EspA demonstrates discrete sequence similarity to flagellin in the carboxyl-terminal region of the protein which is predicted with high probability to adopt a coiled-coil conformation [15, 16]. Similar to the
assembly of flagella from the polymerization of monomeric flagellin , polymerization of EspA to form filaments depends on coiled-coil interactions between EspA subunits . In addition, it has been shown that EspA subunits are added to the tip of the growing filament in a similar manner https://www.selleckchem.com/products/ew-7197.html to a growing flagellum . Although EspA filament diameter (120 Å) is smaller than that of flagella (230 Å), its assembly has a lumen diameter and helical symmetry parameters very similar to those of the flagellar filamentous structure [13, 19, 20].
Despite these structural similarities, PLX-4720 chemical structure to date no functional overlap has been observed between the two protein secretion systems in EPEC. In this study, we observed that FliC was consistently present in the secretome of wild type EPEC E2348/69 or an ΔespADB mutant of E2348/69 but only weakly present in the secretome of a ΔescF (T3SS) mutant of EPEC E2348/69. We determined that FliC could be secreted by the LEE-encoded T3SS of EPEC E2348/69 and that FliC exported in this manner was able to stimulate an inflammatory response via the pathogen-recognition molecule for bacterial flagellin, Toll-like receptor 5 (TLR5). Results Analysis of the EPEC E2348/69 secretome The secretome Liothyronine Sodium of EPEC E2348/69 is dominated by the presence of the translocators, EspA, EspB and EspD [9, 21]. The genes encoding these proteins are located together in the LEE4 operon. To identify less abundant proteins in the EPEC E2348/69 supernatant, we generated an ΔespADB mutant and compared the secreted protein profile of this mutant with that of a ΔescF T3SS mutant EPEC ICC171 by two dimensional gel electrophoresis (2-DGE). escF encodes the needle structure of the LEE-encoded T3SS and mutations in escF abolish secretion of the translocator and effector
proteins [14, 22]. An escF mutant was used in preference to escN, which encodes the T3SS ATPase, as an escN mutant showed greater cell lysis in culture during growth in hDMEM (data not shown). However some cell breakdown was still observed for ICC171 which may click here account for some spots visualized by 2-DGE (Fig. 1). Both the ΔespADB mutant and ICC171 were grown in HEPES buffered DMEM (hDMEM) pH 7.4–7.7 to an OD600 of 1.0 to induce expression of the LEE T3SS. Cultures (20 × 5 ml) were pooled to control for variations in growth and supernatant proteins were collected by trichloroacetic acid (TCA) precipitation. Following 2-DGE, consistent and dominant spots were excised for tryptic in-gel digestion and MALDI-TOF mass spectrometry analysis.