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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

A single nucleotide exchange is sufficient to produce the evolved phenotype.A. Sanger sequencing confirmed the sequence alteration in CHS2 of the Evo strain (identified by whole genome sequencing), which led to an Asn→Lys (WT→Evo) amino acid exchange in the protein. Following a counter-selection experiment, the gene reverted to its original sequence (Rev), concomitant with the reversal to the original yeast growth form. B. This single nucleotide exchange observed in of the Evo strain was introduced into the WT strain by PCR amplification of CHS2 from the Evo strain, and cotransformation of this fragment with an PCR-amplified HIS3 marker including an overlapping (U1) region. C. The resulting strain was called CHSEvo, and correct integration was tested by sequencing. Similarly, CHSWT was created by amplifying the WT CHS2 gene and following the same cloning strategy. D. Morphologies of the WT, Evo, CHSWT and CHSEvo strain. The introduction of the Evo CHS2 gene into CHSEvo resulted in a growth form indistinguishable from the original Evo strain. The reintroduction of the WT gene did not change morphology (CHSWT).
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ppat-1004478-g007: A single nucleotide exchange is sufficient to produce the evolved phenotype.A. Sanger sequencing confirmed the sequence alteration in CHS2 of the Evo strain (identified by whole genome sequencing), which led to an Asn→Lys (WT→Evo) amino acid exchange in the protein. Following a counter-selection experiment, the gene reverted to its original sequence (Rev), concomitant with the reversal to the original yeast growth form. B. This single nucleotide exchange observed in of the Evo strain was introduced into the WT strain by PCR amplification of CHS2 from the Evo strain, and cotransformation of this fragment with an PCR-amplified HIS3 marker including an overlapping (U1) region. C. The resulting strain was called CHSEvo, and correct integration was tested by sequencing. Similarly, CHSWT was created by amplifying the WT CHS2 gene and following the same cloning strategy. D. Morphologies of the WT, Evo, CHSWT and CHSEvo strain. The introduction of the Evo CHS2 gene into CHSEvo resulted in a growth form indistinguishable from the original Evo strain. The reintroduction of the WT gene did not change morphology (CHSWT).

Mentions: The Saccharomyces cerevisiae orthologues of NSP1 and PYC2 encode an essential nucleoporin and a pyruvate carboxylase, respectively. Interestingly, in S. cerevisiae, CHS2 encodes a chitin synthase. Moreover, S. cerevisiae cells lacking CHS2 grew in clumps and exhibited thick septa, which lacked an intact primary septum [31]. Given these notable similarities between S. cerevisiae chs2Δ and our Evo strain, we reasoned that the Asn→Lys exchange in the Chs2 chitin synthase (Fig. 7A) may be responsible for the altered growth phenotype of the Evo strain. Indeed, the corresponding (Asn556) residue in ScChs2 lies within the chitin synthase catalytic domain and is essential for full enzymatic activity in S. cerevisiae[32]. We therefore introduced the evolved Asn→Lys mutation into a C. glabrata wild type strain, replacing the original copy of CHS2 (strain CHSEvo, Fig. 7B and 7C).


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)

A single nucleotide exchange is sufficient to produce the evolved phenotype.A. Sanger sequencing confirmed the sequence alteration in CHS2 of the Evo strain (identified by whole genome sequencing), which led to an Asn→Lys (WT→Evo) amino acid exchange in the protein. Following a counter-selection experiment, the gene reverted to its original sequence (Rev), concomitant with the reversal to the original yeast growth form. B. This single nucleotide exchange observed in of the Evo strain was introduced into the WT strain by PCR amplification of CHS2 from the Evo strain, and cotransformation of this fragment with an PCR-amplified HIS3 marker including an overlapping (U1) region. C. The resulting strain was called CHSEvo, and correct integration was tested by sequencing. Similarly, CHSWT was created by amplifying the WT CHS2 gene and following the same cloning strategy. D. Morphologies of the WT, Evo, CHSWT and CHSEvo strain. The introduction of the Evo CHS2 gene into CHSEvo resulted in a growth form indistinguishable from the original Evo strain. The reintroduction of the WT gene did not change morphology (CHSWT).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004478-g007: A single nucleotide exchange is sufficient to produce the evolved phenotype.A. Sanger sequencing confirmed the sequence alteration in CHS2 of the Evo strain (identified by whole genome sequencing), which led to an Asn→Lys (WT→Evo) amino acid exchange in the protein. Following a counter-selection experiment, the gene reverted to its original sequence (Rev), concomitant with the reversal to the original yeast growth form. B. This single nucleotide exchange observed in of the Evo strain was introduced into the WT strain by PCR amplification of CHS2 from the Evo strain, and cotransformation of this fragment with an PCR-amplified HIS3 marker including an overlapping (U1) region. C. The resulting strain was called CHSEvo, and correct integration was tested by sequencing. Similarly, CHSWT was created by amplifying the WT CHS2 gene and following the same cloning strategy. D. Morphologies of the WT, Evo, CHSWT and CHSEvo strain. The introduction of the Evo CHS2 gene into CHSEvo resulted in a growth form indistinguishable from the original Evo strain. The reintroduction of the WT gene did not change morphology (CHSWT).
Mentions: The Saccharomyces cerevisiae orthologues of NSP1 and PYC2 encode an essential nucleoporin and a pyruvate carboxylase, respectively. Interestingly, in S. cerevisiae, CHS2 encodes a chitin synthase. Moreover, S. cerevisiae cells lacking CHS2 grew in clumps and exhibited thick septa, which lacked an intact primary septum [31]. Given these notable similarities between S. cerevisiae chs2Δ and our Evo strain, we reasoned that the Asn→Lys exchange in the Chs2 chitin synthase (Fig. 7A) may be responsible for the altered growth phenotype of the Evo strain. Indeed, the corresponding (Asn556) residue in ScChs2 lies within the chitin synthase catalytic domain and is essential for full enzymatic activity in S. cerevisiae[32]. We therefore introduced the evolved Asn→Lys mutation into a C. glabrata wild type strain, replacing the original copy of CHS2 (strain CHSEvo, Fig. 7B and 7C).

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