EspA filament-mediated protein translocation into red blood cells

Abstract

SummaryType III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells. EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes. Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro. As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria±RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes. EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane. Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis. Following haemolysis, only EspD was consistently detected in the RBC membrane. This study shows that intimate bacteria±RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane.

Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin

“…Numerous candidate BFP receptors have been proposed, including a variety of oligosaccharides and, most recently, the phospholipid phosphatidylethanolamine (PE) (Nougayrede et al, 2003). This study also confirmed our previous conclusion that EPEC adhesion to RBCs is independent of BFP (Shaw et al, 2001) and indicates a BFP receptor that is lacking on the RBC surface. A PE receptor would be consistent with these RBC data because in the erythrocyte membrane PE is predominantly localized on the cytosolic side of the membrane.…”

“…However, since EspA filaments interact with host cells during the early stages of A/E lesion formation, it has been proposed that they may also function as adhesins. We showed previously that EspA filaments promoted attachment of strains lacking BFP to epithelial cells and to RBCs Shaw et al, 2001) and similar results supporting a role of EspA filaments as initial attachment factors were demonstrated with Shiga toxin-producing E. coli (Ebel et al, 1998) which also lacks BFP. This present study also supports an adhesive role for EspA filaments in EPEC adhesion to brush border cells since adhesion correlated with EspA filament expression and, by immunofluorescence, EspA filaments appeared to connect bacteria to the brush border surface.…”

“…As reported previously, wild-type E2348/69 adhered to RBC monolayers (Fig. 8a) and induced a high level (~80 %) of haemolysis (Shaw et al, 2001). Similar levels of RBC adhesion (and haemolysis) were produced by strains UMD886 (Fig.…”

“…In recent studies we demonstrated EPEC adhesion to RBC monolayers, initially by EspA filaments and subsequently with strains able to translocate Tir, by intimin-Tir intimate interaction (Shaw et al, 2001. In this study we also screened the collection of mutants in bfpA, espA and eae for their ability to adhere to RBC monolayers.…”

“…However, to form an intimate attachment and A/E lesion formation, the LEE-encoded effector protein Tir (translocated intimin receptor) has to be translocated and inserted into the host cell membrane by the TTSS (Kenny et al, 1997); the intimin-Tir interaction triggers the assembly of actin into pedestals beneath intimately attached bacteria (Campellone & Leong, 2003). Whilst Tir is a well characterized intimin receptor, additional uncharacterized host cell receptors have been implicated in intimin-mediated EPEC adhesion (Frankel et al, 2001) and recently a specific host cell protein, nucleolin, was shown to bind a specific intimin subtype, intimin c, expressed by some EPEC and enterohaemorrhagic E. coli strains (Sinclair & O'Brien, 2002).…”