Expression of Recombinant Clostridial Neurotoxin by C. tetani.

IF 4.1 2区 生物学 Q2 MICROBIOLOGY
Brieana M Gregg, Sonal Gupta, William H Tepp, Sabine Pellett
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引用次数: 0

Abstract

Tetanus neurotoxins (TeNT) and botulinum neurotoxins (BoNTs) are closely related ~150 kDa protein toxins that together comprise the group of clostridial neurotoxins (CNTs) expressed by various species of Clostridia. While TeNT is expressed as a single polypeptide, BoNTs are always produced alongside multiple non-toxic proteins that form a stabilizing complex with BoNT and are encoded in a conserved toxin gene cluster. It is unknown how tent evolved without a similar gene cluster and why complex-free TeNT is secreted as a stable and soluble protein by C. tetani, whereas complexing proteins appear to be essential for BoNT stability in culture supernatants of C. botulinum. To assess whether the stability of TeNT is due to an innate property of the toxin or is a result of C. tetani's intra- and extra-cellular environment, both TeNT and complex-free BoNT/A1ERY were expressed recombinantly in atoxic C. tetani and analyzed for expression and stability. The strong clostridial ferredoxin (fdx) promotor resulted in the expression of recombinant TeNT at greater levels and earlier time points than endogenously produced TeNT. Recombinant BoNT/A1ERY was similarly expressed by atoxic C. tetani, although partial degradation was observed. The rBoNT/A1ERY produced in C. tetani was also partially proteolytically processed to the dichain form. Investigations of bacterial growth media and pH conditions found that the stability of rTeNT and rBoNT/A1ERY in spent media of C. tetani or C. botulinum was affected by growth media but not by pH. These data indicate that the distinct metabolism of C. tetani or C. botulinum under various growth conditions is a primary factor in creating a more or less favorable environment for complex-free CNT stability.

重组梭菌神经毒素在破伤风梭菌中的表达。
破伤风神经毒素(TeNT)和肉毒杆菌神经毒素(BoNTs)是密切相关的~150 kDa蛋白毒素,它们共同构成梭状芽胞杆菌表达的梭状芽胞杆菌神经毒素(CNTs)组。虽然TeNT以单一多肽形式表达,但BoNT总是与多种无毒蛋白一起产生,这些蛋白与BoNT形成稳定复合物,并在保守的毒素基因簇中编码。目前尚不清楚在没有类似基因簇的情况下,tent是如何进化的,以及为什么无络合物的tent作为稳定的可溶性蛋白被破伤风杆菌分泌,而在肉毒杆菌培养上清中,络合蛋白似乎对BoNT的稳定性至关重要。为了评估TeNT的稳定性是由于毒素的先天特性还是破伤风杆菌细胞内和细胞外环境的结果,在毒性破伤风杆菌中重组表达了TeNT和无复合物的BoNT/A1ERY,并分析了其表达和稳定性。强梭菌铁氧还蛋白(fdx)启动子导致重组TeNT在更高水平和更早的时间点上表达。重组BoNT/A1ERY在毒性破伤风杆菌中同样表达,尽管观察到部分降解。在破伤风杆菌中产生的rBoNT/A1ERY也被部分蛋白水解加工成双链形式。对细菌生长介质和pH条件的研究发现,在C.破伤风杆菌或C.肉毒杆菌的废培养基中,rTeNT和rBoNT/A1ERY的稳定性受到生长介质的影响,而不受pH的影响。这些数据表明,C.破伤风杆菌或C.肉毒杆菌在不同生长条件下的不同代谢是创造或多或少有利的无络合物碳纳米管稳定性环境的主要因素。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Microorganisms
Microorganisms Medicine-Microbiology (medical)
CiteScore
7.40
自引率
6.70%
发文量
2168
审稿时长
20.03 days
期刊介绍: Microorganisms (ISSN 2076-2607) is an international, peer-reviewed open access journal which provides an advanced forum for studies related to prokaryotic and eukaryotic microorganisms, viruses and prions. It publishes reviews, research papers and communications. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. The full experimental details must be provided so that the results can be reproduced. Electronic files and software regarding the full details of the calculation or experimental procedure, if unable to be published in a normal way, can be deposited as supplementary electronic material.
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