MITOTIC BLOCK AND DELAYED LETHALITY IN HELA EPITHELIAL-CELLS EXPOSED TO ESCHERICHIA-COLI BM2-1 PRODUCING CYTOTOXIC NECROTIZING FACTOR TYPE-1

The cytopathic effect (CPE) of Escherichia coli producing cytotoxic ne crotizing factor type 1 (CNF1) was investigated by using a human epith elial 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 den se network of actin stress fibers, (ii) inhibition of cell division du e to a complete block in the G(2) phase of the cell cycle, and (iii) n ucleus swelling and chromatin fragmentation. These alterations resulte d in cell death starting about 5 days after interaction. The absence o f multinucleation clearly distinguished the CPE from the effect produc ed by free CNF1, which was shown to result in a true endomitosis. More over, the CPE was neither produced by cell-free culture supernatants o f infected cells nor prevented by a CNF1-neutralizing antiserum. Patho genicity was completely abolished after Tn5::phoA insertion mutagenesi s in the cnf-1 structural gene but not restored by trans complementati on 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 h and, transformation of the wild-type strain BM2-1,vith the same recomb inant plasmid leads to a significant increase in both CNF1 activity an d CPE, demonstrating the direct contribution of CNF1 to the CPE. In co nclusion, the pathogenicity of E. coli BM2-1 for HeLa cells results fr om a complex interaction involving cnf-1 and associated genes and poss ibly requiring a preliminary step of binding of bacterial organisms to target cells.