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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.

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Genes regulated by osmotic stress were dysregulated in strains lacking Mak1, Mak2, and Mak3. The real-time qPCR analysis of transcript levels of (a) atf21+, (b) gpd1+, and (c) SPAC22A12.17c genes in strains PJ120 (wt), PJ1640 (mak1Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ). The levels of mRNA transcripts were measured under non-induced conditions (black bars) and upon 15 min of treatment with 1 M sorbitol (grey bars). The transcripts were quantified by real-time qPCR amplification of cDNA and normalised to act1+ mRNA levels and presented as fold change, relative to wt strain (see “Methods”). Graphic data present the average of at least two biological replicates, each tested by at least two technical replicates, and error bars represent standard error of the mean (SEM) (Simon 2003)
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Fig5: Genes regulated by osmotic stress were dysregulated in strains lacking Mak1, Mak2, and Mak3. The real-time qPCR analysis of transcript levels of (a) atf21+, (b) gpd1+, and (c) SPAC22A12.17c genes in strains PJ120 (wt), PJ1640 (mak1Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ). The levels of mRNA transcripts were measured under non-induced conditions (black bars) and upon 15 min of treatment with 1 M sorbitol (grey bars). The transcripts were quantified by real-time qPCR amplification of cDNA and normalised to act1+ mRNA levels and presented as fold change, relative to wt strain (see “Methods”). Graphic data present the average of at least two biological replicates, each tested by at least two technical replicates, and error bars represent standard error of the mean (SEM) (Simon 2003)

Mentions: To determine whether the reduced growth of the strain lacking Mak1 in combination with lack of Mak2 or Mak3 on plates containing sorbitol was due to impaired gene regulation, the expression level of a few representative genes known to be upregulated during osmotic stress was measured. The genes that we chose to examine were: atf21+ (putative transcription factor), gpd1+ (glycerol-3-phosphate dehydrogenase), and SPAC22A12.17c (putative sugar oxidoreductase) (Chen et al. 2003). The mRNA levels were measured using real-time qPCR in six different strains; wild-type, mak1Δ, mak1,2Δ, mak1,3Δ, mak2,3Δ, and mak1,2,3Δ, before and 15 min after the addition of 1 M sorbitol to the growth media. As expected, the atf21+ gene was upregulated, about 5-fold, in the wild-type strain upon incubation with sorbitol (Fig. 5a). Under treatment with sorbitol two mutant strains, mak1Δ and mak2,3Δ, demonstrated even stronger upregulation of the atf21+ gene, reaching around a 3- to 4-fold higher level as compared with the wild-type expression level. In contrast, the mak1,2Δ and mak1,3Δ strains showed mRNA levels similar to the wild-type strain. Finally, expression of the atf21+ gene was weaker in the triple knockout strain, being only about half of the mRNA level, compared with the wild-type strain upon sorbitol treatment (Fig. 5a). In addition, all the mutant strains, except for the triple knockout, displayed a mild upregulation of the atf21+ gene, 1.5–2-fold, in the normal media without sorbitol.Fig. 5


Stress sensitivity of a fission yeast strain lacking histidine kinases is rescued by the ectopic expression of Chk1 from Candida albicans
Genes regulated by osmotic stress were dysregulated in strains lacking Mak1, Mak2, and Mak3. The real-time qPCR analysis of transcript levels of (a) atf21+, (b) gpd1+, and (c) SPAC22A12.17c genes in strains PJ120 (wt), PJ1640 (mak1Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ). The levels of mRNA transcripts were measured under non-induced conditions (black bars) and upon 15 min of treatment with 1 M sorbitol (grey bars). The transcripts were quantified by real-time qPCR amplification of cDNA and normalised to act1+ mRNA levels and presented as fold change, relative to wt strain (see “Methods”). Graphic data present the average of at least two biological replicates, each tested by at least two technical replicates, and error bars represent standard error of the mean (SEM) (Simon 2003)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383687&req=5

Fig5: Genes regulated by osmotic stress were dysregulated in strains lacking Mak1, Mak2, and Mak3. The real-time qPCR analysis of transcript levels of (a) atf21+, (b) gpd1+, and (c) SPAC22A12.17c genes in strains PJ120 (wt), PJ1640 (mak1Δ), PJ1643 (mak1,2Δ), PJ1644 (mak1,3Δ), PJ1645 (mak2,3Δ), and PJ1646 (mak1,2,3Δ). The levels of mRNA transcripts were measured under non-induced conditions (black bars) and upon 15 min of treatment with 1 M sorbitol (grey bars). The transcripts were quantified by real-time qPCR amplification of cDNA and normalised to act1+ mRNA levels and presented as fold change, relative to wt strain (see “Methods”). Graphic data present the average of at least two biological replicates, each tested by at least two technical replicates, and error bars represent standard error of the mean (SEM) (Simon 2003)
Mentions: To determine whether the reduced growth of the strain lacking Mak1 in combination with lack of Mak2 or Mak3 on plates containing sorbitol was due to impaired gene regulation, the expression level of a few representative genes known to be upregulated during osmotic stress was measured. The genes that we chose to examine were: atf21+ (putative transcription factor), gpd1+ (glycerol-3-phosphate dehydrogenase), and SPAC22A12.17c (putative sugar oxidoreductase) (Chen et al. 2003). The mRNA levels were measured using real-time qPCR in six different strains; wild-type, mak1Δ, mak1,2Δ, mak1,3Δ, mak2,3Δ, and mak1,2,3Δ, before and 15 min after the addition of 1 M sorbitol to the growth media. As expected, the atf21+ gene was upregulated, about 5-fold, in the wild-type strain upon incubation with sorbitol (Fig. 5a). Under treatment with sorbitol two mutant strains, mak1Δ and mak2,3Δ, demonstrated even stronger upregulation of the atf21+ gene, reaching around a 3- to 4-fold higher level as compared with the wild-type expression level. In contrast, the mak1,2Δ and mak1,3Δ strains showed mRNA levels similar to the wild-type strain. Finally, expression of the atf21+ gene was weaker in the triple knockout strain, being only about half of the mRNA level, compared with the wild-type strain upon sorbitol treatment (Fig. 5a). In addition, all the mutant strains, except for the triple knockout, displayed a mild upregulation of the atf21+ gene, 1.5–2-fold, in the normal media without sorbitol.Fig. 5

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