Limits...
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

Co-incubation with macrophages led to regained filamentation in the cph1Δ/efg1Δ strain.Morphology of wild type (WT), cph1Δ/efg1Δ and Evo strain after 18 h of incubation in DMEM+10% FBS at 37°C and 5% CO2 demonstrate the re-appearance of filamentation in the Evo strain (scale bar: 100 µm, upper panel). All strains were grown for 4 h for filipin (Fil) staining, and for 2 h or 16 h for calcofluor white (CFW) staining on cover slips, and analyzed by fluorescence microscopy (scale bar: 10 µm, lower panels). Arrow heads highlight septa (true hyphae), while asterisks indicate constrictions (pseudohyphae).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256171&req=5

pgen-1004824-g001: Co-incubation with macrophages led to regained filamentation in the cph1Δ/efg1Δ strain.Morphology of wild type (WT), cph1Δ/efg1Δ and Evo strain after 18 h of incubation in DMEM+10% FBS at 37°C and 5% CO2 demonstrate the re-appearance of filamentation in the Evo strain (scale bar: 100 µm, upper panel). All strains were grown for 4 h for filipin (Fil) staining, and for 2 h or 16 h for calcofluor white (CFW) staining on cover slips, and analyzed by fluorescence microscopy (scale bar: 10 µm, lower panels). Arrow heads highlight septa (true hyphae), while asterisks indicate constrictions (pseudohyphae).

Mentions: To test whether the regained ability to form filaments was restricted to macrophage interactions or observed under additional hypha-inducing conditions, we analyzed the morphology of the Evo strain in the absence of host cells. In the cell culture medium DMEM with 10% serum at 37°C and 5% CO2, clear filament formation of the Evo strain, but not the cph1Δ/efg1Δ strain, was observed (Fig. 1). Filamentous growth is associated with highly polarized ergosterol inclusion in membranes, which can be visualized by filipin staining [25]. As shown in Fig. 1, Evo cells grown in the presence of serum exhibited intense filipin staining at the filament tips, equal to the wild type cells. Consistent with the defect in polarized growth, the cph1Δ/efg1Δ strain showed a more uniform filipin staining. Staining with calcofluor white for morphology analyses showed the expected true hyphae for the wild type and elongated yeasts for the cph1Δ/efg1Δ strain (Fig. 1). Interestingly, the Evo strain showed heterogeneous cell morphologies, i.e. a mixture of pseudohyphae with constrictions at the septa and true hyphae with parallel-sided walls (Fig. 1). The percentage of the different morphological forms was quantified using the morphological index (MI) [26] of individual cells after 4 and 12 hours of growth in serum (S2A Figure). The MI for cph1Δ/efg1Δ was <2.5 at both time points, indicating yeast morphology. In contrast, most cells of the Evo strain grew as pseudohyphae (MI 2.5–3.4) after 4 hours, while after 12 hours true hyphae were evident (MI>3.4) in approx. 50% of the population. Both morphologies will be referred to as filaments.


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)

Co-incubation with macrophages led to regained filamentation in the cph1Δ/efg1Δ strain.Morphology of wild type (WT), cph1Δ/efg1Δ and Evo strain after 18 h of incubation in DMEM+10% FBS at 37°C and 5% CO2 demonstrate the re-appearance of filamentation in the Evo strain (scale bar: 100 µm, upper panel). All strains were grown for 4 h for filipin (Fil) staining, and for 2 h or 16 h for calcofluor white (CFW) staining on cover slips, and analyzed by fluorescence microscopy (scale bar: 10 µm, lower panels). Arrow heads highlight septa (true hyphae), while asterisks indicate constrictions (pseudohyphae).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004824-g001: Co-incubation with macrophages led to regained filamentation in the cph1Δ/efg1Δ strain.Morphology of wild type (WT), cph1Δ/efg1Δ and Evo strain after 18 h of incubation in DMEM+10% FBS at 37°C and 5% CO2 demonstrate the re-appearance of filamentation in the Evo strain (scale bar: 100 µm, upper panel). All strains were grown for 4 h for filipin (Fil) staining, and for 2 h or 16 h for calcofluor white (CFW) staining on cover slips, and analyzed by fluorescence microscopy (scale bar: 10 µm, lower panels). Arrow heads highlight septa (true hyphae), while asterisks indicate constrictions (pseudohyphae).
Mentions: To test whether the regained ability to form filaments was restricted to macrophage interactions or observed under additional hypha-inducing conditions, we analyzed the morphology of the Evo strain in the absence of host cells. In the cell culture medium DMEM with 10% serum at 37°C and 5% CO2, clear filament formation of the Evo strain, but not the cph1Δ/efg1Δ strain, was observed (Fig. 1). Filamentous growth is associated with highly polarized ergosterol inclusion in membranes, which can be visualized by filipin staining [25]. As shown in Fig. 1, Evo cells grown in the presence of serum exhibited intense filipin staining at the filament tips, equal to the wild type cells. Consistent with the defect in polarized growth, the cph1Δ/efg1Δ strain showed a more uniform filipin staining. Staining with calcofluor white for morphology analyses showed the expected true hyphae for the wild type and elongated yeasts for the cph1Δ/efg1Δ strain (Fig. 1). Interestingly, the Evo strain showed heterogeneous cell morphologies, i.e. a mixture of pseudohyphae with constrictions at the septa and true hyphae with parallel-sided walls (Fig. 1). The percentage of the different morphological forms was quantified using the morphological index (MI) [26] of individual cells after 4 and 12 hours of growth in serum (S2A Figure). The MI for cph1Δ/efg1Δ was <2.5 at both time points, indicating yeast morphology. In contrast, most cells of the Evo strain grew as pseudohyphae (MI 2.5–3.4) after 4 hours, while after 12 hours true hyphae were evident (MI>3.4) in approx. 50% of the population. Both morphologies will be referred to as filaments.

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