coli nor by EPEC. TLR5 localization is still controversial because the findings and interpretations are inconsistent [41, 42], and techniques used to detect TLR5 on the surface membrane have been unsatisfactory. Here, we showed TLR5 re-localization by use of permeabilized versus non-permeabilized cells, and antibodies enabling us to detect TLR5 inside and outside of the cells. Besides analyzing TLR5 polarity, it was important
to define if TLR5 was properly exposed at the cell surface because TLRs are not restricted to the plasma membrane, but also found in endosomal/lysosomal see more compartments [43]. Furthermore, EPEC is an extracellular pathogen that disrupts epithelial polarity [17]. According to our results, in non-stimulated HT-29 cells and cells
interacting with non-pathogenic E. coli, TLR5 is mainly intracellular. Interestingly, EPEC infection modifies TLR5 distribution and increases its presence on the cell surface. EPEC flagellum, translocation of effectors and intimate adherence are required to shift TLR5 distribution, although more research is necessary to assert the role of intimin in the recruitment of TLR5 to the cell surface, because of discrepancies in our results from FACS and confocal microscopy studies. However, quantitatively, the FACS experiment takes into account the whole cell population and not only random cells. Therefore, this result could be considered as more reliable. Regardless of their molecular homology, TLRs have different expression and functional patterns [43]. Unlike TLR5, TLR4 ABC294640 clinical trial distribution in HT-29 cells was found to be located primarily at the surface, and EPEC infection did not alter TLR4 distribution. This Oxymatrine result indicates that EPEC-induced TLR5 redistribution is specific for this flagellin receptor. Redistribution of host components during EPEC infection has been described for cytoskeletal proteins and other cell factors [44–46], and now we report for the first time
on re-localization of a pathogen recognition receptor during EPEC infection. Changes in TLR5 distribution have important implications, because TLR5 recruitment enables efficient recognition of extracellular flagellin. The requirement of EPEC virulence to activate TLR5 re-localization could be involved in the physiological tolerance to non-pathogenic bacteria and vigorous response to infection that epithelial cells possess. Flagellin recognition by TLR5 activates ERK1/2 and NF-κB pathways [43]. ERK1/2 phosphorylation during E2348/69 infection was previously reported [28]. Here, we showed that two different EPEC strains (E2348/69 and E22) equally activate ERK1/2 phosphorylation in infected cells. We found that EPEC have opposite modulators of ERK1/2 phosphorylation: flagellin enhances ERK1/2 activation, whereas intimin down-regulates it. FliC role on ERK1/2 activation has previously been shown [25], but the effect of EPEC intimate adherence in phosphorylation was not clear.