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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

A single nucleotide polymorphism in SSN3 is essential for filamentation.(A) Distinct impact on morphology by: deleting either the mutated (SSN3/ssn3mΔ) or the wild type SSN3 allele (ssn3Δ/SSN3m) in the Evo strain, by overexpressing either the wild type SSN3 allele (cph1Δ/efg1ΔSAT1SSN3OE) or the mutated SSN3 allele (cph1Δ/efg1ΔSAT1SSN3mOE) or by expressing the mutated SSN3 allele from its native locus (cph1Δ/efg1ΔSAT1SSN3m) in a newly generated cph1Δ/efg1ΔSAT1 strain. The partial SSN3 sequences demonstrate the homozygosity or heterozygosity of the SSN3 allele (left). Filamentous growth is visible in the Evo ssn3Δ/SSN3m, cph1Δ/efg1ΔSAT1SSN3mOE and cph1Δ/efg1ΔSAT1SSN3m strains after growth for 18 h at 37°C and 5% CO2 in DMEM+10% FBS and during co-incubation with macrophages, but not with the Evo SSN3/ssn3mΔ strain, cph1Δ/efg1ΔSAT1 and cph1Δ/efg1ΔSAT1SSN3OE strains (scale bars: 18 h, 50 µm and MΦ, 20 µm; representative pictures are shown) (right). (B) Cell damage of macrophages caused by the different strains, as determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation. Robust host cell damage depends on the presence of the mutated allele SSN3m. Mean and SD of n = 4 (*p<0.05; compared to cph1Δ/efg1Δ and cph1Δ/efg1ΔSAT1 respectively; LC = low control, medium only).
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pgen-1004824-g007: A single nucleotide polymorphism in SSN3 is essential for filamentation.(A) Distinct impact on morphology by: deleting either the mutated (SSN3/ssn3mΔ) or the wild type SSN3 allele (ssn3Δ/SSN3m) in the Evo strain, by overexpressing either the wild type SSN3 allele (cph1Δ/efg1ΔSAT1SSN3OE) or the mutated SSN3 allele (cph1Δ/efg1ΔSAT1SSN3mOE) or by expressing the mutated SSN3 allele from its native locus (cph1Δ/efg1ΔSAT1SSN3m) in a newly generated cph1Δ/efg1ΔSAT1 strain. The partial SSN3 sequences demonstrate the homozygosity or heterozygosity of the SSN3 allele (left). Filamentous growth is visible in the Evo ssn3Δ/SSN3m, cph1Δ/efg1ΔSAT1SSN3mOE and cph1Δ/efg1ΔSAT1SSN3m strains after growth for 18 h at 37°C and 5% CO2 in DMEM+10% FBS and during co-incubation with macrophages, but not with the Evo SSN3/ssn3mΔ strain, cph1Δ/efg1ΔSAT1 and cph1Δ/efg1ΔSAT1SSN3OE strains (scale bars: 18 h, 50 µm and MΦ, 20 µm; representative pictures are shown) (right). (B) Cell damage of macrophages caused by the different strains, as determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation. Robust host cell damage depends on the presence of the mutated allele SSN3m. Mean and SD of n = 4 (*p<0.05; compared to cph1Δ/efg1Δ and cph1Δ/efg1ΔSAT1 respectively; LC = low control, medium only).

Mentions: To ascertain the impact of the SNP on filamentation induction, we selectively deleted either the mutated or the wild type SSN3 allele in the Evo strain, using the dominant selection marker SAT1[53]. Sanger sequencing confirmed the exclusive presence of either one allele in the genome (Fig. 7A). Strikingly, when incubated in DMEM with 10% serum at 37°C and 5% CO2 only the strain with the mutated allele still present (Evo ssn3Δ/SSN3m) was able to induce and maintain filamentation. The mutant containing only the wild type allele (Evo SSN3/ssn3mΔ) remained in the elongated yeast form, and thus presented the typical ancestral (cph1Δ/efg1Δ) phenotype (Fig. 7A). In addition, only the Evo ssn3Δ/SSN3m strain could escape from macrophages by forming filaments like the wild type (Fig. 7A). The damage capacity correlated with this ability to produce filaments: While Evo and Evo ssn3Δ/SSN3m strains showed the same levels of phagocyte lysis, the Evo SSN3/ssn3mΔ strain caused significantly less damage during co-incubation with macrophages. In fact, damage was indistinguishable from the original cph1Δ/efg1Δ strain (Fig. 7B). In contrast, the deletion of the mutated allele had no influence on the hyphal development defect on solid medium (S5A Figure) and sensitivity to cell wall disturbing agents (S5B Figure).


