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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.


Auxotrophic markers contribute to a stress sensitive phenotype. Cell cultures from strains with the h− mating-type configuration were serially diluted in five steps (5-fold per step) and 5 μl were spotted onto rich YEA plates with or without supplements and grown at 30 °C for three days. a Strains with HKs carrying different auxotrophic markers: 972 h-(wt) PJ278 (ura4-D18), PJ1848 (leu1-32), PJ1933 (ade6-M216), PJ1895 (leu1-32 ura4-D18), PJ239 (leu1-32 ade6-M210), PJ1934 (ura4-D18 ade6-M216), FY368 (leu1-32 ura4-D18 ade6-M216). b Strains lacking Mak1, Mak2 and Mak3 (mak1,2,3Δ), with different auxotrophic markers: PJ1903 (mak1,2,3Δ), PJ1951 (mak1,2,3Δ ura4-D18), PJ1846 (mak1,2,3Δ leu1-32), PJ1948 (mak1,2,3Δ ade6-M216) PJ1949 (mak1,2,3Δ ura4-D18 leu1-32) PJ1947 (mak1,2,3Δ leu1-32 ade6-M216) PJ1950 (mak1,2,3Δ ura4-D18 ade6-M216) PJ1713 (mak1,2,3Δ ura4-D18 leu1-32 ade6-M216)
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Fig7: Auxotrophic markers contribute to a stress sensitive phenotype. Cell cultures from strains with the h− mating-type configuration were serially diluted in five steps (5-fold per step) and 5 μl were spotted onto rich YEA plates with or without supplements and grown at 30 °C for three days. a Strains with HKs carrying different auxotrophic markers: 972 h-(wt) PJ278 (ura4-D18), PJ1848 (leu1-32), PJ1933 (ade6-M216), PJ1895 (leu1-32 ura4-D18), PJ239 (leu1-32 ade6-M210), PJ1934 (ura4-D18 ade6-M216), FY368 (leu1-32 ura4-D18 ade6-M216). b Strains lacking Mak1, Mak2 and Mak3 (mak1,2,3Δ), with different auxotrophic markers: PJ1903 (mak1,2,3Δ), PJ1951 (mak1,2,3Δ ura4-D18), PJ1846 (mak1,2,3Δ leu1-32), PJ1948 (mak1,2,3Δ ade6-M216) PJ1949 (mak1,2,3Δ ura4-D18 leu1-32) PJ1947 (mak1,2,3Δ leu1-32 ade6-M216) PJ1950 (mak1,2,3Δ ura4-D18 ade6-M216) PJ1713 (mak1,2,3Δ ura4-D18 leu1-32 ade6-M216)

Mentions: It was also evident that the ura4-D18 leu1-32 auxotrophic mutations in the wild-type background affected the strains survival on plates with elevated NaCl levels (Fig. 6, bottom left, compare the first two rows). A recent publication reports that the ura4 deletion makes the cell wall of S. pombe less robust (Matsuo et al. 2013). The weakened cell wall results in a higher uptake of Phloxin B on peptone-containing plates, which gives rise to red colonies specifically in the ura4Δ strain (Matsuo et al. 2013). The weaker cell wall in the ura4 deletion strains might explain the increased stress sensitivity in the strains lacking both HKs and Ura4. Therefore, we decided to test how the auxotrophic markers commonly used in S. pombe affects a wild-type strain, as well as strains lacking HKs, with regard to environmental stress and growth on peptone containing YPD plates. To this end, we constructed two additional sets of strains, one set with all combinations of the three commonly used auxotrophic makers in S. pombe, namely; ura4-D18, leu1-32, and ade6-M216, and the other set lacking all three HKs in combination with the same auxotrophic makers. These two sets of strains were serial diluted and spotted onto YEA plates, YEA plates with 1 M sorbitol or 75 mM NaCl or 25 μM CdSO4, and also on YPD plates and YPD with Phloxin B (Fig. 7). It was evident that the auxotrophic markers influenced the otherwise wild-type strains ability to grow on plates with additives. The strain with a deletion of ura4 grew slightly less on plates with added sorbitol or NaCl (Fig. 7a, upper panels, row 2) and the ura4-D18 leu1-32 strain had a strong growth reduction on plates containing NaCl (Fig. 7a, row 5), and finally, the ura4-D18 leu1-32 ade6-M216 strain grew poorly on plates containing NaCl and CdSO4. In addition, the ura4-D18 leu1-32 ade6-M216 strain was almost completely growth inhibited on the YPD plates (Fig. 7a, lower panel, row 6 and 8). Surprisingly, this was also the case for the Ura+ strain, leu1-32 ade6-M216, indicating that it is not only the ura4-D18 auxotrophic marker that influences the growth of S. pombe on YPD plates.Fig. 7


Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans
Auxotrophic markers contribute to a stress sensitive phenotype. Cell cultures from strains with the h− mating-type configuration were serially diluted in five steps (5-fold per step) and 5 μl were spotted onto rich YEA plates with or without supplements and grown at 30 °C for three days. a Strains with HKs carrying different auxotrophic markers: 972 h-(wt) PJ278 (ura4-D18), PJ1848 (leu1-32), PJ1933 (ade6-M216), PJ1895 (leu1-32 ura4-D18), PJ239 (leu1-32 ade6-M210), PJ1934 (ura4-D18 ade6-M216), FY368 (leu1-32 ura4-D18 ade6-M216). b Strains lacking Mak1, Mak2 and Mak3 (mak1,2,3Δ), with different auxotrophic markers: PJ1903 (mak1,2,3Δ), PJ1951 (mak1,2,3Δ ura4-D18), PJ1846 (mak1,2,3Δ leu1-32), PJ1948 (mak1,2,3Δ ade6-M216) PJ1949 (mak1,2,3Δ ura4-D18 leu1-32) PJ1947 (mak1,2,3Δ leu1-32 ade6-M216) PJ1950 (mak1,2,3Δ ura4-D18 ade6-M216) PJ1713 (mak1,2,3Δ ura4-D18 leu1-32 ade6-M216)
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Fig7: Auxotrophic markers contribute to a stress sensitive phenotype. Cell cultures from strains with the h− mating-type configuration were serially diluted in five steps (5-fold per step) and 5 μl were spotted onto rich YEA plates with or without supplements and grown at 30 °C for three days. a Strains with HKs carrying different auxotrophic markers: 972 h-(wt) PJ278 (ura4-D18), PJ1848 (leu1-32), PJ1933 (ade6-M216), PJ1895 (leu1-32 ura4-D18), PJ239 (leu1-32 ade6-M210), PJ1934 (ura4-D18 ade6-M216), FY368 (leu1-32 ura4-D18 ade6-M216). b Strains lacking Mak1, Mak2 and Mak3 (mak1,2,3Δ), with different auxotrophic markers: PJ1903 (mak1,2,3Δ), PJ1951 (mak1,2,3Δ ura4-D18), PJ1846 (mak1,2,3Δ leu1-32), PJ1948 (mak1,2,3Δ ade6-M216) PJ1949 (mak1,2,3Δ ura4-D18 leu1-32) PJ1947 (mak1,2,3Δ leu1-32 ade6-M216) PJ1950 (mak1,2,3Δ ura4-D18 ade6-M216) PJ1713 (mak1,2,3Δ ura4-D18 leu1-32 ade6-M216)
Mentions: It was also evident that the ura4-D18 leu1-32 auxotrophic mutations in the wild-type background affected the strains survival on plates with elevated NaCl levels (Fig. 6, bottom left, compare the first two rows). A recent publication reports that the ura4 deletion makes the cell wall of S. pombe less robust (Matsuo et al. 2013). The weakened cell wall results in a higher uptake of Phloxin B on peptone-containing plates, which gives rise to red colonies specifically in the ura4Δ strain (Matsuo et al. 2013). The weaker cell wall in the ura4 deletion strains might explain the increased stress sensitivity in the strains lacking both HKs and Ura4. Therefore, we decided to test how the auxotrophic markers commonly used in S. pombe affects a wild-type strain, as well as strains lacking HKs, with regard to environmental stress and growth on peptone containing YPD plates. To this end, we constructed two additional sets of strains, one set with all combinations of the three commonly used auxotrophic makers in S. pombe, namely; ura4-D18, leu1-32, and ade6-M216, and the other set lacking all three HKs in combination with the same auxotrophic makers. These two sets of strains were serial diluted and spotted onto YEA plates, YEA plates with 1 M sorbitol or 75 mM NaCl or 25 μM CdSO4, and also on YPD plates and YPD with Phloxin B (Fig. 7). It was evident that the auxotrophic markers influenced the otherwise wild-type strains ability to grow on plates with additives. The strain with a deletion of ura4 grew slightly less on plates with added sorbitol or NaCl (Fig. 7a, upper panels, row 2) and the ura4-D18 leu1-32 strain had a strong growth reduction on plates containing NaCl (Fig. 7a, row 5), and finally, the ura4-D18 leu1-32 ade6-M216 strain grew poorly on plates containing NaCl and CdSO4. In addition, the ura4-D18 leu1-32 ade6-M216 strain was almost completely growth inhibited on the YPD plates (Fig. 7a, lower panel, row 6 and 8). Surprisingly, this was also the case for the Ura+ strain, leu1-32 ade6-M216, indicating that it is not only the ura4-D18 auxotrophic marker that influences the growth of S. pombe on YPD plates.Fig. 7

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.