Limits...
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.

Show MeSH

Related in: MedlinePlus

hsp21Δ/Δ has increased susceptibility to thermal and oxidative stress and has a growth defect under nutrient limitation.Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on agar containing different stressors. (A) Growth of the hsp21Δ/Δ mutant on solid SD minimal medium under different environmental stresses, including thermal stress (42°C), oxidative stress (0.4 mM menadione), osmotic stress (1.5 M NaCl) and cell wall stress (450 µg ml-1 Congo red). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), oxidative, osmotic or cell wall stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial dilutions of the indicated strains on agar containing different compounds as sole carbon and nitrogen sources. Agar containing 0.67% yeast nitrogen base plus ammonium sulphate without amino acids was supplemented with 2% glucose, potassium acetate or citrate as sole carbon source. Yeast nitrogen base agar without ammonium sulphate and amino acids was supplemented with 100 µg ml-1 proline or pantothenate as sole carbon and nitrogen source. Plates were incubated at 37°C for 3–7 days depending on the carbon and nitrogen source. Experiments were repeated at least twice yielding similar results. Representative pictures are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369842&req=5

pone-0038584-g002: hsp21Δ/Δ has increased susceptibility to thermal and oxidative stress and has a growth defect under nutrient limitation.Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on agar containing different stressors. (A) Growth of the hsp21Δ/Δ mutant on solid SD minimal medium under different environmental stresses, including thermal stress (42°C), oxidative stress (0.4 mM menadione), osmotic stress (1.5 M NaCl) and cell wall stress (450 µg ml-1 Congo red). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), oxidative, osmotic or cell wall stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial dilutions of the indicated strains on agar containing different compounds as sole carbon and nitrogen sources. Agar containing 0.67% yeast nitrogen base plus ammonium sulphate without amino acids was supplemented with 2% glucose, potassium acetate or citrate as sole carbon source. Yeast nitrogen base agar without ammonium sulphate and amino acids was supplemented with 100 µg ml-1 proline or pantothenate as sole carbon and nitrogen source. Plates were incubated at 37°C for 3–7 days depending on the carbon and nitrogen source. Experiments were repeated at least twice yielding similar results. Representative pictures are shown.

Mentions: Certain HSPs, such as S. cerevisiae HSP70, are dispensable for surviving short lived exposure to very high temperature, but required for long-term growth under less severe thermal stress [2]. We therefore next examined the role of CaHSP21 in adaptation to prolonged thermal stress. Strikingly, under constant elevated temperature of 39.1°C the hsp21Δ/Δ mutant showed a growth defect, was strongly impaired in growth at a constant temperature of 40.5°C (Figure 1D), and completely unable to grow at 42°C (Figure 2A). Growth was restored by complementation of hsp21Δ/Δ with a single copy of HSP21, albeit not to wild type levels. The phenotypes of the hsp21Δ/Δ mutant – surviving short term exposure to very high temperature, but failing to grow over prolonged periods of thermal stress – is reminiscent of S. cerevisiae hsp70? [2]. Hsps and sHsps not only function in adaptation to heat stress but also to other stresses, such as oxidative, osmotic and cell wall stresses. We therefore investigated growth of the hsp21Δ/Δ mutant under these environmental stresses. Oxidative stress, induced by menadione - a naphthoquinone which exerts its toxic function mainly through the generation of reactive oxygen species (ROS) [67], [68] – led to a severe growth defect of the hsp21Δ/Δ mutant (Figure 2A). This points to a possible role for Hsp21 in preventing non-specific protein aggregation upon exposure of C. albicans to ROS. Interestingly, for osmotic stress induced by high concentrations of NaCl, the hsp21Δ/Δ mutant was found to be slightly more resistant than the wild type and hsp21Δ/Δ::HSP21 complemented strain. Cell wall directed stress elicited by Congo red – a compound which binds nascent chitin chains and thereby inhibits connection of chitin to β-1,3-glucan and β-1,6-glucan [69] – did not affect growth of the hsp21Δ/Δ mutant.


