Louie M, de Azavedo J C, Handelsman M Y, Clark C G, Ally B, Dytoc M, Sherman P, Brunton J
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Infect Immun. 1993 Oct;61(10):4085-92. doi: 10.1128/iai.61.10.4085-4092.1993.
In enteropathogenic Escherichia coli, the eaeA gene produces a 94-kDa outer membrane protein called intimin which has been shown to be necessary but not sufficient to produce the attaching-and-effacing lesion. The purpose of this study was to characterize the intimin specified by the eaeA allele of the enterohemorrhagic E. coli (EHEC) serotype O157:H7 strain CL8 and to determine its role in adherence. The carboxyl-terminal 266 amino acids of the CL8 intimin were expressed as a protein fusion with glutathione S-transferase, which was used to raise antiserum in rabbits. The antiserum reacted in Western immunoblots with a 97-kDa outer membrane protein of EHEC strains of serogroups O5, O26, O111, and O157 and enteropathogenic E. coli strains of serogroups O55 and O127. Surface labelling of CL8 with 125I showed that intimin was surface exposed. An eaeA insertional inactivation mutant of CL8 was produced and was designated CL8-KO1. Total adherence of CL8-KO1 to HEp-2 cells was not significantly different from that of CL8, but CL8-KO1 gave a negative result in the fluorescent actin staining test. The eaeA gene expressed alone in E. coli HB101 also gave a negative fluorescent actin staining test result. The eaeA gene of CL8 was able to complement the eaeA deletion mutation in CVD206. We conclude that the product of the EHEC eaeA gene is a 97-kDa surface-exposed protein and propose that it be designated intiminO157. Sherman et al. described a 94-kDa outer membrane protein which played an important role in adherence of E. coli O157:H7 (Infect. Immun. 59:890-899, 1991). Western immunoblotting and indirect fluorescent antibody studies showed that the protein described by Sherman et al. is not intimin.
在肠致病性大肠杆菌中，eaeA基因产生一种94 kDa的外膜蛋白，称为紧密素，已证明它是产生紧密黏附损伤所必需的，但并不充分。本研究的目的是鉴定肠出血性大肠杆菌（EHEC）血清型O157:H7菌株CL8的eaeA等位基因所指定的紧密素，并确定其在黏附中的作用。CL8紧密素的羧基末端266个氨基酸与谷胱甘肽S-转移酶作为蛋白融合体表达，用于在兔中制备抗血清。该抗血清在Western免疫印迹中与血清群O5、O26、O111和O157的EHEC菌株以及血清群O55和O127的肠致病性大肠杆菌菌株的97 kDa外膜蛋白发生反应。用125I对CL8进行表面标记表明紧密素暴露于表面。产生了CL8的eaeA插入失活突变体，并将其命名为CL8-KO1。CL8-KO1对HEp-2细胞的总黏附与CL8没有显著差异，但CL8-KO1在荧光肌动蛋白染色试验中呈阴性结果。单独在大肠杆菌HB101中表达的eaeA基因在荧光肌动蛋白染色试验中也呈阴性结果。CL8的eaeA基因能够互补CVD206中的eaeA缺失突变。我们得出结论，EHEC eaeA基因的产物是一种97 kDa的表面暴露蛋白，并建议将其命名为intiminO157。Sherman等人描述了一种94 kDa的外膜蛋白，它在大肠杆菌O157:H7的黏附中起重要作用（《感染与免疫》59:890 - 899，1991）。Western免疫印迹和间接荧光抗体研究表明，Sherman等人描述的蛋白不是紧密素。
