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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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Hsp21 regulates intracellular glycerol, glycogen and trehalose homeostasis.(A) Measurement of intracellular glycerol levels in the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after growth for 24 h in SD medium (control) at 30°C, SD medium supplemented with 0.4 mM menadione (+menadione) at 30°C, SD medium supplemented with 1.5 M NaCl (+NaCl) at 30°C, SD medium at 42°C (42°C), or SD medium supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). Glycerol levels are plotted in nM normalized against wet weight (g). Results are the mean ± SD of three independent experiments. **P<0.01 and *P<0.05 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Estimation of glycogen content with iodine vapour for the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after cultivation on SD agar (control) at 37°C, SD agar supplemented with 0.4 mM menadione at 37°C (+menadione), SD agar supplemented with 1.5 M NaCl (+NaCl) at 37°C, SD agar at 42°C (42°C), or SD agar supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). The darker the colour of a colony, the more intracellular glycogen is present. Experiments were performed twice in duplicate yielding similar results. Representative pictures are shown. (C) Measurement of intracellular trehalose levels in the wild type (Wt) and the hsp21Δ/Δ mutant strain. Growth conditions were the same as described for panel (A). Trehalose levels (nmol trehalose per mg total cell protein) are indicated relative to the Wt grown under control conditions. Results are the mean ± SD of five (control; +NaCl; 42°C) or two (+menadione; +NaCl, 42°C) independent experiments. *P<0.05 compared with the wild type strain under the same condition.
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pone-0038584-g006: Hsp21 regulates intracellular glycerol, glycogen and trehalose homeostasis.(A) Measurement of intracellular glycerol levels in the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after growth for 24 h in SD medium (control) at 30°C, SD medium supplemented with 0.4 mM menadione (+menadione) at 30°C, SD medium supplemented with 1.5 M NaCl (+NaCl) at 30°C, SD medium at 42°C (42°C), or SD medium supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). Glycerol levels are plotted in nM normalized against wet weight (g). Results are the mean ± SD of three independent experiments. **P<0.01 and *P<0.05 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Estimation of glycogen content with iodine vapour for the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after cultivation on SD agar (control) at 37°C, SD agar supplemented with 0.4 mM menadione at 37°C (+menadione), SD agar supplemented with 1.5 M NaCl (+NaCl) at 37°C, SD agar at 42°C (42°C), or SD agar supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). The darker the colour of a colony, the more intracellular glycogen is present. Experiments were performed twice in duplicate yielding similar results. Representative pictures are shown. (C) Measurement of intracellular trehalose levels in the wild type (Wt) and the hsp21Δ/Δ mutant strain. Growth conditions were the same as described for panel (A). Trehalose levels (nmol trehalose per mg total cell protein) are indicated relative to the Wt grown under control conditions. Results are the mean ± SD of five (control; +NaCl; 42°C) or two (+menadione; +NaCl, 42°C) independent experiments. *P<0.05 compared with the wild type strain under the same condition.

Mentions: Only very low levels of intracellular glycerol could be measured under non-stress conditions in the wild type (1.6 nM/g wet weight), the hsp21Δ/Δ mutant (0.4 nM/g wet weight) and the hsp21Δ/Δ::HSP21-reconstituted strain (0.2 nM/g wet weight) (Figure 6A). Oxidative stress (0.4 mM menadione) resulted in moderate accumulation of glycerol for the different strains. Osmotic stress, induced by 0.5 M NaCl however, led to a strong accumulation of glycerol in the wild type (36.4 nM/g wet weight). Interestingly, the hsp21Δ/Δ mutant (10.3 nM/g wet weight) produced 72% less glycerol than the wild type upon osmotic stress. Complementation of the mutant with HSP21 restored wild type glycerol production (27.5 nM/g wet weight).


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)

Hsp21 regulates intracellular glycerol, glycogen and trehalose homeostasis.(A) Measurement of intracellular glycerol levels in the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after growth for 24 h in SD medium (control) at 30°C, SD medium supplemented with 0.4 mM menadione (+menadione) at 30°C, SD medium supplemented with 1.5 M NaCl (+NaCl) at 30°C, SD medium at 42°C (42°C), or SD medium supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). Glycerol levels are plotted in nM normalized against wet weight (g). Results are the mean ± SD of three independent experiments. **P<0.01 and *P<0.05 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Estimation of glycogen content with iodine vapour for the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after cultivation on SD agar (control) at 37°C, SD agar supplemented with 0.4 mM menadione at 37°C (+menadione), SD agar supplemented with 1.5 M NaCl (+NaCl) at 37°C, SD agar at 42°C (42°C), or SD agar supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). The darker the colour of a colony, the more intracellular glycogen is present. Experiments were performed twice in duplicate yielding similar results. Representative pictures are shown. (C) Measurement of intracellular trehalose levels in the wild type (Wt) and the hsp21Δ/Δ mutant strain. Growth conditions were the same as described for panel (A). Trehalose levels (nmol trehalose per mg total cell protein) are indicated relative to the Wt grown under control conditions. Results are the mean ± SD of five (control; +NaCl; 42°C) or two (+menadione; +NaCl, 42°C) independent experiments. *P<0.05 compared with the wild type strain under the same condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369842&req=5

pone-0038584-g006: Hsp21 regulates intracellular glycerol, glycogen and trehalose homeostasis.(A) Measurement of intracellular glycerol levels in the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after growth for 24 h in SD medium (control) at 30°C, SD medium supplemented with 0.4 mM menadione (+menadione) at 30°C, SD medium supplemented with 1.5 M NaCl (+NaCl) at 30°C, SD medium at 42°C (42°C), or SD medium supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). Glycerol levels are plotted in nM normalized against wet weight (g). Results are the mean ± SD of three independent experiments. **P<0.01 and *P<0.05 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Estimation of glycogen content with iodine vapour for the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain after cultivation on SD agar (control) at 37°C, SD agar supplemented with 0.4 mM menadione at 37°C (+menadione), SD agar supplemented with 1.5 M NaCl (+NaCl) at 37°C, SD agar at 42°C (42°C), or SD agar supplemented with 1.5 M NaCl at 42°C (+NaCl, 42°C). The darker the colour of a colony, the more intracellular glycogen is present. Experiments were performed twice in duplicate yielding similar results. Representative pictures are shown. (C) Measurement of intracellular trehalose levels in the wild type (Wt) and the hsp21Δ/Δ mutant strain. Growth conditions were the same as described for panel (A). Trehalose levels (nmol trehalose per mg total cell protein) are indicated relative to the Wt grown under control conditions. Results are the mean ± SD of five (control; +NaCl; 42°C) or two (+menadione; +NaCl, 42°C) independent experiments. *P<0.05 compared with the wild type strain under the same condition.
Mentions: Only very low levels of intracellular glycerol could be measured under non-stress conditions in the wild type (1.6 nM/g wet weight), the hsp21Δ/Δ mutant (0.4 nM/g wet weight) and the hsp21Δ/Δ::HSP21-reconstituted strain (0.2 nM/g wet weight) (Figure 6A). Oxidative stress (0.4 mM menadione) resulted in moderate accumulation of glycerol for the different strains. Osmotic stress, induced by 0.5 M NaCl however, led to a strong accumulation of glycerol in the wild type (36.4 nM/g wet weight). Interestingly, the hsp21Δ/Δ mutant (10.3 nM/g wet weight) produced 72% less glycerol than the wild type upon osmotic stress. Complementation of the mutant with HSP21 restored wild type glycerol production (27.5 nM/g wet weight).

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