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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

Increased fitness of the evolved strain in macrophages in direct competition.Macrophages were infected with WT and Evo cells at a ratio of 100∶1 at day 0 and the relative proportion between the two strains monitored daily. The ratio reversed after a few days of coincubation, demonstrating an advantage for the Evo strain during interaction with macrophages. Mean values and standard deviations of three independent experiments are shown.
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ppat-1004478-g002: Increased fitness of the evolved strain in macrophages in direct competition.Macrophages were infected with WT and Evo cells at a ratio of 100∶1 at day 0 and the relative proportion between the two strains monitored daily. The ratio reversed after a few days of coincubation, demonstrating an advantage for the Evo strain during interaction with macrophages. Mean values and standard deviations of three independent experiments are shown.

Mentions: We were interested to know whether the appearance and expansion of the Evo strain in our original co-culture experiment was due to a high selective advantage of this strain in macrophage co-culture. Therefore, we inoculated fresh RAW 246.7 cells with the wild type and the evolved strain at a ratio of 100∶1, and performed daily transfers as in the original co-culture, but with an additional sonification step to ensure similar uptake ratios of WT and Evo strain (see below). After an average of 7 passages (in three independent experiments), the ratio completely inverted to approximately 1∶100 wild type to evolved strain (Fig. 2). This corresponds to a mean competitive fitness ratio of 4.75 d−1 for the wrinkled strain under macrophage exposure.


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)

Increased fitness of the evolved strain in macrophages in direct competition.Macrophages were infected with WT and Evo cells at a ratio of 100∶1 at day 0 and the relative proportion between the two strains monitored daily. The ratio reversed after a few days of coincubation, demonstrating an advantage for the Evo strain during interaction with macrophages. Mean values and standard deviations of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004478-g002: Increased fitness of the evolved strain in macrophages in direct competition.Macrophages were infected with WT and Evo cells at a ratio of 100∶1 at day 0 and the relative proportion between the two strains monitored daily. The ratio reversed after a few days of coincubation, demonstrating an advantage for the Evo strain during interaction with macrophages. Mean values and standard deviations of three independent experiments are shown.
Mentions: We were interested to know whether the appearance and expansion of the Evo strain in our original co-culture experiment was due to a high selective advantage of this strain in macrophage co-culture. Therefore, we inoculated fresh RAW 246.7 cells with the wild type and the evolved strain at a ratio of 100∶1, and performed daily transfers as in the original co-culture, but with an additional sonification step to ensure similar uptake ratios of WT and Evo strain (see below). After an average of 7 passages (in three independent experiments), the ratio completely inverted to approximately 1∶100 wild type to evolved strain (Fig. 2). This corresponds to a mean competitive fitness ratio of 4.75 d−1 for the wrinkled strain under macrophage exposure.

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