The project is characterized by a strong multidisciplinary approach involving researchers expert in biochemistry (protein purification and enzyme characterization), cell biology (studies on pathway of intestinal cell lines treated with protein toxins) and microbiology (effects of toxic enzymes on in vitro gut microbiota). In this framework, the project involves a synergistic contribution of 3 different Research units, listed below:

1- Research Unit “Caserta” - University of Campania “Luigi Vanvitelli” (Caserta-RU)

2- Research Unit “Bologna” – “Alma Mater Studiorum - University of Bologna” (Bologna-RU)

3- Research Unit “Napoli” - University of Naples “Federico II” (Napoli-RU).

1- Objectives achieved by Caserta-RU

The main objective of the Caserta-RU project is to screen, purify and enzymatically characterise protein synthesis inhibitor enzymes (i.e. RIPs and RL-Ps) from edible plants and mushrooms.

In particular, the Caserta-RU achieved: i) purify and characterise both sodin 5 from S. soda seeds and edulitin 2 from B. edulis fruiting bodies; ii) screen, isolate and purify novel protein synthesis inhibitor enzymes from edible mushrooms; iii) determine the digestibility of protein synthesis inhibitor enzymes in vitro to mimic hydrolytic conditions in the stomach and intestine; iv) structurally characterise indicitin, the novel protein synthesis inhibitor that has been purified from Calocybe indica fruiting bodies, and attempt to obtain information on its amino acid sequence and physicochemical properties. These activities were performed in milestones 1, 2, 4, 6 and 7.

2- Objectives achieved by Bologna-RU

The main objective of Bologna-RU in the project is to evaluate the toxic mechanism of protein synthesis inhibitor enzymes (i.e. RIPs and RLPs) on intestinal cells, investigate the pathway that triggers death and evaluate the onset of inflammation.

In particular, Bologna-RU achieved the following: i) evaluation of the toxicity mechanism of sodin 5, edulitin 2 and indicitin on intestinal cells (Caco-2 and HT-29 cell lines), investigation of the triggered pathway of death, and evaluation of the onset of inflammation; ii) evaluation of the involvement of oxidative stress and inflammatory signalling during the effects of sodin 5, edulitin 2 and indicitin; iii) evaluation of the involvement of oxidative stress and inflammation in cytotoxicity in a complex model during the effects of sodin 5, edulitin 2 and indicitin. These activities were performed in milestones 1, 3, 5 and 8.

3- Objectives achieved by Napoli-RU

The main objective of Napoli-RU in the project is to evaluate the effects of protein synthesis inhibitor enzymes (i.e. RIPs and RLPs) on the gut microbial composition.

In particular, Napoli-RU achieved the following: i) the effects of sodin 5, edulitin 2 and indicitin on intestinal bacteria; ii) the in vitro effects of sodin 5, edulitin 2 and indicitin on the intestinal microbiota. These activities were performed in milestones 1, 3, 5 and 8.

Activities of each research unit divided by milestone:

Milestone 1: Set-up of experimental systems and purification of sodin 5

Caserta Research Unit (RU) purified sodin 5, a type 1 ribosome-inactivating protein from Salsola soda seeds. The purification protocol involved protein extraction followed by acid precipitation and two sequential chromatographic separations based on differences in molecular weight and cationic charge. The purified protein was dialysed and concentrated, allowing the recovery of ~20 mg of sodin 5, which were subsequently distributed to the Bologna and Napoli research units for biological assays.

Bologna-RU established the experimental systems required to investigate the effects of ribosome-targeting enzymes on intestinal epithelial cells. Human intestinal cell lines Caco-2 and HT-29 were selected as experimental models, and the culture conditions were optimized for two-dimensional cell culture systems, enabling the subsequent evaluation of cytotoxicity, oxidative stress and epithelial barrier integrity.

Napoli-RU developed the experimental system to study the effects of ribosome-targeting enzymes on intestinal microbiota. Preliminary growth experiments were performed to evaluate the cultivation of anaerobic bacteria in MGAM (Gifu Anaerobic Medium). The growth of intestinal microbial samples under anaerobic conditions was optimized to allow the execution of subsequent in vitro assays in the presence of the selected enzymes.

Milestone 2: Purification and characterization of edulitin 2

Caserta-RU initiated and completed the purification of edulitin 2, a ribotoxin-like protein isolated from the fruiting bodies of Boletus edulis. The purification procedure involved the extraction and enrichment of basic proteins followed by chromatographic separation. The enzymatic activity of the purified protein was verified by evaluating its specific ribonuclease activity on 28S rRNA and its ability to inhibit protein synthesis in vitro. To obtain sufficient quantities of the enzyme for biological assays, a semi-preparative purification procedure was implemented using approximately 2 kg of porcini mushroom fruiting bodies. This approach allowed the recovery of ~20 mg of purified edulitin 2. Aliquots of the purified enzyme were subsequently distributed to Bologna-RU and Napoli-RU for further biological evaluation.

