Study on the virulence factors of enterohemorrhagic Escherichia coli

由肠出血性大肠杆菌（EHEC）产生的滋贺毒素（Stx）包括志贺毒素1（Stx 1）以及志贺毒素2（Stx 2）。Stx1与细胞相关，而Stx2定位于培养上清液。我们通过产生组氨酸标记的StxB（StxBH）分析了Stx2的分泌。尽管Stx1BH和Stx2BH在StxBH过表达的EHEC中均不分泌，但Stx2BH过表达的EHEC显示Stx2分泌受到抑制。另一方面，Stx1BH过表达的EHEC没有显示Stx2分泌的改变。Stx 1和Stx 2的B亚基嵌合体用于鉴定Stx 2分泌系统识别的Stx 2 B的特异性残基。将Stx2BH中的丝氨酸31残基改变为天冬酰胺残基（S31N）使得能够恢复Stx2分泌。另一方面，在Stx1BH中天冬酰胺32残基改变为丝氨酸残基（N32 S）引起EHEC中点突变的组氨酸标记的B亚基的部分分泌。基于这些证据，似乎该残留物可能包含Stx2分泌的分泌相关信息。为了验证这一假设，我们构建了产Stx 1（B亚基，N32 S）和Stx 2（B亚基，S31 N）EHEC的同基因突变株。虽然在同基因突变型EHEC中突变Stx2是细胞相关的，但突变Stx1不是胞外的。然而，当我们使用质粒表达突变体全毒素时，在突变体全毒素基因转化的EHEC的上清液部分中发现过表达的突变体Stx 1，在细胞相关部分中发现过表达的突变体Stx 2。这些结果表明，Stx 2的B亚基的丝氨酸31位残基包含分泌相关信息。

Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) include shiga toxin 1 (Stx1) as well as shiga toxin 2 (Stx2). Stx1 is cell-associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxBH). Although neither Stx1BH nor Stx2BH was secreted in StxBH-overexpressed EHEC, Stx2BH-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, Stx1BH-overexpressed EHEC showed no alteration of Stx2 secretion. B subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of Stx2B that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in Stx2BH enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in Stx1BH caused partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed isogenic mutant strains, Stx1 (B subunit, N32S)-producing EHEC and Stx2 (B subunit, S31N)-producing EHEC. Although the mutant Stx2 was cell-associated in isogenic mutant EHEC, the mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotexins, the overexpressed mutant Stx1 was found in the supernatant fraction and the overexpressed mutant Stx2 was found in the cell-associated fraction in the mutant holotexin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.