Milestone 1 – Purification and characterization of sodin 5 from Salsola soda
During the first phase of the project, the Caserta Research Unit focused on the purification and characterization of sodin 5, a type-1 ribosome-inactivating protein (RIP) from Salsola soda seeds. Crude protein extracts were obtained by homogenizing seeds in phosphate-buffered saline (pH 7.2). The extract displayed polynucleotide:adenosine glycosylase activity and inhibited protein synthesis in a rabbit reticulocyte lysate system. Purification was carried out through acid precipitation followed by stepwise cation exchange chromatography and gel filtration. Fractions corresponding to proteins with molecular weight around 29 kDa were further purified by cation exchange chromatography using a NaCl gradient. Five protein fractions showing RIP activity were detected, confirming the presence of five type-1 RIPs previously identified in S. soda. The major peak corresponded to sodin 5. Pooled fractions were dialysed to obtain homogeneous protein preparations. The purification protocol allowed the recovery of approximately 3 mg of sodin 5 per 100 g of seeds.
Subsequently, the enzymatic activity of purified sodin 5 was verified. The enzyme showed the characteristic N-glycosylase activity of RIPs, releasing adenine residues from nucleic acid substrates and generating the diagnostic β-fragment. In addition, sodin 5 inhibited protein synthesis in rabbit reticulocyte lysate with an IC50 of approximately 4.75 pM, consistent with previously reported values.
To enable biological investigations within the project, 5 mg of purified sodin 5 were distributed to the Bologna and Naples Research Units for cytotoxicity studies on intestinal epithelial models and microbiota experiments, respectively.
Milestone 2 – Purification and characterization of edulitin 2 from Boletus edulis (porcini)
The Caserta Research Unit subsequently focused on the purification of edulitin 2, a ribotoxin-like protein (RL-P) isolated from the fruiting bodies of Boletus edulis (porcini mushroom). Proteins were extracted from mushroom tissues, followed by acid precipitation and cation exchange chromatography. Basic protein fractions were further separated by gel filtration and additional cation exchange chromatography steps, allowing the isolation of homogeneous edulitin 2 with an apparent molecular weight of approximately 14 kDa. The purification procedure yielded approximately 0.8 mg of edulitin 2 per 100 g of fruiting bodies.
The enzymatic activity of purified edulitin 2 was verified through ribonuclease assays. The protein showed the typical activity of ribotoxin-like proteins, releasing the α-fragment from ribosomal RNA and inhibiting protein synthesis in a rabbit reticulocyte lysate system.
To obtain sufficient protein for biological experiments, additional semi-preparative purifications were performed starting from approximately 2 kg of mushroom material, resulting in a total yield of about 20 mg of purified protein.
Aliquots of 5 mg were sent to the Bologna and Naples Research Units for cellular toxicity studies and microbiota analyses.
Milestone 4 – Proteases susceptibility of sodin and edulitin in vitro, mimicking gastro-intestinal conditions
Caserta- RU evaluated the digestibility of sodin 5 and f edulitin. This effect was evaluated using an in vitro pepsin-trypsin digestion model simulating gastrointestinal conditions. In this conditions sodin 5 was completely hydrolysed by pepsin treatment under acidic conditions within 120 minutes, and the resulting peptides were further degraded by trypsin digestion, suggesting that sodin 5 is highly susceptible to gastrointestinal proteolysis.
Unlike sodin 5, edulitin 2 displayed remarkable resistance to proteolytic digestion, remaining largely intact after pepsin and trypsin treatment. This result suggests that part of the protein could reach the intestinal tract in an intact form and potentially interact with gut microbiota.
Milestone 6 – Identification and purification of a novel ribotoxin-like protein from Calocybe indica
During the second year of the project, the Caserta Research Unit investigated the presence of ribotoxin-like proteins in other edible mushrooms. Screening experiments identified inhibitory activity in extracts from Calocybe indica (milky mushroom). An analytical purification procedure led to the identification of a novel ribotoxin-like protein, named indicitin, with an apparent molecular weight of approximately 28 kDa. The protein was purified through sequential chromatographic steps including cation exchange chromatography and gel filtration. Biochemical assays demonstrated that indicitin inhibits protein synthesis in vitro and is capable of releasing the characteristic α-fragment from ribosomal RNA in Endo’s assay, confirming that it belongs to the family of ribotoxin-like proteins.
