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

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.

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.

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.

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.

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.

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.

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.