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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 is a virulence factor.(A) The deletion of HSP21 leads to increased susceptibility of C. albicans to killing by human neutrophils. Wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant cells were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Experiments were performed three times. The bar represents the mean of these single values. *P<0.01 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Hsp21 is required for C. albicans to cause full damage to endothelial and oral epithelial cells in vitro. Monolayers of human-derived endothelial and oral epithelial cells were infected with C. albicans wild type (Wt) and hsp21Δ/Δ mutant strains for 15 or 24 h. Host cell damage was then determined by measuring lactate dehydrogenase (LDH) levels. Results are the mean ± SD of at least three independent experiments, each performed in triplicate. **P<0.01 and ***P<0.001 compared with the wild type strain. (C) The hsp21Δ/Δ mutant is avirulent in a mouse model of hematogenously disseminated candidiasis. Female Balb/C mice (n = 10 mice per C. albicans strain) were challenged intravenously with either the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain via the lateral tail vein. *P<0.0001 compared with mice either infected with the wild type or hsp21Δ/Δ::HSP21 complemented strain. (D) Periodic acid Schiff staining of kidney sections from mice infected with the wild type and hsp21Δ/Δ::HSP21 complemented strain six days, and with the hsp21Δ/Δ mutant strain 21 days post infection. Pictures were taken at 63x (upper panel) and 100x magnification (lower panel). The lower panel of images show magnifications of the white boxed areas from the above images. Arrows point to C. albicans filaments within the tissue.
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pone-0038584-g005: Hsp21 is a virulence factor.(A) The deletion of HSP21 leads to increased susceptibility of C. albicans to killing by human neutrophils. Wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant cells were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Experiments were performed three times. The bar represents the mean of these single values. *P<0.01 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Hsp21 is required for C. albicans to cause full damage to endothelial and oral epithelial cells in vitro. Monolayers of human-derived endothelial and oral epithelial cells were infected with C. albicans wild type (Wt) and hsp21Δ/Δ mutant strains for 15 or 24 h. Host cell damage was then determined by measuring lactate dehydrogenase (LDH) levels. Results are the mean ± SD of at least three independent experiments, each performed in triplicate. **P<0.01 and ***P<0.001 compared with the wild type strain. (C) The hsp21Δ/Δ mutant is avirulent in a mouse model of hematogenously disseminated candidiasis. Female Balb/C mice (n = 10 mice per C. albicans strain) were challenged intravenously with either the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain via the lateral tail vein. *P<0.0001 compared with mice either infected with the wild type or hsp21Δ/Δ::HSP21 complemented strain. (D) Periodic acid Schiff staining of kidney sections from mice infected with the wild type and hsp21Δ/Δ::HSP21 complemented strain six days, and with the hsp21Δ/Δ mutant strain 21 days post infection. Pictures were taken at 63x (upper panel) and 100x magnification (lower panel). The lower panel of images show magnifications of the white boxed areas from the above images. Arrows point to C. albicans filaments within the tissue.

Mentions: Attack by phagocytic cells of the innate immune system represents a significant stress to invading microorganisms. Given the severe stress adaptation defects observed upon HSP21 deletion (above), and the strong transcriptional upregulation of HSP21 upon exposure to both macrophages and neutrophils (Table S1), we hypothesized that Hsp21 may play a role in defending fungal cells from attack by phagocytes. We initially tested the survival of wild type and hsp21Δ/Δ mutant cells following exposure to macrophages derived from the immortalized monocyte cell line, THP1. Overall, fungal killing by THP1 cells was low (10–30%), and although we observed a modest decrease in survival of the hsp21Δ/Δ mutant, this difference was not significant (data not shown). Neutrophils play a crucial role in controlling C. albicans infections [52], [78], [79]. We therefore investigated the survival of C. albicans following a 3 h co-incubation with human neutrophils. As shown in Figure 5A, deletion of HSP21 significantly reduced survival from 45% (wild type) to 32%; complementation of the mutant with a single copy of HSP21 significantly restored survival to 51%.


