Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin.

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24 h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures.

Facial neuromuscular junctions and brainstem nuclei are the target of tetanus neurotoxin in cephalic tetanus.

Cephalic tetanus (CT) is a severe form of tetanus that follows head wounds and the intoxication of cranial nerves by tetanus neurotoxin (TeNT). Hallmarks of CT are cerebral palsy, which anticipates the spastic paralysis of tetanus, and rapid evolution of cardiorespiratory deficit even without generalized tetanus. How TeNT causes this unexpected flaccid paralysis, and how the canonical spasticity then rapidly evolves into cardiorespiratory defects, remain unresolved aspects of CT pathophysiology. Using electrophysiology and immunohistochemistry, we demonstrate that TeNT cleaves its substrate vesicle-associated membrane protein within facial neuromuscular junctions and causes a botulism-like paralysis overshadowing tetanus spasticity. Meanwhile, TeNT spreads among brainstem neuronal nuclei and, as shown by an assay measuring the ventilation ability of CT mice, harms essential functions like respiration. A partial axotomy of the facial nerve revealed a potentially new ability of TeNT to undergo intra-brainstem diffusion, which allows the toxin to spread to brainstem nuclei devoid of direct peripheral efferents. This mechanism is likely to be involved in the transition from local to generalized tetanus. Overall, the present findings suggest that patients with idiopathic facial nerve palsy should be immediately considered for CT and treated with antisera to block the potential progression to a life-threatening form of tetanus.

Pathogenicity and virulence of Clostridium botulinum.

Clostridium botulinum, a polyphyletic Gram-positive taxon of bacteria, is classified purely by their ability to produce botulinum neurotoxin (BoNT). BoNT is the primary virulence factor and the causative agent of botulism. A potentially fatal disease, botulism is classically characterized by a symmetrical descending flaccid paralysis, which is left untreated can lead to respiratory failure and death. Botulism cases are classified into three main forms dependent on the nature of intoxication; foodborne, wound and infant. The BoNT, regarded as the most potent biological substance known, is a zinc metalloprotease that specifically cleaves SNARE proteins at neuromuscular junctions, preventing exocytosis of neurotransmitters, leading to muscle paralysis. The BoNT is now used to treat numerous medical conditions caused by overactive or spastic muscles and is extensively used in the cosmetic industry due to its high specificity and the exceedingly small doses needed to exert long-lasting pharmacological effects. Additionally, the ability to form endospores is critical to the pathogenicity of the bacteria. Disease transmission is often facilitated via the metabolically dormant spores that are highly resistant to environment stresses, allowing persistence in the environment in unfavourable conditions. Infant and wound botulism infections are initiated upon germination of the spores into neurotoxin producing vegetative cells, whereas foodborne botulism is attributed to ingestion of preformed BoNT. C. botulinum is a saprophytic bacterium, thought to have evolved its potent neurotoxin to establish a source of nutrients by killing its host.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets.

Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently labeled C3larvinA. Following binding to the macrophage membrane in a discontinuous punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 h. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for the escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodeling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N-terminal α1 -helix for lysosomal swelling and successful intoxication.

Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins.

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations > 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

Botulinum Neurotoxins beyond Neurons: Interplay with Glial Cells.

In recent years, numerous studies have highlighted the significant use of botulinum neurotoxins (BoNTs) in the human therapy of various motor and autonomic disorders. The therapeutic action is exerted with the selective cleavage of specific sites of the SNARE's protein complex, which plays a key role in the vesicular neuroexocytosis which is responsible for neural transmission. The primary target of the BoNTs' action is the peripheral neuromuscular junction (NMJ), where, by blocking cholinergic neurons releasing acetylcholine (ACh), they interfere with neural transmission. A great deal of experimental evidence has demonstrated that BoNTs are also effective in blocking the release of other neurotransmitters or neuromodulators, such as glutamate, substance-P, and CGRP, and they can interfere with the function of glial cells, both at the peripheral and central level. The purpose of this review is to provide an update on the available experimental data from animal models that suggest or confirm the direct interactions between BoNTs and glial cells. From the data collected, it appears evident that, through mechanisms that are not yet fully understood, BoNTs can block the activation of spinal glial cells and their subsequent release of pro-inflammatory factors. BoNTs are also able to promote peripheral regeneration processes after nerve injury by stimulating the proliferation of Schwann cells. The data will be discussed in consideration of the possible therapeutic implications of the use of BoNTs on those pathological conditions where the contribution of glial cell activation is fundamental, such as in peripheral and central neuropathies.

