The membrane was probed with His-EspA protein, and its interaction with other proteins was detected by Western blotting using anti-EspA antibodies. rod to a hollow needle through which unfolded effectors are secreted (7,C9). It has been shown that this syringe-like, membrane-embedded injectisome functions as a conduit, which CDKI-73 was visualized using heterologous substrates that become caught within the secretion path inside the injectisome conduit while CDKI-73 in action (10). Three proteins (one hydrophilic protein and two hydrophobic proteins) known as translocators are themselves secreted via the T3SS and are required for the transit across the host cell membrane (11). However, the degree to which they are conserved is quite variable in all T3SS proteins, including such translocator proteins. In EPEC and EHEC strains, the hydrophilic translocon component (EspA) is related to LcrV from spp. or IpaD from only distantly, but all of them share the coiled-coil structure (12). EspA binds to the needle protein EscF but does not form a pentameric ring at the needle tip such as LcrV does (13). Instead, EspA apparently tethers the bacterium to the host cell by forming a sheath-like filament extending about 93?nm on average (14, 15). The EspA filament is usually a helical tube with 5.6 subunits per change, an outer diameter of 12?nm, and an inner diameter of CDKI-73 25?? (16). On the other hand, the other two hydrophobic translocators have predicted transmembrane helices. YopD (EspB in EPEC and EHEC) and YopB (EspD) can be considered prototypes of the hydrophobic translocon components thought to form a pore in the host cell membrane through which effector proteins pass (17). In the case of EPEC, the T3SS is located in a 35.6-kb pathogenicity island termed LEE (locus of enterocyte effacement) (18). LEE is usually organized into five polycistronic operons: LEE1, LEE2, and LEE3 encode the T3SS or injectisome; the products encoded by LEE4 comprise the T3SS-secreted translocator proteins EspA, EspB, and EspD. Through this injectisome, a LEE5 effector, Tir, is usually injected directly into the cell and is inserted into the membrane, exposing an extracellular domain name that is recognized by intimin (an EPEC membrane adhesin), also encoded by LEE5 (19). Intimin-Tir interactions lead to elicitation of a histopathologic lesion created at the mucosal intestinal surface that displays a pedestal-like structure, known as an attaching and effacing (AE) lesion (20). Other LEE-encoded effector proteins (EspG, EspZ, EspH, Map, and EspF) are also injected into the cell during contamination (20). Notably, the EPEC T3SS also translocates non-LEE-encoded effectors, including NleA/EspI, EspJ, EspL, EspO, NleB, NleC, NleD, NleE, NleF, NleG, NleH, and Cif (21). All these effectors hamper different aspects of the cell physiology, including Isl1 subverting innate immune pathways, specifically those involved in phagocytosis, host cell survival, apoptotic cell death, and inflammatory signaling, which are all required to cause disease (20, 22). A second pathogenicity island of EPEC that encodes EspC has been recognized in pathogenic EPEC1 strains. Unlike proteins secreted by the T3SS, EspC secretion is usually mediated by the type V secretion system (T5SS) or autotransporter system (23, 24). A recent study showed that is one of the most prevalent genes among those encoding autotransporter proteins in both common and atypical EPEC strains (25). EspC is able to exert cytotoxic effects on epithelial cells, and these effects depend on its serine protease motif (26). EspC protein has to get inside the cells in order to cleave intracellular targets such as fodrin and focal adhesion proteins such as focal adhesion kinase (FAK) and paxillin (27) as well as proteins related to the apoptosis cascade such as pro-caspase 3 (28). The cleavage of these intracellular targets by EspC prospects to cell rounding and detachment followed by cell death through apoptosis and necrosis (28). Interestingly, EspC is not efficiently internalized under nonphysiological conditions (as a purified protein), because no receptor is usually involved in its uptake. However, EspC physiologically secreted by EPEC is usually efficiently internalized during the conversation of EPEC with epithelial cells (29). Thus, a key step for the cell damage.
