Betancourt-Sanchez Miguel, Navarro-Garcia Fernando
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados (CINVESTAV-IPN), Ap. Postal 14-740, 07000 Mexico DF, Mexico.
Microbiology (Reading). 2009 Sep;155(Pt 9):2895-2906. doi: 10.1099/mic.0.029116-0. Epub 2009 Jun 18.
In an in vitro model using HEp-2 cells treated with purified plasmid-encoded toxin (Pet), we have identified morphological changes characterized by cell rounding and detachment after toxin internalization; these changes progress to cell death. However, these effects have not yet been shown to occur during the infection of epithelial cells by enteroaggregative Escherichia coli (EAEC). Here, we show that the secretion of Pet by EAEC is regulated at the transcriptional level, since secretion was inhibited in eukaryotic cell culture medium, although Pet was efficiently secreted in the same medium supplemented with tryptone. Inefficient secretion of Pet by EAEC in DMEM prevented cell detachment, whereas efficient Pet secretion in DMEM/tryptone increased cell detachment in a HEp-2 cell adherence assay. Interestingly, Pet toxin was efficiently delivered to epithelial cells, since it was internalized into epithelial cells infected with EAEC at similar concentrations to those obtained by using 37 microg ml(-1) purified Pet protein. Additionally, Pet was not internalized when the epithelial cells were infected with a pet clone, HB101(pCEFN1), unlike the wild-type strain, which has a high adherence capability. There is a correlation between Pet secretion by EAEC, the internalization of Pet into epithelial cells, cell detachment and cell death in EAEC-infected cells. The ratio between live and dead cells decreased in cells treated with wild-type EAEC in comparison with cells treated with an isogenic mutant in the pet gene, whereas the effects were restored by complementing the mutant with the pet gene. All these data indicate that Pet is an important virulence factor in the pathogenesis of EAEC infection.
在一个体外模型中，我们使用经纯化的质粒编码毒素（Pet）处理的人喉表皮样癌细胞（HEp-2细胞），鉴定出毒素内化后以细胞变圆和脱离为特征的形态学变化；这些变化会发展为细胞死亡。然而，尚未证实这些效应会在肠聚集性大肠杆菌（EAEC）感染上皮细胞期间发生。在此，我们表明EAEC分泌Pet是在转录水平受到调控的，因为在真核细胞培养基中其分泌受到抑制，尽管在添加胰蛋白胨的相同培养基中Pet能有效分泌。在DMEM中EAEC分泌Pet效率低下可防止细胞脱离，而在DMEM/胰蛋白胨中Pet有效分泌则会在HEp-2细胞黏附试验中增加细胞脱离。有趣的是，Pet毒素能有效地递送至上皮细胞，因为它以内化至感染EAEC的上皮细胞中的浓度与使用37μg/ml纯化Pet蛋白时获得的浓度相似。此外，与具有高黏附能力的野生型菌株不同，当上皮细胞感染pet克隆HB101(pCEFN1)时，Pet不会被内化。EAEC分泌Pet、Pet内化至上皮细胞、细胞脱离以及EAEC感染细胞中的细胞死亡之间存在相关性。与用pet基因的同基因突变体处理的细胞相比，用野生型EAEC处理的细胞中活细胞与死细胞的比例降低，而通过用pet基因补充突变体可恢复这些效应。所有这些数据表明Pet是EAEC感染发病机制中的一个重要毒力因子。
