Sanchez J, Solorzano R M, Holmgren J
Centro de Investigación sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, Mexico.
FEBS Lett. 1993 Sep 20;330(3):265-9. doi: 10.1016/0014-5793(93)80885-x.
The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1-19) and Pro (aa 20-53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.
大肠杆菌成熟的由19个氨基酸组成的STa热稳定肠毒素之前有一段由53个氨基酸组成的肽段，该肽段包含两个结构域，分别称为Pre（第1 - 19位氨基酸）和Pro（第20 - 53位氨基酸）序列，据推测这两个结构域对于该宿主细胞外毒素的释放至关重要。然而，Pro序列已被证明对于此过程并非不可或缺，因为Pro缺失突变体能够分泌STa。为了弄清楚Pre和/或其他未去除的天然STa侧翼序列是否对这些突变体中的毒素分泌负责，我们将成熟的STa直接与大肠杆菌周质热不稳定肠毒素B亚基（LTB）的前导肽进行基因融合。该基因融合体的表达导致大肠杆菌能够独立于天然STa相邻基因序列分泌具有生物活性的细胞外STa。此外，这些结果表明，一旦被LTB前导肽引导进入大肠杆菌周质，STa可能只需进入周质就能进入细胞外环境。为了测试这种细胞外分泌方式是否可以扩展到其他富含二硫键的小肽，我们克隆了由13个氨基酸组成的芋螺毒素GI和一个与STa相关的非肠毒素十肽。尽管这两种肽与毒素具有高度的结构同源性，但在培养上清液中均未检测到。未能分泌很可能导致降解，因为在细菌超声裂解物中也未检测到这些肽。我们推测，富含半胱氨酸的肽必须具有与STa更接近的氨基酸长度和/或二硫键数量，才能在大肠杆菌中遵循这种毒素分泌途径。
