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Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans

View Article: PubMed Central - PubMed

ABSTRACT

The development of new drugs against the pathogenic yeast Candida albicans is compelling and the evolution of relevant bioassays is important to achieve this goal. Promising drug targets are proteins that lack human counterparts which are true for the His-to-Asp phosphorelay signal transduction systems, important for stress sensing in bacteria, fungi, and plants. In the pathogenic yeast, Candida albicans, the CaChk1 histidine kinase is a trigger of the pathway that leads to a switch from yeast to hyphal growth necessary for invasion. Intriguingly, the model yeast Schizosaccharomyces pombe has a similar phosphorelay system, with three histidine kinases named Mak1, Mak2, and Mak3, which are important for the prevention of aberrant mating and sporulation on rich media. This study uncovered distinct functions for the three histidine kinases; Mak1 alone or Mak2 and Mak3 together were sufficient for the repression of the meiotic cycle when nutrients were available. Moreover, strains lacking histidine kinase genes were sensitive to various types of stress conditions in an auxotrophic strain background, while the stress sensitivity was lost in prototrophic strains. Finally, the stress sensitivity of a S. pombe strain that lacks endogenous histidine kinases could be complemented by the ectopic expression of the CaChk1 histidine kinase from C. albicans. This finding opens up for the possibility to perform a drug screen with a biological read-out in S. pombe to find inhibitors of CaChk1.

Electronic supplementary material: The online version of this article (doi:10.1007/s00294-016-0644-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Mak1 alone or Mak2 and Mak3 together repress sporulation on rich media. a 20 μl of cell cultures of homothallic, h90, strains PJ1329 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) were spotted onto rich YEA media and grown for 3 days at 20 °C. The plate was stained by iodine vapour, and strains that were stained brown contained spore asci, while strains with vegetatively growing cells stained yellow. b The same cell cultures as in (a) were inspected under the microscope and the number of vegetatively growing cells (white bar), conjugating cells (black bar), and spore asci (grey bar) was counted (n = 3). 200 cells from three independent cultures were counted for each genotype and the average number of vegetatively growing cells (white), conjugating cells (black), and spore asci (grey) ±SD was plotted. Relevant genotypes are indicated in the picture. c 10 μl of log-phase cultures of strain PJ1882 with plasmids pREP3X(−) or pREP3X with Spmak1+ (Mak1), Spmak2+ (Mak2), Spmak3+ (Mak3), CaCHK1 (Chk1), CaNIK1 (Nik1), or CaSLN1 (Sln1) were spotted onto PMG-Leu plates with or w/o thiamine and incubated at 30 °C for 36 or 72 h
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Fig2: Mak1 alone or Mak2 and Mak3 together repress sporulation on rich media. a 20 μl of cell cultures of homothallic, h90, strains PJ1329 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) were spotted onto rich YEA media and grown for 3 days at 20 °C. The plate was stained by iodine vapour, and strains that were stained brown contained spore asci, while strains with vegetatively growing cells stained yellow. b The same cell cultures as in (a) were inspected under the microscope and the number of vegetatively growing cells (white bar), conjugating cells (black bar), and spore asci (grey bar) was counted (n = 3). 200 cells from three independent cultures were counted for each genotype and the average number of vegetatively growing cells (white), conjugating cells (black), and spore asci (grey) ±SD was plotted. Relevant genotypes are indicated in the picture. c 10 μl of log-phase cultures of strain PJ1882 with plasmids pREP3X(−) or pREP3X with Spmak1+ (Mak1), Spmak2+ (Mak2), Spmak3+ (Mak3), CaCHK1 (Chk1), CaNIK1 (Nik1), or CaSLN1 (Sln1) were spotted onto PMG-Leu plates with or w/o thiamine and incubated at 30 °C for 36 or 72 h

