Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity.

Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2-3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold-12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal Select Agent list. In this study, we added four additional mutations in the receptor-binding domain, translocation domain, and enzymatic cleft to further decrease toxicity, creating 7M rBoNT/A1. Due to poor expression in E. coli, 7M rBoNT/A1 was produced in an endogenous C. botulinum expression system. This protein had higher residual toxicity (LD50: 280 ng/mouse) than previously reported for the catalytically inactive rBoNT/A1 containing only three of the mutations (>10 µg/mouse). To investigate this discrepancy, several additional rBoNT/A1 constructs containing individual sets of amino acid substitutions from 7M rBoNT/A1 and related mutations were also endogenously produced. Similarly to endogenously produced 7M rBoNT/A1, all of the endogenously produced mutants had ~100-1000-fold greater toxicity than what was reported for their original heterologous host counterparts. A combination of mutations in multiple functional domains resulted in a greater but not multiplicative reduction in toxicity. This report demonstrates the impact of production systems on residual toxicity of genetically inactivated rBoNTs.

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain.

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a ß-strand peptide (556-564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the ß-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 ß-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 ß-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C ß-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C ß-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.

Multiple Domains of Staphylococcal Superantigen-like Protein 11 (SSL11) Contribute to Neutrophil Inhibition.

Staphylococcus aureus is an opportunistic pathogen producing many immune evasion molecules targeting various components of the host immune defense, including the Staphylococcal superantigen-like protein (SSL 1-14) family. Despite sharing similar structures with the powerful superantigens (SAgs), which cause massive T cell activation, SSLs interfere with a wide range of innate immune defenses. SSLs are divided into two subgroups, SSLs that contain a conserved carbohydrate Sialyl Lewis X [Neu5Acα2-3Galß1-4(Fucα1-3) GlcNAcß, SLeX] binding site and SSLs that lack the SLeX binding site. SSL2-6 and SSL11 possess the SLeX binding site. Our previous studies showed that SSL11 arrests cell motility by inducing cell adhesion in differentiated HL60 (dHL60) cells, while SSL7 did not bind dHL60 cells. SSL7-based chimeras were engineered by exchanging the SSL7 sequence with the corresponding SSL11 sequence and assaying for a gain of SSL11 function, namely, the induction of cell spreading and motility arrest. In addition to the SLeX-binding site, we observed that three beta-strands ß6, ß7, and ß9 and the N-terminal residues, Y16 and Y17, transitioned SSL7 to gain SSL11 activities. These studies define the structure-function properties of SSL11 that may allow SSL11 to inhibit S. aureus clearance by the host innate immune system, allowing S. aureus to maintain a carrier state in humans, an understudied aspect of S. aureus pathogenesis.

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1-A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.

When Escherichia coli doesn't fit the mold: A pertussis-like toxin with altered specificity.

Bacterial toxins introduce protein modifications such as ADP-ribosylation to manipulate host cell signaling and physiology. Several general mechanisms for toxin function have been established, but the extent to which previously uncharacterized toxins utilize these mechanisms is unknown. A study of an Escherichia coli pertussis-like toxin demonstrates that this protein acts on a known toxin substrate but displays distinct and dual chemoselectivity, suggesting this E. coli pertussis-like toxin may serve as a unique tool to study G-protein signaling in eukaryotic cells.

Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons.

Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans but also are common therapies for neurological diseases. BoNTs are dichain toxins, comprising an N-terminal catalytic domain (LC) disulfide bond linked to a C-terminal heavy chain (HC) which includes a translocation domain (HN) and a receptor binding domain (HC). Recently, the BoNT serotype A (BoNT/A) subtypes A1 and A2 were reported to possess similar potencies but different rates of cellular intoxication and pathology in a mouse model of botulism. The current study measured HCA1 and HCA2 entry into rat primary neurons and cultured Neuro2A cells. We found that there were two sequential steps during the association of BoNT/A with neurons. The initial step was ganglioside dependent, while the subsequent step involved association with synaptic vesicles. HCA1 and HCA2 entered the same population of synaptic vesicles and entered cells at similar rates. The primary difference was that HCA2 had a higher degree of receptor occupancy for cells and neurons than HcA1. Thus, HCA2 and HCA1 share receptors and entry pathway but differ in their affinity for receptor. The initial interaction of HCA1 and HCA2 with neurons may contribute to the unique pathologies of BoNT/A1 and BoNT/A2 in mouse models.

Multiple domains of tetanus toxin direct entry into primary neurons.

