Effects of indicitin, a novel ribosome-targeting enzyme from milky mushrooms, on four human cell lines and the gut microbiota

Nowadays, increasing research attention is being devoted to edible mushrooms, both for their use as nutraceutical foods and as an alternative source of income for rural communities. On the other hand, several edible mushrooms contain enzymes that can damage ribosomes, thereby inhibiting protein synthesis [e.g., ribotoxin-like proteins (RL-Ps) and ribosome-inactivating proteins (RIPs)]. These enzymes can have toxic effects, putting human health at risk. In this context, we report the characterization of a new toxin, named indicitin, isolated from the fruiting bodies of the edible mushroom Calocybe indica. This well-known tropical species is renowned for its delicate flavour and medicinal properties. This enzyme (similar to 28 kDa) is a specific ribonuclease able to inhibit protein synthesis in vitro (IC50 = 1.05 x 10-6 M) by selectively cleaving the alpha-sarcin-ricin loop of 28S rRNA in animal ribosomes. In addition, the N-terminal amino acid sequence and structural features highlight that indicitin belongs to the thaumatin-like protein family. Indicitin exhibits a dose- and time-dependent cytotoxic activity against Raji cells, a human B lymphoblastoid cell line, triggering mainly the apoptotic pathway, as evaluated through annexin V/propidium iodide staining. However, no effects were observed on the viability of indicitin-treated HeLa, NB100 and Caco-2 cells. Moreover, indicitin does not affect the growth of the gut microbiota, as it is unable to damage fungal and prokaryotic ribosomes. Overall, these findings confirm that indicitin could represent a novel biotechnological tool that has low toxicity toward several cell types and is non-harmful to the gut microbiota.