Zheng Liu

Botulinum neurotoxins (BoNTs) are potent bacterial toxins. Distinct BoNT-like toxins, including BoNT/X, BoNT/En, and PMP1, form a distinct lineage. Here we characterize two additional members of this group, PG-toxin 1 and 2 (PG1 and PG2). Their light-chain (LC) and heavy-chain (HC) domains are encoded by adjacent genes. PG1 and PG2 LCs cleave Drosophila SNAP-25 at distinct sites (N196-E197 and R191-K193, respectively); PG1 is more potent, while PG2 activity depends on pH and ionic strength. Both LCs enter Sf9 cells via clathrin-mediated endocytosis. PG1 LC is toxic to Drosophila and northern armyworm independently of its HC, whereas PG2 LC toxicity is enhanced by its HC. Cryo-EM shows that PG1 adopts a distinct compact di-chain architecture with a door-hinge-like “belt.” These findings expand the structural and functional diversity of the BoNT family and support the potential of PG-toxins as insecticidal agents and research tools.

Insecticidal toxins from Bacillus thuringiensis (Bt) have been extensively and successfully used in genetically engineered crops for decades but continue to face challenges from the adaptive resistance in the insect population. The Bt binary toxin Vip1Ad1(Vip1) and Vip2Ag1(Vip2), a promising next-generation candidate gene combination for transgenic crops, have demonstrated high efficacy against the destructive coleopteran pest white grubs, however, their mode of action remains largely elusive. In this study, we report cryo-EM structures of the heptameric Vip1-pore and Vip2-bound Vip1-pore complex, capturing a series of putative assembly-related intermediates that suggest a binary toxin assembly and translocation pathway. Together with structure-guided mutagenesis, these data provide insights into a sequential assembly of binary complex and a sequence-independent translocation mechanism. Proof-of-principle experiments showed successful delivery of a desired protein cargo into host cells based on the mini-Vip2-Vip1 pore system, paving the way for developing much needed extracellular pesticidal protein delivery platforms. These findings not only clarify the assembly and translocation mechanism of the binary insecticidal toxin pair but also offer an excellent alternative model to investigate human-pathogenic pore-forming toxins because of its similarity and biosafety.

Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (H_CE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between H_CE and SV2 provide the crucial location and specificity information for H_CE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, H_CE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.

Diverse TcdB variants use a flexible structural platform to tune their specificity toward different GTPase substrates.

Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB–antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.