Anne Favel

Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis

BackgroundFilamentous fungi are potent biomass degraders due to their ability to thrive in ligno(hemi)cellulose-rich environments. During the last decade, fungal genome sequencing initiatives have yielded abundant information on the genes that are putatively involved in lignocellulose degradation. At present, additional experimental studies are essential to provide insights into the fungal secreted enzymatic pools involved in lignocellulose degradation.ResultsIn this study, we performed a wide analysis of 20 filamentous fungi for which genomic data are available to investigate their biomass-hydrolysis potential. A comparison of fungal genomes and secretomes using enzyme activity profiling revealed discrepancies in carbohydrate active enzymes (CAZymes) sets dedicated to plant cell wall. Investigation of the contribution made by each secretome to the saccharification of wheat straw demonstrated that most of them individually supplemented the industrial Trichoderma reesei CL847 enzymatic cocktail. Unexpectedly, the most striking effect was obtained with the phytopathogen Ustilago maydis that improved the release of total sugars by 57% and of glucose by 22%. Proteomic analyses of the best-performing secretomes indicated a specific enzymatic mechanism of U. maydis that is likely to involve oxido-reductases and hemicellulases.ConclusionThis study provides insight into the lignocellulose-degradation mechanisms by filamentous fungi and allows for the identification of a number of enzymes that are potentially useful to further improve the industrial lignocellulose bioconversion process.

Colony morphology switching of Candida lusitaniae and acquisition of multidrug resistance during treatment of a renal infection in a newborn: case report and review of the literature.

Candida lusitaniae is an emerging opportunistic pathogen which exhibits an unusual antifungal susceptibility pattern. We describe a case of fatal renal infection due to C. lusitaniae in a very low birth weight neonate who was treated with short courses of fluconazole given alternately with amphotericin B. A colony morphology switching was detected on the standard primary culture medium by changes in colony size. Switching was shown to affect deeply the susceptibility to amphotericin B. Afterwards, the switched phenotype developed a cross resistance to fluconazole and itraconazole. Several issues raised by this case are discussed in the light of an extensive review of the literature. Our observations point out the importance of both the detection of colony morphology switching and the close monitoring of antifungal susceptibility in the management of infections due to C. lusitaniae. A judicious therapeutic strategy should prevent the acquisition of multidrug resistance during antifungal therapy.

Exploring the Natural Fungal Biodiversity of Tropical and Temperate Forests toward Improvement of Biomass Conversion

ABSTRACT In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.

Alternative Identification Test Relying upon Sexual Reproductive Abilities of Candida lusitaniae Strains Isolated from Hospitalized Patients

ABSTRACT The in vitro mating ability of Candidalusitaniae (teleomorph Clavisporalusitaniae) clinical isolates has been investigated. Studying the effects of culture conditions, we showed that ammonium ion depletion in the medium is a major trigger of the sexual cycle. Moreover, a solid support is required for mating, suggesting a role for adhesion factors in addition to the mating type gene recognition function. Monitoring of mating and meiosis efficiency with auxotrophic strains showed great variations in ascospore yields, which appeared to be strain and temperature dependent, with an optimal range of 18 to 28°C. The morphogenetic events taking place from mating to ascospore release were studied by scanning and electron microscopy, and the ultrastructure of the conjugation canal, through which intercellular nuclear exchanges occur, was revealed. Labeling experiments with a lectin-fluorochrome system revealed that the nuclear transfer was predominantly polarized, thus allowing a distinction between the nucleus donor and the nucleus acceptor strains. The direction of the transfer depended on the strain combination used, rather than on the genotypes of the strains, and did not appear to be controlled by the mating type genes. Finally, we demonstrated that all of the 76 clinical isolates used in this study were able to reproduce sexually when mated with an opposite mating type strain, and we identified a 1:1MATa/MATα ratio in the collection. These results support the idea that there is no anamorph state in C. lusitaniae. Accordingly, the mating type test, which is easy to use and can usually be completed within 48 h, is a reliable alternative identification system forC. lusitaniae.

Pycnoporus laccase-mediated bioconversion of rutin to oligomers suitable for biotechnology applications.

The Pycnoporus fungi are white-rot basidiomycetes listed as food- and cosmetic-grade microorganisms. Three high redox potential laccases from Pycnoporus coccineus and Pycnoporus sanguineus were tested and compared, with the commercial Suberase® as reference, for their ability to synthesise natural active oligomers from rutin (quercetin-3-rutinoside, one of the best-known naturally occurring flavonoid glycosides). The aim of this work was to develop a process with technical parameters (solvent, temperature, reaction time and raw materials) that were easy to scale up for industrial production and compatible with cosmetic and pharmaceutical formulation guidelines. The aqueous mixture of glycerol/ethanol/buffer described in this study met this requirement and allowed the solubilisation of rutin and its oxidative bioconversion into oligomers. The four flavonoid oligomer mixtures synthesised using laccases as catalysts were analysed by high performance liquid chromatography–diode array detection–negative electrospray ionisation–multistage mass spectrometry. Their chromatographic elution profiles were compared and 16 compounds were characterised and identified as dimers and trimers of rutin. The oligorutins were different in Suberase® and Pycnoporus laccase reaction mixtures. They were evaluated for their antioxidant, anti-inflammatory and anti-ageing activities on specific enzymatic targets such as cyclooxygenase (COX-2) and human matrix metalloproteinase 3 (MMP-3). Expressed in terms of IC50, the flavonoid oligomers displayed a 2.5- to 3-fold higher superoxide scavenging activity than monomeric rutin. Pycnoporus laccase and Suberase® oligorutins led to an inhibition of COX-2 of about 35% and 70%, respectively, while monomeric rutin showed a near-negligible inhibition effect, less than about 10%. The best results on MMP-3 activity were obtained with rutin oligomers from P. sanguineus IMB W006-2 laccase and Suberase® with about 70–75% inhibition.

