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Fig. 7 | Gut Pathogens

Fig. 7

From: Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA

Fig. 7

HtrA cleaves occludin during C. jejuni infection in vivo and in cleavage assays in vitro. a Immunoblotting of protein extracts from polarized Caco-2 cell monolayers infected with C. jejuni wild-type (wt) or ΔhtrA mutant. The blots were stained with polyclonal α-occludin antibodies recognizing the C-terminus of the protein. The α-HtrA and α-GAPDH blots served as controls. Besides the full-length protein (65 kDa), a cleaved C-terminal fragment (37 kDa) was visualized with α-occludin antibodies upon infection with wt bacteria. The asterisk marks a double band at ~ 45–50 kDa, presumably corresponding to two isoforms of occludin recognized by this antibody. b Recombinant GST-tagged human occludin was incubated with purified HtrA in in vitro cleavage assays. Cleavage of full-length GST-occludin (monomer at 100 kDa) resulted in the production of the same 37 kDa C-terminal fragment as detected upon infection. Reprobing of the blot with α-GST antibodies resulted in the detection of the corresponding N-terminal cleavage fragment of 63 kDa. The asterisks mark the position of GST-occludin dimers migrating at ~ 200 kDa. c Mapping of the occludin cleavage fragments. An HtrA cleavage site was localized in the second extracellular loop between the transmembrane (TM) domains 3 and 4 as indicated. Bioinformatical investigation revealed the exact cleavage position as indicated on the bottom. d Secondary structure prediction for the second extracellular loop. The predicted type of secondary structure (Helix ‘H’, Coil ‘C’, and Strand ‘E’) is shown above the sequence. The top line gives the confidence of the predictions, with high bars indicating a more reliable prediction

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