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Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to horhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhorhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

International audience; Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

International audience; Electrochemical determination of paracetamol (PCT) was successfully performed using carbon paste electrodes (CPEs) modified with treated coffee husks (CHt) or cellulose powder (Ce). Scanning electron microscopy was used to characterize unmodified or modified CPEs prior to their use. The electrochemical oxidation of PCT was investigated using square wave voltammetry (SWV) and cyclic voltammetry (CV). The oxidation current density of PCT was two-fold higher with the CPE-CHt sensor and 30% higher with CPE-Ce in comparison with the unmodified CPE, and this correlated with the higher hydrophilicity of the modified electrodes. Using SWV for the electrochemical analysis of PCT, carbon paste electrode modified with raw coffee husks (CPE-CHr) showed the presence of impurities at +0.27 V/SCE, showing the interest in using pure cellulose for the present analytical application. Furthermore, CPE-Ce presented a higher real area compared to CPE-CHr, which explains the increase in the limit of saturation from 400 mg/L to 950 mg/L. The better saturation limit exhibited by CPE-Ce justifies its choice for electroanalysis of PCT in commercialized tablets. The proposed method was successfully applied in the determination of PCT in commercialized tablets (Doliprane® 500) with a recovery rate close to 100%, and no interference with the excipients contained in the tablets analyzed was observed.

International audience; Patients with cystic fibrosis (CF) are at high risk of colonization of the respiratory tract by filamentous fungi, mainly Aspergillus fumigatus, but also other Aspergillus species including A. terreus. In our experience, this last fungus ranks the third among the filamentous fungi colonizing the respiratory tract of CF patients. Additionally, although uncommon, infections caused by A. terreus present a high mortality rate due to its usually low susceptibility to systemic antifungals. The recent development of a microsatellite typing system allowed us to investigate the molecular epidemiology of the airway colonization by this fungus in CF. Nine short tandem repeats of A. terreus were used to genotype multiple and sequential isolates from sputum samples from CF patients followed-up in Angers and Giens hospitals (France). Sputum samples were collected over a 2 month to 7 year period, and for each sample, all the obtained isolates were studied, with a maximum of five per sample. Thus a total of 122 isolates was studied, corresponding to 47 samples collected from 5 distinct patients. Three colonization patterns were observed. The first one consisted of a chronic colonization (presence of the same genotype in at least two successive samples collected over a minimum period of two months) by a largely dominant genotype associated with two or three other genotypes found occasionally (patient 1) or over a short period (patient 2). The second pattern consisted of a chronic colonization by two distinct genotypes simultaneously detected (patients 3 and 4). For the last patient (patient 5), 16 isolates recovered from 6 sputum samples collected during four years were analyzed, corresponding to 8 distinct genotypes which succeeded to each other. Numerous questions rise from these different colonization patterns, relatively uncommon environmental fungus; and (ii) differences between genotypes in their ability to chronically colonize the airways of the CF patients or differences between CF patients in their individual susceptibility to same genotypes. Strikingly, some genotypes shared by some patients were responsible for a chronic colonization in some patients while they rapidly disappear in other patients. A multicenter study should be conducted combining genetic study of the host and genotyping of fungal isolates, searching for predisposing factors to the airway colonization by A. terreus.

International audience; Clinical reports have established that mucormycosis, mainly caused by Rhizopus spp., frequently occurs in patients treated with deferoxamine B (DFO, Desferal®) which is misappropriated by these fungi. Siderophore production by twenty mucoralean isolates was therefore investigated using a commercial iron-depleted culture medium. Siderophore production was detected for most of the isolates. Our experiments confirmed that feroxamine B (iron chelate of DFO) promoted in vitro growth of Rhizopus arrhizus. Electrophoretic analysis of somatic extracts revealed iron-regulated proteins of 60 and 32kDa which were lacking in iron-depleted culture conditions. Using a fluorescent derivative of deferoxamine B, we showed by fluorescence microscopy the entry of the siderophore within the fungal cells, thus suggesting a shuttle mechanism encompassing the uptake of the entire siderophore-ion complex into the cell. This useful tool renders possible a better understanding of iron metabolism in Mucorales which could lead to the development of new diagnostic method or new antifungal therapy using siderophores as imaging contrast agents or active drug vectors.

International audience; As various new sibling species within the Pseudallescheria boydii/Scedosporium apiospermum complex have been described recently with differences in their susceptibility to antifungals, this study was conducted in order to determine their respective frequency in cystic fibrosis. Results indicated that P. boydii largely predominated (62%), followed by S. apiospermum (24%), Scedosporium aurantiacum (10%) and Pseudallescheria minutispora (4%). Scedosporium dehoogii was not recovered in this study. The multiple correspondence factor analysis highlighted geographical discrepancies within species distribution: P. boydii was rarely encountered in Northern France, while S. apiospermum was less represented in the west of the country. Additionally, we demonstrated that all species encountered in the cystic fibrosis context were capable to chronically colonize the respiratory tract of patients. Molecular typing of a large set of environmental and clinical isolates should be conducted to delineate the epidemiology of each sibling species in the complex.

