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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

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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.

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Complementation of the stress sensitive phenotype in a strain lacking endogenous HKs by the Chk1 HK from C. albicans. The wild-type strain FY368 (h−) transformed with empty vector pREP3X and the strain PJ1713 (h−, mak1,2,3Δ) transformed with pREP3X or pREP3X-mak1+, pREP3X-mak2+, pREP3X-mak3+, pREP3X-CaCHK1, pREP3X-CaNIK1, or pREP3X-CaSLN1 were serial diluted in five steps (5-fold per step) and 5 μl were spotted onto YEA plates and AA plates with (+T, left panels) or without (−T, right panels) thiamine. a Non-selective YEA plates, AA plates without leucine to select for the plasmid (AA-Leu), b AA-Leu with 1 M sorbitol, and c AA-Leu with 75 mM NaCl
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Fig4: Complementation of the stress sensitive phenotype in a strain lacking endogenous HKs by the Chk1 HK from C. albicans. The wild-type strain FY368 (h−) transformed with empty vector pREP3X and the strain PJ1713 (h−, mak1,2,3Δ) transformed with pREP3X or pREP3X-mak1+, pREP3X-mak2+, pREP3X-mak3+, pREP3X-CaCHK1, pREP3X-CaNIK1, or pREP3X-CaSLN1 were serial diluted in five steps (5-fold per step) and 5 μl were spotted onto YEA plates and AA plates with (+T, left panels) or without (−T, right panels) thiamine. a Non-selective YEA plates, AA plates without leucine to select for the plasmid (AA-Leu), b AA-Leu with 1 M sorbitol, and c AA-Leu with 75 mM NaCl

Mentions: To investigate whether the overexpression of any of the HKs could rescue the stress sensitive phenotype of the mak1,2,3Δ strain, the S. pombe triple knockout strain was supplemented with one of the three HKs from S. pombe or one of the HKs from C. albicans. The empty vector, pREP3X, was transformed into a heterothallic, h−, wild-type strain as well as a strain lacking all HKs, mak1,2,3Δ, both strains with the same auxotrophic markers, ura4-D18 leu1-32 ade6-M216. In addition, the mak1,2,3Δ strain was transformed with a pREP3X plasmids expressing one of the six different HKs genes. Log-phase cultures in defined AA media lacking leucine (to select for the plasmid) were serial diluted and spotted onto unselective YEA plates and selective AA plates without leucine, with or w/o thiamine, containing 1 M sorbitol or 75 mM NaCl and onto control plates without supplements. All the strains grew fine on the rich YEA plates, but on selective plates, the result was different. First, the wild-type strain had full growth on all plates in contrast to the strain lacking the HKs (Fig. 4a, compare the first and second rows). Second, the mak1,2,3Δ strain was severely inhibited on the plates containing sorbitol or sodium chloride (Fig. 4b, c, second row). The expression of mak1+ was slightly beneficial for the mak1,2,3Δ strain (Fig. 4, compare rows 2 and 3); however, the effect was only partial. Ectopic expression of mak2+ or mak3+ was neutral and high expression, under induced conditions, of mak2+ resulted in poor growth (Fig. 4, compare row 2 with row 4 or 5). To our surprise, the growth reduction of the mak1,2,3Δ strain was completely rescued by the ectopic expression of the CaCHK1 gene (Fig. 4, compare rows 2 and 6). The rescue was dependent on the presence of thiamine (+T) in the media, i.e., under a basal expression level (Fig. 4, left panels). The plates without thiamine (−T) most likely resulted in a too high expression level of the CaCHK1 gene, since there was almost no rescue of the poor growth phenotype on these plates, underscoring the importance of a correct expression level of the HKs (Fig. 4, right panels, compare rows 2 and 6). Finally, the expression of the CaNik1 or CaSln1 HKs was neutral for the mak1,2,3Δ strain (Fig. 4, two last rows). Similar results were obtained using a set of homothallic strains with the same auxotrophic markers and carrying the same plasmids (Fig. S3).Fig. 4


Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans
Complementation of the stress sensitive phenotype in a strain lacking endogenous HKs by the Chk1 HK from C. albicans. The wild-type strain FY368 (h−) transformed with empty vector pREP3X and the strain PJ1713 (h−, mak1,2,3Δ) transformed with pREP3X or pREP3X-mak1+, pREP3X-mak2+, pREP3X-mak3+, pREP3X-CaCHK1, pREP3X-CaNIK1, or pREP3X-CaSLN1 were serial diluted in five steps (5-fold per step) and 5 μl were spotted onto YEA plates and AA plates with (+T, left panels) or without (−T, right panels) thiamine. a Non-selective YEA plates, AA plates without leucine to select for the plasmid (AA-Leu), b AA-Leu with 1 M sorbitol, and c AA-Leu with 75 mM NaCl
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Related In: Results  -  Collection

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Fig4: Complementation of the stress sensitive phenotype in a strain lacking endogenous HKs by the Chk1 HK from C. albicans. The wild-type strain FY368 (h−) transformed with empty vector pREP3X and the strain PJ1713 (h−, mak1,2,3Δ) transformed with pREP3X or pREP3X-mak1+, pREP3X-mak2+, pREP3X-mak3+, pREP3X-CaCHK1, pREP3X-CaNIK1, or pREP3X-CaSLN1 were serial diluted in five steps (5-fold per step) and 5 μl were spotted onto YEA plates and AA plates with (+T, left panels) or without (−T, right panels) thiamine. a Non-selective YEA plates, AA plates without leucine to select for the plasmid (AA-Leu), b AA-Leu with 1 M sorbitol, and c AA-Leu with 75 mM NaCl
Mentions: To investigate whether the overexpression of any of the HKs could rescue the stress sensitive phenotype of the mak1,2,3Δ strain, the S. pombe triple knockout strain was supplemented with one of the three HKs from S. pombe or one of the HKs from C. albicans. The empty vector, pREP3X, was transformed into a heterothallic, h−, wild-type strain as well as a strain lacking all HKs, mak1,2,3Δ, both strains with the same auxotrophic markers, ura4-D18 leu1-32 ade6-M216. In addition, the mak1,2,3Δ strain was transformed with a pREP3X plasmids expressing one of the six different HKs genes. Log-phase cultures in defined AA media lacking leucine (to select for the plasmid) were serial diluted and spotted onto unselective YEA plates and selective AA plates without leucine, with or w/o thiamine, containing 1 M sorbitol or 75 mM NaCl and onto control plates without supplements. All the strains grew fine on the rich YEA plates, but on selective plates, the result was different. First, the wild-type strain had full growth on all plates in contrast to the strain lacking the HKs (Fig. 4a, compare the first and second rows). Second, the mak1,2,3Δ strain was severely inhibited on the plates containing sorbitol or sodium chloride (Fig. 4b, c, second row). The expression of mak1+ was slightly beneficial for the mak1,2,3Δ strain (Fig. 4, compare rows 2 and 3); however, the effect was only partial. Ectopic expression of mak2+ or mak3+ was neutral and high expression, under induced conditions, of mak2+ resulted in poor growth (Fig. 4, compare row 2 with row 4 or 5). To our surprise, the growth reduction of the mak1,2,3Δ strain was completely rescued by the ectopic expression of the CaCHK1 gene (Fig. 4, compare rows 2 and 6). The rescue was dependent on the presence of thiamine (+T) in the media, i.e., under a basal expression level (Fig. 4, left panels). The plates without thiamine (−T) most likely resulted in a too high expression level of the CaCHK1 gene, since there was almost no rescue of the poor growth phenotype on these plates, underscoring the importance of a correct expression level of the HKs (Fig. 4, right panels, compare rows 2 and 6). Finally, the expression of the CaNik1 or CaSln1 HKs was neutral for the mak1,2,3Δ strain (Fig. 4, two last rows). Similar results were obtained using a set of homothallic strains with the same auxotrophic markers and carrying the same plasmids (Fig. S3).Fig. 4

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