Oligomer assembly of Bacillus thuringiensis Cyt2Aa2 on lipid membranes reveals a thread-like structure

<i>Bacillus thuringiensis</i>, a well-known insecticidal bacterium, produces several insecticidal proteins, including cytolytic (Cyt) proteins. Cyt proteins bind directly to the lipid membrane and form large protein complexes. In addition to the protein ladder bands, information on the oligomeric structure in lipid membranes is necessary to understand the mechanism of Cyt proteins on target cells. In this work, we have investigated the oligomeric Cyt2Aa2 complex with synthetic lipid and with erythrocyte membranes. When the activated Cyt2Aa2 protein was incubated with these lipid membranes, the protein ladder pattern relevant to hemolytic activity was detected in SDS-PAGE. Moreover, AFM topographic images revealed a fusilli-like structure and a ring-like structure for synthetic POPC and POPC/Chol, respectively. Furthermore, TEM micrographs provided an additional information on the oligomeric structure of Cyt2Aa2 in erythrocytes. Cyt2Aa2 appears to oligomerise/aggregate into mixed structures between the filamentous structure and small protein complexes in erythrocytes. In addition, a nanopore was found to be a substructure of the filamentous structure. These results strengthen the understanding of Cyt2Aa2 behavior in these two membrane systems, the fusilli and ring-like structures, depending on the type of lipid membrane. Furthermore, the structure of Cyt2Aa2 in insect target membranes remains to be investigated.