Following the analytical purification, a scale-up purification protocol was developed to obtain larger amounts of the protein. The optimized preparative protocol included fast gel filtration followed by cation exchange chromatography and allowed the recovery of approximately 2.2 mg of indicitin per 100 g of lyophilized mushroom tissue.
Milestone 7 – Digestibility and structural characterization of indicitin
The gastrointestinal digestibility of indicitin was evaluated by Cserta-RU using the same in vitro digestion system described in Milestone 4. The protein showed high susceptibility to pepsin under acidic conditions, leading to almost complete degradation within 120 minutes. Subsequent trypsin treatment resulted in the complete digestion of remaining peptides. These findings indicate that only limited amounts of intact indicitin are likely to reach the small intestine. Further studies were carried out to investigate the structural and physico-chemical properties of indicitin.
Amino acid composition analysis revealed a high proportion of aliphatic residues (approximately 48%), together with significant amounts of glycine and proline. The protein contains approximately 16 cysteine residues per molecule, suggesting the presence of multiple disulfide bonds. Circular dichroism spectroscopy indicated that indicitin is mainly composed of β-sheet secondary structure elements. Thermal denaturation experiments revealed a high melting temperature (Tm ≈ 86 °C), indicating strong structural stability, likely due to the extensive disulfide bond network. Additional analyses confirmed that indicitin is a non-glycosylated protein.
Partial amino acid sequences obtained through Edman degradation were used for database searches. Sequence homology analysis revealed similarities with thaumatin-like proteins (TLPs) belonging to the pathogenesis-related protein family PR-5, suggesting that indicitin may represent a new member of this protein family with ribotoxin-like activity.
Further studies are ongoing to determine the complete primary structure and better define the biological role of this novel protein.
Overall conclusions of Caserta Research Unit
The results obtained enabled us to develop methodologies for purifying ribosome-targeting enzymes. Furthermore, a new ribosome-targeting enzyme was identified and purified from Calocybe indica (milky mushroom), and its partial structural and enzymatic characterisation was achieved.
Milestone 1 – Set-up of intestinal epithelial cell models
During the initial phase of the project, the Bologna Research Unit established in vitro intestinal epithelial cell models to evaluate the biological effects of ribosome-inactivating proteins and ribotoxin-like proteins isolated from edible plants and mushrooms.
Two widely used intestinal epithelial cell lines were selected: Caco-2 and HT29. To optimize the experimental conditions, preliminary experiments were carried out using saporin, a well-characterized type-1 ribosome-inactivating protein isolated from Saponaria officinalis, which shares structural and enzymatic similarities with sodin 5.
Cells were seeded as monolayers and treated with increasing concentrations of saporin to determine dose-response and time-response relationships. Cytotoxicity was evaluated through cell viability assays and flow cytometric analyses. Apoptotic and necrotic cell death were distinguished using Annexin V/propidium iodide staining, and apoptosis was further confirmed through caspase 3/7 activation assays.
These experiments allowed the identification of the appropriate experimental conditions and toxicity ranges for the subsequent investigations with sodin 5 and edulitin 2.
The intestinal models were further optimized by promoting cell differentiation and polarization. Caco-2 cells were cultured on collagen supports or permeable membranes, allowing the formation of polarized epithelial monolayers. Differentiation was enhanced by supplying fetal bovine serum only to the basolateral compartment. HT29 cell differentiation was induced by culturing cells in glucose-free medium supplemented with galactose.
These procedures enabled the establishment of differentiated intestinal epithelial models suitable for the study of toxin-induced damage to the intestinal barrier.
Milestone 3 – Evaluation of sodin 5 effects on intestinal epithelial cells
The Bologna-RU investigated the biological activity of sodin 5, purified by the Caserta Research Unit, using both undifferentiated and differentiated intestinal epithelial cell models.
Caco-2 and HT29 cells were exposed to increasing concentrations of sodin 5 and cell viability was assessed through metabolic assays and dye exclusion tests. The toxin showed a strong cytotoxic effect in both cell lines, with a progressive decrease in cell viability over time. After prolonged exposure, viable cells dropped below 15%. Dose-response analyses indicated EC50 values in the range of 0.01–0.1 µM, comparable to those previously reported for saporin.