Tetanus toxin elicits spastic paralysis by cleaving VAMP-2 to inhibit neurotransmitter release in inhibitory neurons of the central nervous system. As the retrograde transport of tetanus neurotoxin (TeNT) from endosomes has been described, the initial steps that define how TeNT initiates trafficking to the retrograde system are undefined. This study examines TeNT entry into primary cultured cortical neurons by total internal reflection fluorescence (TIRF) microscopy. The initial association of TeNT with the plasma membrane was dependent upon ganglioside binding, but segregated from synaptophysin1 (Syp1), a synaptic vesicle (SV) protein. TeNT entry was unaffected by membrane depolarization and independent of SV cycling, whereas entry of the receptor-binding domain of TeNT (HCR/T) was stimulated by membrane depolarization and inhibited by blocking SV cycling. Measurement of the incidence of colocalization showed that TeNT segregated from Syp1, whereas HCR/T colocalized with Syp1. These studies show that while the HCR defines the initial association of TeNT with the cell membrane, regions outside the HCR define how TeNT enters neurons independent of SV cycling. This provides a basis for the unique entry of botulinum toxin and tetanus toxin into neurons.

Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes.

The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E were insoluble, prohibiting studies towards understanding the mechanisms of toxin action and substrate recognition. In this work, the molecular basis of insolubility of the recombinant LCs of two representative subtypes of BoNT/E, E1(Beluga) and E3 (Alaska), was determined. Hydrophobicity profile and structural modeling predicted a C-terminal candidate region responsible for the insolubility of LC/Es. Deletion of C-terminal 19 residues of LC/E(1-400) resulted in enhanced solubility, from 2 to ∼50% for LC/EAlaska and from 16 to ∼95% for LC/EBeluga. In addition, resides 230-236 were found to contribute to a different solubility level of LC/EAlaska when compared to LC/EBeluga. Substituting residues (230)TCI(232) in LC/EAlaska to the corresponding residues of (230)KYT(232) in LC/EBeluga enhanced the solubility of LC/EAlaska to a level approaching that of LC/EBeluga. Among these LC/Es and their derivatives, LC/EBeluga 1-400 was the most soluble and stable protein. Each LC/E derivative possessed similar catalytic activity, suggesting that the C-terminal region of LC/Es contributed to protein solubility, but not catalytic activity. In conclusion, this study generated a soluble and stable recombinant LC/E and provided insight into the structural components that govern the solubility and stability of the LCs of other BoNT serotypes and Tetanus toxin.

Nonenzymatic conversion of ADP-ribosylated arginines to ornithine alters the biological activities of human neutrophil peptide-1.

Activated neutrophils, recruited to the airway of diseased lung, release human neutrophil peptides (HNP1-4) that are cytotoxic to airway cells as well as microbes. Airway epithelial cells express arginine-specific ADP ribosyltransferase (ART)-1, a GPI-anchored ART that transfers ADP-ribose from NAD to arginines 14 and 24 of HNP-1. We previously reported that ADP-ribosyl-arginine is converted nonenzymatically to ornithine and that ADP-ribosylated HNP-1 and ADP-ribosyl-HNP-(ornithine) were isolated from bronchoalveolar lavage fluid of a patient with idiopathic pulmonary fibrosis, indicating that these reactions occur in vivo. To determine effects of HNP-ornithine on the airway, three analogs of HNP-1, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn), were tested for their activity against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus; their cytotoxic effects on A549, NCI-H441, small airway epithelial-like cells, and normal human lung fibroblasts; and their ability to stimulate IL-8 and TGF-ß1 release from A549 cells, and to serve as ART1 substrates. HNP and the three analogs had similar effects on IL-8 and TGF-ß1 release from A549 cells and were all cytotoxic for small airway epithelial cells, NCI-H441, and normal human lung fibroblasts. HNP-(R14,24orn), when compared with HNP-1 and HNP-1 with a single ornithine substitution for arginine 14 or 24, exhibited reduced cytotoxicity, but it enhanced proliferation of A549 cells and had antibacterial activity. Thus, arginines 14 and 24, which can be ADP ribosylated by ART1, are critical to the regulation of the cytotoxic and antibacterial effects of HNP-1. The HNP analog, HNP-(R14,24orn), lacks the epithelial cell cytotoxicity of HNP-1, but partially retains its antibacterial activity and thus may have clinical applications in airway disease.

Bacillus cereus Certhrax ADP-ribosylates vinculin to disrupt focal adhesion complexes and cell adhesion.

Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton.

The chaperone protein SmgGDS interacts with small GTPases entering the prenylation pathway by recognizing the last amino acid in the CAAX motif.

