Florence Chapeland-Leclerc

Acquisition of Flucytosine, Azole, and Caspofungin Resistance in Candida glabrata Bloodstream Isolates Serially Obtained from a Hematopoietic Stem Cell Transplant Recipient

ABSTRACT We describe the acquisition of flucytosine, azole, and caspofungin resistance in sequential Candida glabrata bloodstream isolates collected from a bone marrow transplant patient with clinical failure. Point mutations in C. glabrata FUR1 (CgFUR1) and CgFKS2 and overexpression of CgCDR1 and CgCDR2 were observed in resistant isolates.

Molecular Mechanism of Flucytosine Resistance in Candida lusitaniae: Contribution of the FCY2, FCY1, and FUR1 Genes to 5-Fluorouracil and Fluconazole Cross-Resistance

ABSTRACT Inactivation of the FCY2 (cytosine permease), FCY1 (cytosine deaminase), and FUR1 (uracil phosphoribosyltransferase) genes in Candida lusitaniae produced two patterns of resistance to flucytosine. Mutant fur1 demonstrated resistance to 5-fluorouracil, whereas mutants fcy1 and fcy2 demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.

Inactivation of the FCY2 Gene Encoding Purine-Cytosine Permease Promotes Cross-Resistance to Flucytosine and Fluconazole in Candida lusitaniae

ABSTRACT In a previous work, we described the possible relationship between a defect of purine-cytosine permease and the acquisition of a cross-resistance to the antifungal combination flucytosine (5FC) and fluconazole (FLC) in Candida lusitaniae (T. Noël, F. François, P. Paumard, C. Chastin, D. Brethes, and J. Villard, Antimicrob. Agents Chemother. 47:1275-1284, 2003). Using degenerate PCR and chromosome walking, we cloned two FCY2-like genes in C. lusitaniae. Northern blot analysis revealed that only one gene was expressed; it was named FCY2. The other one behaved as a pseudogene and was named FCY21. In order to better characterize the possible role of FCY2 in cross-resistance to 5FC-FLC, disruption experiments with auxotrophic strain 6936 ura3(D95V) FCY2 with an integrative vector carrying the URA3 gene and a partial sequence of the C. lusitaniae FCY2 gene were undertaken. Southern blot analysis revealed that homologous recombination events occurred in all transformants analyzed at rates of 50% at resident locus FCY2 and 50% at resident locus URA3, resulting in the genotypes ura3 fcy2::URA3 and ura3::URA3 FCY2, respectively. It was then demonstrated that only transformants harboring a disrupted fcy2 gene were resistant to 5FC, susceptible to FLC, and resistant to the 5FC-FLC combination. Finally, complementation experiments with a functional FCY2 gene restored 5FC and FLC susceptibilities to the wild-type levels. The results of this study provide molecular evidence that inactivation of the sole FCY2 gene promotes cross-resistance to the antifungal association 5FC-FLC in C. lusitaniae.

Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae.

ABSTRACT The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae. We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2(C505T) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC- and 5FC/FLC-resistant fcy1Δ strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.

Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker

The nucleotide sequence of the URA3 gene encoding orotidine‐5′‐phosphate decarboxylase (OMP DCase) of the human opportunistic pathogen yeast Candida lusitaniae was determined by degenerate PCR and chromosome walking. Deduced amino acid sequence showed strong homologies (59–85% identity) with OMP DCases of different Saccharomycetales and allowed identification of the known conserved domains. Very close upstream from the URA3 gene, the 3′‐end of a gene encoding a Gea2‐like protein was identified. A non‐revertible C. lusitaniae ura3 mutant was selected on the basis of 5‐fluoroorotic acid resistance. The mutation was a single point mutation resulting in the amino acid substitution D95V in a highly conserved domain, and in a concomitant EcoRV restriction site polymorphism. The mutant strain was successfully transformed to prototrophy following electroporation with the URA3 gene cloned in an integrative vector, with frequencies of 100–200 transformants per µg of DNA. Southern blot analysis revealed that almost all transformants were derived from homologous recombination events at the resident locus. The GeneBank Accession No. for C. lusitaniae URA3 gene is AF450297. Copyright

Differential Involvement of Histidine Kinase Receptors in Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast Candida lusitaniae