Specific Isolation of Clostridium botulinum Group I Cells by Phage Lysin Cell Wall Binding Domain with the Aid of S-Layer Disruption.

Clostridium botulinum is a notorious pathogen that raises health and food safety concerns by producing the potent botulinum neurotoxin and causing botulism, a potentially fatal neuroparalytic disease in humans and animals. Efficient methods for the identification and isolation of C. botulinum are warranted for laboratory diagnostics of botulism and for food safety risk assessment. The cell wall binding domains (CBD) of phage lysins are recognized by their high specificity and affinity to distinct types of bacteria, which makes them promising for the development of diagnostic tools. We previously identified CBO1751, which is the first antibotulinal phage lysin showing a lytic activity against C. botulinum Group I. In this work, we assessed the host specificity of the CBD of CBO1751 and tested its feasibility as a probe for the specific isolation of C. botulinum Group I strains. We show that the CBO1751 CBD specifically binds to C. botulinum Group I sensu lato (including C. sporogenes) strains. We also demonstrate that some C. botulinum Group I strains possess an S-layer, the disruption of which by an acid glycine treatment is required for efficient binding of the CBO1751 CBD to the cells of these strains. We further developed CBO1751 CBD-based methods using flow cytometry and magnetic separation to specifically isolate viable cells of C. botulinum Group I. These methods present potential for applications in diagnostics and risk assessment in order to control the botulism hazard.

Requirement of Peptidyl-Prolyl Cis/Trans isomerases and chaperones for cellular uptake of bacterial AB-type toxins.

Bacterial AB-type toxins are proteins released by the producing bacteria and are the causative agents for several severe diseases including cholera, whooping cough, diphtheria or enteric diseases. Their unique AB-type structure enables their uptake into mammalian cells via sophisticated mechanisms exploiting cellular uptake and transport pathways. The binding/translocation B-subunit facilitates binding of the toxin to a specific receptor on the cell surface. This is followed by receptor-mediated endocytosis. Then the enzymatically active A-subunit either escapes from endosomes in a pH-dependent manner or the toxin is further transported through the Golgi to the endoplasmic reticulum from where the A-subunit translocates into the cytosol. In the cytosol, the A-subunits enzymatically modify a specific substrate which leads to cellular reactions resulting in clinical symptoms that can be life-threatening. Both intracellular uptake routes require the A-subunit to unfold to either fit through a pore formed by the B-subunit into the endosomal membrane or to be recognized by the ER-associated degradation pathway. This led to the hypothesis that folding helper enzymes such as chaperones and peptidyl-prolyl cis/trans isomerases are required to assist the translocation of the A-subunit into the cytosol and/or facilitate their refolding into an enzymatically active conformation. This review article gives an overview about the role of heat shock proteins Hsp90 and Hsp70 as well as of peptidyl-prolyl cis/trans isomerases of the cyclophilin and FK506 binding protein families during uptake of bacterial AB-type toxins with a focus on clostridial binary toxins Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridioides difficile CDT toxin, as well as diphtheria toxin, pertussis toxin and cholera toxin.

Botulinum neurotoxin accurately separates tonic vs. phasic transmission and reveals heterosynaptic plasticity rules in Drosophila.

In developing and mature nervous systems, diverse neuronal subtypes innervate common targets to establish, maintain, and modify neural circuit function. A major challenge towards understanding the structural and functional architecture of neural circuits is to separate these inputs and determine their intrinsic and heterosynaptic relationships. The Drosophila larval neuromuscular junction is a powerful model system to study these questions, where two glutamatergic motor neurons, the strong phasic-like Is and weak tonic-like Ib, co-innervate individual muscle targets to coordinate locomotor behavior. However, complete neurotransmission from each input has never been electrophysiologically separated. We have employed a botulinum neurotoxin, BoNT-C, that eliminates both spontaneous and evoked neurotransmission without perturbing synaptic growth or structure, enabling the first approach that accurately isolates input-specific neurotransmission. Selective expression of BoNT-C in Is or Ib motor neurons disambiguates the functional properties of each input. Importantly, the blended values of Is+Ib neurotransmission can be fully recapitulated by isolated physiology from each input. Finally, selective silencing by BoNT-C does not induce heterosynaptic structural or functional plasticity at the convergent input. Thus, BoNT-C establishes the first approach to accurately separate neurotransmission between tonic vs. phasic neurons and defines heterosynaptic plasticity rules in a powerful model glutamatergic circuit.

