Tan Xiqian, Xiao Huazhi, Han Ye, Hong Xiaodi, Cui Qiang, Zhou Zhijiang
School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR China.
School of Chemical Engineering and Technology, Tianjin University, Tianjin, PR China.
Microbiol Res. 2014 Nov;169(11):855-61. doi: 10.1016/j.micres.2014.03.001. Epub 2014 Mar 12.
Adhesion is one of the significant virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. It is regulated by specific loci in the genome sequence. This study mainly focused on investigating the influence of ycbR gene and its encoded YCBR protein on the adhesion of EHEC O157:H7 to HEp-2 cells. In the first part, mutants of EHEC O157:H7 were constructed through TnphoA mutagenesis and assayed for adherent ability. Six mutant strains with lost adherence to HEp-2 cells were isolated and then sequenced using a primer that hybridized to phoA sequence downstream of the fusion joint. The sequencing results indicated that the gene of eae and ycbR, between the initiation codon and the -10 sequence of Z4182, yciI, ARAC-type regulator protein, and high-affinity gluconate transporter of EHEC were all possibly related to adhesion. Of the six genes, the ycbR gene was cloned to the pET28a vector to analyze its function further. Recombinant YCBR protein fused to a His tag (YCBR-His) was expressed under IPTG induction and purified by Ni-NTA column. The purified protein was subcutaneously injected to rabbits to prepare antisera. The results of an adherence assay in the presence of anti-YCBR-His antibodies indicated that antibodies blocked the adherence of EHEC O157:H7 to HEp-2 cells. These observations suggested that ycbR encoded a novel adherence-associated determinant of EHEC O157:H7, which could contribute to the adhesive capacity of the bacteria.
黏附是肠出血性大肠杆菌（EHEC）O157:H7致病过程中的重要毒力因子之一。它受基因组序列中特定基因座的调控。本研究主要聚焦于探究ycbR基因及其编码的YCBR蛋白对EHEC O157:H7黏附人喉表皮样癌细胞（HEp-2细胞）的影响。在第一部分中，通过TnphoA诱变构建了EHEC O157:H7突变体，并检测其黏附能力。分离出6株对HEp-2细胞失去黏附能力的突变菌株，然后使用与融合接头下游的phoA序列杂交的引物进行测序。测序结果表明，EHEC的eae和ycbR基因、Z4182起始密码子与-10序列之间的基因、yciI、ARAC型调节蛋白以及高亲和力葡萄糖酸盐转运蛋白均可能与黏附有关。在这6个基因中，ycbR基因被克隆到pET28a载体中以进一步分析其功能。在异丙基-β-D-硫代半乳糖苷（IPTG）诱导下表达了融合有His标签的重组YCBR蛋白（YCBR-His），并通过镍-次氮基三乙酸（Ni-NTA）柱进行纯化。将纯化后的蛋白皮下注射到兔子体内制备抗血清。在抗YCBR-His抗体存在下进行黏附试验的结果表明，抗体可阻断EHEC O157:H7对HEp-2细胞的黏附。这些观察结果表明，ycbR编码一种新型的EHEC O157:H7黏附相关决定簇，其可能有助于该细菌的黏附能力。
