De Rycke J, Mazars P, Nougayrede J P, Tasca C, Boury M, Herault F, Valette A, Oswald E
Laboratoire Associé de Microbiologie Moléculaire, Institut National de la Recherche Agronomique, Ecole Nationale Vétérinaire, Toulouse, France.
Infect Immun. 1996 May;64(5):1694-705. doi: 10.1128/iai.64.5.1694-1705.1996.
The cytopathic effect (CPE) of Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) was investigated by using a human epithelial cell (HeLa) model of infection with CNF1-producing E. coli BM2-1. This strain was shown to bind loosely, but massively, to HeLa cells. A 4-h interaction between bacteria and eukaryotic cells triggered the delayed appearance of a progressive dose-dependent CPE characterized by (i) intense swelling of cells accompanied by the formation of a dense network of actin stress fibers, (ii) inhibition of cell division due to a complete block in the G2 phase of the cell cycle, and (iii) nucleus swelling and chromatin fragmentation. These alterations resulted in cell death starting about 5 days after interaction. The absence of multinucleation clearly distinguished the CPE from the effect produced by cell-free culture supernatants of infected cells nor prevented by a CNF1-neutralizing antiserum. Pathogenicity was completely abolished after Tn5::phoA insertion mutagenesis in the cnf-1 structural gene but not restored by trans complementation with a recombinant plasmid containing intact cnf-1 and its promoter. These results suggest that a gene downstream of cnf-1, essential to the induction of the CPE, was affected by the mutation. On the other hand, transformation of the wild-type strain BM2-1 with the same recombinant plasmid leads to a significant increase in both CNF1 activity and CPE, demonstrating the direct contribution of CNF1 to the CPE. In conclusion, the pathogenicity of E. coli BM2-1 for HeLa cells results from a complex interaction involving cnf-1 and associated genes and possibly requiring a preliminary step of binding of bacterial organisms to target cells.
利用产细胞毒素坏死因子1（CNF1）的大肠杆菌感染人上皮细胞（HeLa）模型，研究了产CNF1大肠杆菌的细胞病变效应（CPE）。结果显示，该菌株与HeLa细胞松散但大量结合。细菌与真核细胞4小时的相互作用引发了渐进性剂量依赖性CPE的延迟出现，其特征为：（i）细胞强烈肿胀，伴有肌动蛋白应力纤维致密网络的形成；（ii）由于细胞周期G2期的完全阻滞导致细胞分裂抑制；（iii）细胞核肿胀和染色质碎片化。这些改变导致相互作用后约5天开始细胞死亡。未出现多核现象，这明显将CPE与感染细胞的无细胞培养上清液产生的效应区分开来，且不受CNF1中和抗血清的影响。在cnf - 1结构基因中进行Tn5::phoA插入诱变后，致病性完全丧失，但用含有完整cnf - 1及其启动子的重组质粒进行反式互补未能恢复致病性。这些结果表明，cnf - 1下游的一个对诱导CPE至关重要的基因受到了突变的影响。另一方面，用相同的重组质粒转化野生型菌株BM2 - 1会导致CNF1活性和CPE均显著增加，证明了CNF1对CPE的直接作用。总之，大肠杆菌BM2 - 1对HeLa细胞的致病性源于涉及cnf - 1和相关基因的复杂相互作用，可能需要细菌与靶细胞结合的初步步骤。