Regulatory Networks Controlling Neurotoxin Synthesis in Clostridium botulinum and Clostridium tetani.

Clostridium botulinum and Clostridium tetani are Gram-positive, spore-forming, and anaerobic bacteria that produce the most potent neurotoxins, botulinum toxin (BoNT) and tetanus toxin (TeNT), responsible for flaccid and spastic paralysis, respectively. The main habitat of these toxigenic bacteria is the environment (soil, sediments, cadavers, decayed plants, intestinal content of healthy carrier animals). C. botulinum can grow and produce BoNT in food, leading to food-borne botulism, and in some circumstances, C. botulinum can colonize the intestinal tract and induce infant botulism or adult intestinal toxemia botulism. More rarely, C. botulinum colonizes wounds, whereas tetanus is always a result of wound contamination by C. tetani. The synthesis of neurotoxins is strictly regulated by complex regulatory networks. The highest levels of neurotoxins are produced at the end of the exponential growth and in the early stationary growth phase. Both microorganisms, except C. botulinum E, share an alternative sigma factor, BotR and TetR, respectively, the genes of which are located upstream of the neurotoxin genes. These factors are essential for neurotoxin gene expression. C. botulinum and C. tetani share also a two-component system (TCS) that negatively regulates neurotoxin synthesis, but each microorganism uses additional distinct sets of TCSs. Neurotoxin synthesis is interlocked with the general metabolism, and CodY, a master regulator of metabolism in Gram-positive bacteria, is involved in both clostridial species. The environmental and nutritional factors controlling neurotoxin synthesis are still poorly understood. The transition from amino acid to peptide metabolism seems to be an important factor. Moreover, a small non-coding RNA in C. tetani, and quorum-sensing systems in C. botulinum and possibly in C. tetani, also control toxin synthesis. However, both species use also distinct regulatory pathways; this reflects the adaptation of C. botulinum and C. tetani to different ecological niches.

Survey of immunopharmacological effects of botulinum toxin in cell signaling of bronchial smooth muscle cells in allergic asthma.

BACKGROUND: Asthma is a lung disease that has influenced more than 350 million people worldwide. Airway smooth muscle (ASM) spasm leads to airway hyperresponsiveness (AHR) and bronchial obstruction, which are clinical manifestations of an asthma attack. Botulinum toxin (BTX) is a bacteria toxin that acts as muscle relaxant and may have therapeutic effects on AHR and asthma. OBJECTIVE: In this study, the effect of BTX on AHR and related gene expressions was evaluated. MATERIAL AND METHODS: An asthma mice model was developed which was treated with BTX in two ways: intranasally (IN) and via nebulization (N) (0.01, 0.1, and 1 U/mL and 10 U/mL, respectively) on days 25, 27 and 29. AHR was evaluated on days 24, 26, 28, and 30, and gene expressions were evaluated for TrkA, TrkB, M1-M5, α7nAChR, TNF-α, and extracellular signal-regulated kinase 2 (ERK2) proteins. For histopathology of the lungs, perivascular and peribronchial inflammation, production of mucus, and goblet cell hyperplasia were studied. RESULTS: On day 24, treatment with BTX (for all doses) had no significant effect on AHR, but on days 26 and 28, AHR was decreased and this continued up to day 30 for all treated groups. Treatment with BTX had no significant effect on the gene expressions of TrkA, TrkB, M1-M5, α7nAChR, TNF-α, and ERK2 proteins, perivascular inflammation, peribronchial inflammation, hyperplasia of the goblet cell and production of mucus. Besides, mice administered with 10 mg/mL BTX perished. The BTX therapy controlled asthma attacks by decreasing AHR and relaxation of ASMs. CONCLUSION: However, BTX had no significant effect on airway inflammation and production of mucus. While using BTX, it is necessary to prescribe safe doses in order to prevent adverse reactions.