The nonpathogenic filamentous fungus Scedosporium dehoogii was used for the first time to study the electrochemical biodegradation of acetaminophen (APAP). A carbon fiber microelectrode (CFME) modified by nickel tetrasulfonated phthalocyanine (p-NiTSPc) and a carbon paste electrode (CPE) modified with coffee husks (CH) were prepared to follow the kinetics of APAP biodegradation. The electrochemical response of APAP at both electrodes was studied by cyclic voltammetry and square wave voltammetry. p-NiTSPc-CFME was suitable to measure high concentrations of APAP, whereas CH-CPE gave rise to high current densities but was subject to the passivation phenomenon. p-NiTSPc-CFME was then successfully applied as a sensor to describe the kinetics of APAP biodegradation: this was found to be of first order with a kinetics constant of 0.11 day(-1) (at 25 °C) and a half-life of 6.30 days. APAP biodegradation by the fungus did not lead to the formation of p-aminophenol (PAP) and hydroquinone (HQ) that are carcinogenic, mutagenic, and reprotoxic (CMR). Graphical Abstract The kinetics of APAP biodegradation, followed by a poly-nickel tetrasulfonated phtalocyanine modified carbon fiber microelectrode.

Objectives: Cystic fibrosis is the most common genetic inherited disease in the European Caucasian population (frequency 1 out of 2500 births). Disease causes are mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene, which encodes a chloride channel in the plasma membrane of several epithelial cells. However, prognosis essentially depends on the severity of the lesions in the lungs. The defective mucociliary clearance and the thickening of the bronchial mucus resulting from these mutations facilitate the entrapment of airborne microbes. Patients with such chronic pulmonary defect are at high risk for colonizations/infections by fungi, including the emergent pathogens of the Scedoporium apiospermum complex. This species complex ranks the second among the filamentous fungi colonizing the airways of CF patients. Recent molecular studies supported by morphological, physiological and genetic observations showed that the S. apiospermum complex comprises at least five distinct species. Antifungal susceptibility profiles and virulence of these sibling species showed differences. This study aims to determine the prevalence of the different species of S. apiospermum complex in the CF context and to analyze the chronic trait of these infections. Methods: A set of 50 epidemiologically unrelated isolates was analyzed to estimate the prevalence, all isolates having been recovered from distinct patients with CF followed up in different university hospitals from France and initially identified as belonging to the S. apiospermum complex. The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal RNA gene, a fragment of the nuclear gene calmodulin (CAL) and two regions (BT2 and TUB) within the s-tubulin gene were amplified and sequenced as described by Gilgado et al. Additionally, sequential (from successive sputum samples from the same patient) and multiple (from the same clinical sample) isolates were genotyped by random amplification of polymorphic DNA (RAPD) using primers GC70, UBC-701 and UBC-703. Results and conclusion: Pseudallescheria boydii was the most common specie encountered (62% of the isolates) in the CF context, followed by S. apiospermum sensu stricto (24%) and S. aurantiacum (10%). These results contrast with the natural occurrence of these species in the environment, reinforcing the need for further studies aiming to elucidate the pathogenic mechanisms of these fungi. As reported by Kalsteis et al., S. apiospermum sensu stricto is the most prevalent specie in the environment, accounting for 58.7–77.7% of the isolates (depending on the habitat studied), while low prevalence were found for S. aurantiacum and P. boydii, which represented 4.8 (industrial areas) to 7.7% (parks and playgrounds) and 1.9–2.3% of the isolates, respectively. Likewise, Scedosporium dehoogii, which represented about 13.8–28.8% of the isolates from environmental samples, has not been described in a clinical context as yet. Moreover, our results suggest a clustering according to the geographic origin of the isolates: CF patients from the West of France were predominantly colonized by P. boydii. Events of chronic colonization were observed for each specie of S. apiospermum complex encountered in CF context. The RAPD genotyping demonstrated also that two different species could concomitantly colonize the same patient.

We report eight cases of airway colonization by Geosmithia argillacea in patients with cystic fibrosis. This filamentous fungus, resembling members of the genera Penicillium and Paecilomyces , was identified by molecular analysis. All patients carried a mutation on each CFTR (cystic fibrosis transmembrane conductance regulator) allele, with at least one copy of the F508del mutation. The first isolation of this fungus occurred from F508del-homozygous patients at a younger age than in F508del-heterozygous patients. Before recovery of G. argillacea , all patients were treated with itraconazole; two of them had also received voriconazole for an Aspergillus fumigatus infection. However, antifungal susceptibility patterns showed high MICs of voriconazole for all isolates, and high MICs of amphotericin B and itraconazole for the majority of them, but mostly low minimum effective concentrations (MECs) of caspofungin. The appearance and persistence of G. argillacea in the airways were not associated with exacerbation of the disease. However, the clinical implications of G. argillacea , particularly in immunocompromised patients, remain a concern, particularly given recent observations suggesting that this fungus may also cause disseminated infections.