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One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

Brunke S, Seider K, Fischer D, Jacobsen ID, Kasper L, Jablonowski N, Wartenberg A, Bader O, Enache-Angoulvant A, Schaller M, d'Enfert C, Hube B - PLoS Pathog. (2014)

Bottom Line: Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure.Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type.These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

View Article: PubMed Central - PubMed

Affiliation: Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC), Universitätsklinikum Jena, Jena, Germany; Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Jena, Germany.

ABSTRACT
Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

No MeSH data available.


Related in: MedlinePlus

No large-scale genomic changes, but single SNP differences can be detected between WT and Evo strains.Following alignment of Solexa/Illumina reads for the genomes of strains ATCC2001 and Evo on the C. glabrata reference genome, an average coverage score was calculated for each 1 kb region and normalized to the coverage obtained across the whole genome. These coverage ratio are shown in log2 scale. C. glabrata chromosomes A to M are shown in alternating black and grey colors. The location of SNPs identified in both strains relative to the reference genome is shown with green diamonds. The location of SNPs that distinguish the two strains is shown with red diamonds, the SNP on chromosome I responsible for the phenotype of strain Evo being shown in larger size. Note that several 1 kb regions harbored more than one SNP and are nevertheless represented using a single diamond.
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ppat-1004478-g006: No large-scale genomic changes, but single SNP differences can be detected between WT and Evo strains.Following alignment of Solexa/Illumina reads for the genomes of strains ATCC2001 and Evo on the C. glabrata reference genome, an average coverage score was calculated for each 1 kb region and normalized to the coverage obtained across the whole genome. These coverage ratio are shown in log2 scale. C. glabrata chromosomes A to M are shown in alternating black and grey colors. The location of SNPs identified in both strains relative to the reference genome is shown with green diamonds. The location of SNPs that distinguish the two strains is shown with red diamonds, the SNP on chromosome I responsible for the phenotype of strain Evo being shown in larger size. Note that several 1 kb regions harbored more than one SNP and are nevertheless represented using a single diamond.

Mentions: Complete genome sequences were then obtained by Solexa/Illumina technology from the parental strain and the Evo strain. The 36 bp single-end reads were aligned to the ATCC2001 reference genome [28], with 98.5% of the reference genome covered for the two strains and an average sequencing depth of 69.6-fold and 73.1-fold for the parental strain and Evo strain, respectively (Table S2). Sequencing depth was plotted over the chromosomes to detect duplication or deletion events. Sequencing depth for both strains was homogeneous across all chromosomes, except for chromosome K that showed a duplication of ca. 130 kb on its left arm, consistent with previous karyotyping [chromosome K* in 7], and chromosomes C and L that showed over-covered regions corresponding to a tandem array of genes encoding adhesin-like proteins and rDNA, respectively (Fig. 6). Additionally, several under-covered regions were detected; these were mainly located at subtelomeres and telomeres, known to harbor gene families and tandem repeats [29], [30]. Importantly, no obvious difference could be observed between the wild type and the evolved strain with respect to regions showing increased or decreased sequencing depth (Fig. 6). Similarly, when the sequencing depth over individual ORFs was analyzed, no obvious difference was observed between the two strains (Fig. S7). Taken together, these results suggest that no major regional amplifications or deletions had occurred during the microevolution process. This is in agreement with results obtained from karyotyping via PFGE and microsatellite analysis (Fig. S6).


One small step for a yeast--microevolution within macrophages renders Candida glabrata hypervirulent due to a single point mutation.

Brunke S, Seider K, Fischer D, Jacobsen ID, Kasper L, Jablonowski N, Wartenberg A, Bader O, Enache-Angoulvant A, Schaller M, d'Enfert C, Hube B - PLoS Pathog. (2014)

No large-scale genomic changes, but single SNP differences can be detected between WT and Evo strains.Following alignment of Solexa/Illumina reads for the genomes of strains ATCC2001 and Evo on the C. glabrata reference genome, an average coverage score was calculated for each 1 kb region and normalized to the coverage obtained across the whole genome. These coverage ratio are shown in log2 scale. C. glabrata chromosomes A to M are shown in alternating black and grey colors. The location of SNPs identified in both strains relative to the reference genome is shown with green diamonds. The location of SNPs that distinguish the two strains is shown with red diamonds, the SNP on chromosome I responsible for the phenotype of strain Evo being shown in larger size. Note that several 1 kb regions harbored more than one SNP and are nevertheless represented using a single diamond.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214790&req=5

ppat-1004478-g006: No large-scale genomic changes, but single SNP differences can be detected between WT and Evo strains.Following alignment of Solexa/Illumina reads for the genomes of strains ATCC2001 and Evo on the C. glabrata reference genome, an average coverage score was calculated for each 1 kb region and normalized to the coverage obtained across the whole genome. These coverage ratio are shown in log2 scale. C. glabrata chromosomes A to M are shown in alternating black and grey colors. The location of SNPs identified in both strains relative to the reference genome is shown with green diamonds. The location of SNPs that distinguish the two strains is shown with red diamonds, the SNP on chromosome I responsible for the phenotype of strain Evo being shown in larger size. Note that several 1 kb regions harbored more than one SNP and are nevertheless represented using a single diamond.
Mentions: Complete genome sequences were then obtained by Solexa/Illumina technology from the parental strain and the Evo strain. The 36 bp single-end reads were aligned to the ATCC2001 reference genome [28], with 98.5% of the reference genome covered for the two strains and an average sequencing depth of 69.6-fold and 73.1-fold for the parental strain and Evo strain, respectively (Table S2). Sequencing depth was plotted over the chromosomes to detect duplication or deletion events. Sequencing depth for both strains was homogeneous across all chromosomes, except for chromosome K that showed a duplication of ca. 130 kb on its left arm, consistent with previous karyotyping [chromosome K* in 7], and chromosomes C and L that showed over-covered regions corresponding to a tandem array of genes encoding adhesin-like proteins and rDNA, respectively (Fig. 6). Additionally, several under-covered regions were detected; these were mainly located at subtelomeres and telomeres, known to harbor gene families and tandem repeats [29], [30]. Importantly, no obvious difference could be observed between the wild type and the evolved strain with respect to regions showing increased or decreased sequencing depth (Fig. 6). Similarly, when the sequencing depth over individual ORFs was analyzed, no obvious difference was observed between the two strains (Fig. S7). Taken together, these results suggest that no major regional amplifications or deletions had occurred during the microevolution process. This is in agreement with results obtained from karyotyping via PFGE and microsatellite analysis (Fig. S6).

Bottom Line: Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure.Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type.These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

View Article: PubMed Central - PubMed

Affiliation: Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC), Universitätsklinikum Jena, Jena, Germany; Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Jena, Germany.

ABSTRACT
Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures - a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.

No MeSH data available.


Related in: MedlinePlus