Peng Chen

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 neurotoxins (BoNTs) rank among the most potent toxins and many of them are produced by bacteria carrying the orfX gene cluster that also encodes four nontoxic proteins (OrfX1, OrfX2, OrfX3 and P47). The orfX gene cluster is also found in the genomes of many non-BoNT-producing bacteria, often alongside genes encoding oral insecticidal toxins. However, the functions of these OrfXs and P47 remain elusive. Here, we demonstrate that the combined action of all four components (OrfXs and P47) drastically boosts the oral toxicity of BoNT in mice, following proteolytic activation by digestive proteases that oral toxins regularly confront. In particular, OrfX2 adopts a self-inhibiting state, engaging with BoNT through another clostridial protein, nontoxic non-hemagglutinin (NTNH), only after proteolytic activation. Cryo-electron microscopy studies unveil that two molecules of protease-activated OrfX2 simultaneously associate with NTNH, a binding mode crucial for boosting BoNT oral toxicity. Collectively, these studies offer novel insights into the physiological functions and regulatory mechanisms of OrfXs and P47 of BoNTs, shedding light on the pathogenesis of other bacterial toxins associated with homologous OrfXs and P47.

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.

Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843–T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.

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

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.

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.

Toxic hijack of a cell signaling pathway: The pathogen Clostridium difficile colonizes the human colon when the normal microbiota is disrupted, often after antibiotic treatment. It is a leading cause of hospital-acquired diarrhea, especially among elderly patients. Chen et al. describe a 2.5-Å-resolution crystal structure that shows how a major virulence factor in C. difficile , toxin B (TcdB), binds to the G protein–coupled receptor Frizzled (FZD). This receptor activates the Wnt signaling pathway, which regulates homeostasis of the colonic epithelium. Surprisingly, TcdB uses a lipid cofactor to recognize FZD. This cofactor replaces a lipid normally associated with the Wnt ligand that binds FZD to activate signaling. Inhibiting the Wnt pathway likely plays a role in C. difficile pathology. Science , this issue p. 664