Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157:H7
Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157:H7
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DOI：
10.1111/j.1432-1033.1996.00594.x
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发表时间：
1996-10-15
期刊：
影响因子：
--
通讯作者：
Benz, R
中科院分区：
文献类型：
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作者：
Schmidt, H;Maier, E;Benz, R
以肠出血性大肠杆菌(EHEC)O157：H7菌株EDL933编码的溶血素为底物，进行脂质双层实验。EHEC-溶血素通过整合到由asolectin、二油酰甘油磷乙醇胺和磷酸丝氨酸组成的脂质双层膜中而不是二植酰甘油磷胆碱，导致瞬时离子通透通道的形成。用EDL-933或HB101/pEO40菌株的培养上清液、沉淀或纯化的EHEC-溶血素进行通道形成的实验也显示出相同的特征。EHEC-溶血素通道有两种不同的状态，即小跨膜电位(20 MV)：预态TAI代表第一步或通道形成(单通道电导在0.15M KCl中为40ps)和开放状态(在0.15MKCl中为550pS，pH为6.0)。不同盐类的实验表明，EHEC-溶血素诱导的通道在中性pH下具有阳离子选择性。通道内阳离子的迁移率序列类似于它们在水相中的迁移率序列。单通道数据与EHEC溶血素形成的宽的、充满水的通道一致。单通道电导强烈地依赖于pH，在pH为5-8的范围内增加了2.5倍以上。用Renkin校正因子对单通道数据的分析表明，EHEC-溶血素形成了平均直径为2.6 nm的通道。这一大小可以通过渗透保护实验的结果来证实。蔗糖和棉子糖都不能抑制毒素依赖的溶血，而葡聚糖4(分子质量，4 kDa)不能抑制溶血。我们的结果表明，EHEC-溶血素可以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-溶血素相似但不相同。
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 al small transmembrane potential (20 mV): a prestate thai represented the first step or 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.