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)

hsp21Δ/Δ has increased susceptibility to thermal and oxidative stress and has a growth defect under nutrient limitation.Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on agar containing different stressors. (A) Growth of the hsp21Δ/Δ mutant on solid SD minimal medium under different environmental stresses, including thermal stress (42°C), oxidative stress (0.4 mM menadione), osmotic stress (1.5 M NaCl) and cell wall stress (450 µg ml-1 Congo red). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), oxidative, osmotic or cell wall stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial dilutions of the indicated strains on agar containing different compounds as sole carbon and nitrogen sources. Agar containing 0.67% yeast nitrogen base plus ammonium sulphate without amino acids was supplemented with 2% glucose, potassium acetate or citrate as sole carbon source. Yeast nitrogen base agar without ammonium sulphate and amino acids was supplemented with 100 µg ml-1 proline or pantothenate as sole carbon and nitrogen source. Plates were incubated at 37°C for 3–7 days depending on the carbon and nitrogen source. Experiments were repeated at least twice yielding similar results. Representative pictures are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038584-g002: hsp21Δ/Δ has increased susceptibility to thermal and oxidative stress and has a growth defect under nutrient limitation.Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on agar containing different stressors. (A) Growth of the hsp21Δ/Δ mutant on solid SD minimal medium under different environmental stresses, including thermal stress (42°C), oxidative stress (0.4 mM menadione), osmotic stress (1.5 M NaCl) and cell wall stress (450 µg ml-1 Congo red). Plates subjected to thermal stress were incubated for 4–5 days, cells grown under non-stress (control), oxidative, osmotic or cell wall stress for 2–3 days at 37°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial dilutions of the indicated strains on agar containing different compounds as sole carbon and nitrogen sources. Agar containing 0.67% yeast nitrogen base plus ammonium sulphate without amino acids was supplemented with 2% glucose, potassium acetate or citrate as sole carbon source. Yeast nitrogen base agar without ammonium sulphate and amino acids was supplemented with 100 µg ml-1 proline or pantothenate as sole carbon and nitrogen source. Plates were incubated at 37°C for 3–7 days depending on the carbon and nitrogen source. Experiments were repeated at least twice yielding similar results. Representative pictures are shown.
Mentions: Certain HSPs, such as S. cerevisiae HSP70, are dispensable for surviving short lived exposure to very high temperature, but required for long-term growth under less severe thermal stress [2]. We therefore next examined the role of CaHSP21 in adaptation to prolonged thermal stress. Strikingly, under constant elevated temperature of 39.1°C the hsp21Δ/Δ mutant showed a growth defect, was strongly impaired in growth at a constant temperature of 40.5°C (Figure 1D), and completely unable to grow at 42°C (Figure 2A). Growth was restored by complementation of hsp21Δ/Δ with a single copy of HSP21, albeit not to wild type levels. The phenotypes of the hsp21Δ/Δ mutant – surviving short term exposure to very high temperature, but failing to grow over prolonged periods of thermal stress – is reminiscent of S. cerevisiae hsp70? [2]. Hsps and sHsps not only function in adaptation to heat stress but also to other stresses, such as oxidative, osmotic and cell wall stresses. We therefore investigated growth of the hsp21Δ/Δ mutant under these environmental stresses. Oxidative stress, induced by menadione - a naphthoquinone which exerts its toxic function mainly through the generation of reactive oxygen species (ROS) [67], [68] – led to a severe growth defect of the hsp21Δ/Δ mutant (Figure 2A). This points to a possible role for Hsp21 in preventing non-specific protein aggregation upon exposure of C. albicans to ROS. Interestingly, for osmotic stress induced by high concentrations of NaCl, the hsp21Δ/Δ mutant was found to be slightly more resistant than the wild type and hsp21Δ/Δ::HSP21 complemented strain. Cell wall directed stress elicited by Congo red – a compound which binds nascent chitin chains and thereby inhibits connection of chitin to β-1,3-glucan and β-1,6-glucan [69] – did not affect growth of the hsp21Δ/Δ mutant.

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