Gregg Brieana M, Gupta Sonal, Tepp William H, Pellett Sabine
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 27157, USA.
Microorganisms. 2024 Dec 17;12(12):2611. doi: 10.3390/microorganisms12122611.
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 . 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 evolved without a similar gene cluster and why complex-free TeNT is secreted as a stable and soluble protein by , whereas complexing proteins appear to be essential for BoNT stability in culture supernatants of . To assess whether the stability of TeNT is due to an innate property of the toxin or is a result of 's intra- and extra-cellular environment, both TeNT and complex-free BoNT/A1 were expressed recombinantly in atoxic and analyzed for expression and stability. The strong clostridial ferredoxin () promotor resulted in the expression of recombinant TeNT at greater levels and earlier time points than endogenously produced TeNT. Recombinant BoNT/A1 was similarly expressed by atoxic , although partial degradation was observed. The rBoNT/A1 produced in 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/A1 in spent media of or was affected by growth media but not by pH. These data indicate that the distinct metabolism of or under various growth conditions is a primary factor in creating a more or less favorable environment for complex-free CNT stability.
破伤风神经毒素（TeNT）和肉毒杆菌神经毒素（BoNTs）是密切相关的约150 kDa蛋白质毒素，它们共同构成了由各种梭菌属物种表达的梭菌神经毒素（CNTs）组。虽然TeNT以单一多肽形式表达，但BoNTs总是与多种无毒蛋白质一起产生，这些无毒蛋白质与BoNT形成稳定复合物，并在一个保守的毒素基因簇中编码。目前尚不清楚 是如何在没有类似基因簇的情况下进化的，以及为什么无复合物的TeNT会被 分泌为稳定且可溶的蛋白质，而复合物形成蛋白似乎对BoNT在 的培养上清液中的稳定性至关重要。为了评估TeNT的稳定性是由于毒素的固有特性还是 的细胞内和细胞外环境的结果，TeNT和无复合物的BoNT/A1都在无毒的 中进行重组表达，并分析其表达和稳定性。强梭菌铁氧化还原蛋白（ ）启动子导致重组TeNT的表达水平高于内源性产生的TeNT，且表达时间更早。无毒的 同样表达重组BoNT/A1，尽管观察到部分降解。在 中产生的rBoNT/A1也部分被蛋白水解加工成双链形式。对细菌生长培养基和pH条件的研究发现，rTeNT和rBoNT/A1在 或 的用过培养基中的稳定性受生长培养基影响，但不受pH影响。这些数据表明， 在各种生长条件下的独特代谢是为无复合物的CNT稳定性创造或多或少有利环境的主要因素。
