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 exhibits reduced invasive growth and hyphal formation.(A) Formation of hyphae was induced by embedding fungal cells in YPS (2% saccharose) agar or by plating them on solid water agar supplemented with 10% fetal bovine serum, SLAD agar or on solid Spider medium. Serum agar plates were incubated for 2, SLAD agar plates for 4, and Spider agar plates for 10 days at 37°C. Embedded plates were incubated at 25°C for 5 days. Experiments were performed twice in duplicate. Representative pictures are shown. Scale bar: 100 µm. (B) Hyphal elongation in RPMI1640 and 10% serum. Wild type, hsp21Δ/Δ mutant or hsp21Δ/Δ::HSP21 complemented mutant cells were grown overnight in SD medium. After washing twice with water, 104 cells were incubated in RPMI1640 or water supplemented with 10% serum in 24-well cell culture plates at 37°C for 4 hours in the presence of 5% CO2. Hyphal lengths were then determined using an Inverse microscope (Leica). Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 100 cells measured per strain and experiment. *P<0.0001 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. Pictures of representative hyphae were taken using a 40x-magnification. Scale bar: 10 µm. (C) Hyphal formation on epithelial monolayers. TR146 epithelial cells were cultured to confluency and infected with C. albicans cells for three hours. Fungal cells were then stained with Calcofluor white (stains invaded and non-invaded fungal elements) and hyphal lengths were determined by fluorescence microscopy. Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 200 cells measured per strain and experiment. *P<0.0001 compared with the wild type strain. (D) Representative pictures of wild type and hsp21Δ/Δ hyphae are shown. Scale bar: 20 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369842&req=5

pone-0038584-g004: hsp21Δ/Δ exhibits reduced invasive growth and hyphal formation.(A) Formation of hyphae was induced by embedding fungal cells in YPS (2% saccharose) agar or by plating them on solid water agar supplemented with 10% fetal bovine serum, SLAD agar or on solid Spider medium. Serum agar plates were incubated for 2, SLAD agar plates for 4, and Spider agar plates for 10 days at 37°C. Embedded plates were incubated at 25°C for 5 days. Experiments were performed twice in duplicate. Representative pictures are shown. Scale bar: 100 µm. (B) Hyphal elongation in RPMI1640 and 10% serum. Wild type, hsp21Δ/Δ mutant or hsp21Δ/Δ::HSP21 complemented mutant cells were grown overnight in SD medium. After washing twice with water, 104 cells were incubated in RPMI1640 or water supplemented with 10% serum in 24-well cell culture plates at 37°C for 4 hours in the presence of 5% CO2. Hyphal lengths were then determined using an Inverse microscope (Leica). Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 100 cells measured per strain and experiment. *P<0.0001 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. Pictures of representative hyphae were taken using a 40x-magnification. Scale bar: 10 µm. (C) Hyphal formation on epithelial monolayers. TR146 epithelial cells were cultured to confluency and infected with C. albicans cells for three hours. Fungal cells were then stained with Calcofluor white (stains invaded and non-invaded fungal elements) and hyphal lengths were determined by fluorescence microscopy. Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 200 cells measured per strain and experiment. *P<0.0001 compared with the wild type strain. (D) Representative pictures of wild type and hsp21Δ/Δ hyphae are shown. Scale bar: 20 µm.

Mentions: It has recently been shown by Cowen and colleagues that the molecular chaperone and heat-shock protein Hsp90 acts as physiological link between fungal morphogenesis and temperature [74], [75]. We therefore postulated that Hsp21 may also play a role in morphogenesis. Hyphal formation was induced by embedding fungal cells in yeast peptone saccharose agar, by plating cells on agar supplemented with 10% fetal bovine serum, on SLAD agar or on spider medium agar [76] (Figure 4A). The hsp21Δ/Δ mutant cells formed filamentous colonies under embedded conditions and on serum-containing agar, however, colonies appeared to be smaller than those of the wild type. The reduced colony size appeared to be mainly due to shorter radial filaments produced by the hsp21Δ/Δ mutant in comparison to the wild type. An even more striking phenotype caused by deletion of HSP21 was observed on SLAD and Spider agar. On SLAD agar the hsp21Δ/Δ mutant formed aberrant colonies which completely lacked the peripheral filaments observed for the wild type. When grown on Spider agar the wild type typically forms colonies with a central wrinkled area consisting of yeast, hyphae and pseudohyphae and a peripheral area consisting mainly of agar-invading filaments [77]. In contrast, the hsp21Δ/Δ mutant developed wrinkled colonies that completely lacked peripheral hyphae. We conclude that C. albicans HSP21 is required for optimal invasive growth in agar. In order to further characterize the hyphal formation defect of the hsp21Δ/Δ mutant, we next investigated filamentation on a single cell level in liquid hyphae inducing media (Figure 4B). While wild type and revertant cells formed hyphae with a mean length of around 60 µm, the hsp21Δ/Δ mutant filaments only reached a mean length of around 40 µm upon exposure to RPMI or 10% serum for 4 h at 37°C and 5% CO2. In order to more closely mimic an in vivo situation, we also induced hyphal formation by incubation on oral epithelial cells for 3 h at 37°C and 5% CO2 (Figure 4C and 4D). Here, wild type cells reached a length of about 40 µm. Again, hsp21Δ/Δ hyphae were significantly shorter, reaching only about 25 µm. Taken together, these results indicate that Hsp21 contributes to hyphal formation in C. albicans. Together with the prominent stress phenotypes of the hsp21Δ/Δ mutant (above) we concluded that HSP21 represents a promising virulence factor candidate and continued by investigating the role of HSP21 during infection.


