Nature-Inspired Chemistry of Complex Alkaloids: Combining Targeted Molecular Networking Approach and Semisynthetic Strategy to Access Rare Communesins in a Marine-Derived Penicillium expansum.

Communesins are rare alkaloids isolated from fungi of the genus Penicillium. In this work, the extract of a marine-derived Penicillium expansum strain was studied using targeted molecular networking approach allowing to detect 65 communesins including 55 new ones. A fragmentation pattern for dimethylvinyl communesins was established and a script was implemented allowing to predict the structure and map all communesins in a global molecular network. A semisynthetic strategy was carried out to obtain some minor congeners from the two isolated communesins A and B. Nine communesins were then synthetised: two of them were already described as produced by the studied strain; four are new natural products which occurrence in the extracts was confirmed; three are new semi-synthetic analogues never described so far. These communesins were evaluated for their cytotoxicity on two human cancer cell lines KB and MCF-7 leading to a preliminary study of their structure-activity relationships.

Development and validation of a UHPLC-ESI-QTOF mass spectrometry method to analyze opines, plant biomarkers of crown gall or hairy root diseases.

Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts.

Impact of Paraburkholderia phytofirmans PsJN on Grapevine Phenolic Metabolism.

Phenolic compounds are implied in plant-microorganisms interaction and may be induced in response to plant growth-promoting rhizobacteria (PGPRs). Among PGPR, the beneficial bacterium Paraburkholderia phytofirmans PsJN was previously described to stimulate the growth of plants and to induce a better adaptation to both abiotic and biotic stresses. This study aimed to investigate the impact of PsJN on grapevine secondary metabolism. For this purpose, gene expression (qRT-PCR) and profiling of plant secondary metabolites (UHPLC-UV/DAD-MS QTOF) from both grapevine root and leaves were compared between non-bacterized and PsJN-bacterized grapevine plantlets. Our results showed that PsJN induced locally (roots) and systemically (leaves) an overexpression of PAL and STS and specifically in leaves the overexpression of all the genes implied in phenylpropanoid and flavonoid pathways. Moreover, the metabolomic approach revealed that relative amounts of 32 and 17 compounds in roots and leaves, respectively, were significantly modified by PsJN. Once identified to be accumulated in response to PsJN by the metabolomic approach, antifungal properties of purified molecules were validated in vitro for their antifungal effect on Botrytis cinerea spore germination. Taking together, our findings on the impact of PsJN on phenolic metabolism allowed us to identify a supplementary biocontrol mechanism developed by this PGPR to induce plant resistance against pathogens.

Ecological Conditions and Molecular Determinants Involved in Agrobacterium Lifestyle in Tumors.

The study of pathogenic agents in their natural niches allows for a better understanding of disease persistence and dissemination. Bacteria belonging to the Agrobacterium genus are soil-borne and can colonize the rhizosphere. These bacteria are also well known as phytopathogens as they can cause tumors (crown gall disease) by transferring a DNA region (T-DNA) into a wide range of plants. Most reviews on Agrobacterium are focused on virulence determinants, T-DNA integration, bacterial and plant factors influencing the efficiency of genetic transformation. Recent research papers have focused on the plant tumor environment on the one hand, and genetic traits potentially involved in bacterium-plant interactions on the other hand. The present review gathers current knowledge about the special conditions encountered in the tumor environment along with the Agrobacterium genetic determinants putatively involved in bacterial persistence inside a tumor. By integrating recent metabolomic and transcriptomic studies, we describe how tumors develop and how Agrobacterium can maintain itself in this nutrient-rich but stressful and competitive environment.

Regulation of Hydroxycinnamic Acid Degradation Drives Agrobacterium fabrum Lifestyles.

Regulatory factors are key components for the transition between different lifestyles to ensure rapid and appropriate gene expression upon perceiving environmental cues. Agrobacterium fabrum C58 (formerly called A. tumefaciens C58) has two contrasting lifestyles: it can interact with plants as either a rhizosphere inhabitant (rhizospheric lifestyle) or a pathogen that creates its own ecological niche in a plant tumor via its tumor-inducing plasmid (pathogenic lifestyle). Hydroxycinnamic acids are known to play an important role in the pathogenic lifestyle of Agrobacterium spp. but can be degraded in A. fabrum species. We investigated the molecular and ecological mechanisms involved in the regulation of A. fabrum species-specific genes responsible for hydroxycinnamic acid degradation. We characterized the effectors (feruloyl-CoA and p-coumaroyl-CoA) and the DNA targets of the MarR transcriptional repressor, which we named HcaR, which regulates hydroxycinnamic acid degradation. Using an hcaR-deleted strain, we further revealed that hydroxycinnamic acid degradation interfere with virulence gene expression. The HcaR deletion mutant shows a contrasting competitive colonization ability, being less abundant than the wild-type strain in tumors but more abundant in the rhizosphere. This supports the view that A. fabrum C58 HcaR regulation through ferulic and p-coumaric acid perception is important for the transition between lifestyles.

Essential oils of Origanum compactum and Thymus vulgaris exert a protective effect against the phytopathogen Allorhizobium vitis.

