Downloaded from www.microbiologyresearch.org by IP: 54.159.222.200 On: Wed, 01 Jun 2016 07:06:39 Intoxication of epithelial cells by plasmid-encoded toxin requires clathrin-mediated endocytosis Fernando Navarro-Garcı ´a, Adria ´ n Canizalez-Roman, Jorge E. Vidal and Ma. Isabel Salazar Correspondence Fernando Navarro-Garcı ´a fnavarro@cell.cinvestav.mx Department of Cell Biology, Centro de Investigacio ´ n y de Estudios Avanzados (Cinvestav-Zacatenco), Ap. Postal 14-740, 07000 Me ´ xico, DF, Mexico Received 14 February 2007 Revised 1 May 2007 Accepted 14 May 2007 It has been shown that the autotransporter plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) produces cytotoxic and enterotoxic effects. Both effects can be explained by the proteolytic activity of Pet on its intracellular target a-fodrin (aII spectrin). In addition, Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, and on its internalization into epithelial cells. However, the mechanisms of Pet uptake by epithelial cells are unknown. Here, we show that Pet interacts with the plasma membrane of epithelial cells, and afterwards is detected inside the cells. Furthermore, Pet was internalized via clathrin-mediated endocytosis, since its internalization was inhibited by monodansylcadaverine and sucrose, but not by filipin or methyl-b-cyclodextrin, which are drugs that interfere with protein entry via a clathrin-independent pathway. Additionally, Pet was immunoprecipitated by anti-clathrin antibodies, but not by anti-caveolin antibodies. Moreover, small interfering RNA (siRNA), designed to knock out clathrin gene expression in HEp-2 cells, prevented Pet internalization, and thereby the Pet-induced cytotoxic effect. However, the use of siRNA to knock out caveolin expression had no effect on Pet internalization, and the cytotoxic effect was clearly observed. Together, these data indicate that Pet secreted by EAEC binds to the cell surface via an unknown receptor, to be taken up by clathrin-mediated endocytosis, and exert its toxic effect in the cytoplasm. INTRODUCTION Enteroaggregative Escherichia coli (EAEC) has been asso- ciated with persistent infant diarrhoea, especially in developing countries (Bhan et al., 1989; Cravioto et al., 1991). The pathogenesis of EAEC infection is not completely understood (Harrington et al., 2006), although histopathological alterations to the intestinal epithelium from patients and animal models infected with EAEC have been reported (Nataro et al., 1995; Tzipori et al., 1992). Similar histological alterations have been observed in autopsy samples of the ileum from children who had died as a consequence of persistent diarrhoea caused by an EAEC infection (Eslava et al., 1993). We have shown that a 104 kDa EAEC protein, termed Pet (plasmid-encoded toxin) (Eslava et al., 1998), is required for EAEC-induced damage to human intestinal mucosa (Henderson et al., 1999). We have also found that Pet bears nucleotide homology to a class of serine protease autotransporter proteins from E. coli and Shigella (Henderson et al., 2004). Pet encompasses the three classical domains (signal sequence, passenger domain and translocation unit) of autotransporters that are needed for its autosecretion (Henderson et al., 2004). Pet also has a conserved serine protease motif responsible for cleaving its target (Canizalez-Roman & Navarro-Garcı´a, 2003; Eslava et al., 1998). Pet appears to be a cytoskeleton-altering toxin, since it induces contraction of the cytoskeleton, loss of actin stress fibres, and release of focal contacts in HEp-2 and HT29/C1 cells, followed by cell rounding and detachment (Navarro- Garcı´a et al., 1999). Pet cytotoxicity and enterotoxicity depend on Pet serine protease activity, since both effects are inhibited by PMSF, and are not induced by Pet S260I, which is mutated in the catalytic serine (Navarro-Garcı´a et al., 1999). Recently, we have shown that Pet enters the eukaryotic cell by a retrograde transport (Navarro-Garcı´a et al., 2007), and that this step is required for the induction of the cytotoxic effect (Navarro-Garcı´a et al., 2001). In addition, we found an intracellular target for Pet: fodrin, which is cleaved by Pet in vivo. The cleavage site is localized within the 11th repetitive unit of fodrin, in the helix C between M 1198 and V 1199 , which is inside the calmodulin- binding domain. Pet-treated HEp-2 cells have revealed intracellular redistribution of fodrin that leads to Abbreviations: CHC, clathrin heavy chain; CT, cholera toxin; EAEC, enteroaggregative Escherichia coli; FAS, fluorescence actin staining; Pet, plasmid-encoded toxin; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate. A supplementary figure showing inhibition of CT and transferrin internalization by filipin or cadaverine is available with the online version of this paper. Microbiology (2007), 153, 2828–2838 DOI 10.1099/mic.0.2007/007088-0 2828 2007/007088 G 2007 SGM Printed in Great Britain