ABSTRACT
Botulinum neurotoxins (BoNTs) are the most deadly biological substances, including seven BoNT serotypes (A-G), and characterized by persistent flaccid paralysis of peripheral never terminals with high specificity called botulism. Due to their easy production and well-defined biological mechanism, BoNTs are wildly used in cosmetics and as very particular biopharmaceuticals in clinical therapy, so there is the risk of poisoning caused by accidental overdose. Also, because of their high toxicity, they are potential bioterrorism weapons. Thus, there is an urgent need for the development of BoNT inhibitors. In this review, based on the structure of BoNTs and the mechanism of botulism, we summarize recent advances in small molecule inhibitors targeting the Zn2+ active site of BoNT/A, such as 8-hydroxyquinoline and hydroxamic acid, or exosite of BoNT/A, small molecule inhibitors of BoNT/A through covalent binding that are irreversible, as well as small molecule inhibitors of targeting BoNT/B/ E light chain (LC).
ABSTRACT
Background & objectives: Botulism, a potentially fatal paralytic illness, is caused by the botulinum neurotoxins (BoNTs) secreted by Clostridium botulinum. It is an obligate anaerobic, Gram-positive, spore-forming bacterium. BoNTs are classified into seven serotypes based on the serological properties. Among these seven serotypes, A, B, E and, rarely, F are responsible for human botulism. The present study was undertaken to develop an enzyme-linked immunosorbent assay (ELISA)-based detection system for the detection of BoNT/E. Methods: The synthetic gene coding the light chain of BoNT serotype E (BoNT/E LC) was constructed using the polymerase chain reaction primer overlapping method, cloned into pQE30UA vector and then transformed into Escherichia coli M15 host cells. Recombinant protein expression was optimized using different concentrations of isopropyl-?-D-1-thiogalactopyranoside (IPTG), different temperature and the rBoNT/E LC protein was purified in native conditions using affinity column chromatography. The purified recombinant protein was checked by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and further confirmed by western blot and matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF). Polyclonal antibodies were generated against rBoNT/E LC using Freund's adjuvant in BALB/c mice and rabbit. Sandwich ELISA was optimized for the detection of rBoNT/E LC and native crude BoNT/E, and food matrix interference was tested. The developed antibodies were further evaluated for their specificity/cross-reactivity with BoNT serotypes and other bacterial toxins. Results: BoNT/E LC was successfully cloned, and the maximum expression was achieved in 16 h of post-induction using 0.5 mM IPTG concentration at 25癈. Polyclonal antibodies were generated in BALB/c mice and rabbit and the antibody titre was raised up to 128,000 after the 2nd booster dose. The developed polyclonal antibodies were highly specific and sensitive with a detection limit about 50 ng/ml for rBoNT/E LC and 2.5�[3] MLD50 of native crude BoNT/E at a dilution of 1:3000 of mouse (capturing) and rabbit (revealing) antibodies. Further, different liquid, semisolid and solid food matrices were tested, and rBoNT/E LC was detected in almost all food samples, but different levels of interference were detected in different food matrices. Interpretation & conclusions: There is no immune detection system available commercially in India to detect botulism. The developed system might be useful for the detection of botulinum toxin in food and clinical samples. Further work is in progress.
ABSTRACT
Objective To investigate the effect of recombinant botulinum neurotoxin serotype A heavy chain (BoNT/A heavy chain)on local proteins which are related to nerve growth after spinal cord injury in rats,and to get some experimental evidence to explain the mechanism of BoNT/A heavy chain in stimulating neuritogenesis. Methods Recombinant botulinum neurotoxin serotype A heavy chain was applied locally or intrathecally to rats with ipsilateral semi-dissociated lumbar spinal injury. Local spinal tissue was extracted for general protein expression by two dimension electrophoresis plus nitrate silver staining after different time period of injury. Based on the results of 2-D gel electrophoresis,growth-associated protein 43(GAP-43)and of superior cervical ganglion 10(SCG 10)were selected to examine the changes of their expression and distribution features under BoNT/A heavy chain administration using SDS-PAGE,western blot and immunofluorescence. Results (1)The model of spinal cord injury(SCI)in this study was an ipsilateral semi-dissociated lumbar SCI in rat. The rats showed obvious motor and sensory dysfunction in the ipsilateral hind limb.(2)The results from 2-D gel electrophoresis plus nitrate silver staining showed that the administration of BoNT/A heavy chain based on SCI altered the local protein expression pattern. The decrease or increase in the expression of some protein dots /dots group was clearly seen after single SCI. However, these changes were transformed by BoNT/A heavy chain treatment,which appeared as a reversed pattern turning toward that in control group or further increased expression upon SCI,such as the dots located respectively at 35-45 kDa and 18-25 kDa level,pI between 5-7. In addition,the expression of the two dots located at the level as above increased after SCI only, and showed further increase in their expression with BoNT/A heavy chain intervention.(3)The changes of selective GAP-43 and SCG 10 expression and distribution by western blot and immunofluorescence indicated that the administration of BONT/A heavy chain based on SCI amplified the expression of GAP-43 and SCG 10(P < 0.05). Meanwhile,the positive immuonfluorescent staining for both GAP-43 and SCG 10 mainly distributed nearby the proximal area of injury, both cytoplasm and neuronal processes were positively stained. Conclusions Intrathecal delivery of BoNT/A heavy chain increases the expression of growth-associated proteins GAP 43 and SCG 10 after SCI in rats.