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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Apr 26;91(9):3814–3818. doi: 10.1073/pnas.91.9.3814

Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers.

E Oswald 1, M Sugai 1, A Labigne 1, H C Wu 1, C Fiorentini 1, P Boquet 1, A D O'Brien 1
PMCID: PMC43672  PMID: 8170993

Abstract

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.

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Selected References

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