NTNH protein: more than a bodyguard for botulinum neurotoxins.

As one of the most fatal substances, botulinum neurotoxins (BoNTs) have never acted solo to accomplish their formidable missions. Most notably, nontoxic nonhemagglutinin (NTNH), a protein co-secreted with BoNT by bacteria, plays critical roles to stabilize and protect BoNT by tightly associating with it to form the minimal progenitor toxin complex (M-PTC). A new cryo-EM structure of the M-PTC of a BoNT-like toxin from Weissella oryzae (BoNT/Wo) reveals similar assembly modes between M-PTC/Wo and that of other BoNTs, yet also reveals some unique structural features of NTNH/Wo. These findings shed new light on the potential versatile roles of NTNH during BoNT intoxication.

Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2.

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 (HCE) 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 HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE 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.

Crystal structures of OrfX1, OrfX2 and the OrfX1-OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1.

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.

Receptor binding mechanisms of Clostridioides difficile toxin B and implications for therapeutics development.

Clostridioides difficile is classified as an urgent antibiotic resistance threat by the Centers for Disease Control and Prevention (CDC). C. difficile infection (CDI) is mainly caused by the C. difficile exotoxin TcdB, which invades host cells via receptor-mediated endocytosis. However, many natural variants of TcdB have been identified including some from the hypervirulent strains, which pose significant challenges for developing effective CDI therapies. Here, we review the recent research progress on the molecular mechanisms by which TcdB recognizes Frizzed proteins (FZDs) and chondroitin sulfate proteoglycan 4 (CSPG4) as two major host receptors. We suggest that the receptor-binding sites and several previously identified neutralizing epitopes on TcdB are ideal targets for the development of broad-spectrum inhibitors to protect against diverse TcdB variants.

Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A.

While covalent drug discovery is reemerging as an important route to small-molecule therapeutic leads, strategies for the discovery and engineering of protein-based irreversible binding agents remain limited. Here, we describe the use of yeast display in combination with noncanonical amino acids (ncAAs) to identify irreversible variants of single-domain antibodies (sdAbs), also called VHHs and nanobodies, targeting botulinum neurotoxin light chain A (LC/A). Starting from a series of previously described, structurally characterized sdAbs, we evaluated the properties of antibodies substituted with reactive ncAAs capable of forming covalent bonds with nearby groups after UV irradiation (when using 4-azido-l-phenylalanine) or spontaneously (when using O-(2-bromoethyl)-l-tyrosine). Systematic evaluations in yeast display format of more than 40 ncAA-substituted variants revealed numerous clones that retain binding function while gaining either UV-mediated or spontaneous crosslinking capabilities. Solution-based analyses indicate that ncAA-substituted clones exhibit site-dependent target specificity and crosslinking capabilities uniquely conferred by ncAAs. Interestingly, not all ncAA substitution sites resulted in crosslinking events, and our data showed no apparent correlation between detected crosslinking levels and distances between sdAbs and LC/A residues. Our findings highlight the power of yeast display in combination with genetic code expansion in the discovery of binding agents that covalently engage their targets. This platform streamlines the discovery and characterization of antibodies with therapeutically relevant properties that cannot be accessed in the conventional genetic code.

Neutralizing epitopes on Clostridioides difficile toxin A revealed by the structures of two camelid VHH antibodies.

Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile, which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.

Identification of TFPI as a receptor reveals recombination-driven receptor switching in Clostridioides difficile toxin B variants.

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.

Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition.

Clostridioides difficile is one of the most common causes of antibiotic-associated diarrhea in developed countries. As key virulence factors of C. difficile, toxin A (TcdA) and toxin B (TcdB) act by glucosylating and inactivating Rho and Ras family small GTPases in host cells, which leads to actin cytoskeleton disruption, cell rounding, and ultimately cell death. Here we present the co-crystal structure of the glucosyltransferase domain (GTD) of TcdA in complex with its substrate human RhoA at 2.60-angstrom resolution. This structure reveals that TcdA GTD grips RhoA mainly through its switch I and switch II regions, which is complemented by interactions involving RhoA's pre-switch I region. Comprehensive structural comparisons between the TcdA GTD-RhoA complex and the structures of TcdB GTD in complex with Cdc42 and R-Ras reveal both the conserved and divergent features of these two toxins in terms of substrate recognition. Taken together, these findings establish the structural basis for TcdA recognition of small GTPases and advance our understanding of the substrates selectivity of large clostridial toxins.

Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer.

CDC42 family GTPases (RHOJ, RHOQ, CDC42) are upregulated but rarely mutated in cancer and control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. Here, we use computer-aided drug design to discover a chemical entity (ARN22089) that has broad activity against a panel of cancer cell lines, inhibits S6 phosphorylation and MAPK activation, activates pro-inflammatory and apoptotic signaling, and blocks tumor growth and angiogenesis in 3D vascularized microtumor models (VMT) in vitro. Additionally, ARN22089 has a favorable pharmacokinetic profile and can inhibit the growth of BRAF mutant mouse melanomas and patient-derived xenografts in vivo. ARN22089 selectively blocks CDC42 effector interactions without affecting the binding between closely related GTPases and their downstream effectors. Taken together, we identify a class of therapeutic agents that influence tumor growth by modulating CDC42 signaling in both the tumor cell and its microenvironment.

Structure and conformational dynamics of Clostridioides difficile toxin A.

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.

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.

Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies.

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

Structural basis for selective modification of Rho and Ras GTPases by Clostridioides difficile toxin B.

Toxin B (TcdB) is a primary cause of Clostridioides difficile infection (CDI). This toxin acts by glucosylating small GTPases in the Rho/Ras families, but the structural basis for TcdB recognition and selectivity of specific GTPase substrates remain unsolved. Here, we report the cocrystal structures of the glucosyltransferase domain (GTD) of two distinct TcdB variants in complex with human Cdc42 and R-Ras, respectively. These structures reveal a common structural mechanism by which TcdB recognizes Rho and R-Ras. Furthermore, we find selective clustering of adaptive residue changes in GTDs that determine their substrate preferences, which helps partition all known TcdB variants into two groups that display distinct specificities toward Rho or R-Ras. Mutations that selectively disrupt GTPases binding reduce the glucosyltransferase activity of the GTD and the toxicity of TcdB holotoxin. These findings establish the structural basis for TcdB recognition of small GTPases and reveal strategies for therapeutic interventions for CDI.

Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection.

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.

Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells.

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Camelid VHH Antibodies that Neutralize Botulinum Neurotoxin Serotype E Intoxication or Protease Function.

Botulinum neurotoxin (BoNT) serotype E is one of three serotypes that cause the preponderance of human botulism cases and is a Tier 1 Select Agent. BoNT/E is unusual among BoNT serotypes for its rapid onset and short duration of intoxication. Here we report two large panels of unique, unrelated camelid single-domain antibodies (VHHs) that were selected for their ability to bind to BoNT/E holotoxin and/or to the BoNT/E light chain protease domain (LC/E). The 19 VHHs which bind to BoNT/E were characterized for their subunit specificity and 8 VHHs displayed the ability to neutralize BoNT/E intoxication of neurons. Heterodimer antitoxins consisting of two BoNT/E-neutralizing VHHs, including one heterodimer designed using structural information for simultaneous binding, were shown to protect mice against co-administered toxin challenges of up to 500 MIPLD50. The 22 unique VHHs which bind to LC/E were characterized for their binding properties and 9 displayed the ability to inhibit LC/E protease activity. Surprisingly, VHHs selected on plastic-coated LC/E were virtually unable to recognize soluble or captured LC/E while VHHs selected on captured LC/E were poorly able to recognize LC/E coated to a plastic surface. This panel of anti-LC/E VHHs offer insight into BoNT/E function, and some may have value as components of therapeutic antidotes that reverse paralysis following BoNT/E exposures.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.