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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

Single nucleotide polymorphism in SSN3 of the Evo strain and location of the mutated amino acid.(A) Partial SSN3 sequence for cph1Δ/efg1Δ and Evo strains flanking SNP 1055 (marked with an arrow). Notice the heterozygosity in the Evo strain. (B) Schematic view of the catalytic domain of Ssn3 (STK = serine/threonine kinase) with the position of the activation segment highlighted in brown and the amino acid exchange indicated by an arrow (top). Sequence alignment of the Ssn3 activation segment in different species (H. s. Homo sapiens [NP_001251.1], M. m. Mus musculus [NP_705827.2], C. n. Cryptococcus neoformans [XP_568416.1], C. a. C. albicans [XP_720918.1] and S. c. Saccharomyces cerevisiae [NP_015283.1]). The arrow indicates the amino acid exchange in the Evo strain. The Mg-binding loop is highlighted in yellow, the activation loop in blue and the P+1 loop in purple. Amino acids that are known to abrogate kinase activity when mutated are colored in green [51], [98]. Asterisks underneath the alignment indicate positions with conserved amino acids and colons indicate highly similar residues (bottom). The mutated arginine (red) is part of the highly conserved P+1 substrate recognition loop.
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pgen-1004824-g006: Single nucleotide polymorphism in SSN3 of the Evo strain and location of the mutated amino acid.(A) Partial SSN3 sequence for cph1Δ/efg1Δ and Evo strains flanking SNP 1055 (marked with an arrow). Notice the heterozygosity in the Evo strain. (B) Schematic view of the catalytic domain of Ssn3 (STK = serine/threonine kinase) with the position of the activation segment highlighted in brown and the amino acid exchange indicated by an arrow (top). Sequence alignment of the Ssn3 activation segment in different species (H. s. Homo sapiens [NP_001251.1], M. m. Mus musculus [NP_705827.2], C. n. Cryptococcus neoformans [XP_568416.1], C. a. C. albicans [XP_720918.1] and S. c. Saccharomyces cerevisiae [NP_015283.1]). The arrow indicates the amino acid exchange in the Evo strain. The Mg-binding loop is highlighted in yellow, the activation loop in blue and the P+1 loop in purple. Amino acids that are known to abrogate kinase activity when mutated are colored in green [51], [98]. Asterisks underneath the alignment indicate positions with conserved amino acids and colons indicate highly similar residues (bottom). The mutated arginine (red) is part of the highly conserved P+1 substrate recognition loop.

Mentions: As the SNP at nucleotide position 1,055 in the SSN3 ORF (Fig. 6A) was detected in both analyses, we focused our investigation on this specific mutation. Ssn3 has been well characterized in Saccharomyces cerevisiae as an RNA polymerase II holoenzyme-associated cyclin-dependent kinase of the Mediator complex contributing to transcriptional control [50]. It was shown that Ssn3 promotes the degradation of the transcription factor Ste12 by phosphorylation and thereby regulates S. cerevisiae filamentous growth [51]. As depicted in Fig. 6B, the heterozygous Arg352Gln mutation of Ssn3 in the Evo strain is located within the activation segment of the protein kinase catalytic domain. An amino acid sequence comparison of C. albicans Ssn3 to sequences from S. cerevisiae, Cryptococcus neoformans, Mus musculus and Homo sapiens demonstrated this arginine residue to be conserved from fungi to mammals. The activation segment comprises several conserved structural features: the magnesium binding loop, the activation loop and the P+1 loop, in which the mutation occurred. While the activation loop is the site of regulatory phosphorylation in many kinases, the P+1 loop forms a pocket that recognizes the substrate protein [52].


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)

Single nucleotide polymorphism in SSN3 of the Evo strain and location of the mutated amino acid.(A) Partial SSN3 sequence for cph1Δ/efg1Δ and Evo strains flanking SNP 1055 (marked with an arrow). Notice the heterozygosity in the Evo strain. (B) Schematic view of the catalytic domain of Ssn3 (STK = serine/threonine kinase) with the position of the activation segment highlighted in brown and the amino acid exchange indicated by an arrow (top). Sequence alignment of the Ssn3 activation segment in different species (H. s. Homo sapiens [NP_001251.1], M. m. Mus musculus [NP_705827.2], C. n. Cryptococcus neoformans [XP_568416.1], C. a. C. albicans [XP_720918.1] and S. c. Saccharomyces cerevisiae [NP_015283.1]). The arrow indicates the amino acid exchange in the Evo strain. The Mg-binding loop is highlighted in yellow, the activation loop in blue and the P+1 loop in purple. Amino acids that are known to abrogate kinase activity when mutated are colored in green [51], [98]. Asterisks underneath the alignment indicate positions with conserved amino acids and colons indicate highly similar residues (bottom). The mutated arginine (red) is part of the highly conserved P+1 substrate recognition loop.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004824-g006: Single nucleotide polymorphism in SSN3 of the Evo strain and location of the mutated amino acid.(A) Partial SSN3 sequence for cph1Δ/efg1Δ and Evo strains flanking SNP 1055 (marked with an arrow). Notice the heterozygosity in the Evo strain. (B) Schematic view of the catalytic domain of Ssn3 (STK = serine/threonine kinase) with the position of the activation segment highlighted in brown and the amino acid exchange indicated by an arrow (top). Sequence alignment of the Ssn3 activation segment in different species (H. s. Homo sapiens [NP_001251.1], M. m. Mus musculus [NP_705827.2], C. n. Cryptococcus neoformans [XP_568416.1], C. a. C. albicans [XP_720918.1] and S. c. Saccharomyces cerevisiae [NP_015283.1]). The arrow indicates the amino acid exchange in the Evo strain. The Mg-binding loop is highlighted in yellow, the activation loop in blue and the P+1 loop in purple. Amino acids that are known to abrogate kinase activity when mutated are colored in green [51], [98]. Asterisks underneath the alignment indicate positions with conserved amino acids and colons indicate highly similar residues (bottom). The mutated arginine (red) is part of the highly conserved P+1 substrate recognition loop.
Mentions: As the SNP at nucleotide position 1,055 in the SSN3 ORF (Fig. 6A) was detected in both analyses, we focused our investigation on this specific mutation. Ssn3 has been well characterized in Saccharomyces cerevisiae as an RNA polymerase II holoenzyme-associated cyclin-dependent kinase of the Mediator complex contributing to transcriptional control [50]. It was shown that Ssn3 promotes the degradation of the transcription factor Ste12 by phosphorylation and thereby regulates S. cerevisiae filamentous growth [51]. As depicted in Fig. 6B, the heterozygous Arg352Gln mutation of Ssn3 in the Evo strain is located within the activation segment of the protein kinase catalytic domain. An amino acid sequence comparison of C. albicans Ssn3 to sequences from S. cerevisiae, Cryptococcus neoformans, Mus musculus and Homo sapiens demonstrated this arginine residue to be conserved from fungi to mammals. The activation segment comprises several conserved structural features: the magnesium binding loop, the activation loop and the P+1 loop, in which the mutation occurred. While the activation loop is the site of regulatory phosphorylation in many kinases, the P+1 loop forms a pocket that recognizes the substrate protein [52].

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