Differentiation between Atypical Isolates of Candida lusitaniae and Candida pulcherrima by Determination of Mating Type

ABSTRACT We report on five clinical isolates routinely identified as Candida lusitaniae that the ID 32C system was unable to discriminate from the closely related species Candida pulcherrima. When additional tests did not allow accurate identification, the less usual mating type test identified all of them as Clavispora lusitaniae. Mating type testing appears to be a valuable tool for assessing the true incidence of this emerging non-albicans Candida species.

Molecular phylogeny of Trametes and related genera, and description of a new genus Leiotrametes

A phylogenetic analysis of Trametes and related taxa is proposed, based on a wide sampling of temperate and tropical species. Concatenate sequences of ribosomal DNA (ITS1-5.8S-ITS2) and RPB2 gene from mycelia cultures were analyzed by Maximum Likelihood and Bayesian methods, whilst morphological features were documented from the corresponding herbarium vouchers. Congruent results were obtained from analyses of ribosomal LSU sequences downloaded from Genbank. The Bayesian analysis of ITS + RPB2 sequences gave the best resolution for the phylogenetic reconstructions and revealed the existence of three main lineages in the Trametes-clade: 1) a monospecific lineage represented by Artolenzites elegans 2) a lineage including the genus Pycnoporus in its traditional sense and several species usually classified in the genus Trametes (T. cingulata, T. lactinea, T. ljubarskyi, T. menziesii) & 3) a lineage corresponding to the core genus Trametes, including type species of Trametes, Coriolopsis and Lenzites. The presence of a pseudostipe, aspect and structure of the abhymenial surface, colour change with 5% aqueous solutions of potassium hydroxide and topography of pigments on skeletal hyphae gave relevant morphological support to these clades. When the structure of the hymenial surface, presence of a black line below the tomentum and color of context (except for genus Pycnoporus) usually used in traditional polypore-classifications did not reveal any phylogenetic significance. A partial systematic arrangement of the Trametes clade is proposed, with the introduction of a new genus: Leiotrametes Welti & Courtec. gen. nov.. Two new combinations: Leiotrametes lactinea (Berk.) Welti & Courtec. comb. nov. and L. menziesii (Berk.) Welti & Courtec. comb. nov. are proposed.

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.

Phylogeographic relationships in the polypore fungus Pycnoporus inferred from molecular data.

The genus Pycnoporus forms a group of four species known especially for producing high redox potential laccases suitable for white biotechnology. A sample of 36 Pycnoporus strains originating from different geographical areas was studied to seek informative molecular markers for the typing of new strains in laboratory culture conditions and to analyse the phylogeographic relationships in this cosmopolitan group. ITS1-5.8S-ITS2 ribosomal DNA and partial regions of β-tubulin and laccase lac3-1 gene were sequenced. Phylogenetic trees inferred from these sequences clearly differentiated the group of Pycnoporus cinnabarinus strains from the group of Pycnoporus puniceus strains into strongly supported clades (100% bootstrap value). Molecular clustering based on lac 3-1 sequences enabled the distribution of Pycnoporus sanguineus and Pycnoporus coccineus through four distinct, well supported clades and sub-clades. A neotropical sub-clade, grouping the P. sanguineus strains from French Guiana and Venezuela, corresponded to P. sanguineus sensu stricto. A paleotropical sub-clade, clustering the strains from Madagascar, Vietnam and New Caledonia, was defined as Pycnoporus cf. sanguineus. The Australian clade corresponded to P. coccineus sensu stricto. The Eastern Asian region clade, clustering the strains from China and Japan, formed a P. coccineus-like group. Laccase gene (lac 3-1) analysis within the Pycnoporus species can highlight enzyme functional diversity associated with biogeographical origin.

Trametes polyzona, an emerging filamentous basidiomycete in Réunion Island

We describe two serious Trametes polyzona pulmonary infections, which occurred in Réunion Island, in critically ill patients. The identification was performed using sequencing of the internal transcribed spacer region of ribosomal DNA and D1/D2 region of 28S rDNA. In one case, the significance of T. polyzona in the pathological process was certain, proven by histopathological evidence of fungal lung infection. T. polyzona, an emerging filamentous basidiomycete, prevalent in tropical areas, has not been described so far in human infections.