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The Cytotoxic Necrotizing Factors from Yersinia pseudotuberculosis and from Escherichia coli Bind to Different Cellular Receptors but Take the Same Route to the Cytosol

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albert-Str. 25, 79104 Freiburg, Germany,¹ Institut für Medizinische Mikrobiologie der Otto-von-Guericke-Universität Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany²

Received 8 December 2006/ Returned for modification 31 January 2007/ Accepted 5 April 2007

The cytotoxic necrotizing factors CNF1 and CNF2 produced by pathogenic Escherichia coli strains and CNF_Y of Yersinia pseudotuberculosis constitutively activate small GTPases of the Rho family. They deamidate a glutamine (Gln63 in RhoA), which is crucial for GTP hydrolysis. CNF1 and CNF_Y exhibit 61% identity on the amino acid level, with equal distribution over the whole molecule. Although the two toxins are homologous in the receptor binding domain, we show that they bind to different cellular receptors. CNF_Y does not enter Caco-2 and CHO-K1 cells, which are responsive to CNF1. In contrast, HeLa, Hep-2, and HEK 293 cells do respond to both toxins. Competition studies with catalytically inactive mutants of the toxins revealed that binding of CNF1 has no influence on the uptake of CNF_Y into HeLa cells. In contrast, uptake of CNF1 is retarded after preincubation of HeLa cells with the catalytically inactive mutant of CNF_Y, suggesting that the toxin receptors overlap. Moreover, we compared the pathways of the toxins from receptor binding into the cytosol and showed that both toxins are taken up independent of the presence of clathrin or lipid rafts and are released into the cytosol from acidified endosomes.

Published ahead of print on 16 April 2007.

Editor: D. L. Burns

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