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

Abstract

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.Cytotoxic necrotizing factor 1 (CNF1) is an AB-type toxin expressed by pathogenic Escherichia coli strains which cause urinary tract infections and neonatal meningitis. CNF1 is a 115-kDa single-chain molecule comprising an N-terminal receptor binding domain and a C-terminal catalytic domain, which contains deamidase activity (for a review, see reference 9). These two domains are separated by a putative translocation domain, which contains two hydrophobic helices involved in membrane translocation (16). CNF1 deamidates small GTPbinding proteins of the Rho family at glutamine 63/61, leading to constitutive activation of the small GTPases by blocking their intrinsic and GTPase activating protein-stimulated GTP hydrolysis (5, 17).CNF1 has been shown to enter cells by receptor-mediated endocytosis independent of clathrin and independent of sphingolipid-cholesterol-rich membrane microdomains (lipid rafts), including caveolae (4). Based on interaction studies, using a yeast two-hybrid system, it has been suggested that cell binding of the toxin is mediated by the laminin receptor precursor p37, a subunit of the mature nonintegrin 67-kDa laminin receptor (3, 11). Moreover, it was shown that CNF1 contributes to E. coli K1 invasion of human brain microvascular endothelial cells via the 67-kDa laminin receptor. Following internalization, CNF1 is delivered to late endosomes by microtubule-dependent transport. From there, it is released into the cytosol in an acidic pH-dependent manner. Specific acidic residues located in the hydrophilic loop, connecting the two hydrophobic helices of the translocation domain, appear to be involved in the translocation process (16).More recently, a cytotoxic necrotizing factor (CNF) produced by Yersinia pseudotuberculosis (CNF Y ) was discovered, which catalyzes the same deamidation reac...

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