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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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Osmotic stress bypasses the Hsp21-dependent thermal stress tolerance.Simultaneous osmotic and thermal stress lead to growth of the heat-sensitive hsp21Δ/Δ mutant. (A) Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on SD agar or SD agar containing 1.5 M NaCl. Plates were incubated at 37°C or 42°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial 10-fold dilutions of the wild type and hsp21Δ/Δ mutant on SD agar containing 1.5 M sorbitol, 2% glycerol, or 30 mM trehalose. Plates were incubated at 37°C or 42°C. Experiments were repeated twice yielding similar results. Representative pictures are shown. (C) Growth curves for the wild type and hsp21Δ/Δ mutant in SD medium and SD medium supplemented with 1.5 M NaCl at 37°C or 42°C. Experiments were repeated twice yielding similar results. Results are the mean of two measurements per strain and time point. Representative growth curves are shown.
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pone-0038584-g003: Osmotic stress bypasses the Hsp21-dependent thermal stress tolerance.Simultaneous osmotic and thermal stress lead to growth of the heat-sensitive hsp21Δ/Δ mutant. (A) Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on SD agar or SD agar containing 1.5 M NaCl. Plates were incubated at 37°C or 42°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial 10-fold dilutions of the wild type and hsp21Δ/Δ mutant on SD agar containing 1.5 M sorbitol, 2% glycerol, or 30 mM trehalose. Plates were incubated at 37°C or 42°C. Experiments were repeated twice yielding similar results. Representative pictures are shown. (C) Growth curves for the wild type and hsp21Δ/Δ mutant in SD medium and SD medium supplemented with 1.5 M NaCl at 37°C or 42°C. Experiments were repeated twice yielding similar results. Results are the mean of two measurements per strain and time point. Representative growth curves are shown.

Mentions: During growth within a host, C. albicans must adapt to a variety of stresses and it is likely that some of these stresses occur simultaneously. We therefore sought to characterize the role of HSP21 in adaptation to multiple stresses. The hsp21Δ/Δ mutant grew normally at 37°C on solid SD medium, exhibited wild type resistance to osmotic stress and did not grow at 42°C as described above (Figure 3A). Strikingly, the combination of NaCl-induced osmotic stress and thermal stress fully bypassed Hsp21-dependence for growth under thermal stress (Figure 3A). We observed the same phenomenon for growth under thermal stress combined with sorbitol- and potassium chloride-induced osmotic stress (Figure 3B and data not shown). Osmotic stress is known to elicit a protective intracellular glycerol accumulation. It has been shown for Escherichia coli that osmolytes such as glycerol and trehalose not only stabilize the medium under osmotic stress, but, importantly, also can act as chemical chaperones by stabilizing native proteins, preventing protein aggregation and helping in refolding unfolded polypeptides under thermal stress [72], [73]. We therefore also tested growth of the mutant at 42°C in the presence of exogenously added glycerol or trehalose, however, this did not restore growth of the hsp21Δ/Δ mutant (Figure 3B).


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)

Osmotic stress bypasses the Hsp21-dependent thermal stress tolerance.Simultaneous osmotic and thermal stress lead to growth of the heat-sensitive hsp21Δ/Δ mutant. (A) Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on SD agar or SD agar containing 1.5 M NaCl. Plates were incubated at 37°C or 42°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial 10-fold dilutions of the wild type and hsp21Δ/Δ mutant on SD agar containing 1.5 M sorbitol, 2% glycerol, or 30 mM trehalose. Plates were incubated at 37°C or 42°C. Experiments were repeated twice yielding similar results. Representative pictures are shown. (C) Growth curves for the wild type and hsp21Δ/Δ mutant in SD medium and SD medium supplemented with 1.5 M NaCl at 37°C or 42°C. Experiments were repeated twice yielding similar results. Results are the mean of two measurements per strain and time point. Representative growth curves are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369842&req=5

pone-0038584-g003: Osmotic stress bypasses the Hsp21-dependent thermal stress tolerance.Simultaneous osmotic and thermal stress lead to growth of the heat-sensitive hsp21Δ/Δ mutant. (A) Drop test analysis with serial dilutions of C. albicans wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant on SD agar or SD agar containing 1.5 M NaCl. Plates were incubated at 37°C or 42°C. Experiments were repeated at least twice yielding similar results. Representative pictures are shown. (B) Drop test analysis with serial 10-fold dilutions of the wild type and hsp21Δ/Δ mutant on SD agar containing 1.5 M sorbitol, 2% glycerol, or 30 mM trehalose. Plates were incubated at 37°C or 42°C. Experiments were repeated twice yielding similar results. Representative pictures are shown. (C) Growth curves for the wild type and hsp21Δ/Δ mutant in SD medium and SD medium supplemented with 1.5 M NaCl at 37°C or 42°C. Experiments were repeated twice yielding similar results. Results are the mean of two measurements per strain and time point. Representative growth curves are shown.
Mentions: During growth within a host, C. albicans must adapt to a variety of stresses and it is likely that some of these stresses occur simultaneously. We therefore sought to characterize the role of HSP21 in adaptation to multiple stresses. The hsp21Δ/Δ mutant grew normally at 37°C on solid SD medium, exhibited wild type resistance to osmotic stress and did not grow at 42°C as described above (Figure 3A). Strikingly, the combination of NaCl-induced osmotic stress and thermal stress fully bypassed Hsp21-dependence for growth under thermal stress (Figure 3A). We observed the same phenomenon for growth under thermal stress combined with sorbitol- and potassium chloride-induced osmotic stress (Figure 3B and data not shown). Osmotic stress is known to elicit a protective intracellular glycerol accumulation. It has been shown for Escherichia coli that osmolytes such as glycerol and trehalose not only stabilize the medium under osmotic stress, but, importantly, also can act as chemical chaperones by stabilizing native proteins, preventing protein aggregation and helping in refolding unfolded polypeptides under thermal stress [72], [73]. We therefore also tested growth of the mutant at 42°C in the presence of exogenously added glycerol or trehalose, however, this did not restore growth of the hsp21Δ/Δ mutant (Figure 3B).

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