Gwenaël Ruprich-Robert

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.

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.

Combination of different molecular mechanisms leading to fluconazole resistance in a Candida lusitaniae clinical isolate.

We report on the underlying molecular mechanisms likely responsible for the high-level fluconazole resistance in a Candida lusitaniae clinical isolate. Fluconazole resistance correlated with overexpression of ERG11 and of several efflux pump genes, in particular, the orthologs of the Candida albicans MDR1, PDR16, CDR1, CDR2, and YOR1.

Insight into the Role of HOG Pathway Components Ssk2p, Pbs2p, and Hog1p in the Opportunistic Yeast Candida lusitaniae

ABSTRACT In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.

Bilirubin oxidase‐like proteins from Podospora anserina: promising thermostable enzymes for application in transformation of plant biomass

Plant biomass degradation by fungi is a critical step for production of biofuels, and laccases are common ligninolytic enzymes envisioned for ligninolysis. Bilirubin oxidases (BODs)-like are related to laccases, but their roles during lignocellulose degradation have not yet been fully investigated. The two BODs of the ascomycete fungus Podospora anserina were characterized by targeted gene deletions. Enzymatic assay revealed that the bod1(Δ) and bod2(Δ) mutants lost partly a thermostable laccase activity. A triple mutant inactivated for bod1, bod2 and mco, a previously investigated multicopper oxidase gene distantly related to laccases, had no thermostable laccase activity. The pattern of fruiting body production in the bod1(Δ) bod2(Δ) double mutant was changed. The bod1(Δ) and bod2(Δ) mutants were reduced in their ability to grow on ligneous and cellulosic materials. Furthermore, bod1(Δ) and bod2(Δ) mutants were defective towards resistance to phenolic substrates and H2 O2 , which may also impact lignocellulose breakdown. Double and triple mutants were more affected than single mutants, evidencing redundancy of function among BODs and mco. Overall, the data show that bod1, bod2 and mco code for non-canonical thermostable laccases that participate in the degradation of lignocellulose. Thanks to their thermal stability, these enzymes may be more promising candidate for biotechnological application than canonical laccases.

Characterization of three multicopper oxidases in the filamentous fungus Podospora anserina : A new role of an ABR1-like protein in fungal development?

The Podospora anserina genome contains a large family of 15 multicopper oxidases (MCOs), including three genes encoding a FET3-like protein, an ABR1-like protein and an ascorbate oxidase (AO)-like protein. FET3, ABR1 and AO1 are involved in global laccase-like activity since deletion of the relevant genes led to a decrease of activity when laccase substrate (ABTS) was used as substrate. However, contrary to the P. anserina MCO proteins previously characterized, none of these three MCOs seemed to be involved in lignocellulose degradation and in resistance to phenolic compounds and oxidative stress. We showed that the bulk of ferroxidase activity was clearly due to ABR1, and only in minor part to FET3, although ABR1 does not contain all the residues typical of FET3 proteins. Moreover, we showed that ABR1, related to the Aspergillus fumigatus ABR1 protein, was clearly and specifically involved in pigmentation of ascospores. Surprisingly, phenotypes were more severe in mutants lacking both abr1 and ao1. Deletion of the ao1 gene led to an almost total loss of AO activity. No direct involvement of AO1 in fungal developmental process in P. anserina was evidenced, except in a abr1Δ background. Overall, unlike other previously characterized MCOs, we thus evidence a clear involvement of ABR1 protein in fungal development.

Inositol-phosphate signaling as mediator for growth and sexual reproduction in Podospora anserina

The molecular pathways involved in the development of multicellular fruiting bodies in fungi are still not well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe PM154, a new strain of the model ascomycetes Podospora anserina able to mate with itself and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of spod1, one of the new mutants, we identified an inositol phosphate polykinase gene as essential, especially for fruiting body development. A factor present in the wild type and diffusible in mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in spod1, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in spod1. Overall, the data indicated that inositol and inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. The same effect of inositol was seen in two other fungi, Sordaria macrospora and Chaetomium globosum. Key role of the inositol polyphosphate pathway during fruiting body maturation appears thus conserved during the evolution of Sordariales fungi.