Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157:H7 to H Ep-2 cells
Positive effects of multiple pch genes on expression of the locus of enterocyte effacement genes and adherence of enterohaemorrhagic Escherichia coli O157:H7 to H Ep-2 cells
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DOI：
10.1099/mic.0.27100-0
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发表时间：
2004-07-01
期刊：
影响因子：
2.8
通讯作者：
Watanabe, H
中科院分区：
文献类型：
--
作者：
Iyoda, S;Watanabe, H
肠源性和肠出血性大肠埃希菌(EPEC和EHEC)基因组分别含有一个致病岛，称为肠细胞消失点(Lee)，它编码与上皮细胞附着和消失性病变形成有关的基因。为了阐明EHEC中Lee基因的调控机制，从EHEC O157基因组文库中筛选了调控Lee基因表达的克隆。从5000多个克隆中，获得了一个含有PERC同源物的DNA片段，作为Lee基因的正调控因子。在EPEC中，已知PerC与PerA和PERB一起是PER操纵子的一部分，位于EPEC黏附因子质粒上，而在EHEC中没有发现。然而，EHEC O157酒井株的全基因组序列显示，在染色体上有5个类似perc的序列，但没有perA和perb。对这五个perC同源物进行了鉴定，发现其中三个同源物(重命名为perc同源物pchA、pchB和pchC)编码104个蛋白质，并且当在多拷贝质粒上表达时，增强了Lee基因的表达。相反，编码89和90aa蛋白的perc同源物，分别被重新命名为pchD和pchE，没有显著的作用。构建PCH基因缺失突变体，检测其对Lee编码的III型效应蛋白EspA、B和D的表达及对Hep-2细胞黏附表型的影响。PchA或pchB的缺失，而不是pchC的缺失，降低了ESP蛋白的表达和与Hep-2细胞的粘附性。这种影响在pchA/pchB或pchA/pchC双重缺失的突变体中更为明显，这表明pchA/B/C对于Lee基因的充分表达和与Hep-2细胞的黏附都是必需的。进一步的研究表明，pchA/B/C的积极作用是通过增强Lee编码的调控基因LER的转录而产生的。携带每个pchA/B/C基因的多拷贝质粒可显著诱导EHEC O157在Hep-2细胞上形成微集落。这些结果表明，pchABC基因是EHEC O157完全毒力所必需的。
Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) genomes contain a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes genes involved in the formation of attaching and effacing lesions on epithelial cells. To elucidate the regulatory mechanism of the LEE genes in EHEC, an EHEC O157 genomic library was screened for clones which modulated expression of the LEE genes. From more than 5000 clones, a DNA fragment was obtained containing a perC homologue as a positive regulator for the LEE genes. In EPEC, perC is known to be part of the per operon, along with perA and perB, located on the EPEC adherence factor plasmid, which is not found in EHEC. However, the complete genome sequence of EHEC O157 Sakai strain reveals that there are five perC-like sequences, but no perA and perB, on the chromosome. These five perC homologues were characterized, and it was found that three of the homologues (renamed perC homologue pchA, pchB and pchC) encoded 104 as proteins, and when expressed on a multicopy plasmid enhanced the expression of LEE genes. In contrast, perC homologues encoding proteins of 89 and 90 aa, renamed pchD and pchE, respectively, had no significant effect. Deletion mutants of the pch genes were constructed, and the effect on the expression of LEE-encoded type III effector proteins, such as EspA, B and D, and adhesion phenotype to HEp-2 cells was examined. Deletion of pchA or pchB, but not pchC, decreased the expression of Esp proteins and adhesion to HEp-2 cells. Such effects were more apparent with mutants carrying double deletions of pchA/pchB or pchA/pchC, suggesting that pchA/B/C are all necessary for full expression of the LEE genes and adhesion to HEp-2 cells. Further study demonstrated that the positive effect of pchA/B/C was caused by enhanced transcription of the LEE-encoded regulatory gene, ler. Introduction of a multicopy plasmid carrying each pchA/B/C gene significantly induced microcolony formation by EHEC O157 on HEp-2 cells. These results suggest that the pchABC genes are necessary for full virulence of EHEC O157.