Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.

The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologues of all the genes necessary for sexual reproduction and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically engineered strains of N. delphensis, including by CRISPR-Cas 9 gene editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid-type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life cycle.

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

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.

Comparative genomics of emerging pathogens in the Candida glabrata clade.

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces. SEQUENCE ACCESSION NUMBERS: Nakaseomyces delphensis: CAPT01000001 to CAPT01000179Candida bracarensis: CAPU01000001 to CAPU01000251Candida nivariensis: CAPV01000001 to CAPV01000123Candida castellii: CAPW01000001 to CAPW01000101Nakaseomyces bacillisporus: CAPX01000001 to CAPX01000186.

Candidal abscess of the parotid gland due to Candida glabrata: report of a case and literature review.

We report the case of an immunocompetent woman who developed a Candida glabrata abscess of the parotid gland and present a review of similar cases from the literature. Diagnosis was based on the isolation of C. glabrata in pure culture from the abscess pus. Examination of stained smears of the same material demonstrated small sized yeast cells, some being intra-macrophagic. Combination of a local drainage and oral fluconazole proved to be an efficient therapeutic strategy. Candidal abscesses are rare in immunocompetent patients and salivary gland localization has only been reported in five cases.

Invasive Saccharomyces infection: a comprehensive review.

BACKGROUND: Saccharomyces cerevisiae (also known as "baker's yeast" or "brewer's yeast") is mostly considered to be an occasional digestive commensal. However, since the 1990s, there have been a growing number of reports about its implication as an etiologic agent of invasive infection. A particular feature of such infections is their association with a probiotic preparation of Saccharomyces boulardii (a subtype of S. cerevisiae) for treatment various diarrheal disorders. METHODS: We collected published case reports, through May 2005, of invasive Saccharomyces infection by use of a Medline query. Epidemiological and clinical charts and therapeutic strategies were analyzed. RESULTS: We found 92 cases of Saccharomyces invasive infection. Predisposing factors were similar to those of invasive candidiasis, with intravascular catheter and antibiotic therapy being the most frequent. Blood was the most frequent site of isolation (for 72 patients). S. boulardii accounted for 51.3% of fungemias and was exclusively isolated from blood. Compared with patients infected with S. cerevisiae, patients infected with S. boulardii were more frequently immunocompetent and had a better prognosis. Saccharomyces invasive infection was clinically indistinguishable from an invasive candidiasis. Overall, S. cerevisiae clinical isolates exhibited low susceptibility to amphotericin B and azole derivatives. However, global outcome was favorable in 62% of the cases. Treatment with intravenous amphotericin B and fluconazole, in combination with central vascular catheter removal, were effective therapeutic options. CONCLUSION: Saccharomyces organisms should now be added to the growing list of emerging fungal pathogens. Special caution should be taken regarding the use of S. boulardii probiotic preparations.

Efficient Mating-Type Switching in Candida glabrata Induces Cell Death.

Candida glabrata is an apparently asexual haploid yeast that is phylogenetically closer to Saccharomyces cerevisiae than to Candida albicans. Its genome contains three MAT-like cassettes, MAT, which encodes either MATa or MATalpha information in different strains, and the additional loci, HML and HMR. The genome also contains an HO gene homolog, but this yeast has never been shown to switch mating-types spontaneously, as S. cerevisiae does. We have recently sequenced the genomes of the five species that, together with C. glabrata, make up the Nakaseomyces clade. All contain MAT-like cassettes and an HO gene homolog. In this work, we express the HO gene of all Nakaseomyces and of S. cerevisiae in C. glabrata. All can induce mating-type switching, but, despite the larger phylogenetic distance, the most efficient endonuclease is the one from S. cerevisiae. Efficient mating-type switching in C. glabrata is accompanied by a high cell mortality, and sometimes results in conversion of the additional cassette HML. Mortality probably results from the cutting of the HO recognition sites that are present, in HML and possibly HMR, contrary to what happens naturally in S. cerevisiae. This has implications in the life-cycle of C. glabrata, as we show that efficient MAT switching is lethal for most cells, induces chromosomal rearrangements in survivors, and that the endogenous HO is probably rarely active indeed.

Hepatic and pulmonary cystic echinococcosis in a patient from the Central African Republic.

Apical lung opacity was diagnosed in an asymptomatic 30 year-old woman native of Central African Republic by routine chest X-ray. CT scan demonstrated an excavated pulmonary mass and revealed a simple hepatic cyst. Tuberculosis was suspected but mycobacterial cultures remained negative. Three months later, ultrasonography showed septations within the hepatic lesion suggestive of cystic echinococcosis. The detection of seric anti-Echinococcus antibodies was positive. Hepatic and pulmonary cysts were removed surgically and association with three-month course of albendazole resulted in a favorable outcome. Cystic echinococcosis is exceptional in Central Africa and to our knowledge never reported from the Central African Republic.