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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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C. albicans orf19.822 encodes a predicted sHsp required for adaptation to long-term thermal stress.(A) Structural organization of orf19.822 with a conserved central α-crystallin domain (red) flanked by variable N- and C-terminal domains (grey), based on results from http://www.expasy.ch/prosite/database. Numbers below the structural elements represent amino acid position. (B) Alignment of the orf19.822 protein sequence with orthologues from other organisms (generated with ClustalW2). The conserved α-crystallin-domain sequence is shown in red characters. Identical residues are marked with (*), residues with the same size and hydropathy are marked by (:), residues with the same size or hydropathy are marked by (.). (C) Short-term heat shock and endoplasmic reticulum (ER)-stress. Cells of YPD-overnight cultures of the wild type (Wt), hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) were serially diluted from 106 to 101 cells (left to right), either exposed to heat shock (50°C, 15 min) or not (control), plated on YPD and incubated for 2 days at 37°C. ER-stress was induced by growing the cells on YPD agar plates supplemented with 30 mM dithiothreitol (DTT). (D) Growth of the Wt, hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) on solid SD minimal medium at temperatures ranging from 30°C to 40.5°C.
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pone-0038584-g001: C. albicans orf19.822 encodes a predicted sHsp required for adaptation to long-term thermal stress.(A) Structural organization of orf19.822 with a conserved central α-crystallin domain (red) flanked by variable N- and C-terminal domains (grey), based on results from http://www.expasy.ch/prosite/database. Numbers below the structural elements represent amino acid position. (B) Alignment of the orf19.822 protein sequence with orthologues from other organisms (generated with ClustalW2). The conserved α-crystallin-domain sequence is shown in red characters. Identical residues are marked with (*), residues with the same size and hydropathy are marked by (:), residues with the same size or hydropathy are marked by (.). (C) Short-term heat shock and endoplasmic reticulum (ER)-stress. Cells of YPD-overnight cultures of the wild type (Wt), hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) were serially diluted from 106 to 101 cells (left to right), either exposed to heat shock (50°C, 15 min) or not (control), plated on YPD and incubated for 2 days at 37°C. ER-stress was induced by growing the cells on YPD agar plates supplemented with 30 mM dithiothreitol (DTT). (D) Growth of the Wt, hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) on solid SD minimal medium at temperatures ranging from 30°C to 40.5°C.

Mentions: orf19.822 was first identified and chosen for detailed investigation according to two criteria. Firstly, orf19.822 was found to be amongst the most strongly upregulated genes in multiple transcriptional profiles of different C. albicans infection models as well as in transcriptional profiles for C. albicans subjected to different stress conditions (Table S1). Upregulation of orf19.822 was detected during ex vivo liver infection (up to 20-fold) [51], interaction with whole blood (up to 4.8-fold) as well as interaction with neutrophils (up to 6.2-fold) [52]. Furthermore, the gene was found to be upregulated under mild oxidative stress (up to 3.2-fold) [53], during interaction with macrophages (up to 29.6-fold) [54] and upon weak acid induced stress (up to 88.7-fold) [55]. Finally, orf19.822 was shown to be highly expressed during heat shock, induced by a shift from either 23–37°C (up to 10.9-fold) [53], 30–42°C (up to 19-fold) [56] or 30–45°C (up to 25-fold) [12]. Secondly, to the best of our knowledge, this gene was of completely unknown function prior to our investigations and a preliminary in silico analysis of the protein sequence identified interesting structural features (see below). Using ExPASy PROSITE, we identified a sHsp-typical α-crystallin domain and N- and C-terminal regions within the deduced amino acid sequence of orf19.822 (Figure 1A). A BLASTp search analysis of the amino acid sequence revealed sequence similarities to proteins of unknown function in Candida dubliniensis (96% identity), Candida tropicalis (51%) and Candida parapsilosis (40%). C. albicans orf19.822 also displayed significant sequence similarity to the Pichia stipitis small heat shock protein Hsp18 (39% identity over the full length of these proteins) (Figures 1B and S1A). The similarity was higher within the α-crystallin domains (42%). No orf19.822 orthologues were identified in the non-pathogenic yeast Saccharomyces cerevisiae or, indeed, in any other species outside the CUG clade of fungi [57]. Analysis of the orf19.822 promoter region revealed the presence of two characteristic Hsp heat shock elements (HSEs) [11], [58] as well as one non-standard HSE (nHSE) motif [59]. Furthermore, a Hsp-typical stress-responsive element (STRE) [60], [61], [62] was detected within the orf19.822 promoter region (Figure S1B). Together with the presence of the sHsp-family-defining α-crystallin domain within the amino acid sequence, its transcriptional upregulation under thermal stress, the occurrence of HSEs and STRE in the promoter region, the significant homology to P. stipitis Hsp18 and the predicted molecular mass of 21.487 kDa, we refer to C. albicans orf19.822 as Hsp21 (heat shock protein 21). Next, we were interested in elucidating whether HSP21 does indeed play a role in stress adaptation in C. albicans. For this purpose we constructed a hsp21Δ/Δ homozygous deletion mutant (Figure S2).


