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Small but crucial: the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans.

Mayer FL, Wilson D, Jacobsen ID, Miramón P, Slesiona S, Bohovych IM, Brown AJ, Hube B - PLoS ONE (2012)

Bottom Line: Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions.Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines.Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenicity Mechanisms, Hans-Knoell-Institute, Jena, Germany.

ABSTRACT
Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen.

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Mutants defective in trehalose synthesis phenocopy HSP21 deletion.(A) Drop test analysis with serial dilutions of the wild type (Wt) and the indicated mutant strains on SD minimal medium under different environmental stresses, including osmotic stress (1.5 M NaCl), thermal stress (42°C) and oxidative stress (0.4 mM menadione). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), osmotic or oxidative stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Capacity of the indicated strains to damage oral epithelial cells. Monolayers of epithelial cells were infected with the different strains for 15 h and host cell damage was then quantified by measuring LDH levels. Results are the mean ± SD of two independent experiments, each performed in septuplicate. ***P<0.0001 compared with the wild type strain. (C) Neutrophil killing assay. Cells of the indicated strains were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Wild type survival was set to 100%. BWP17+CIp30 was used as wild type control for gpp1Δ/Δ and gpd2Δ/Δ, and CAI4+CIp10 was used as wild type control for tps1Δ/Δ and tps2Δ/Δ. Experiments were performed at least three times. The bar represents the mean of the single values. *P<0.01 compared with the wild type.
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pone-0038584-g007: Mutants defective in trehalose synthesis phenocopy HSP21 deletion.(A) Drop test analysis with serial dilutions of the wild type (Wt) and the indicated mutant strains on SD minimal medium under different environmental stresses, including osmotic stress (1.5 M NaCl), thermal stress (42°C) and oxidative stress (0.4 mM menadione). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), osmotic or oxidative stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Capacity of the indicated strains to damage oral epithelial cells. Monolayers of epithelial cells were infected with the different strains for 15 h and host cell damage was then quantified by measuring LDH levels. Results are the mean ± SD of two independent experiments, each performed in septuplicate. ***P<0.0001 compared with the wild type strain. (C) Neutrophil killing assay. Cells of the indicated strains were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Wild type survival was set to 100%. BWP17+CIp30 was used as wild type control for gpp1Δ/Δ and gpd2Δ/Δ, and CAI4+CIp10 was used as wild type control for tps1Δ/Δ and tps2Δ/Δ. Experiments were performed at least three times. The bar represents the mean of the single values. *P<0.01 compared with the wild type.

Mentions: In order to confirm the role of Hsp21 in the homeostasis of glycerol and trehalose levels, we next used C. albicans mutant strains with deletions in GPP1 (encoding a putative glycerol 3-phosphatase) [82], GPD2 (encoding a predicted glycerol 3-phosphate dehydrogenase) [82], TPS1 (encoding a trehalose-6-phosphate synthase) [88] or TPS2 (encoding a trehalose-6-phosphate phosphatase) [89]. First, the gpp1Δ/Δ, gpd2Δ/Δ, tps1Δ/Δ and tps2Δ/Δ mutants were investigated using drop dilution assays on solid SD minimal medium under osmotic, thermal or oxidative stress (Figure 7A). As previously described, gpp1Δ/Δ exhibited a severe growth defect under osmotic stress [90]. gpd2Δ/Δ also displayed a strong defect in growth under this condition. The tps1Δ/Δ mutant had a severe growth defect at 42°C while tps2Δ/Δ and gpp1Δ/Δ had a moderate growth defect under elevated temperature. Oxidative stress, elicited by treatment with menadione resulted in impaired growth for all strains.


Small but crucial: the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans.

Mayer FL, Wilson D, Jacobsen ID, Miramón P, Slesiona S, Bohovych IM, Brown AJ, Hube B - PLoS ONE (2012)

Mutants defective in trehalose synthesis phenocopy HSP21 deletion.(A) Drop test analysis with serial dilutions of the wild type (Wt) and the indicated mutant strains on SD minimal medium under different environmental stresses, including osmotic stress (1.5 M NaCl), thermal stress (42°C) and oxidative stress (0.4 mM menadione). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), osmotic or oxidative stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Capacity of the indicated strains to damage oral epithelial cells. Monolayers of epithelial cells were infected with the different strains for 15 h and host cell damage was then quantified by measuring LDH levels. Results are the mean ± SD of two independent experiments, each performed in septuplicate. ***P<0.0001 compared with the wild type strain. (C) Neutrophil killing assay. Cells of the indicated strains were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Wild type survival was set to 100%. BWP17+CIp30 was used as wild type control for gpp1Δ/Δ and gpd2Δ/Δ, and CAI4+CIp10 was used as wild type control for tps1Δ/Δ and tps2Δ/Δ. Experiments were performed at least three times. The bar represents the mean of the single values. *P<0.01 compared with the wild type.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369842&req=5

pone-0038584-g007: Mutants defective in trehalose synthesis phenocopy HSP21 deletion.(A) Drop test analysis with serial dilutions of the wild type (Wt) and the indicated mutant strains on SD minimal medium under different environmental stresses, including osmotic stress (1.5 M NaCl), thermal stress (42°C) and oxidative stress (0.4 mM menadione). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), osmotic or oxidative stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Capacity of the indicated strains to damage oral epithelial cells. Monolayers of epithelial cells were infected with the different strains for 15 h and host cell damage was then quantified by measuring LDH levels. Results are the mean ± SD of two independent experiments, each performed in septuplicate. ***P<0.0001 compared with the wild type strain. (C) Neutrophil killing assay. Cells of the indicated strains were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Wild type survival was set to 100%. BWP17+CIp30 was used as wild type control for gpp1Δ/Δ and gpd2Δ/Δ, and CAI4+CIp10 was used as wild type control for tps1Δ/Δ and tps2Δ/Δ. Experiments were performed at least three times. The bar represents the mean of the single values. *P<0.01 compared with the wild type.
Mentions: In order to confirm the role of Hsp21 in the homeostasis of glycerol and trehalose levels, we next used C. albicans mutant strains with deletions in GPP1 (encoding a putative glycerol 3-phosphatase) [82], GPD2 (encoding a predicted glycerol 3-phosphate dehydrogenase) [82], TPS1 (encoding a trehalose-6-phosphate synthase) [88] or TPS2 (encoding a trehalose-6-phosphate phosphatase) [89]. First, the gpp1Δ/Δ, gpd2Δ/Δ, tps1Δ/Δ and tps2Δ/Δ mutants were investigated using drop dilution assays on solid SD minimal medium under osmotic, thermal or oxidative stress (Figure 7A). As previously described, gpp1Δ/Δ exhibited a severe growth defect under osmotic stress [90]. gpd2Δ/Δ also displayed a strong defect in growth under this condition. The tps1Δ/Δ mutant had a severe growth defect at 42°C while tps2Δ/Δ and gpp1Δ/Δ had a moderate growth defect under elevated temperature. Oxidative stress, elicited by treatment with menadione resulted in impaired growth for all strains.

Bottom Line: Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions.Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines.Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenicity Mechanisms, Hans-Knoell-Institute, Jena, Germany.

ABSTRACT
Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen.

Show MeSH
Related in: MedlinePlus