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 exhibits reduced invasive growth and hyphal formation.(A) Formation of hyphae was induced by embedding fungal cells in YPS (2% saccharose) agar or by plating them on solid water agar supplemented with 10% fetal bovine serum, SLAD agar or on solid Spider medium. Serum agar plates were incubated for 2, SLAD agar plates for 4, and Spider agar plates for 10 days at 37°C. Embedded plates were incubated at 25°C for 5 days. Experiments were performed twice in duplicate. Representative pictures are shown. Scale bar: 100 µm. (B) Hyphal elongation in RPMI1640 and 10% serum. Wild type, hsp21Δ/Δ mutant or hsp21Δ/Δ::HSP21 complemented mutant cells were grown overnight in SD medium. After washing twice with water, 104 cells were incubated in RPMI1640 or water supplemented with 10% serum in 24-well cell culture plates at 37°C for 4 hours in the presence of 5% CO2. Hyphal lengths were then determined using an Inverse microscope (Leica). Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 100 cells measured per strain and experiment. *P<0.0001 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. Pictures of representative hyphae were taken using a 40x-magnification. Scale bar: 10 µm. (C) Hyphal formation on epithelial monolayers. TR146 epithelial cells were cultured to confluency and infected with C. albicans cells for three hours. Fungal cells were then stained with Calcofluor white (stains invaded and non-invaded fungal elements) and hyphal lengths were determined by fluorescence microscopy. Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 200 cells measured per strain and experiment. *P<0.0001 compared with the wild type strain. (D) Representative pictures of wild type and hsp21Δ/Δ hyphae are shown. Scale bar: 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038584-g004: hsp21Δ/Δ exhibits reduced invasive growth and hyphal formation.(A) Formation of hyphae was induced by embedding fungal cells in YPS (2% saccharose) agar or by plating them on solid water agar supplemented with 10% fetal bovine serum, SLAD agar or on solid Spider medium. Serum agar plates were incubated for 2, SLAD agar plates for 4, and Spider agar plates for 10 days at 37°C. Embedded plates were incubated at 25°C for 5 days. Experiments were performed twice in duplicate. Representative pictures are shown. Scale bar: 100 µm. (B) Hyphal elongation in RPMI1640 and 10% serum. Wild type, hsp21Δ/Δ mutant or hsp21Δ/Δ::HSP21 complemented mutant cells were grown overnight in SD medium. After washing twice with water, 104 cells were incubated in RPMI1640 or water supplemented with 10% serum in 24-well cell culture plates at 37°C for 4 hours in the presence of 5% CO2. Hyphal lengths were then determined using an Inverse microscope (Leica). Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 100 cells measured per strain and experiment. *P<0.0001 compared with the wild type and hsp21Δ/Δ::HSP21 complemented strain. Pictures of representative hyphae were taken using a 40x-magnification. Scale bar: 10 µm. (C) Hyphal formation on epithelial monolayers. TR146 epithelial cells were cultured to confluency and infected with C. albicans cells for three hours. Fungal cells were then stained with Calcofluor white (stains invaded and non-invaded fungal elements) and hyphal lengths were determined by fluorescence microscopy. Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 200 cells measured per strain and experiment. *P<0.0001 compared with the wild type strain. (D) Representative pictures of wild type and hsp21Δ/Δ hyphae are shown. Scale bar: 20 µm.
Mentions: It has recently been shown by Cowen and colleagues that the molecular chaperone and heat-shock protein Hsp90 acts as physiological link between fungal morphogenesis and temperature [74], [75]. We therefore postulated that Hsp21 may also play a role in morphogenesis. Hyphal formation was induced by embedding fungal cells in yeast peptone saccharose agar, by plating cells on agar supplemented with 10% fetal bovine serum, on SLAD agar or on spider medium agar [76] (Figure 4A). The hsp21Δ/Δ mutant cells formed filamentous colonies under embedded conditions and on serum-containing agar, however, colonies appeared to be smaller than those of the wild type. The reduced colony size appeared to be mainly due to shorter radial filaments produced by the hsp21Δ/Δ mutant in comparison to the wild type. An even more striking phenotype caused by deletion of HSP21 was observed on SLAD and Spider agar. On SLAD agar the hsp21Δ/Δ mutant formed aberrant colonies which completely lacked the peripheral filaments observed for the wild type. When grown on Spider agar the wild type typically forms colonies with a central wrinkled area consisting of yeast, hyphae and pseudohyphae and a peripheral area consisting mainly of agar-invading filaments [77]. In contrast, the hsp21Δ/Δ mutant developed wrinkled colonies that completely lacked peripheral hyphae. We conclude that C. albicans HSP21 is required for optimal invasive growth in agar. In order to further characterize the hyphal formation defect of the hsp21Δ/Δ mutant, we next investigated filamentation on a single cell level in liquid hyphae inducing media (Figure 4B). While wild type and revertant cells formed hyphae with a mean length of around 60 µm, the hsp21Δ/Δ mutant filaments only reached a mean length of around 40 µm upon exposure to RPMI or 10% serum for 4 h at 37°C and 5% CO2. In order to more closely mimic an in vivo situation, we also induced hyphal formation by incubation on oral epithelial cells for 3 h at 37°C and 5% CO2 (Figure 4C and 4D). Here, wild type cells reached a length of about 40 µm. Again, hsp21Δ/Δ hyphae were significantly shorter, reaching only about 25 µm. Taken together, these results indicate that Hsp21 contributes to hyphal formation in C. albicans. Together with the prominent stress phenotypes of the hsp21Δ/Δ mutant (above) we concluded that HSP21 represents a promising virulence factor candidate and continued by investigating the role of HSP21 during infection.

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