Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III-secreted proteins EspB an d EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the ta rget cell membrane. Together with EspA, these proteins are supposed to cons titute a molecular syringe, channelling other effector proteins into the ho st cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the fo rmation of a putative pore resulting in haemolysis has not been demonstrate d so far. Here, we show that (i) diffusely adhering (DA)-EPEC strains exhib it a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC str ains, does not require bacterial contact; and (iii) pores are introduced in to the target cell membrane. Osmoprotection revealed a minimal pore size of 3-5 nm. The pores induced by type III-secreted proteins of DA-EPEC were ch aracterized by electron microscopy techniques. Analysis by atomic force mic roscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55-65 nm and to be raised 15-20 nm above the membra ne plane. We could also demonstrate an association of EspB and EspD with er ythrocyte membranes and an interaction of both proteins with each other in vitro. These results, together with the homologies of EspB and EspD to prop osed functional domains of other pore-forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation , which might represent the type III secretion system translocon.