Joint research project

Nutri-C@rgo: Characterization of plant secreted nanovesicles

Project leaders
Gabriellakatalin Pocsfalvi, Karoly Vekey
UNGHERIA - HAS (MTA) - Accademia Ungherese delle Scienze
CNR/HAS (MTA) 2016-2018
Biology, agriculture and food sciences
Thematic area
Biology, agriculture and food sciences
Status of the project

Research proposal

Introduction and study aims
Discovered 30 years ago, mammalian extracellular vesicles (EVs), including exosomes, microsomes and apoptotic bodies have shown to be implicated in a novel form of cell-cell communication; and thus represent an important and rapidly growing research field in biology and translational sciences.(1) More recently, plants were also shown to secrete membrane-bound particles.(2) Today, two classes of EVs have been isolated from plants: apoplastic vesicles from rice, sunflower and tomato; and exosome-like particles from fruit juices such as grapes and grapefruits. It is well established that a plant-derived diet has great influence on regulation of homeostasis of the cells lining the digestive system. Deregulation of intestinal epithelial cell homeostasis, for example, mediates chronic inflammation (like inflammatory bowel diseases), increased propensity to develop infections and cancer. In a recent study, Mu et al reported that edible plant derived exosome-like nanoparticles can be taken up by intestinal macrophages and stem cells where they induce anti-inflammatory processes.(3) Interspecies communication between plant and mammalian cells through nanoparticles is a new and very interesting field which opens up new avenues for further studies. In contrast to the mammalian EVs there is a limited information available about the bio-cargo of plant derives nanoparticles. Here we propose to study the molecular cargo of EVs isolated from fruit juices of the citrus family. The molecular data will be used to answer the following questions: i.) what is the molecular cargo of citrus derived EVs? ii) what are the differences among EVs isolated from different citrus species? iii.) how much does the protein and lipid cargo of plant EVs differ from that of mammalian cells? and, iv.) what are the possible interlocutors involved in the crosstalk between plant EVs and mammalian cells? The obtained data sets will be deposited into two manually curated web-based databases, EVpedia (4) and Vesiclepedia (5), that are publicly available and at the moment lack such data.

Motivation for co-operation
The two research groups, Mass Spectrometry and Proteomics Laboratory at IBBR-OUS-Napoli and Core Technologies Centre of the Hungarian Academy of Sciences (CTC) are working in complementary fields, and the principal investigators (PIs) have a long-standing collaboration. PIs already led joint projects in the past and have published together.(6) Recently, they have been collaborating in the characterization of mammalian EVs using mass spectrometry (6) and chromatography-based techniques. They both have relevant expertise in the food sector as well.(7,8) PIs recently launched collaborations with the research group at IBBR-OUS Perugia, Italy and the research group of Cell- and Immunobiology of the Department of Genetics, Semmelweis University, Budapest, Hungary on the exploitation of extracellular vesicles. The expertise of the principal and partner investigators and their collaborators in the exosome field is well-documented by an existing patent application,(9) scientific papers and book chapters published in the last five years. The relevant expertise and the available instrumentation put the CNR-MTA team, the proponent of this project, in an excellent position to establish joint research collaboration through NutriC@rgo. The ultimate aim of the project is to set up a collaborative network in order to prepare successful applications of European collaborative research projects under Horizon2020 collaborative Research and Innovation projects and research networks (COST actions).

State-of-the-art and preliminary results
EV research is proceeding at a fast pace; now they start to appear as promising therapeutic targets, diagnostic tools and drug delivery systems. Isolation and analysis of EVs are prerequisites for understanding their biological roles and their clinical exploitation. In this process chromatography and MS-based strategies have rapidly gained importance. Mass spectrometry-based proteomics and lipidomics led to an exponential increase in molecular data that pertain to EVs.Plant-derived exosomes, on the other hand, only recently have attracted interest. From a nutraceutical point of view, fruit juice seems to be an especially promising source of nanovesicles. Citrus fruit-derived nanovesicles have shown to inhibit cancer cell proliferation and suppress xenograft growth by inducing apoptosis (Raimondo S, et al. (2015). This suggests that these EVs may have important biological influence, worth exploiting.
Recently we have developed a protocol for isolation of nanovesicles with discrete size distributions from fruit juice. The method is based on gradient ultracentrifugation using double sucrose/D2O cushion, and yields high quality preparations suitable for downstream proteomic and lipidomic characterization. The method was tested to isolate EVs from Citrus clementina (clementine) juice. Clementine is almost exclusively grown in Mediterranean countries and became an important species for citrus production. The recently published genome of citrus facilitates the genome and proteome-wide analysis of these species. SDS-PAGE and proteomic analysis of the lysed vesicles resulted in the identification of proteins, which were functionally annotated. The developed protocol forms a promising base for the success of the present proposal.

1.Yáñez-Mó M, et al. (2015) J Extracell Vesicles 4.
2.Regente M, et al. (2012) Plant Signal Behav 7(5):544-546.
3.Mu J, et al. (2014) Mol Nutr Food Res 58(7):1561-1573.
4.Kim DK, et al. (2015) Bioinformatics 31(6):933-939.
5.Kalra H, et al. (2012 PLoS Biol 10(12):e1001450.
6.Pocsfalvi G, et al. (2015) Mass Spectrom Rev 20(10):21457.
7.Vekey K, et al. (1997) J Agricultural and Food Chem 45(7):2447-2451.
8.Rossano EC, et al. (2007) J Agricultural and Food Chem55(2):311-317.
9.Pocsfalvi G, Fiume I, Raj DAA, & Capasso G (2011) Brevetto UIBM.

Research goals

The objectives of this proposal are threefold:
O1.Basic research: In the framework of NutriC@rgo we will undertake chromatography-mass spectrometry based molecular characterization of two different types of plant EV biocargo: proteins and lipids. Proteins of EVs will be investigated by proteomics. The plant vesicular proteome will be a promising source for discovery of novel nutraceuticals, and will also provide information on novel form(s) of inter species communication and on the mechanism(s) of vesicle biogenesis. Lipids of EVs will be analysed by lipidomics. We will develop GC-MS and/or LC-MS-based protocols for lipid analysis.
O2.Applied research: we will analyse plants from the Mediterranean with high agricultural value and great potential. The following citrus taxa will be used to isolate EVs: citron (C. medica), pummelo (C. maxima), mandarine (C. reticulata) and clementina (C. clementina). The proposal aims to increase the value of these fruits and point to possible ways for their biotechnological exploitation.
O3.Training of young researchers/students: during the study trips we will put particular emphasis on the training of young researchers and students in chromatography and mass spectrometry, isolation, physiochemical and molecular characterization of EVs. During the visits, the visiting scientist will give a lecture and round table meetings will be held to organize the works and discuss results.

Last update: 27/09/2020