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Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

Wartenberg A, Linde J, Martin R, Schreiner M, Horn F, Jacobsen ID, Je S, Wolf T, Kuchler K, Guthke R, Kurzai O, Forche A, d'Enfert C, Brunke S, Hube B - PLoS Genet. (2014)

Bottom Line: In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation.We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery.These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Jena, Germany.

ABSTRACT
Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

No MeSH data available.


Related in: MedlinePlus

Analysis of hyphae-associated gene expression, Als3 surface expression and response to farnesol.(A) The Evo strain expresses hyphae-associated genes after growth for 1 h at 37°C at 5% CO2 on a plastic surface similar to WT. Relative gene expression of filament-inducing conditions was compared to yeast promoting conditions (YPD, 30°C) for three independent experiments. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1) and data are shown as mean+SD of three biological experiments (*p<0.05). (B) Immunofluorescence micrographs of cells immuno-stained for Als3 after growth in DMEM+10% FBS at 37°C and 5% CO2 on cover slips. Wild type (WT) and Evo cells are Als3-positive, while cph1Δ/efg1Δ cells show no signal (representative samples). (C) Morphogenetic response to farnesol treatment alone or in combination with exogenous dibutyryl-cyclic AMP (db-cAMP). All strains were exposed to either methanol (control), 1 µM farnesol or 1 µM farnesol+10 mM db-cAMP and incubated at 37°C and 5% CO2 for 18 h (representative pictures from three independent experiments are shown). Note that in Evo cells inhibition of filamentation by farnesol treatment was completely abrogated when db-cAMP was added. (D) Repression of hyphae-associated gene expression in the Evo strain by 10 µM farnesol. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1). The fold change in expression relative to filament-inducing conditions alone is shown as mean+SD of three biological experiments.
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pgen-1004824-g003: Analysis of hyphae-associated gene expression, Als3 surface expression and response to farnesol.(A) The Evo strain expresses hyphae-associated genes after growth for 1 h at 37°C at 5% CO2 on a plastic surface similar to WT. Relative gene expression of filament-inducing conditions was compared to yeast promoting conditions (YPD, 30°C) for three independent experiments. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1) and data are shown as mean+SD of three biological experiments (*p<0.05). (B) Immunofluorescence micrographs of cells immuno-stained for Als3 after growth in DMEM+10% FBS at 37°C and 5% CO2 on cover slips. Wild type (WT) and Evo cells are Als3-positive, while cph1Δ/efg1Δ cells show no signal (representative samples). (C) Morphogenetic response to farnesol treatment alone or in combination with exogenous dibutyryl-cyclic AMP (db-cAMP). All strains were exposed to either methanol (control), 1 µM farnesol or 1 µM farnesol+10 mM db-cAMP and incubated at 37°C and 5% CO2 for 18 h (representative pictures from three independent experiments are shown). Note that in Evo cells inhibition of filamentation by farnesol treatment was completely abrogated when db-cAMP was added. (D) Repression of hyphae-associated gene expression in the Evo strain by 10 µM farnesol. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1). The fold change in expression relative to filament-inducing conditions alone is shown as mean+SD of three biological experiments.

Mentions: Hyphal-associated virulence of C. albicans is not only due to filamentation per se, but also to the expression of hyphae-associated genes. In order to monitor the expression of typical hyphae-associated genes in the Evo strain, we measured the mRNA levels of HWP1, ECE1 and ALS3, all encoding hyphal cell surface proteins, and of EED1, a gene that is associated with hyphal cell elongation [27]. An upregulation of all four genes in the Evo strain was confirmed by qRT-PCR after 1 hour of incubation in DMEM+10% FBS at 37°C and 5% CO2 (Fig. 3A). HWP1 expression was similar in the Evo and WT strain, whereas ECE1 and ALS3 were higher expressed in the WT strain, and EED1 was more strongly upregulated in the Evo strain. Furthermore, we observed Als3 exposure on the surface of wild type and Evo cells by immunofluorescence, but not on the cph1Δ/efg1Δ strain (Fig. 3B). This regained cell-surface exposure of the Als3 adhesin [28] is in accordance with the increased adhesion potential of the Evo strain.


Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

Wartenberg A, Linde J, Martin R, Schreiner M, Horn F, Jacobsen ID, Je S, Wolf T, Kuchler K, Guthke R, Kurzai O, Forche A, d'Enfert C, Brunke S, Hube B - PLoS Genet. (2014)

Analysis of hyphae-associated gene expression, Als3 surface expression and response to farnesol.(A) The Evo strain expresses hyphae-associated genes after growth for 1 h at 37°C at 5% CO2 on a plastic surface similar to WT. Relative gene expression of filament-inducing conditions was compared to yeast promoting conditions (YPD, 30°C) for three independent experiments. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1) and data are shown as mean+SD of three biological experiments (*p<0.05). (B) Immunofluorescence micrographs of cells immuno-stained for Als3 after growth in DMEM+10% FBS at 37°C and 5% CO2 on cover slips. Wild type (WT) and Evo cells are Als3-positive, while cph1Δ/efg1Δ cells show no signal (representative samples). (C) Morphogenetic response to farnesol treatment alone or in combination with exogenous dibutyryl-cyclic AMP (db-cAMP). All strains were exposed to either methanol (control), 1 µM farnesol or 1 µM farnesol+10 mM db-cAMP and incubated at 37°C and 5% CO2 for 18 h (representative pictures from three independent experiments are shown). Note that in Evo cells inhibition of filamentation by farnesol treatment was completely abrogated when db-cAMP was added. (D) Repression of hyphae-associated gene expression in the Evo strain by 10 µM farnesol. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1). The fold change in expression relative to filament-inducing conditions alone is shown as mean+SD of three biological experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256171&req=5

pgen-1004824-g003: Analysis of hyphae-associated gene expression, Als3 surface expression and response to farnesol.(A) The Evo strain expresses hyphae-associated genes after growth for 1 h at 37°C at 5% CO2 on a plastic surface similar to WT. Relative gene expression of filament-inducing conditions was compared to yeast promoting conditions (YPD, 30°C) for three independent experiments. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1) and data are shown as mean+SD of three biological experiments (*p<0.05). (B) Immunofluorescence micrographs of cells immuno-stained for Als3 after growth in DMEM+10% FBS at 37°C and 5% CO2 on cover slips. Wild type (WT) and Evo cells are Als3-positive, while cph1Δ/efg1Δ cells show no signal (representative samples). (C) Morphogenetic response to farnesol treatment alone or in combination with exogenous dibutyryl-cyclic AMP (db-cAMP). All strains were exposed to either methanol (control), 1 µM farnesol or 1 µM farnesol+10 mM db-cAMP and incubated at 37°C and 5% CO2 for 18 h (representative pictures from three independent experiments are shown). Note that in Evo cells inhibition of filamentation by farnesol treatment was completely abrogated when db-cAMP was added. (D) Repression of hyphae-associated gene expression in the Evo strain by 10 µM farnesol. Expression was normalized against three housekeeping genes (ACT1, EFB1 and PMA1). The fold change in expression relative to filament-inducing conditions alone is shown as mean+SD of three biological experiments.
Mentions: Hyphal-associated virulence of C. albicans is not only due to filamentation per se, but also to the expression of hyphae-associated genes. In order to monitor the expression of typical hyphae-associated genes in the Evo strain, we measured the mRNA levels of HWP1, ECE1 and ALS3, all encoding hyphal cell surface proteins, and of EED1, a gene that is associated with hyphal cell elongation [27]. An upregulation of all four genes in the Evo strain was confirmed by qRT-PCR after 1 hour of incubation in DMEM+10% FBS at 37°C and 5% CO2 (Fig. 3A). HWP1 expression was similar in the Evo and WT strain, whereas ECE1 and ALS3 were higher expressed in the WT strain, and EED1 was more strongly upregulated in the Evo strain. Furthermore, we observed Als3 exposure on the surface of wild type and Evo cells by immunofluorescence, but not on the cph1Δ/efg1Δ strain (Fig. 3B). This regained cell-surface exposure of the Als3 adhesin [28] is in accordance with the increased adhesion potential of the Evo strain.

Bottom Line: In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation.We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery.These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Jena, Germany.

ABSTRACT
Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

No MeSH data available.


Related in: MedlinePlus