T. Ide et al., Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli, CELL MICROB, 3(10), 2001, pp. 669-679
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.