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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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Diverse functions of the three histidine kinases under various types of stress. Cell cultures of strains with h− mating-type configuration, PJ120 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) 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 for the indicated number of days (d). All plates were grown at 30 °C, except when cells were subjected to heat stress at 37 °C
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Fig3: Diverse functions of the three histidine kinases under various types of stress. Cell cultures of strains with h− mating-type configuration, PJ120 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) 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 for the indicated number of days (d). All plates were grown at 30 °C, except when cells were subjected to heat stress at 37 °C

Mentions: A function for S. pombe Mak2 and Mak3 in oxidative stress has been established in one of two redundant pathways (Buck et al. 2001), but the possible involvement of the three HKs in other types of stress responses has not been thoroughly investigated. We decided to examine the possible contribution of the three HKs in response to osmotic, salt, heavy metal, and heat stress. To this end, we crossed the triple knockout strain (mak1,2,3Δ) to a heterothallic h− strain to obtain a set of seven mutant strains in the h− mating configuration. These strains along with the wild-type control strain were serial diluted and spotted onto non-selective rich YEA media and onto YEA plates containing either 1 M sorbitol (osmotic stress), 75 mM NaCl (salt stress), 25 μM CdSO4 (heavy metal stress), or 1.5 mM H2O2 (oxidative stress). There was very little difference between any of the strains during oxidative stress. The growth of the wild-type strain was inhibited as well as all of the strains lacking one or more of the HKs, consistent with previous reports (Fig. 3b, right panel) (Buck et al. 2001). Surprisingly, the two strains that grew least were the wild-type and the triple knockout strain (mak1,2,3Δ) on H2O2 containing plates (Fig. 3b, top and bottom row). Under all other stress conditions that were tested, strains lacking Mak1 in combination with lack of Mak2 or Mak3 grew poorly. On the other hand, strains with Mak1, but lacking Mak3, mak3Δ and mak2,3Δ, survived slightly better during heat, salt, heavy metal, and oxidative stress conditions (Fig. 3). We obtained very similar results with the homothallic, h90, strains used for the sporulation assay, as with the strains with an h− mating type (Fig. S1). Finally, on plates with defined AA media, the sensitivity to sorbitol by the strains lacking Mak1 in combination with a deletion of mak2+ or mak3+ was enhanced, resulting in complete growth inhibition (Fig. S2).Fig. 3


Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans
Diverse functions of the three histidine kinases under various types of stress. Cell cultures of strains with h− mating-type configuration, PJ120 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) 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 for the indicated number of days (d). All plates were grown at 30 °C, except when cells were subjected to heat stress at 37 °C
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: Diverse functions of the three histidine kinases under various types of stress. Cell cultures of strains with h− mating-type configuration, PJ120 (wt), PJ1640 (mak1Δ), PJ1641 (mak2Δ), PJ1642 (mak3Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ) 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 for the indicated number of days (d). All plates were grown at 30 °C, except when cells were subjected to heat stress at 37 °C
Mentions: A function for S. pombe Mak2 and Mak3 in oxidative stress has been established in one of two redundant pathways (Buck et al. 2001), but the possible involvement of the three HKs in other types of stress responses has not been thoroughly investigated. We decided to examine the possible contribution of the three HKs in response to osmotic, salt, heavy metal, and heat stress. To this end, we crossed the triple knockout strain (mak1,2,3Δ) to a heterothallic h− strain to obtain a set of seven mutant strains in the h− mating configuration. These strains along with the wild-type control strain were serial diluted and spotted onto non-selective rich YEA media and onto YEA plates containing either 1 M sorbitol (osmotic stress), 75 mM NaCl (salt stress), 25 μM CdSO4 (heavy metal stress), or 1.5 mM H2O2 (oxidative stress). There was very little difference between any of the strains during oxidative stress. The growth of the wild-type strain was inhibited as well as all of the strains lacking one or more of the HKs, consistent with previous reports (Fig. 3b, right panel) (Buck et al. 2001). Surprisingly, the two strains that grew least were the wild-type and the triple knockout strain (mak1,2,3Δ) on H2O2 containing plates (Fig. 3b, top and bottom row). Under all other stress conditions that were tested, strains lacking Mak1 in combination with lack of Mak2 or Mak3 grew poorly. On the other hand, strains with Mak1, but lacking Mak3, mak3Δ and mak2,3Δ, survived slightly better during heat, salt, heavy metal, and oxidative stress conditions (Fig. 3). We obtained very similar results with the homothallic, h90, strains used for the sporulation assay, as with the strains with an h− mating type (Fig. S1). Finally, on plates with defined AA media, the sensitivity to sorbitol by the strains lacking Mak1 in combination with a deletion of mak2+ or mak3+ was enhanced, resulting in complete growth inhibition (Fig. S2).Fig. 3

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