The mechanism of cell death was investigated by flow cytometric analysis. Sodin 5 induced predominantly apoptotic cell death, with more than 75% of treated cells undergoing apoptosis after 24 hours. Only a limited fraction of necrotic cells was observed.
To further clarify the death pathways involved, cells were pretreated with the pan-caspase inhibitor Z-VAD and the necroptosis inhibitor necrostatin. Both inhibitors significantly reduced cytotoxicity, indicating that sodin 5 toxicity involves both apoptotic and necroptotic pathways.
The effects of sodin 5 on intestinal barrier integrity were then investigated using differentiated epithelial monolayers. Co-culture systems composed of Caco-2 and HT29 cells were grown on Transwell membranes to reproduce the structural properties of the intestinal barrier. Barrier integrity was monitored through transepithelial electrical resistance (TEER) measurements.
Exposure to sodin 5 caused a significant reduction in TEER values, indicating increased epithelial permeability. After 24 hours of treatment, TEER values decreased by approximately 30%, suggesting disruption of tight junction structures and impairment of epithelial barrier function.
Milestone 5 – Evaluation of edulitin 2 effects on intestinal epithelial cells
The Bologna-RU investigated the cytotoxic effects of edulitin 2, a ribotoxin-like protein purified from Boletus edulis, using both undifferentiated and differentiated intestinal epithelial models. Time- and dose-response experiments demonstrated that edulitin 2 exerted a moderate cytotoxic effect compared to sodin 5. In both Caco-2 and HT29 cells, only limited effects were observed after 24 hours, while stronger reductions in cell viability occurred after longer incubation times (48–72 hours). The calculated EC50 values after 72 hours were approximately 2.85 × 10⁻⁷ M in Caco-2 cells and 4.98 × 10⁻⁷ M in HT29 cells.
The effect of edulitin 2 on epithelial barrier integrity was evaluated using differentiated cell models grown on permeable supports. TEER measurements revealed a time-dependent decrease in barrier integrity, indicating increased epithelial permeability after prolonged exposure. Similar effects were observed in both monoculture and co-culture systems.
To further investigate the structural basis of barrier disruption, tight junction proteins were analyzed by immunofluorescence. A reduction in the signal associated with the tight junction protein claudin-3 was observed in treated cells, suggesting that edulitin 2 affects epithelial barrier integrity through disruption of tight junction complexes.
Cell death mechanisms induced by edulitin 2 were investigated through flow cytometry. The results indicated that apoptosis was the predominant mechanism of cell death, with no significant necrotic component detected.
Pretreatment with apoptosis and necroptosis inhibitors partially protected cells from edulitin 2 toxicity, indicating that multiple pathways may contribute to the cytotoxic effects of the protein.
Additional experiments suggested the involvement of oxidative stress in edulitin 2 toxicity. Pretreatment with reactive oxygen species scavengers partially protected cells from toxin-induced cytotoxicity, indicating that oxidative stress contributes to the mechanism of cell damage.
Milestone 8 – Biological effects of the novel enzyme indicitin
During the later stages of the project, the Bologna Research Unit investigated the biological activity of indicitin, a novel ribosome-targeting enzyme identified by the Caserta Research Unit in the edible mushroom Calocybe indica. Initial experiments confirmed that purified indicitin was able to inhibit protein synthesis in a rabbit reticulocyte lysate system, with an IC50 value of approximately 1.05 × 10⁻⁶ M. The cytotoxic potential of indicitin was subsequently evaluated in intestinal epithelial cell models. In contrast to sodin 5 and edulitin 2, indicitin did not show significant toxicity toward Caco-2 or HT29 cells within the tested concentration range.
To further investigate its biological activity, additional experiments were performed on other human cell lines previously shown to be sensitive to ribosome-inactivating proteins, including NB100 neuroblastoma cells, HeLa cervical carcinoma cells, and Raji Burkitt lymphoma cells.
Among the tested models, Raji cells showed the highest sensitivity to indicitin, displaying a reduction in cell viability of approximately 60% at the highest tested concentration after prolonged exposure. In contrast, no significant cytotoxic effects were observed in NB100 or HeLa cells.
