Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

<i>Candida albicans</i> is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). <i>In vitro</i> susceptibility profiling experiments identified 33 NPs with activity against <i>C. albicans</i> (MIC <sub>50</sub> s ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato ( <i>Solanum lycopersicum</i> ) and exhibited high levels of fungistatic activity against <i>Candida</i> species (MIC <sub>50</sub> s ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated <i>C. albicans</i> cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in <i>Saccharomyces cerevisiae</i> coupled with whole-genome sequencing identified 2 nonsynonymous mutations in <i>ERG6</i> (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the <i>in vivo</i> efficacy of tomatidine in a mouse model of <i>C. albicans</i> systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent

Orchids and their mycorrhizal fungi

Orchids are associated with diverse fungal taxa, including non-mycorrhizal endophytic fungi and mycorrhizas. Orchid mycorrhiza (OM) symbiosis is an excellent model for investigating the biological interactions between plants and fungi due to their high dependency on these symbionts for growth and survival. To capture the complexity of OM interactions, significant genomic, numerous transcriptomic, and proteomics studies have been performed, unravelling partly the role of each partner. In this review, the orchid and mycorrhizal fungus relationship will be described summarizing the recent published literature on OM with special attention to the nutrient exchange model, the correlation on fitness and distribution of orchid populations, and finally the chemical communication and defense mechanisms. Based on the recent finding on orchids endophytes, OM relationship, arbuscular mycorrhiza (AM) and OM similarities [1], [2], a putative model representing the different strategies that OM fungi might employ to establish this symbiosis is proposed. It is hypothesized here that (i) orchids would excrete signaling molecules such as strigolactones and flavonoids to facilitate the establishment of the symbiosis. In response, (ii) OM fungi would secrete mycorrhizal (Myc) factors to activate the common symbiosis genes, (iii) evade the pathogen associated molecular patterns triggered immunity and secrete effectors to overcome the defense mechanism (iv) and finally secrete phytohormones to help the colonization or disrupt the crosstalk of plant defense phytohormones. To challenge this supposed model, metabolomics studies with special attention to each partner contribution are encouraged and some technical approaches are proposed

ANTIFUNGAL COMPOUNDS ISOLATED FROM DIOSPYROS BIPINDENSIS

Diospyros bipindensis (Gürke) stem barks are used in Cameroon by pygmies Baka for the treatment of pulmonary diseases. One hypothesis was that this type of diseases could be caused by fungi. Thus the antifungal activity of the traditional preparation as well as that of different extracts was tested against Candida albicans. Some extracts presented a significant antifungal activity. The bioguided isolation was undertaken using HPLC-microfractionation in 96 well plates combined with bioautography in order to localize the active compounds in the HPLC profiling of the extracts. In a second step, medium pressure chromatography was used to isolate the active compounds. Using this approach seventeen compounds were isolated, nine of them are new natural products. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR and HR-MS

Extensive phytochemical investigation of the polar constituents of Diospyros bipindensis Gürke traditionally used by Baka pygmies

The water maceration and methanolic extract of the stem barks of Diospyros bipindensis, which is a medicinal plant used in Cameroon by Baka pygmies, revealed a complex high-performance liquid chromatography (HPLC) profile primarily composed of coumarin and naphthoquinone glycosides. The methanolic and apolar extracts also exhibited significant antifungal activity on a TLC bioautography assay against Candida albicans. HPLC-microfractionation in 96-well plates combined with bioautography enabled the rapid localization of the antifungal compound that was identified by HPLC-PDA and HPLC-MS analysis as plumbagin. These on-line structural information were also used to dereplicate four known compounds. The isolation of the polar constituents from the methanolic extract enabled the identification of eleven other compounds also present in the traditional preparation, nine of which are reported for the first time. The structures of those compounds were elucidated by UV, NMR and HR-MS analysis

Anti-Candida Cassane-Type Diterpenoids from the Root Bark of Swartzia simplex.

A dichloromethane extract of the roots from the Panamanian plant Swartzia simplex exhibited a strong antifungal activity in a bioautography assay against a genetically modified hypersusceptible strain of Candida albicans. At-line HPLC activity based profiling of the crude extract enabled a precise localization of the antifungal compounds, and dereplication by UHPLC-HRESIMS indicated the presence of potentially new metabolites. Transposition of the HPLC reversed-phase analytical conditions to medium-pressure liquid chromatography (MPLC) allowed an efficient isolation of the major constituents. Minor compounds of interest were isolated from the MPLC fractions using semipreparative HPLC. Using this strategy, 14 diterpenes (1-14) were isolated, with seven (5-10, 14) being new antifungal natural products. The new structures were elucidated using NMR spectroscopy and HRESIMS analysis. The absolute configurations of some of the compounds were elucidated by electronic circular dichroism spectroscopy. The antifungal properties of these compounds were evaluated as their minimum inhibitory concentrations in a dilution assay against both hypersusceptible and wild-type strains of C. albicans and by assessment of their antibiofilm activities. The potential cytological effects on the ultrastructure of C. albicans of the antifungal compounds isolated were evaluated on thin sections by transmission electron microscopy

Antifungal Quinoline Alkaloids from Waltheria indica.

Chemical investigation of a dichloromethane extract of the aerial parts of Waltheria indica led to the isolation and characterization of five polyhydroxymethoxyflavonoids, namely, oxyanin A (1), vitexicarpin (3), chrysosplenol E (4), flindulatin (5), 5-hydroxy-3,7,4'-trimethoxyflavone (6), and six quinolone alkaloids, waltheriones M-Q (2, 7, 8, 10, 11) and 5(R)-vanessine (9). Among these, compounds 2, 7, 8, 10, and 11 have not yet been described in the literature. Their chemical structures were established by means of spectroscopic data interpretation including (1)H and (13)C, HSQC, HMBC, COSY, and NOESY NMR experiments and UV, IR, and HRESIMS. The absolute configurations of the compounds were established by ECD. The isolated constituents and 10 additional quinoline alkaloids previously isolated from the roots of the plant were evaluated for their in vitro antifungal activity against the human fungal pathogen Candida albicans, and 10 compounds (7, 9, 11-16, 18, 21) showed growth inhibitory activity on both planktonic cells and biofilms (MIC ≤ 32 μg/mL). Their spectrum of activity against other pathogenic Candida species and their cytotoxicity against human HeLa cells were also determined. In addition, the cytological effect of the antifungal isolated compounds on the ultrastructure of C. albicans was evaluated by transmission electron microscopy

Antifungals and acetylcholinesterase inhibitors from the stem bark of Croton heliotropiifolius

The ethanolic extract of the stem bark of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant in vitro inhibition of acetylcholinesterase using a dilution spectrophotometric assay and antifungal activity against Candida albicans with a thin layer chromatography (TLC) bioautographic assay. In order to isolate the active compounds, bioassay-guided fractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to acetylcholinesterase inhibition or to antifungal activities. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and HPLC-TOF-HRMS were used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and further semi-preparative HPLC. Using this approach, nine compounds were isolated, one of them being a new indole alkaloid derivative. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS. (C) 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq