Schmidt H, Maier E, Karch H, Benz R
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Germany.
Eur J Biochem. 1996 Oct 15;241(2):594-601. doi: 10.1111/j.1432-1033.1996.00594.x.
Lipid bilayer experiments were performed with the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933. EHEC-hemolysin caused the formation of transient ion-permeable channels by integration in lipid bilayer membranes composed of asolectin, dioleoylglycerophosphoethanolamine and phosphoserine but not of diphytanoylglycerophosphocholine. Channel formation showed the same characteristics when culture supernatants of E. coli strains EDL 933 or HB101/pEO40, precipitated or purified EHEC-hemolysin were used for these experiments. The EHEC-hemolysin channels had two different states at small transmembrane potential (20 mV): a prestate that represented the first step of channel formation (single-channel conductance 40 pS in 0.15 M KCl) and an open state (550 pS in 0.15 M KCl at pH 6.0). Experiments with different salts suggested that the EHEC-hemolysin-induced channels were cation-selective at neutral pH. The mobility sequence of the cations within the channels resembles their mobility sequence in the aqueous phase. The single-channel data were consistent with the formation of wide, water-filled channels by the EHEC hemolysin. The single channel conductance was strongly pH dependent and increased over 2.5-fold in the pH range 5-8. The analysis of the single-channel data using the Renkin correction factor suggested that the EHEC-hemolysin formed channels with an average diameter of 2.6 nm. This size could be confirmed by the results of osmotic-protection experiments. Neither sucrose nor raffinose inhibited toxin-dependent hemolysis, whereas hemolysis did not occur in the presence of dextran 4 (molecular mass, 4 kDa). Our results demonstrate that EHEC-hemolysin can be considered to be a highly active repeats-in-toxin (RTX)-toxin with a similar but not identical pore-forming capacity as the chromosomal encoded E. coli alpha-hemolysin.
利用肠出血性大肠杆菌（EHEC）O157:H7菌株EDL933的质粒编码溶血素进行了脂质双层实验。EHEC溶血素通过整合到由大豆卵磷脂、二油酰甘油磷酸乙醇胺和磷酸丝氨酸组成而非二植烷酰甘油磷酸胆碱组成的脂质双层膜中，导致形成瞬时离子通透通道。当使用大肠杆菌菌株EDL 933或HB101/pEO40的培养上清液、沉淀或纯化的EHEC溶血素进行这些实验时，通道形成表现出相同的特征。EHEC溶血素通道在小跨膜电位（20 mV）时有两种不同状态：一种预状态代表通道形成的第一步（在0.15 M KCl中，单通道电导为40 pS）和一种开放状态（在pH 6.0的0.15 M KCl中为550 pS）。用不同盐进行的实验表明，EHEC溶血素诱导的通道在中性pH下对阳离子具有选择性。通道内阳离子的迁移顺序与其在水相中的迁移顺序相似。单通道数据与EHEC溶血素形成宽的、充满水的通道一致。单通道电导强烈依赖于pH，在pH 5 - 8范围内增加超过2.5倍。使用伦金校正因子对单通道数据进行分析表明，EHEC溶血素形成的通道平均直径为2.6 nm。这个大小可以通过渗透保护实验的结果得到证实。蔗糖和棉子糖均不抑制毒素依赖性溶血，而在存在葡聚糖4（分子量4 kDa）的情况下不发生溶血。我们的结果表明，EHEC溶血素可被认为是一种高活性的毒素重复序列（RTX）毒素，其成孔能力与染色体编码的大肠杆菌α溶血素相似但不完全相同。