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 is a virulence factor.(A) The deletion of HSP21 leads to increased susceptibility of C. albicans to killing by human neutrophils. Wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant cells were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Experiments were performed three times. The bar represents the mean of these single values. *P<0.01 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Hsp21 is required for C. albicans to cause full damage to endothelial and oral epithelial cells in vitro. Monolayers of human-derived endothelial and oral epithelial cells were infected with C. albicans wild type (Wt) and hsp21Δ/Δ mutant strains for 15 or 24 h. Host cell damage was then determined by measuring lactate dehydrogenase (LDH) levels. Results are the mean ± SD of at least three independent experiments, each performed in triplicate. **P<0.01 and ***P<0.001 compared with the wild type strain. (C) The hsp21Δ/Δ mutant is avirulent in a mouse model of hematogenously disseminated candidiasis. Female Balb/C mice (n = 10 mice per C. albicans strain) were challenged intravenously with either the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain via the lateral tail vein. *P<0.0001 compared with mice either infected with the wild type or hsp21Δ/Δ::HSP21 complemented strain. (D) Periodic acid Schiff staining of kidney sections from mice infected with the wild type and hsp21Δ/Δ::HSP21 complemented strain six days, and with the hsp21Δ/Δ mutant strain 21 days post infection. Pictures were taken at 63x (upper panel) and 100x magnification (lower panel). The lower panel of images show magnifications of the white boxed areas from the above images. Arrows point to C. albicans filaments within the tissue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038584-g005: Hsp21 is a virulence factor.(A) The deletion of HSP21 leads to increased susceptibility of C. albicans to killing by human neutrophils. Wild type (Wt), hsp21Δ/Δ mutant and hsp21Δ/Δ::HSP21 complemented mutant cells were exposed to human neutrophils for three hours and viability was then determined by plating on YPD agar. Experiments were performed three times. The bar represents the mean of these single values. *P<0.01 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. (B) Hsp21 is required for C. albicans to cause full damage to endothelial and oral epithelial cells in vitro. Monolayers of human-derived endothelial and oral epithelial cells were infected with C. albicans wild type (Wt) and hsp21Δ/Δ mutant strains for 15 or 24 h. Host cell damage was then determined by measuring lactate dehydrogenase (LDH) levels. Results are the mean ± SD of at least three independent experiments, each performed in triplicate. **P<0.01 and ***P<0.001 compared with the wild type strain. (C) The hsp21Δ/Δ mutant is avirulent in a mouse model of hematogenously disseminated candidiasis. Female Balb/C mice (n = 10 mice per C. albicans strain) were challenged intravenously with either the wild type (Wt), the hsp21Δ/Δ mutant or the hsp21Δ/Δ::HSP21 complemented strain via the lateral tail vein. *P<0.0001 compared with mice either infected with the wild type or hsp21Δ/Δ::HSP21 complemented strain. (D) Periodic acid Schiff staining of kidney sections from mice infected with the wild type and hsp21Δ/Δ::HSP21 complemented strain six days, and with the hsp21Δ/Δ mutant strain 21 days post infection. Pictures were taken at 63x (upper panel) and 100x magnification (lower panel). The lower panel of images show magnifications of the white boxed areas from the above images. Arrows point to C. albicans filaments within the tissue.
Mentions: Attack by phagocytic cells of the innate immune system represents a significant stress to invading microorganisms. Given the severe stress adaptation defects observed upon HSP21 deletion (above), and the strong transcriptional upregulation of HSP21 upon exposure to both macrophages and neutrophils (Table S1), we hypothesized that Hsp21 may play a role in defending fungal cells from attack by phagocytes. We initially tested the survival of wild type and hsp21Δ/Δ mutant cells following exposure to macrophages derived from the immortalized monocyte cell line, THP1. Overall, fungal killing by THP1 cells was low (10–30%), and although we observed a modest decrease in survival of the hsp21Δ/Δ mutant, this difference was not significant (data not shown). Neutrophils play a crucial role in controlling C. albicans infections [52], [78], [79]. We therefore investigated the survival of C. albicans following a 3 h co-incubation with human neutrophils. As shown in Figure 5A, deletion of HSP21 significantly reduced survival from 45% (wild type) to 32%; complementation of the mutant with a single copy of HSP21 significantly restored survival to 51%.

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