Characterization of a mosquitocidal botulinum toxin-like neurotoxin complex

Paraclostridial mosquitocidal protein 1 (PMP1), a botulinum-like toxin from Paraclostridium bifermentans, belongs to the clostridial neurotoxin (CNT) family, which includes botulinum and tetanus neurotoxins. PMP1 is the first member of this family shown to target insects, and presents high selectivity for Anopheles mosquitoes, the main vector of malaria which is a huge healthcare burden worldwide. All the other BoNTs are only known to target vertebrates so far. PMP1 is encoded on an operon that contains the orfX gene cluster, which in addition to the toxin, contains the genes for five toxin-associated proteins (OrfX1, OrfX2, OrfX3, p47 and NTNH). They are expected to form an assembly that is defined as the large progenitor toxin complex (L-PTC), which displays an increased toxicity compared to the minimal progenitor complex (M-PTC), whose components are the PMP1 toxin and NTNH (non-toxic non-hemagglutinin) protein. The function of the OrfX proteins remains to be discovered. OrfX1 and OrfX3 were recently identified to form a complex which was solved by X-ray crystallography. Here, we aim to solve the structures of OrfX2 and p47, and to identify the optimal conditions to obtain the L-PTC in order to determine its structure by cryo-EM. This should help us understand the function of OrfX proteins, and provide the basis to design novel biotechnological tools to control anopheles mosquito populations.