These results suggest that indicitin may display cell-type selective cytotoxicity, with lymphoid cells appearing more sensitive than epithelial or neuronal cell lines.
Oxidative stress, inflammation and complex intestinal models
In the final phase of the project, the Bologna Research Unit investigated the molecular mechanisms underlying toxin-induced intestinal damage, focusing on oxidative stress, inflammation and barrier disruption in more complex cellular models.
Reactive oxygen species (ROS) production was measured in Caco-2 and HT29 cells after exposure to sodin 5. Flow cytometry analyses demonstrated a clear increase in intracellular ROS levels. The involvement of oxidative stress was confirmed by experiments using ROS scavengers, which significantly improved cell survival following toxin exposure.
The inflammatory response triggered by sodin 5 and edulitin 2 was investigated by analysing the expression of pro-inflammatory cytokines in Caco-2 cells through quantitative PCR. Sodin 5 induced a strong inflammatory response, particularly characterized by the marked up-regulation of IL-6 and IL-8, while TNF-α showed moderate induction and IL-1β remained largely unchanged. In contrast, edulitin 2 induced only a mild inflammatory response, mainly involving a moderate increase in IL-6 expression. Finally, more complex intestinal models were established to better reproduce the interaction between the epithelial barrier and immune cells. Co-culture systems including epithelial cells and Raji B lymphocytes were used to evaluate toxin-induced barrier damage and immune cell responses.
Both sodin 5 and edulitin 2 reduced TEER values in these models, indicating disruption of epithelial barrier integrity. In addition, a significant decrease in the viability of Raji cells located in the basolateral compartment was observed, suggesting that both toxins were able to cross the epithelial layer and affect immune cells.
Overall conclusions of Bologna Research Unit
These findings highlight the cytotoxic potential of ribosome-targeting toxins from edible organisms on tumoral cell lines. Furthermore, they demonstrate that these enzymes can alter intestinal barrier function and trigger inflammatory responses, thereby contributing to a better understanding of their potential biological effects following dietary exposure.
Milestone 1 – Set-up of an in vitro intestinal microbiota model
During the initial phase of the project, the Napoli Research Unit established an in vitro experimental model of intestinal microbiota to evaluate the effects of ribosome-targeting enzymes on gut microbial communities. The system was based on the use of Gifu Anaerobic Medium (MGAM), a complex culture medium widely used for the cultivation of intestinal bacteria. This medium supports the growth of a large proportion of microbial taxa present in human faecal samples, allowing the establishment of simplified in vitro models of the gut microbial ecosystem. Preliminary experiments were carried out to optimize the growth conditions of intestinal microorganisms in MGAM under anaerobic conditions. Both bacterial and fungal microorganisms were included in the experimental design to represent major components of the intestinal microbiota. The initial microbial panel included Gram-positive bacteria (Bacillus subtilis, Bacillus cereus, Lactobacillus rhamnosus), Gram-negative bacteria (Escherichia coli, Salmonella enterica), and the yeast Candida albicans. These microorganisms were selected because they can grow efficiently in MGAM and represent relevant intestinal microbial groups. This experimental system provided the basis for subsequent studies aimed at evaluating the effects of the enzymes sodin 5, edulitin 2, and indicitin on both individual microorganisms and complex microbial consortia.
Milestone 3 – Effects of sodin 5 on selected intestinal bacteria and on a faecal microbial consortium
The Napoli-RU first evaluated the effects of sodin 5, purified by the Caserta Research Unit, on a collection of intestinal microorganisms grown in vitro. Different concentrations of the protein were added to microbial cultures either at the beginning of growth or during the exponential phase. Growth analyses showed that sodin 5 did not affect the growth of any of the tested microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and Candida albicans. To determine whether digestion products of the protein could display antimicrobial activity, pepsin-digested sodin 5 peptides were also tested. No antimicrobial activity was observed. The absence of growth inhibition suggested that sodin 5 may not be able to enter microbial cells. To test this hypothesis, fluorescein-labelled sodin 5 was used in fluorescence microscopy experiments. No fluorescence signal was detected inside bacterial cells, indicating that the protein was unable to cross the microbial cell envelope.
Additional experiments performed on permeabilized bacterial cells demonstrated that sodin 5 could enter the cytoplasm only after membrane permeabilization. These results confirmed that the lack of antimicrobial activity is likely due to the inability of sodin 5 to penetrate bacterial cells and reach the ribosomal target.
In parallel, experiments were initiated to evaluate the effects of sodin 5 on biofilm formation and on complex microbial consortia derived from fecal samples.
Milestone 5 – Effects of edulitin 2 on selected intestinal bacteria and on a faecal microbial consortium
The effects of edulitin 2, a ribotoxin-like protein isolated from Boletus edulis, were subsequently investigated by Napoli-RU using the same experimental models set-up for sodin 5 during Milestone 3.
Similarly to sodin 5, edulitin 2 did not affect the growth of Gram-positive or Gram-negative bacteria at any of the tested concentrations. A minor effect was observed only in the microscopic fungus Candida albicans, which showed a modest increase in generation time at the highest protein concentration tested.
Based on the limited effects observed in bacteria, additional experiments were carried out on other intestinal fungal species isolated from faecal samples, including Candida parapsilosis, Candida albicans, and Candida glabrata. These analyses confirmed that edulitin 2 did not significantly affect the growth of intestinal microorganisms, including both bacterial and fungal species.
To investigate the possible impact of the two ribosome-targeting enzymes sodin 5 and edulitin 2 on complex microbial communities, an in vitro faecal consortium model was established by Napoli-RU.
Faecal samples from three healthy children not undergoing antibiotic or probiotic treatment were collected. Microbial suspensions were serially diluted and cultured in MGAM under anaerobic conditions. A dilution showing stable microbial growth was selected to inoculate fresh cultures either without supplementation or supplemented with sodin 5 or edulitin 2.
After incubation, total microbial DNA was extracted and used for 16S rRNA gene sequencing to analyse microbial composition.
The results showed that the cultivation conditions allowed the recovery of approximately 58% of the bacterial phyla and families originally present in the fecal samples. Although some strict anaerobes were lost during cultivation, this recovery rate was consistent with previously reported in vitro gut microbiota cultivation systems. Comparative analyses between untreated cultures and enzyme-treated cultures showed only minimal differences in microbial composition. At the phylum level, small increases in Proteobacteria were observed in sodin 5-treated samples, while Patescibacteria showed a modest increase in cultures treated with edulitin 2. At the genus level, slight increases in Bacteroides and Desulfotomaculum were detected following treatment with sodin 5 and edulitin 2, respectively.
However, global diversity analyses using standard alpha diversity indices (Observed ASVs, Chao1 and Shannon index) did not reveal significant differences between treated and untreated cultures. These results indicate that neither sodin 5 nor edulitin 2 substantially alters the overall diversity or composition of the intestinal microbial community in the in vitro model.
Milestone 8 – Effects of the novel enzyme indicitin on intestinal microbiota
In the final phase of the project, the Napoli Research Unit investigated the biological effects of indicitin, a novel ribosome-targeting enzyme identified and purified by the Caserta Research Unit from the edible mushroom Calocybe indica. First, the effects of indicitin were evaluated on pure cultures of intestinal microorganisms, including Gram-positive and Gram-negative bacteria as well as fungal species of the Candida genus. No growth inhibition was observed in any of the tested microorganisms, even at the highest protein concentrations.
The effects of indicitin were then evaluated using the in vitro faecal consortium model previously established. Microbial cultures were grown in MGAM under anaerobic conditions either in the absence or presence of indicitin, followed by DNA extraction and 16S rRNA gene sequencing.
The sequencing analysis showed that more than 50% of the bacterial phyla originally present in fecal samples were recovered after cultivation, consistent with previous results obtained using this experimental system. Comparative analyses between treated and untreated cultures revealed no statistically significant differences in microbial composition at either the phylum or genus level. Similarly, analyses of alpha diversity (Observed ASVs, Chao1 and Shannon index) and beta diversity did not reveal any detectable differences between the two conditions.
Overall, these findings indicate that indicitin does not significantly affect the growth or diversity of intestinal microorganisms, either in pure cultures or in complex microbial communities.
Overall conclusions of Napoli Research Unit
The studies conducted by the Napoli Research Unit suggest that ribosome-targeting proteins present in edible plants and mushrooms are unlikely to exert major direct effects on gut microbiota, contributing to a better understanding of their potential biological impact following dietary exposure.