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)

A single nucleotide polymorphism in SSN3 is essential for filamentation.(A) Distinct impact on morphology by: deleting either the mutated (SSN3/ssn3mΔ) or the wild type SSN3 allele (ssn3Δ/SSN3m) in the Evo strain, by overexpressing either the wild type SSN3 allele (cph1Δ/efg1ΔSAT1SSN3OE) or the mutated SSN3 allele (cph1Δ/efg1ΔSAT1SSN3mOE) or by expressing the mutated SSN3 allele from its native locus (cph1Δ/efg1ΔSAT1SSN3m) in a newly generated cph1Δ/efg1ΔSAT1 strain. The partial SSN3 sequences demonstrate the homozygosity or heterozygosity of the SSN3 allele (left). Filamentous growth is visible in the Evo ssn3Δ/SSN3m, cph1Δ/efg1ΔSAT1SSN3mOE and cph1Δ/efg1ΔSAT1SSN3m strains after growth for 18 h at 37°C and 5% CO2 in DMEM+10% FBS and during co-incubation with macrophages, but not with the Evo SSN3/ssn3mΔ strain, cph1Δ/efg1ΔSAT1 and cph1Δ/efg1ΔSAT1SSN3OE strains (scale bars: 18 h, 50 µm and MΦ, 20 µm; representative pictures are shown) (right). (B) Cell damage of macrophages caused by the different strains, as determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation. Robust host cell damage depends on the presence of the mutated allele SSN3m. Mean and SD of n = 4 (*p<0.05; compared to cph1Δ/efg1Δ and cph1Δ/efg1ΔSAT1 respectively; LC = low control, medium only).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256171&req=5

pgen-1004824-g007: A single nucleotide polymorphism in SSN3 is essential for filamentation.(A) Distinct impact on morphology by: deleting either the mutated (SSN3/ssn3mΔ) or the wild type SSN3 allele (ssn3Δ/SSN3m) in the Evo strain, by overexpressing either the wild type SSN3 allele (cph1Δ/efg1ΔSAT1SSN3OE) or the mutated SSN3 allele (cph1Δ/efg1ΔSAT1SSN3mOE) or by expressing the mutated SSN3 allele from its native locus (cph1Δ/efg1ΔSAT1SSN3m) in a newly generated cph1Δ/efg1ΔSAT1 strain. The partial SSN3 sequences demonstrate the homozygosity or heterozygosity of the SSN3 allele (left). Filamentous growth is visible in the Evo ssn3Δ/SSN3m, cph1Δ/efg1ΔSAT1SSN3mOE and cph1Δ/efg1ΔSAT1SSN3m strains after growth for 18 h at 37°C and 5% CO2 in DMEM+10% FBS and during co-incubation with macrophages, but not with the Evo SSN3/ssn3mΔ strain, cph1Δ/efg1ΔSAT1 and cph1Δ/efg1ΔSAT1SSN3OE strains (scale bars: 18 h, 50 µm and MΦ, 20 µm; representative pictures are shown) (right). (B) Cell damage of macrophages caused by the different strains, as determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation. Robust host cell damage depends on the presence of the mutated allele SSN3m. Mean and SD of n = 4 (*p<0.05; compared to cph1Δ/efg1Δ and cph1Δ/efg1ΔSAT1 respectively; LC = low control, medium only).
Mentions: To ascertain the impact of the SNP on filamentation induction, we selectively deleted either the mutated or the wild type SSN3 allele in the Evo strain, using the dominant selection marker SAT1[53]. Sanger sequencing confirmed the exclusive presence of either one allele in the genome (Fig. 7A). Strikingly, when incubated in DMEM with 10% serum at 37°C and 5% CO2 only the strain with the mutated allele still present (Evo ssn3Δ/SSN3m) was able to induce and maintain filamentation. The mutant containing only the wild type allele (Evo SSN3/ssn3mΔ) remained in the elongated yeast form, and thus presented the typical ancestral (cph1Δ/efg1Δ) phenotype (Fig. 7A). In addition, only the Evo ssn3Δ/SSN3m strain could escape from macrophages by forming filaments like the wild type (Fig. 7A). The damage capacity correlated with this ability to produce filaments: While Evo and Evo ssn3Δ/SSN3m strains showed the same levels of phagocyte lysis, the Evo SSN3/ssn3mΔ strain caused significantly less damage during co-incubation with macrophages. In fact, damage was indistinguishable from the original cph1Δ/efg1Δ strain (Fig. 7B). In contrast, the deletion of the mutated allele had no influence on the hyphal development defect on solid medium (S5A Figure) and sensitivity to cell wall disturbing agents (S5B Figure).

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