Abstract
Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81–176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells.
Highlights
Campylobacter jejuni are Gram-negative, motile bacteria with a spirally shaped body that commensally colonize the intestines of birds and mammals
Since HtrA can be incorporated as cargo into released outer membrane vesicles (OMVs) or secreted as soluble protein, we investigated whether tight junction proteins are targeted by the protease activity of C. jejuni HtrA
The results presented here suggest that occludin is a direct cleavage target of C. jejuni HtrA, which results in opening of tight junctions followed by paracellular transmigration of the bacteria
Summary
Campylobacter jejuni are Gram-negative, motile bacteria with a spirally shaped body that commensally colonize the intestines of birds and mammals. HtrA of Escherichia coli is the best studied model, and this species contains three homologs called DegP, DegQ and DegS Their main function is to protect E. coli against heat and other stresses, and to remove misfolded proteins [19, 21, 22]. C. jejuni contains only one HtrA homolog, and this periplasmic protein can be secreted into the extracellular space, where it is able to cleave the extracellular domain of the adherens junction protein E-cadherin [10]. This helps C. jejuni to transmigrate between neighbouring cells to reach the basal side the polarized epithelium, a process that depends on HtrA activity [11, 23]
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