Combined Immunodeficiency Caused by a Novel De Novo Gain-of-Function RAC2 Mutation.

Ras-related C3 botulinum toxin substrate 2 (RAC2) is a GTPase exclusively expressed in hematopoietic cells that acts as a pivotal regulator of several aspects of cell behavior via various cellular processes. RAC2 undergoes a tightly regulated GTP-binding/GTP-hydrolysis cycle, enabling it to function as a molecular switch. Mutations in RAC2 have been identified in 18 patients with different forms of primary immunodeficiency, ranging from phagocyte defects caused by dominant negative mutations to common variable immunodeficiency resulting from autosomal recessive loss-of-function mutations, or severe combined immunodeficiency due to dominant activating gain-of-function mutations. Here, we describe an 11-year-old girl with combined immunodeficiency presenting with recurrent respiratory infections and bronchiectasis. Immunological investigations revealed low T-cell receptor excision circle/K-deleting recombination excision circles numbers, lymphopenia, and low serum immunoglobulin G. Targeted next-generation sequencing identified a novel heterozygous mutation in RAC2, c.86C > G (p.P29R), located in the highly conserved Switch I domain. The mutation resulted in enhanced reactive oxygen species production, elevated F-actin content, and increased RAC2 protein expression in neutrophils, as well as increased cytokine production and a dysregulated phenotype in T lymphocytes. Furthermore, the dominant activating RAC2 mutation led to accelerated apoptosis with augmented intracellular active caspase 3, impaired actin polarization in lymphocytes and neutrophils, and diminished RAC2 polarization in neutrophils. We present a novel RAC2 gain-of-function mutation with implications for immunodeficiency and linked to functional dysregulation, including abnormal apoptosis and cell polarization arising from altered RAC2 expression. Thus, our findings broaden the spectrum of known RAC2 mutations and their underlying mechanisms.

Advanced Glycation End Products (AGEs) Inhibit Macrophage Efferocytosis of Apoptotic ß Cells through Binding to the Receptor for AGEs.

Pancreatic ß cell apoptosis is important in the pathogenesis of type 2 diabetes mellitus (T2DM). Generally, apoptotic ß cells are phagocytosed by macrophages in a process known as "efferocytosis." Efferocytosis is critical to the resolution of inflammation and is impaired in T2DM. Advanced glycation end products (AGEs), which are increased in T2DM, are known to suppress phagocytosis function in macrophages. In this study, we found that AGEs inhibited efferocytosis of apoptotic ß cells by primary peritoneal macrophages in C57BL/6J mice or mouse macrophage cell line Raw264.7. Mechanistically, AGEs inhibit efferocytosis by blocking Ras-related C3 botulinum toxin substrate 1 activity and cytoskeletal rearrangement through receptor for advanced glycation end products/ras homolog family member A/Rho kinase signaling in macrophages. Furthermore, it was observed that AGEs decreased the secretion of anti-inflammatory factors and promoted the proinflammatory ones to modulate the inflammation function of efferocytosis. Taken together, our results indicate that AGEs inhibit efferocytosis through binding to receptor for advanced glycation end products and activating ras homolog family member A/Rho kinase signaling, thereby inhibiting the anti-inflammatory function of efferocytosis.

Multi-dimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry for detection of Clostridium botulinum type C and the produced botulinum neurotoxin type C complex.

