Cylindracin, a Fruiting Body-Specific Protein of Cyclocybe cylindracea, Represses the Egg-Laying and Development of Caenorhabditis elegans and Drosophila melanogaster

Mushrooms are a valuable source of bioactive compounds to develop efficient, secure medicines and environmentally friendly agrochemicals. Cylindracin is a small cysteine-rich protein that is specifically expressed in the immature fruiting body of the edible mushroom <i>Cyclocybe cylindracea</i>. Recombinant protein (rCYL), comprising the C-terminal cysteine-rich domain of cylindracin, inhibits the hyphal growth and conidiogenesis of filamentous fungi. Here, we show that rCYL represses the egg-laying and development of <i>Caenorhabditis elegans</i> and <i>Drosophila melanogaster</i>. The feeding of rCYL at 16 µM reduced the body volume of <i>C. elegans</i> larvae to approximately 60% when compared to the control. At the same concentration, rCYL repressed the frequencies of pupation and emergence of <i>D. melanogaster</i> to 74% and 40%, respectively, when compared to the control. In virgin adult flies, feeding of rCYL at 47 µM substantially repressed the frequency of egg-laying, and the pupation and emergence of the next generation, especially for females. These inhibitory effects of rCYL gradually disappeared after ceasing the ingestion of rCYL. The use of fluorescence-labeled rCYL revealed that the protein accumulates specifically at the pharynx cuticles of <i>C. elegans</i>. In <i>D. melanogaster</i>, fluorescence-labeled rCYL was detected primarily in the midguts and to a lesser degree in the hindguts, ovaries, testes, and malpighian tubules. rCYL was stable against trypsin, chymotrypsin, and pepsin, whereas it did not inhibit proteolytic and glycolytic enzymes in vitro. rCYL oligomerized and formed amyloid-like aggregates through the binding to heparin and heparan sulfate in vitro. These results suggest that rCYL has potential as a new biocontrol agent against pests.