Interaction of Escherichia coli producing cytotoxic necrotizing factor with HeLa epithelial cells.
Cytotoxic necrotizing factors (CNF) constitute a group a cell-associated proteic toxins of 110-115 kDa produced by some clinical isolates of Escherichia coli from man and animals. Purified CNFs are known to exacerbate actin polymerization in exposed cells, a property that has been ascribed to their ability to modify rho a small GTP-protein involved in the regulation of the cytoskeleton. We speculated that, in spite of their lack of excretion in broth culture supernatants, CNF might be expressed upon direct interaction of organisms with infected cells. To test this hypothesis, we set up a model of interaction using epithelial cell line HeLa and the CNF1-producing strain BM2-1, which is adherent to Hela cells. An interaction of four hour duration triggers the progressive development of a dose-dependent cytopathic effect (CPE) with following characteristics: (1) intense cell enlargement with formation of a dense network of stress fibers, (2) inhibition of cell mitosis due to an irreversible block in G2/M transition phase, (3) nucleus swelling and fragmentation, and (4) cell death starting five days after infection. The three last features clearly differentiate CPE from the effect produced by CNF1 alone. In addition CPE, was not produced by cell-free culture supernatants nor abolished by an antiserum neutralizing CNF1. Tn5::PhoA insertion in the 3' end of cnf1 structural gene abolished CPE, which was not restored by trans complementation with cloned cnf1. These results demonstrate that CNF1-producing E. coli exert a specific pathogenic effect in HeLa cells, which is determined by cnf1 and at least one additional gene, located downstream cnf1.
De Rycke
J
J
Laboratoire INRA Associé de Microbiologie Moléculaire, Toulouse, France.
Nougayrede
J P
JP
Oswald
E
E
Mazars
P
P
D001422:Bacterial Adhesion; D001427:Bacterial Toxins / Q000633:toxicity*; D003588:Cytopathogenic Effect, Viral; D003603:Cytotoxins / Q000633:toxicity*; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans
D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; C060296:cytotoxic necrotizing factor type 1