Cellular Microbiology (2001) 3(), 1±18 Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli Isabelle Peiffer, 1 Marie-Franc Ë oise Bernet-Camard, 1 Monique Rousset 2 and Alain L. Servin 1 * 1 Institut National de la Sante  et de la Recherche Me  dicale (INSERM), Unite  510, Faculte  de Pharmacie Paris XI, F-92296 Cha à tenay-Malabry Cedex, France. 2 Unite  505, Centre des Cordeliers, F-75006 Paris, France. Summary Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco- 2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal mole- cules, previously found to be active against the Afa/ Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme bio- synthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity. Introduction Diffusely adhering Escherichia coli (DAEC) belonging to the Afa/Dr family are a recently isolated category of uropathogenic and diarrhoeagenic E. coli strains. The only virulence factors identified so far are the Afa/Dr adhesins, which show very similar genetic organization (Labigne et al., 1985; Bilge et al., 1989; Swanson et al., 1991; Le Bougue Ânec et al., 1993). All these adhesins recognized the same receptor, i.e. the glycosylphospha- tidylinositol (GPI)-anchored protein decay-accelerating factor (DAF-CD55) (Nowicki et al., 1993). Moreover, Afa/Dr DAEC adhesins recognized in parallel a second GPI-anchored protein as a receptor, the carcinoembryo- nic antigen (CEA, CD66e) (Guignot et al., 2000). We have recently provided insights about the mechanism of pathogenicity by which these E. coli promoted structural and functional injuries in intestinal cells (Peiffer et al., 1998; 2000a, b). Some members of the Afa/Dr family of E. coli, including strains harbouring the fimbrial adhesin F1845, the haemagglutinin Dr or the afimbrial adhesin Afa-I, were able to infect the human intestinal epithelial Caco-2 cells (Kerne Âis et al., 1994). The brush border intimate attachment of C1845 bacteria harbouring the F1845 adhesin is followed by microvillus injury in fully differentiated Caco-2 cells (Bernet-Camard et al., 1996). Brush border lesions result from dramatic rearrangements in apical cytoskeleton proteins, such as F-actin, villin and fimbrin, proteins that play a pivotal role in the organization and maintenance of brush border integrity (Peiffer et al., 2000b). In parallel, an alteration in the distribution of functional brush border-associated proteins controlling the absorption/secretion function has also been observed. Cytoskeleton rearrangements were reported that resulted in a Ca 21 -dependent signalling through the activation of a CD55 GPI-associated signal transduction cascade invol- ving protein tyrosine kinase, phospholipase Cg and phosphatidylinositol 3-kinase (Peiffer et al., 1998). More- over, lesions in tight junctions (TJs) have been observed and were shown to result from rearrangements in at least Q 2001 Blackwell Science Ltd Received 16 October, 2000; revised 20 December, 2000; accepted 3 January, 2001. *For correspondence. E-mail alain.servin@cep.u- psud.fr; Tel. (133) 1 46 83 56 61; Fax (133) 1 46 83 56 61. Cellular Microbiology 121 CMI CJ6033 MT 27/2/1 14:32 A LDEN