Ras family small GTPases localize at the plasma membrane, where they can activate oncogenic signaling pathways. Understanding the mechanisms that promote membrane localization of GTPases will aid development of new therapies to inhibit oncogenic signaling. We previously reported that SmgGDS splice variants promote prenylation and trafficking of GTPases containing a C-terminal polybasic region and demonstrated that SmgGDS-607 interacts with nonprenylated GTPases, whereas SmgGDS-558 interacts with prenylated GTPases in cells. The mechanism that SmgGDS-607 and SmgGDS-558 use to differentiate between prenylated and nonprenylated GTPases has not been characterized. Here, we provide evidence that SmgGDS-607 associates with GTPases through recognition of the last amino acid in the CAAX motif. We show that SmgGDS-607 forms more stable complexes in cells with nonprenylated GTPases that will become geranylgeranylated than with nonprenylated GTPases that will become farnesylated. These binding relationships similarly occur with nonprenylated SAAX mutants. Intriguingly, farnesyltransferase inhibitors increase the binding of WT K-Ras to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. Using recombinant proteins and prenylated peptides corresponding to the C-terminal sequences of K-Ras and Rap1B, we found that both SmgGDS-607 and SmgGDS-558 directly bind the GTPase C-terminal region, but the specificity of the SmgGDS splice variants for prenylated versus nonprenylated GTPases is diminished in vitro. Finally, we present structural homology models and data from functional prediction software to define both similar and unique features of SmgGDS-607 when compared with SmgGDS-558.

A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation.

Botulinum neurotoxins (BoNTs) and tetanus toxin (TeNT) are the most potent toxins for humans and elicit unique pathologies due to their ability to traffic within motor neurons. BoNTs act locally within motor neurons to elicit flaccid paralysis, while retrograde TeNT traffics to inhibitory neurons within the central nervous system (CNS) to elicit spastic paralysis. BoNT and TeNT are dichain proteins linked by an interchain disulfide bond comprised of an N-terminal catalytic light chain (LC) and a C-terminal heavy chain (HC) that encodes an LC translocation domain (HCT) and a receptor-binding domain (HCR). LC translocation is the least understood property of toxin action, but it involves low pH, proteolysis, and an intact interchain disulfide bridge. Recently, Pirazzini et al. (FEBS Lett 587:150-155, 2013, http://dx.doi.org/10.1016/j.febslet.2012.11.007) observed that inhibitors of thioredoxin reductase (TrxR) blocked TeNT and BoNT action in cerebellar granular neurons. In the current study, an atoxic TeNT LC translocation reporter was engineered by fusing ß-lactamase to the N terminus of TeNT [ßlac-TeNT(RY)] to investigate LC translocation in primary cortical neurons and Neuro-2a cells. ßlac-TeNT(RY) retained the interchain disulfide bond, showed ganglioside-dependent binding to neurons, required acidification to promote ßlac translocation, and was sensitive to auranofin, an inhibitor of thioredoxin reductase. Mutation of ßlac-TeNT(RY) at C439S and C467S eliminated the interchain disulfide bond and inhibited ßlac translocation. These data support the requirement of an intact interchain disulfide for LC translocation and imply that disulfide reduction is a prerequisite for LC delivery into the host cytosol. The data also support a model that LC translocation proceeds from the C to the N terminus. ßlac-TeNT(RY) is the first reporter system to measure translocation by an AB single-chain toxin in intact cells.

Exoenzyme S ADP-ribosylates Rab5 effector sites to uncouple intracellular trafficking.

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained Arg→Ala mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.

Entry of a recombinant, full-length, atoxic tetanus neurotoxin into Neuro-2a cells.

Tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) are clostridial neurotoxins (CNTs) responsible for the paralytic diseases tetanus and botulism, respectively. CNTs are AB toxins with an N-terminal zinc-metalloprotease light chain that is linked by a disulfide bond to a C-terminal heavy chain that includes a translocation domain and a receptor-binding domain (HCR). Current models predict that the HCR defines how CNTs enter and traffic in neurons. Recent studies implicate that domains outside the HCR contribute to CNT trafficking in neurons. In the current study, a recombinant, full-length TeNT derivative, TeNT(RY), was engineered to analyze TeNT cell entry. TeNT(RY) was atoxic in a mouse challenge model. Using Neuro-2a cells, a mouse neuroblastoma cell line, TeNT HCR (HCR/T) and TeNT(RY) were found to bind gangliosides with similar affinities and specificities, consistent with the HCR domain containing receptor binding function. Temporal studies showed that HCR/T and TeNT(RY) entered Neuro-2a cells slower than the HCR of BoNT/A (HCR/A), transferrin, and cholera toxin B. Intracellular localization showed that neither HCR/T nor TeNT(RY) localized with HCR/A or synaptic vesicle protein 2, the protein receptor for HCR/A. HCR/T and TeNT(RY) exhibited only partial intracellular colocalization, indicating that regions outside the HCR contribute to the intracellular TeNT trafficking. TeNT may require this complex functional entry organization to target neurons in the central nervous system.

Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor.

The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kD(a) and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer.

Host cell cytotoxicity and cytoskeleton disruption by CerADPr, an ADP-ribosyltransferase of Bacillus cereus G9241.

Bacillus cereus G9241 was isolated from a welder suffering from an anthrax-like inhalation illness. B. cereus G9241 encodes two megaplasmids, pBCXO1 and pBC210, which are analogous to the toxin- and capsule-encoding virulence plasmids of Bacillus anthracis. Protein modeling predicted that the pBC210 LF homologue contained an ADP-ribosyltransferase (ADPr) domain. This putative bacterial ADP-ribosyltransferase domain was denoted CerADPr. Iterative modeling showed that CerADPr possessed several conserved ADP-ribosyltransferase features, including an α-3 helix, an ADP-ribosyltransferase turn-turn loop, and a "Gln-XXX-Glu" motif. CerADPr ADP-ribosylated an ~120 kDa protein in HeLa cell lysates and intact cells. EGFP-CerADPr rounded HeLa cells, elicited cytoskeletal changes, and yielded a cytotoxic phenotype, indicating that CerADPr disrupts cytoskeletal signaling. CerADPr(E431D) did not possess ADP-ribosyltransferase or NAD glycohydrolase activities and did not elicit a phenotype in HeLa cells, implicating Glu431 as a catalytic residue. These experiments identify CerADPr as a cytotoxic ADP-ribosyltransferase that disrupts the host cytoskeleton.

Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.

BACKGROUND: How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear. RESULTS: BoNT/C utilizes dual gangliosides as host cell receptors. CONCLUSION: BoNT/C accesses gangliosides on the plasma membrane. SIGNIFICANCE: Plasma membrane accessibility of the dual ganglioside receptors suggests synaptic vesicle exocytosis may not be necessary to expose BoNT/C receptors. Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A-G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.

Enhancing the protective immune response against botulism.

The need for a vaccine against botulism has increased since the discontinuation of the pentavalent (ABCDE) botulinum toxoid vaccine by the Centers for Disease Control and Prevention. The botulinum toxins (BoNTs) are the primary virulence factors and vaccine components against botulism. BoNTs comprise three domains which are involved in catalysis (LC), translocation (HCT), and host receptor binding (HCR). Recombinant HCR subunits have been used to develop the next generation of BoNT vaccines. Using structural studies and the known entry properties of BoNT/A, an HCR subunit vaccine against BoNT/A that contained the point mutation W1266A within the ganglioside binding pocket was designed. HCR/A(W1266A) did not enter primary neurons, and the crystal structure of HCR/A(W1266A) was virtually identical to that of wild-type HCR/A. Using a mouse model, experiments were performed using a high-dose vaccine and a low-dose vaccine. At a high vaccine dose, HCR/A and HCR/A(W1266A) elicited a protective immune response to BoNT/A challenge. At the low-dose vaccination, HCR/A(W1266A) was a more protective vaccine than HCR/A. α-HCR IgG titers correlated with protection from BoNT challenge, although titers to block HCR/A entry were greater in serum in HCR/A-vaccinated mice than in HCR/A(W1266A)-vaccinated mice. This study shows that removal of receptor binding capacity enhances potency of the subunit HCR vaccine. Vaccines that lack receptor binding capacity have the added property of limited off-target toxicity.

Testing a Recombinant Form of Tetanus Toxoid as a Carrier Protein for Glycoconjugate Vaccines.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

Insights into the different catalytic activities of Clostridium neurotoxins.

The clostridial neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of botulinum neurotoxins (A-G) and tetanus toxin showed different toxicities and cleave their substrates with different efficiencies. However, the molecular basis of their different catalytic activities with respect to their substrates is not clear. BoNT/B light chain (LC/B) and TeNT light chain (LC/T) cleave vesicle-associated membrane protein 2 (VAMP2) at the same scissile bond but possess different catalytic activities and substrate requirements, which make them the best candidates for studying the mechanisms of their different catalytic activities. The recognition of five major P sites of VAMP2 (P7, P6, P1, P1', and P2') and fine alignment of sites P2 and P3 and sites P2 and P4 by LC/B and LC/T, respectively, contributed to their substrate recognition and catalysis. Significantly, we found that the S1 pocket mutation LC/T(K(168)E) increased the rate of native VAMP2 cleavage so that it approached the rate of LC/B, which explains the molecular basis for the lower k(cat) that LC/T possesses for VAMP2 cleavage relative to that of LC/B. This analysis explains the molecular basis underlying the VAMP2 recognition and cleavage by LC/B and LC/T and provides insight that may extend the pharmacologic utility of these neurological reagents.