Mentions: Previously, it was reported that the three HKs were essential for repressing the mating and sporulation pathway when cells are grown on rich media, containing nitrogen and glucose. This was observed, since a strain lacking all three HKs entered the sexual cycle unrestrained when grown on rich media. All the HKs acted redundantly for this function meaning that all three HKs had to be deleted to obtain this phenotype (Nakamichi et al. 2002). To find out whether our precise deletions strains had the same phenotype, cell suspensions of the eight strains; wild-type, single deletions; mak1Δ, mak2Δ, mak3Δ, double deletions; mak1Δ mak2Δ (mak1,2Δ), mak1Δ mak3Δ (mak1,3Δ), mak2Δ mak3Δ (mak2,3Δ), and, finally, the triple deletion; mak1Δ mak2Δ mak3Δ (mak1,2,3Δ) strain were spotted on rich YEA plates and incubated at 20 or 30 °C for 3 days. At 30 °C, there was no difference in sporulation between the strains, but at the lower temperature of 20 °C, the double deletion strains lacking Mak1 and Mak2 (mak1,2Δ) or Mak3 (mak1,3Δ) as well as the triple deletion strain (mak1,2,3Δ) displayed mating and sporulation even in the presence of nitrogen and glucose. The sporulation can be observed by staining the yeast with iodine vapours, since only the spore asci stain brown, while vegetatively growing cells appear yellow. Spots from the wild-type, the single knockout and the double deletion (mak2,3Δ) strains were yellow after treatment with iodine vapours, whereas the other double deletion strains (mak1,2Δ and mak1,3Δ) and the triple deletion strain (mak1,2,3Δ) stained brown (Fig. 2a). To further characterise this phenotype, the number of conjugating cells and spore asci were counted using light microscopy (Fig. 2b). It was evident that the single knockout strains had the same number of conjugating cells and spore asci as the wild-type strain, whereas the strain with deletions of mak2+ and mak3+ (mak2,3Δ) showed only a slight increase in both these parameters. The other double deletion strains, mak1,2Δ and mak1,3Δ, and the strain lacking all three HKs, mak1,2,3Δ, had clearly increased number of both conjugating cells and spore asci with 25 % conjugating cells in the mak1,3Δ double deletion strains and the triple deletion as compared with 5 % in the wild-type strain. Furthermore, the number of spore asci was up to 35 % in the triple knockout as compared with around 1 % in the wild-type strain (Fig. 2b).Fig. 2


Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans
Mak1 alone or Mak2 and Mak3 together repress sporulation on rich media. a 20 μl of cell cultures of homothallic, h90, strains PJ1329 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) were spotted onto rich YEA media and grown for 3 days at 20 °C. The plate was stained by iodine vapour, and strains that were stained brown contained spore asci, while strains with vegetatively growing cells stained yellow. b The same cell cultures as in (a) were inspected under the microscope and the number of vegetatively growing cells (white bar), conjugating cells (black bar), and spore asci (grey bar) was counted (n = 3). 200 cells from three independent cultures were counted for each genotype and the average number of vegetatively growing cells (white), conjugating cells (black), and spore asci (grey) ±SD was plotted. Relevant genotypes are indicated in the picture. c 10 μl of log-phase cultures of strain PJ1882 with plasmids pREP3X(−) or pREP3X with Spmak1+ (Mak1), Spmak2+ (Mak2), Spmak3+ (Mak3), CaCHK1 (Chk1), CaNIK1 (Nik1), or CaSLN1 (Sln1) were spotted onto PMG-Leu plates with or w/o thiamine and incubated at 30 °C for 36 or 72 h
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Fig2: Mak1 alone or Mak2 and Mak3 together repress sporulation on rich media. a 20 μl of cell cultures of homothallic, h90, strains PJ1329 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) were spotted onto rich YEA media and grown for 3 days at 20 °C. The plate was stained by iodine vapour, and strains that were stained brown contained spore asci, while strains with vegetatively growing cells stained yellow. b The same cell cultures as in (a) were inspected under the microscope and the number of vegetatively growing cells (white bar), conjugating cells (black bar), and spore asci (grey bar) was counted (n = 3). 200 cells from three independent cultures were counted for each genotype and the average number of vegetatively growing cells (white), conjugating cells (black), and spore asci (grey) ±SD was plotted. Relevant genotypes are indicated in the picture. c 10 μl of log-phase cultures of strain PJ1882 with plasmids pREP3X(−) or pREP3X with Spmak1+ (Mak1), Spmak2+ (Mak2), Spmak3+ (Mak3), CaCHK1 (Chk1), CaNIK1 (Nik1), or CaSLN1 (Sln1) were spotted onto PMG-Leu plates with or w/o thiamine and incubated at 30 °C for 36 or 72 h
Mentions: Previously, it was reported that the three HKs were essential for repressing the mating and sporulation pathway when cells are grown on rich media, containing nitrogen and glucose. This was observed, since a strain lacking all three HKs entered the sexual cycle unrestrained when grown on rich media. All the HKs acted redundantly for this function meaning that all three HKs had to be deleted to obtain this phenotype (Nakamichi et al. 2002). To find out whether our precise deletions strains had the same phenotype, cell suspensions of the eight strains; wild-type, single deletions; mak1Δ, mak2Δ, mak3Δ, double deletions; mak1Δ mak2Δ (mak1,2Δ), mak1Δ mak3Δ (mak1,3Δ), mak2Δ mak3Δ (mak2,3Δ), and, finally, the triple deletion; mak1Δ mak2Δ mak3Δ (mak1,2,3Δ) strain were spotted on rich YEA plates and incubated at 20 or 30 °C for 3 days. At 30 °C, there was no difference in sporulation between the strains, but at the lower temperature of 20 °C, the double deletion strains lacking Mak1 and Mak2 (mak1,2Δ) or Mak3 (mak1,3Δ) as well as the triple deletion strain (mak1,2,3Δ) displayed mating and sporulation even in the presence of nitrogen and glucose. The sporulation can be observed by staining the yeast with iodine vapours, since only the spore asci stain brown, while vegetatively growing cells appear yellow. Spots from the wild-type, the single knockout and the double deletion (mak2,3Δ) strains were yellow after treatment with iodine vapours, whereas the other double deletion strains (mak1,2Δ and mak1,3Δ) and the triple deletion strain (mak1,2,3Δ) stained brown (Fig. 2a). To further characterise this phenotype, the number of conjugating cells and spore asci were counted using light microscopy (Fig. 2b). It was evident that the single knockout strains had the same number of conjugating cells and spore asci as the wild-type strain, whereas the strain with deletions of mak2+ and mak3+ (mak2,3Δ) showed only a slight increase in both these parameters. The other double deletion strains, mak1,2Δ and mak1,3Δ, and the strain lacking all three HKs, mak1,2,3Δ, had clearly increased number of both conjugating cells and spore asci with 25 % conjugating cells in the mak1,3Δ double deletion strains and the triple deletion as compared with 5 % in the wild-type strain. Furthermore, the number of spore asci was up to 35 % in the triple knockout as compared with around 1 % in the wild-type strain (Fig. 2b).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

The development of new drugs against the pathogenic yeast Candida albicans is compelling and the evolution of relevant bioassays is important to achieve this goal. Promising drug targets are proteins that lack human counterparts which are true for the His-to-Asp phosphorelay signal transduction systems, important for stress sensing in bacteria, fungi, and plants. In the pathogenic yeast, Candida albicans, the CaChk1 histidine kinase is a trigger of the pathway that leads to a switch from yeast to hyphal growth necessary for invasion. Intriguingly, the model yeast Schizosaccharomyces pombe has a similar phosphorelay system, with three histidine kinases named Mak1, Mak2, and Mak3, which are important for the prevention of aberrant mating and sporulation on rich media. This study uncovered distinct functions for the three histidine kinases; Mak1 alone or Mak2 and Mak3 together were sufficient for the repression of the meiotic cycle when nutrients were available. Moreover, strains lacking histidine kinase genes were sensitive to various types of stress conditions in an auxotrophic strain background, while the stress sensitivity was lost in prototrophic strains. Finally, the stress sensitivity of a S. pombe strain that lacks endogenous histidine kinases could be complemented by the ectopic expression of the CaChk1 histidine kinase from C. albicans. This finding opens up for the possibility to perform a drug screen with a biological read-out in S. pombe to find inhibitors of CaChk1.

Electronic supplementary material: The online version of this article (doi:10.1007/s00294-016-0644-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus