Limits...
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

Long-term co-incubation of C. glabrata with RAW 264.7 macrophages yields a C. glabrata strain with pseudohyphae-like morphology.A. Daily passage of C. glabrata with RAW 264.7 macrophages led to the formation of a pseudohyphae-like growth phenotype. The left panel shows the C. glabrata Evo strain interacting with RAW 264.7 cells. In contrast to the parental strain (WT; middle picture) the evolved strain (Evo) formed clumps (right picture) in liquid media. B. The parental wild type (WT) formed smooth colonies on YPD agar; colonies of the evolved strain (Evo) grew with a strongly wrinkled morphology. Both strains appeared purple colored on CHROMagar plates (right picture), characteristic for C. glabrata. C. Transmission electron micrographs indicate a cell separation defect of the evolved (Evo) strain in comparison to the WT, and show enlarged septa between mother and daughter cells, as well as an increased thickness of the outer cell wall layer.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214790&req=5

ppat-1004478-g001: Long-term co-incubation of C. glabrata with RAW 264.7 macrophages yields a C. glabrata strain with pseudohyphae-like morphology.A. Daily passage of C. glabrata with RAW 264.7 macrophages led to the formation of a pseudohyphae-like growth phenotype. The left panel shows the C. glabrata Evo strain interacting with RAW 264.7 cells. In contrast to the parental strain (WT; middle picture) the evolved strain (Evo) formed clumps (right picture) in liquid media. B. The parental wild type (WT) formed smooth colonies on YPD agar; colonies of the evolved strain (Evo) grew with a strongly wrinkled morphology. Both strains appeared purple colored on CHROMagar plates (right picture), characteristic for C. glabrata. C. Transmission electron micrographs indicate a cell separation defect of the evolved (Evo) strain in comparison to the WT, and show enlarged septa between mother and daughter cells, as well as an increased thickness of the outer cell wall layer.

Mentions: We continued the experiment for over six months. After nearly one month of daily transfers of 106–107 yeasts each, C. glabrata cells with an unusual morphology appeared, and then were enriched over the following weeks. (Fig. 1A, left). This evolved strain of C. glabrata (here called ‘Evo’ strain) showed a striking alteration in growth form: it changed from the typical single spherical yeasts (Fig. 1A, middle) to chains of tightly connected yeasts, reminiscent of pseudohyphal growth (Fig. 1A, right). Colonies on agar plates appeared strongly wrinkled instead of smooth (Fig. 1B, left). We verified the strain to be C. glabrata by genetic tests and the color reaction on CHROMagar (Fig. 1B, right).


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)

Long-term co-incubation of C. glabrata with RAW 264.7 macrophages yields a C. glabrata strain with pseudohyphae-like morphology.A. Daily passage of C. glabrata with RAW 264.7 macrophages led to the formation of a pseudohyphae-like growth phenotype. The left panel shows the C. glabrata Evo strain interacting with RAW 264.7 cells. In contrast to the parental strain (WT; middle picture) the evolved strain (Evo) formed clumps (right picture) in liquid media. B. The parental wild type (WT) formed smooth colonies on YPD agar; colonies of the evolved strain (Evo) grew with a strongly wrinkled morphology. Both strains appeared purple colored on CHROMagar plates (right picture), characteristic for C. glabrata. C. Transmission electron micrographs indicate a cell separation defect of the evolved (Evo) strain in comparison to the WT, and show enlarged septa between mother and daughter cells, as well as an increased thickness of the outer cell wall layer.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004478-g001: Long-term co-incubation of C. glabrata with RAW 264.7 macrophages yields a C. glabrata strain with pseudohyphae-like morphology.A. Daily passage of C. glabrata with RAW 264.7 macrophages led to the formation of a pseudohyphae-like growth phenotype. The left panel shows the C. glabrata Evo strain interacting with RAW 264.7 cells. In contrast to the parental strain (WT; middle picture) the evolved strain (Evo) formed clumps (right picture) in liquid media. B. The parental wild type (WT) formed smooth colonies on YPD agar; colonies of the evolved strain (Evo) grew with a strongly wrinkled morphology. Both strains appeared purple colored on CHROMagar plates (right picture), characteristic for C. glabrata. C. Transmission electron micrographs indicate a cell separation defect of the evolved (Evo) strain in comparison to the WT, and show enlarged septa between mother and daughter cells, as well as an increased thickness of the outer cell wall layer.
Mentions: We continued the experiment for over six months. After nearly one month of daily transfers of 106–107 yeasts each, C. glabrata cells with an unusual morphology appeared, and then were enriched over the following weeks. (Fig. 1A, left). This evolved strain of C. glabrata (here called ‘Evo’ strain) showed a striking alteration in growth form: it changed from the typical single spherical yeasts (Fig. 1A, middle) to chains of tightly connected yeasts, reminiscent of pseudohyphal growth (Fig. 1A, right). Colonies on agar plates appeared strongly wrinkled instead of smooth (Fig. 1B, left). We verified the strain to be C. glabrata by genetic tests and the color reaction on CHROMagar (Fig. 1B, right).

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