Botulinum neurotoxin types C, D and their mosaic forms C/D and D/C produced mainly by Clostridium botulinum types C and D cause botulism in animals and belong to the most toxic substances for poultry and fish. In addition to intoxications, also toxoinfections with C. botulinum types C and D play a role that should not be underestimated, especially in veterinary medicine. Contrary to other botulinum neurotoxin complexes (BT x), the biosynthesis of these types is phage-encoded. Currently, the gold standard for neurotoxin detection in cases of clinical botulism is the mouse bioassay. In the last few years, alternatives for replacing this mouse bioassay have become increasingly interesting for the detection and characterisation of botulinum neurotoxins. Therefore, immunological techniques based mainly on antibodies, PCR or mass spectral methods have been developed. In this context, the most promising development is that of different endopeptidase assays. In our study, we were able to show that the 2D-nano-LC-MS/MS method presented by Klaubert et al. 2009 especially for detecting BT x A, B, E and F in complex culture media can also be used for detecting BT x C. The focus was therefore on transferring this method to detecting BT x C and pointing out necessary modifications of this current method. For method development, we used different culture preparations and sample conditions. To find out whether BT x C is just as stable against acetic peptic pretreatment as other BT x, we used sample preparations with and without peptic pretreatment. The decisive difference to previous publications is the detection of produced BT x C directly from culture supernatant of different strains of C. botulinum type C. In addition, we present a new approach of detecting protein fragments from C3 and C2 toxin and some specific host cell proteins of the bacterium Clostridium spp. in order to specify the carrier bacterium, therefore verifying the presence of an intact neurotoxin-encoding phage also without directly detecting BT x C and thus the possibility to produce neurotoxin. Herein, we describe a new method to examine environmental samples or suspected feed samples in cases of toxoinfections as well as finding out the causes of clinical botulism. This new approach is particularly interesting for veterinary medicine, especially for diseases like chronic botulism in cows or equine grass sickness.

Knockin mouse models demonstrate differential contributions of synaptotagmin-1 and -2 as receptors for botulinum neurotoxins.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are also utilized to treat a wide range of disorders including muscle spasm, overactive bladder, and pain. BoNTs' ability to target neurons determines their specificity, potency, and therapeutic efficacy. Homologous synaptic vesicle membrane proteins synaptotagmin-1 (Syt1) and synaptotagmin-2 (Syt2) have been identified as receptors for BoNT family members including BoNT/B, DC, and G, but their contributions at physiologically relevant toxin concentrations in vivo have yet to be validated and established. Here we generated two knockin mutant mouse models containing three designed point-mutations that specifically disrupt BoNT binding in endogenous Syt1 or Syt2, respectively. Utilizing digit abduction score assay by injecting toxins into the leg muscle, we found that Syt1 mutant mice showed similar sensitivity as the wild type mice, whereas Syt2 mutant mice showed reduced sensitivity to BoNT/B, DC, and G, demonstrating that Syt2 is the dominant receptor at skeletal neuromuscular junctions. We further developed an in vivo bladder injection assay for analyzing BoNT action on bladder tissues and demonstrated that Syt1 is the dominant toxin receptor in autonomic nerves controlling bladder tissues. These findings establish the critical role of protein receptors for the potency and specificity of BoNTs in vivo and demonstrate the differential contributions of Syt1 and Syt2 in two sets of clinically relevant target tissues.

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E.

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228-1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides.

Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes.

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.

Lentiviral vector-mediated expression of C3 transferase attenuates retinal ischemia and reperfusion injury in rats.

AIMS: Our previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury. MAIN METHODS: The LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay. KEY FINDINGS: No obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R. SIGNIFICANCE: This study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies.

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

Engineering Botulinum Neurotoxins for Enhanced Therapeutic Applications and Vaccine Development.

Botulinum neurotoxins (BoNTs) show increasing therapeutic applications ranging from treatment of locally paralyzed muscles to cosmetic benefits. At first, in the 1970s, BoNT was used for the treatment of strabismus, however, nowadays, BoNT has multiple medical applications including the treatment of muscle hyperactivity such as strabismus, dystonia, movement disorders, hemifacial spasm, essential tremor, tics, cervical dystonia, cerebral palsy, as well as secretory disorders (hyperhidrosis, sialorrhea) and pain syndromes such as chronic migraine. This review summarizes current knowledge related to engineering of botulinum toxins, with particular emphasis on their potential therapeutic applications for pain management and for retargeting to non-neuronal tissues. Advances in molecular biology have resulted in generating modified BoNTs with the potential to act in a variety of disorders, however, in addition to the modifications of well characterized toxinotypes, the diversity of the wild type BoNT toxinotypes or subtypes, provides the basis for innovative BoNT-based therapeutics and research tools. This expanding BoNT superfamily forms the foundation for new toxins candidates in a wider range of therapeutic options.