, Weber AM, Shabro A, Sunnerhagen P - PLoS ONE (2015)

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.
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pone.0119297.g003: 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.
Mentions: 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].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

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.

No MeSH data available.