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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

Enhanced virulence and altered organ tropism of the evolved strain.A. Mice were intravenously infected with 5×107C. glabrata cells on day 0. Body weight of animals was monitored daily. During the first four days, infection with the evolved strain (Evo) led to severe loss in body weight, in contrast to mice infected with the parental strain (WT). B. Fungal burden was determined by culture from tissue homogenates of five animals per treatment group and time point. The distribution of fungal burden differed significantly between the wild type (WT) and the evolved strain (Evo) at the early time point. In the brain, the burden on day 2 p.i. was more than 100× higher for the evolved strain than for the wild type (median 1.1×107 cfu/ml vs. 4.9×104 cfu/ml). This difference was not present at later time points (days 7 and 21 p.i.), and both strains persisted at comparable levels in the organ. C. Representative histological images of brain tissue of infected mice. Brains infected with the evolved strain (Evo) showed large and numerous microcolonies (upper right picture, microcolonies are indicated by black arrows). Evo cells formed larger clumps of cells; the wild type (WT) formed only few small microcolonies in the brain (upper and lower left picture, a microcolony is indicated by a black arrow). Please note that the PAS stain used does not allow reliable differentiation between neuronal and endothelial cells.
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ppat-1004478-g004: Enhanced virulence and altered organ tropism of the evolved strain.A. Mice were intravenously infected with 5×107C. glabrata cells on day 0. Body weight of animals was monitored daily. During the first four days, infection with the evolved strain (Evo) led to severe loss in body weight, in contrast to mice infected with the parental strain (WT). B. Fungal burden was determined by culture from tissue homogenates of five animals per treatment group and time point. The distribution of fungal burden differed significantly between the wild type (WT) and the evolved strain (Evo) at the early time point. In the brain, the burden on day 2 p.i. was more than 100× higher for the evolved strain than for the wild type (median 1.1×107 cfu/ml vs. 4.9×104 cfu/ml). This difference was not present at later time points (days 7 and 21 p.i.), and both strains persisted at comparable levels in the organ. C. Representative histological images of brain tissue of infected mice. Brains infected with the evolved strain (Evo) showed large and numerous microcolonies (upper right picture, microcolonies are indicated by black arrows). Evo cells formed larger clumps of cells; the wild type (WT) formed only few small microcolonies in the brain (upper and lower left picture, a microcolony is indicated by a black arrow). Please note that the PAS stain used does not allow reliable differentiation between neuronal and endothelial cells.

Mentions: As expected, mice infected with the C. glabrata wild type strain remained clinically healthy throughout the experiment. In contrast, animals infected with the Evo strain displayed weight loss and unspecific symptoms of illness (ruffled fur and moderate lethargy) for 48 h after infection, but then recovered (Fig. 4A). Coinciding with differences in clinical presentation of infected mice, the distribution of fungal burdens differed significantly between the wild type and the evolved strain at the early time point (Fig. 4B). In the brain, the burden on day 2 post infection (p.i.) was more than 100× higher for the Evo strain than for the wild type (median 1.1×107 cfu/ml vs. 4.9×104 cfu/ml, respectively; p<0.005). This difference disappeared at later time points (days 7 and 21 p.i.), and both strains persisted at comparable levels in the organ.


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)

Enhanced virulence and altered organ tropism of the evolved strain.A. Mice were intravenously infected with 5×107C. glabrata cells on day 0. Body weight of animals was monitored daily. During the first four days, infection with the evolved strain (Evo) led to severe loss in body weight, in contrast to mice infected with the parental strain (WT). B. Fungal burden was determined by culture from tissue homogenates of five animals per treatment group and time point. The distribution of fungal burden differed significantly between the wild type (WT) and the evolved strain (Evo) at the early time point. In the brain, the burden on day 2 p.i. was more than 100× higher for the evolved strain than for the wild type (median 1.1×107 cfu/ml vs. 4.9×104 cfu/ml). This difference was not present at later time points (days 7 and 21 p.i.), and both strains persisted at comparable levels in the organ. C. Representative histological images of brain tissue of infected mice. Brains infected with the evolved strain (Evo) showed large and numerous microcolonies (upper right picture, microcolonies are indicated by black arrows). Evo cells formed larger clumps of cells; the wild type (WT) formed only few small microcolonies in the brain (upper and lower left picture, a microcolony is indicated by a black arrow). Please note that the PAS stain used does not allow reliable differentiation between neuronal and endothelial cells.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004478-g004: Enhanced virulence and altered organ tropism of the evolved strain.A. Mice were intravenously infected with 5×107C. glabrata cells on day 0. Body weight of animals was monitored daily. During the first four days, infection with the evolved strain (Evo) led to severe loss in body weight, in contrast to mice infected with the parental strain (WT). B. Fungal burden was determined by culture from tissue homogenates of five animals per treatment group and time point. The distribution of fungal burden differed significantly between the wild type (WT) and the evolved strain (Evo) at the early time point. In the brain, the burden on day 2 p.i. was more than 100× higher for the evolved strain than for the wild type (median 1.1×107 cfu/ml vs. 4.9×104 cfu/ml). This difference was not present at later time points (days 7 and 21 p.i.), and both strains persisted at comparable levels in the organ. C. Representative histological images of brain tissue of infected mice. Brains infected with the evolved strain (Evo) showed large and numerous microcolonies (upper right picture, microcolonies are indicated by black arrows). Evo cells formed larger clumps of cells; the wild type (WT) formed only few small microcolonies in the brain (upper and lower left picture, a microcolony is indicated by a black arrow). Please note that the PAS stain used does not allow reliable differentiation between neuronal and endothelial cells.
Mentions: As expected, mice infected with the C. glabrata wild type strain remained clinically healthy throughout the experiment. In contrast, animals infected with the Evo strain displayed weight loss and unspecific symptoms of illness (ruffled fur and moderate lethargy) for 48 h after infection, but then recovered (Fig. 4A). Coinciding with differences in clinical presentation of infected mice, the distribution of fungal burdens differed significantly between the wild type and the evolved strain at the early time point (Fig. 4B). In the brain, the burden on day 2 post infection (p.i.) was more than 100× higher for the Evo strain than for the wild type (median 1.1×107 cfu/ml vs. 4.9×104 cfu/ml, respectively; p<0.005). This difference disappeared at later time points (days 7 and 21 p.i.), and both strains persisted at comparable levels in the organ.

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