Allorhizobium (Agrobacterium) vitis is a host-specific pathogenic bacterium that causes grapevine crown gall disease, affecting vine growth and production worldwide. The antibacterial activities of different aromatic plant essential oils were tested in vitro and in planta against A. vitis. Among the essential oils tested, those of Origanum compactum and Thymus vulgaris showed the most significant in vitro antibacterial activities, with a MIC of 0.156 and 0.312 mg/mL, respectively. A synergistic effect of these two essential oils (1:1) was observed and confirmed by the checkerboard test. Carvacrol (61.8%) and thymol (47.8%) are, respectively, the major compounds in the essential oils of O. compactum and T. vulgaris and they have been shown to be largely responsible for the antibacterial activities of their corresponding essential oils. Results obtained in vitro were reinforced by an in planta pathogenicity test. A mixture of O. compactum and T. vulgaris essential oils (1:1), inoculated into the injured stem of a tomato plant and a grapevine at 0.312 mg/mL as a preventive treatment, reduced both the number of plants developing gall symptoms and the size of the tumors.

Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-ß-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-ß-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-ß-ketopropionic acid (HMPKP)-CoA ß-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ß-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

Patulin and secondary metabolite production by marine-derived Penicillium strains.

Genus Penicillium represents an important fungal group regarding to its mycotoxin production. Secondary metabolomes of eight marine-derived strains belonging to subgenera Furcatum and Penicillium were investigated using dereplication by liquid chromatography (LC)-Diode Array Detector (DAD)-mass spectrometry (MS)/MS. Each strain was grown on six different culture media to enhance the number of observable metabolites. Thirty-two secondary metabolites were detected in crude extracts with twenty first observations for studied species. Patulin, a major mycotoxin, was classically detected in extracts of Penicillium expansum, and was also isolated from Penicillium antarcticum cultures, whose secondary metabolome is still to be done. These detections constituted the first descriptions of patulin in marine strains of Penicillium, highlighting the risk for shellfish and their consumers due to the presence of these fungi in shellfish farming areas. Patulin induced acute neurotoxicity on Diptera larvae, indicating the interest of this bioassay as an additional tool for detection of this major mycotoxin in crude extracts.

Enhancement of domoic acid neurotoxicity on Diptera larvae bioassay by marine fungal metabolites.

Peptaibols are small linear fungal peptides which are produced in the marine environment. They exhibit neurotoxicity by forming pores in neuronal membranes. This work describes their combine effect with domoic acid, a neurotoxic phycotoxin, on Diptera larvae. The Acute toxicity bioassay on this biological model was tested with a panel of different toxins (microbial, algal or fungal). It allowed the discrimination of neurotoxins and non-neurotoxic toxins, and an evaluation of the toxicity level (MED and ED(50)) which were correlated with published LD(50) in mice for neurotoxins tested. The highest activities on this test were found for Na(+) channel blockers tetrodotoxin (ED(50) = 0.026 mg/kg) and saxitoxin (ED(50) = 0.18 mg/kg). Domoic acid was less active with an ED(50) = 7.6 mg/kg. For synergism study, longibrachin-A-I, a 20-mer peptaibol isolated from cultures of a marine-derived strain of Trichoderma longibrachiatum Rifai was chosen. Bioassay results confirmed its neuroactivity. Its level of toxicity (ED(50) = 270 mg/kg) was lower than those of phycotoxins tested but higher than mycotoxin ones. Injected together, longibrachin-A-I and domoic acid exhibited an increase of their activities. With doses of longibrachin-A-I below its Minimal Effective Dose (MED), the synergism factor which expresses the enhancement of domoic acid toxicity could reach 34.5. Both domoic acid and longibrachin-A-I are acting on ion channels and pores in neuronal membranes which contribute to the intake of Ca(2+) into cells.

Structural investigation and elucidation of new communesins from a marine-derived Penicillium expansum Link by liquid chromatography/electrospray ionization mass spectrometry.

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio-guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and 1H and 13C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI-MS/MS) and high-resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics.

Effects of seawater on growth and gliotoxin excretion of marine strains of Aspergillus fumigatus Fres.

In order to enhance the knowledge of the putative toxinic risk linked to mycotoxin excretion in shellfish farming areas, the influence of seawater salinity was studied on 2 marine-derived Aspergillus fumigatus strains. This fungal species produces gliotoxin, an epipolythiodioxopiperazine immunosuppressive mycotoxin that can be accumulated in the meat of cultured blue mussel (Mytilus edulis), and could be responsible for disease when ingested. Two marine strains were grown in vitro both on a non-saline and a saline culture media and were compared with 13 terrestrial strains to observe the effects of seawater on fungal growth and gliotoxin excretion in the exudate produced. Daily measurement of the colony areas showed that the seawater salinity significantly reduced the rate of growth of all the strains. Marine and terrestrial strains appeared to be almost similar as regards the appearance, growth and gliotoxin excretion, but the marine strains exudation seemed to be less influenced by seawater salinity than the terrestrial strains. Seawater salinity, however, enhanced exudation and gliotoxin excretion by A. fumigatus, and thus seems to be an aggravating factor for the toxicity of this species in the marine environment.

A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi.

Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.