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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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Cek1 phosphorylation in response to thermal stress is Hsp21-dependent.Western blot analysis of phosphorylated Cek1, Mkc1 or Hog1. The wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented strain were incubated under non-stress conditions (control), conditions of cell wall stress (Congo red), osmotic stress (NaCl), oxidative stress (menadione), thermal stress (42°C) or a combination of thermal and osmotic stress (42°C+NaCl) for 4 hours at 30°C or 42°C. Equal amounts of protein extracts were blotted and probed for phosphorylated Cek1 (Cek1-P) and Mkc1 (Mkc1-P). Blots were then stripped and re-probed for α-tubulin (loading control). Hog1 phosphorylation (Hog1-P) was investigated in separate blots and, after stripping, blots were probed for total Hog1 (phosphorylated plus un-phosphorylated) as loading control. Note that thermal stress induces Cek1 phosphorylation in a Hsp21-dependent manner and that simultaneous osmotic stress bypasses Hsp21-dependence.
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pone-0038584-g008: Cek1 phosphorylation in response to thermal stress is Hsp21-dependent.Western blot analysis of phosphorylated Cek1, Mkc1 or Hog1. The wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented strain were incubated under non-stress conditions (control), conditions of cell wall stress (Congo red), osmotic stress (NaCl), oxidative stress (menadione), thermal stress (42°C) or a combination of thermal and osmotic stress (42°C+NaCl) for 4 hours at 30°C or 42°C. Equal amounts of protein extracts were blotted and probed for phosphorylated Cek1 (Cek1-P) and Mkc1 (Mkc1-P). Blots were then stripped and re-probed for α-tubulin (loading control). Hog1 phosphorylation (Hog1-P) was investigated in separate blots and, after stripping, blots were probed for total Hog1 (phosphorylated plus un-phosphorylated) as loading control. Note that thermal stress induces Cek1 phosphorylation in a Hsp21-dependent manner and that simultaneous osmotic stress bypasses Hsp21-dependence.

Mentions: We therefore undertook a systematic analysis of the three main mitogen-activated protein (MAP) kinase signaling pathways (Mkc1, Cek1 and Hog1) [91], [92] using western blotting in C. albicans wild type, hsp21Δ/Δ and hsp21Δ/Δ+HSP21 strains. We investigated phosphorylation of Mkc1, Cek1 and Hog1 under five different environmental stress conditions, including cell wall, osmotic, oxidative, thermal, and combined thermal and osmotic stress (Figure 8).


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)

Cek1 phosphorylation in response to thermal stress is Hsp21-dependent.Western blot analysis of phosphorylated Cek1, Mkc1 or Hog1. The wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented strain were incubated under non-stress conditions (control), conditions of cell wall stress (Congo red), osmotic stress (NaCl), oxidative stress (menadione), thermal stress (42°C) or a combination of thermal and osmotic stress (42°C+NaCl) for 4 hours at 30°C or 42°C. Equal amounts of protein extracts were blotted and probed for phosphorylated Cek1 (Cek1-P) and Mkc1 (Mkc1-P). Blots were then stripped and re-probed for α-tubulin (loading control). Hog1 phosphorylation (Hog1-P) was investigated in separate blots and, after stripping, blots were probed for total Hog1 (phosphorylated plus un-phosphorylated) as loading control. Note that thermal stress induces Cek1 phosphorylation in a Hsp21-dependent manner and that simultaneous osmotic stress bypasses Hsp21-dependence.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038584-g008: Cek1 phosphorylation in response to thermal stress is Hsp21-dependent.Western blot analysis of phosphorylated Cek1, Mkc1 or Hog1. The wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented strain were incubated under non-stress conditions (control), conditions of cell wall stress (Congo red), osmotic stress (NaCl), oxidative stress (menadione), thermal stress (42°C) or a combination of thermal and osmotic stress (42°C+NaCl) for 4 hours at 30°C or 42°C. Equal amounts of protein extracts were blotted and probed for phosphorylated Cek1 (Cek1-P) and Mkc1 (Mkc1-P). Blots were then stripped and re-probed for α-tubulin (loading control). Hog1 phosphorylation (Hog1-P) was investigated in separate blots and, after stripping, blots were probed for total Hog1 (phosphorylated plus un-phosphorylated) as loading control. Note that thermal stress induces Cek1 phosphorylation in a Hsp21-dependent manner and that simultaneous osmotic stress bypasses Hsp21-dependence.
Mentions: We therefore undertook a systematic analysis of the three main mitogen-activated protein (MAP) kinase signaling pathways (Mkc1, Cek1 and Hog1) [91], [92] using western blotting in C. albicans wild type, hsp21Δ/Δ and hsp21Δ/Δ+HSP21 strains. We investigated phosphorylation of Mkc1, Cek1 and Hog1 under five different environmental stress conditions, including cell wall, osmotic, oxidative, thermal, and combined thermal and osmotic stress (Figure 8).

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