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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

Microevolution led to decreased sensitivity of the Evo strain to different cell wall perturbing agents.(A) Resistance of analyzed strains against different cell wall stressors. The cph1Δ/efg1Δ strain was sensitive to all stresses while the Evo strain regained WT resistance (representative pictures of three experiments are shown). (B) Flow cytometry analysis of mannan and β-glucan exposure on the surface of live cells. Differences in fluorescence intensity between cph1Δ/efg1Δ strain and Evo strain point to an altered cell wall composition. Mean fluorescence intensity+SD of n = 3 (*p<0.05). (C) Western blot analysis to identify phosphorylated Mkc1 and Cek1 in C. albicans strains grown under non-stress conditions (control) or conditions of cell wall stress (450 µg/ml congo red) for 4 hours. Cell wall stress triggered phosphorylation of Mkc1 and Cek1 in the Evo strain, but not in the cph1Δ/efg1Δ strain. Tubulin served as loading control.
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pgen-1004824-g005: Microevolution led to decreased sensitivity of the Evo strain to different cell wall perturbing agents.(A) Resistance of analyzed strains against different cell wall stressors. The cph1Δ/efg1Δ strain was sensitive to all stresses while the Evo strain regained WT resistance (representative pictures of three experiments are shown). (B) Flow cytometry analysis of mannan and β-glucan exposure on the surface of live cells. Differences in fluorescence intensity between cph1Δ/efg1Δ strain and Evo strain point to an altered cell wall composition. Mean fluorescence intensity+SD of n = 3 (*p<0.05). (C) Western blot analysis to identify phosphorylated Mkc1 and Cek1 in C. albicans strains grown under non-stress conditions (control) or conditions of cell wall stress (450 µg/ml congo red) for 4 hours. Cell wall stress triggered phosphorylation of Mkc1 and Cek1 in the Evo strain, but not in the cph1Δ/efg1Δ strain. Tubulin served as loading control.

Mentions: Our data indicated that the Evo strain regained the potential to produce hyphae, showed upregulation of transcription factor genes involved in filamentous growth and other hyphal associated genes, and regained a high virulence potential. The reduced virulence of the cph1Δ/efg1Δ strain is likely predominantly caused by the filamentation defects, however, Efg1 has also an important role in cell wall architecture [32] and the cell wall is essential for adhesion and invasive growth and thus for pathogenicity [33]. We therefore tested the Evo strain for cell wall defects by treatment with cell wall perturbing agents, i.e. congo red (CR), calcofluor white (CFW) and sodium dodecyl sulfate (SDS). As shown in Fig. 5A, the cph1Δ/efg1Δ strain was hypersensitive to all tested agents. In contrast, the Evo strain was as resistant as the wild type to CR and CFW, agents that disturb glucan and chitin architecture, respectively. The same phenotypic reversal was observed for the cell membrane disturbing agent SDS, suggesting a loose structure of the cell wall only in the cph1Δ/efg1Δ 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)

Microevolution led to decreased sensitivity of the Evo strain to different cell wall perturbing agents.(A) Resistance of analyzed strains against different cell wall stressors. The cph1Δ/efg1Δ strain was sensitive to all stresses while the Evo strain regained WT resistance (representative pictures of three experiments are shown). (B) Flow cytometry analysis of mannan and β-glucan exposure on the surface of live cells. Differences in fluorescence intensity between cph1Δ/efg1Δ strain and Evo strain point to an altered cell wall composition. Mean fluorescence intensity+SD of n = 3 (*p<0.05). (C) Western blot analysis to identify phosphorylated Mkc1 and Cek1 in C. albicans strains grown under non-stress conditions (control) or conditions of cell wall stress (450 µg/ml congo red) for 4 hours. Cell wall stress triggered phosphorylation of Mkc1 and Cek1 in the Evo strain, but not in the cph1Δ/efg1Δ strain. Tubulin served as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004824-g005: Microevolution led to decreased sensitivity of the Evo strain to different cell wall perturbing agents.(A) Resistance of analyzed strains against different cell wall stressors. The cph1Δ/efg1Δ strain was sensitive to all stresses while the Evo strain regained WT resistance (representative pictures of three experiments are shown). (B) Flow cytometry analysis of mannan and β-glucan exposure on the surface of live cells. Differences in fluorescence intensity between cph1Δ/efg1Δ strain and Evo strain point to an altered cell wall composition. Mean fluorescence intensity+SD of n = 3 (*p<0.05). (C) Western blot analysis to identify phosphorylated Mkc1 and Cek1 in C. albicans strains grown under non-stress conditions (control) or conditions of cell wall stress (450 µg/ml congo red) for 4 hours. Cell wall stress triggered phosphorylation of Mkc1 and Cek1 in the Evo strain, but not in the cph1Δ/efg1Δ strain. Tubulin served as loading control.
Mentions: Our data indicated that the Evo strain regained the potential to produce hyphae, showed upregulation of transcription factor genes involved in filamentous growth and other hyphal associated genes, and regained a high virulence potential. The reduced virulence of the cph1Δ/efg1Δ strain is likely predominantly caused by the filamentation defects, however, Efg1 has also an important role in cell wall architecture [32] and the cell wall is essential for adhesion and invasive growth and thus for pathogenicity [33]. We therefore tested the Evo strain for cell wall defects by treatment with cell wall perturbing agents, i.e. congo red (CR), calcofluor white (CFW) and sodium dodecyl sulfate (SDS). As shown in Fig. 5A, the cph1Δ/efg1Δ strain was hypersensitive to all tested agents. In contrast, the Evo strain was as resistant as the wild type to CR and CFW, agents that disturb glucan and chitin architecture, respectively. The same phenotypic reversal was observed for the cell membrane disturbing agent SDS, suggesting a loose structure of the cell wall only in the cph1Δ/efg1Δ 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