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)

C. albicans orf19.822 encodes a predicted sHsp required for adaptation to long-term thermal stress.(A) Structural organization of orf19.822 with a conserved central α-crystallin domain (red) flanked by variable N- and C-terminal domains (grey), based on results from http://www.expasy.ch/prosite/database. Numbers below the structural elements represent amino acid position. (B) Alignment of the orf19.822 protein sequence with orthologues from other organisms (generated with ClustalW2). The conserved α-crystallin-domain sequence is shown in red characters. Identical residues are marked with (*), residues with the same size and hydropathy are marked by (:), residues with the same size or hydropathy are marked by (.). (C) Short-term heat shock and endoplasmic reticulum (ER)-stress. Cells of YPD-overnight cultures of the wild type (Wt), hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) were serially diluted from 106 to 101 cells (left to right), either exposed to heat shock (50°C, 15 min) or not (control), plated on YPD and incubated for 2 days at 37°C. ER-stress was induced by growing the cells on YPD agar plates supplemented with 30 mM dithiothreitol (DTT). (D) Growth of the Wt, hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) on solid SD minimal medium at temperatures ranging from 30°C to 40.5°C.
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Related In: Results  -  Collection

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pone-0038584-g001: C. albicans orf19.822 encodes a predicted sHsp required for adaptation to long-term thermal stress.(A) Structural organization of orf19.822 with a conserved central α-crystallin domain (red) flanked by variable N- and C-terminal domains (grey), based on results from http://www.expasy.ch/prosite/database. Numbers below the structural elements represent amino acid position. (B) Alignment of the orf19.822 protein sequence with orthologues from other organisms (generated with ClustalW2). The conserved α-crystallin-domain sequence is shown in red characters. Identical residues are marked with (*), residues with the same size and hydropathy are marked by (:), residues with the same size or hydropathy are marked by (.). (C) Short-term heat shock and endoplasmic reticulum (ER)-stress. Cells of YPD-overnight cultures of the wild type (Wt), hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) were serially diluted from 106 to 101 cells (left to right), either exposed to heat shock (50°C, 15 min) or not (control), plated on YPD and incubated for 2 days at 37°C. ER-stress was induced by growing the cells on YPD agar plates supplemented with 30 mM dithiothreitol (DTT). (D) Growth of the Wt, hsp21Δ/Δ mutant (Δ/Δ) and hsp21Δ/Δ::HSP21 complemented mutant (Δ/Δ+) on solid SD minimal medium at temperatures ranging from 30°C to 40.5°C.
Mentions: orf19.822 was first identified and chosen for detailed investigation according to two criteria. Firstly, orf19.822 was found to be amongst the most strongly upregulated genes in multiple transcriptional profiles of different C. albicans infection models as well as in transcriptional profiles for C. albicans subjected to different stress conditions (Table S1). Upregulation of orf19.822 was detected during ex vivo liver infection (up to 20-fold) [51], interaction with whole blood (up to 4.8-fold) as well as interaction with neutrophils (up to 6.2-fold) [52]. Furthermore, the gene was found to be upregulated under mild oxidative stress (up to 3.2-fold) [53], during interaction with macrophages (up to 29.6-fold) [54] and upon weak acid induced stress (up to 88.7-fold) [55]. Finally, orf19.822 was shown to be highly expressed during heat shock, induced by a shift from either 23–37°C (up to 10.9-fold) [53], 30–42°C (up to 19-fold) [56] or 30–45°C (up to 25-fold) [12]. Secondly, to the best of our knowledge, this gene was of completely unknown function prior to our investigations and a preliminary in silico analysis of the protein sequence identified interesting structural features (see below). Using ExPASy PROSITE, we identified a sHsp-typical α-crystallin domain and N- and C-terminal regions within the deduced amino acid sequence of orf19.822 (Figure 1A). A BLASTp search analysis of the amino acid sequence revealed sequence similarities to proteins of unknown function in Candida dubliniensis (96% identity), Candida tropicalis (51%) and Candida parapsilosis (40%). C. albicans orf19.822 also displayed significant sequence similarity to the Pichia stipitis small heat shock protein Hsp18 (39% identity over the full length of these proteins) (Figures 1B and S1A). The similarity was higher within the α-crystallin domains (42%). No orf19.822 orthologues were identified in the non-pathogenic yeast Saccharomyces cerevisiae or, indeed, in any other species outside the CUG clade of fungi [57]. Analysis of the orf19.822 promoter region revealed the presence of two characteristic Hsp heat shock elements (HSEs) [11], [58] as well as one non-standard HSE (nHSE) motif [59]. Furthermore, a Hsp-typical stress-responsive element (STRE) [60], [61], [62] was detected within the orf19.822 promoter region (Figure S1B). Together with the presence of the sHsp-family-defining α-crystallin domain within the amino acid sequence, its transcriptional upregulation under thermal stress, the occurrence of HSEs and STRE in the promoter region, the significant homology to P. stipitis Hsp18 and the predicted molecular mass of 21.487 kDa, we refer to C. albicans orf19.822 as Hsp21 (heat shock protein 21). Next, we were interested in elucidating whether HSP21 does indeed play a role in stress adaptation in C. albicans. For this purpose we constructed a hsp21Δ/Δ homozygous deletion mutant (Figure S2).

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