Milestone 3: Biological effects of sodin 5 on intestinal systems

Bologna-RU investigated the cytotoxic activity of sodin 5 using the intestinal epithelial cell lines Caco-2 and HT-29. Cell death mechanisms were analysed by flow cytometry through annexin V/propidium iodide staining, allowing the discrimination between apoptotic and necrotic pathways. In addition, the effects of sodin 5 on epithelial barrier integrity were evaluated by measuring transepithelial electrical resistance (TEER) in differentiated Caco-2 monolayers and in Caco-2/HT29 co-culture systems. Treatment with sodin 5 caused a significant reduction in TEER values, indicating an increase in epithelial permeability. Further mechanistic investigations demonstrated the involvement of oxidative stress in sodin 5-induced cytotoxicity. Pre-treatment with reactive oxygen species (ROS) scavengers partially protected the cells from the toxic effects of the enzyme, suggesting that multiple cell death pathways contribute to the observed cytotoxicity.

Napoli-RU evaluated the effects of sodin 5 on representative microorganisms of the intestinal microbiota. The microorganisms tested included Gram-positive bacteria (Bacillus subtilis, Bacillus cereus and Lactobacillus rhamnosus), Gram-negative bacteria (Escherichia coli and Salmonella enterica) and the yeast Candida albicans. The assays were conducted using a range of enzyme concentrations (5-200 μg/mL). Additional experiments evaluated the effect of sodin 5 on biofilm formation. The permeability of bacterial cells to sodin 5 was also investigated using fluorescein-labelled sodin 5 provided by Caserta-RU. The uptake of the labelled protein by bacterial cells was analysed by fluorescence microscopy using Bacillus subtilis as a model microorganism. In addition, Napoli-RU also initiated experiments on complex microbial communities. DNA was extracted from three faecal samples obtained from healthy donors, and the purity of the extracted DNA was verified. The microbial composition of the samples was analysed by sequencing the 16S rRNA gene, allowing the establishment of microbial consortia suitable for in vitro microbiota studies.

Milestone 4: Gastrointestinal digestibility of sodin 5 and edulitin 2

Caserta-RU evaluated the susceptibility of sodin 5 and edulitin 2 to proteolytic degradation using in vitro digestibility systems simulating gastrointestinal conditions. These experiments were performed to assess the stability of the enzymes in the presence of digestive proteases and to estimate their potential persistence during gastrointestinal digestion.

Milestone 5: Biological effects of edulitin 2 on intestinal systems

Bologna-RU evaluated the cytotoxic activity of edulitin 2 in Caco-2 and HT-29 cells. Time-response experiments demonstrated that the enzyme produced only limited effects on cell viability after 24 hours of incubation, whereas more pronounced reductions were observed after 48 and 72 hours. The impact of edulitin 2 on epithelial barrier integrity was assessed by TEER measurements in differentiated Caco-2 monolayers and in Caco-2/HT29 co-culture models. The results indicated that edulitin 2 induced a decrease in TEER values, suggesting an increase in epithelial permeability, although its effects were generally weaker than those observed for sodin 5. To better reproduce the physiological organization of intestinal tissue, additional experiments were performed using more complex co-culture models composed of epithelial cells, collagen matrices and fibroblasts.

Napoli-RU evaluated the effects of edulitin 2 on selected intestinal microorganisms as well as on microbial consortia derived from faecal samples of healthy children who had not received antibiotic or probiotic treatments for at least three months. These experiments allowed the investigation of potential interactions between the enzyme and intestinal microbial communities.

Milestone 6: Identification and purification of indicitin

Caserta-RU conducted a screening of protein extracts obtained from edible plants and mushrooms in order to identify new ribosome-targeting enzymes. The screening was based on the ability of the extracts to inhibit protein synthesis in a rabbit reticulocyte lysate system. Among the analysed extracts, those obtained from the edible mushroom Calocybe indica showed a strong inhibitory activity. This observation led to the purification and identification of a previously undescribed ribotoxin-like protein, named indicitin.

The enzymatic activity of indicitin was confirmed using Endo’s assay, which demonstrated the release of the characteristic α-fragment of rRNA, a hallmark of ribotoxin-like protein activity. A scale-up purification procedure was subsequently optimized, allowing the production of approximately 20–30 mg of purified indicitin. Homogeneous preparations of the enzyme were then distributed to Bologna-RU and Napoli-RU for biological evaluation.

Milestone 7: Digestibility and structural characterization of indicitin

Caserta-RU performed an extensive structural and biochemical characterization of indicitin. Amino acid composition analysis was conducted to determine the relative abundance of amino acid residues and to verify the presence of cysteine residues following oxidation with performic acid. Circular dichroism spectroscopy was used to obtain information on the secondary structure of the protein, while thermal denaturation experiments allowed the determination of its melting temperature. In addition, an in-gel colorimetric assay following SDS-PAGE was performed to verify whether indicitin is a glycoprotein. Finally, partial sequence information was obtained by Edman degradation performed on the N-terminal region of S-pyridylethylated indicitin as well as on peptides generated by tryptic digestion. The susceptibility of indicitin to proteolytic digestion was also evaluated using in vitro digestibility systems.

Milestone 8: Biological effects of indicitin

Bologna-RU investigated the effects of indicitin on intestinal epithelial cells, continuing the analysis of cytotoxicity, oxidative stress and inflammatory responses in Caco-2 and HT-29 models, including more complex co-culture systems designed to better reproduce the interaction between the epithelial barrier and the underlying stromal compartment.

Napoli-RU evaluated the effects of indicitin on intestinal microorganisms, both in pure cultures of selected bacterial and fungal strains and in microbial consortia simulating the intestinal microbiota. The results indicated that indicitin does not significantly interfere with protein synthesis in intestinal microorganisms.