Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

Aegerolysins are remarkable proteins. They are distributed over the tree of life, being relatively widespread in bacteria and fungi, but also present in some insects, plants, protozoa, and viruses. Despite their abundance in cells of certain developmental stages and their presence in secretomes, only a few aegerolysins have been studied in detail. Their function, in particular, is intriguing. Here, we summarize previously published findings on the distribution, molecular interactions, and function of these versatile aegerolysins. They have very diverse protein sequences but a common fold. The machine learning approach of the AlphaFold2 algorithm, which incorporates physical and biological knowledge of protein structures and multisequence alignments, provides us new insights into the aegerolysins and their pore-forming partners, complemented by additional genomic support. We hypothesize that aegerolysins are involved in the mechanisms of competitive exclusion in the niche.

The lipid raft hypothesis emerged as a need to explain the lateral organization and behavior of lipids in the environment of biological membranes. The idea, that lipids segregate in biological membranes to form liquid-disordered and liquid-ordered states, was faced with a challenge: to show that lipid-ordered domains, enriched in sphingomyelin and cholesterol, actually exist in vivo. A great deal of indirect evidence and the use of lipid-binding probes supported this idea, but there was a lack of tools to demonstrate the existence of such domains in living cells. A whole new toolbox had to be invented to biochemically characterize lipid rafts and to define how they are involved in several cellular functions. A potential solution came from basic biochemical experiments in the late 1970s, showing that some mushroom extracts exert hemolytic activities. These activities were later assigned to aegerolysin-based sphingomyelin/cholesterol-specific cytolytic protein complexes. Recently, six sphingomyelin/cholesterol binding proteins from different mushrooms have been identified and have provided some insight into the nature of sphingomyelin/cholesterol-rich domains in living vertebrate cells. In this review, we dissect the accumulated knowledge and introduce the mushroom lipid raft binding proteins as molecules of choice to study the dynamics and origins of these liquid-ordered domains in mammalian cells.