ABSTRACT Fungal histidine kinase receptors (HKRs) sense and transduce many extracellular signals. We investigated the role of HKRs in morphogenetic transition, osmotolerance, oxidative stress response, and mating ability in the opportunistic yeast Candida lusitaniae. We isolated three genes, SLN1, NIK1, and CHK1, potentially encoding HKRs of classes VI, III, and X, respectively. These genes were disrupted by a transformation system based upon the “URA3 blaster” strategy. Functional analysis of disruptants was undertaken, except for the sln1 nik1 double mutant and the sln1 nik1 chk1 triple mutant, which are not viable in C. lusitaniae. The sln1 mutant revealed a high sensitivity to oxidative stress, whereas both the nik1 and chk1 mutants exhibited a more moderate sensitivity to peroxide. We also showed that the NIK1 gene was implicated in phenylpyrrole and dicarboximide compound susceptibility while HKRs seem not to be involved in resistance toward antifungals of clinical relevance. Concerning mating ability, all disruptants were still able to reproduce sexually in vitro in unilateral or bilateral crosses. The most important result of this study was that the sln1 mutant displayed a global defect of pseudohyphal differentiation, especially in high-osmolarity and oxidative-stress conditions. Thus, the SLN1 gene could be crucial for the C. lusitaniae yeast-to-pseudohypha morphogenetic transition. This implication is strengthened by a high level of SLN1 mRNAs revealed by semiquantitative reverse transcription-PCR when the yeast develops pseudohyphae. Our findings highlight a differential contribution of the three HKRs in osmotic and oxidant adaptation during the morphological transition in C. lusitaniae.

Role of Sho1p adaptor in the pseudohyphal development, drugs sensitivity, osmotolerance and oxidant stress adaptation in the opportunistic yeast Candida lusitaniae.

In yeast, external signals such as high osmolarity or oxidant conditions activate the high osmolarity glycerol (HOG) mitogen‐activated protein kinase (MAPK) cascade pathway, which consists of two upstream branches, i.e. Sho1p and Sln1p and common downstream elements, including the Pbs2p MAPK kinase and the Hog1p MAPK. We recently showed that the Candida lusitaniae SLN1 gene, potentially encoding a histidine kinase receptor, is crucial for oxidative stress adaptation when the fungus grows as budding yeast and during the early steps of pseudohyphal development. In the current study, we characterized the SHO1 gene of this opportunistic fungus. Complete loss of SHO1 function causes profound defects in pseudohyphal differentiation, especially in high osmolarity and oxidative stress conditions, suggesting a crucial role of SHO1 in the pseudohyphae morphogenetic transitions. Moreover, when grown as budding yeast, the sho1Δ mutant revealed a sensitivity to compounds that interfere with the cell wall assembly, pointing to a potential role of Sho1p in cell wall biogenesis. However, the sho1Δ mutant does not display evident cell‐wall architecture modifications, such as aggregation phenotypes. Although not hypersusceptible to antifungals of clinical relevance, the sho1Δ mutants are susceptible to the filamentous fungi‐specific antifungals dicarboximides and phenylpyrroles. Finally, our findings highlight some significant phenotypic differences when the C. lusitaniae sho1Δ mutant is compared with the corresponding mutants described in Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus. The GeneBank Accession No. for C. lusitaniae SHO1 gene is EU797514. Copyright

Contributions of the Response Regulators Ssk1p and Skn7p in the Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast Candida lusitaniae

ABSTRACT We recently characterized the histidine kinase receptor genes of Candida lusitaniae. For the present study, we have further investigated the role of SSK1 and SKN7, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.

Systematic gene deletions evidences that laccases are involved in several stages of wood degradation in the filamentous fungus Podospora anserina

Transformation of plant biomass into biofuels may supply environmentally friendly alternative biological sources of energy. Laccases are supposed to be involved in the lysis of lignin, a prerequisite step for efficient breakdown of cellulose into fermentable sugars. The role in development and plant biomass degradation of the nine canonical laccases belonging to three different subfamilies and one related multicopper oxidase of the Ascomycota fungus Podospora anserina was investigated by targeted gene deletion. The 10 genes were inactivated singly, and multiple mutants were constructed by genetic crosses. lac6(Δ), lac8(Δ) and mco(Δ) mutants were significantly reduced in their ability to grow on lignin-containing materials, but also on cellulose and plastic. Furthermore, lac8(Δ), lac7(Δ), mco(Δ) and lac6(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and multiple mutants were generally more affected than single mutants, evidencing redundancy of function among laccases. Our study provides the first genetic evidences that laccases are major actors of wood utilization in a fungus and that they have multiple roles during this process apart from participation in lignin lysis.

Disruption of Bcchs4, Bcchs6 or Bcchs7 chitin synthase genes in Botrytis cinerea and the essential role of class VI chitin synthase (Bcchs6)

Chitin synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulié et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI chitin synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target.