Infections with pathogenic food-borne bacteria constitute one of the leading causes of morbidity and mortality in humans. The World Health Organization (WHO) suggests that the human population worldwide suffers from about 4.5 billion incidences of gastroenteritis annually, causing approximately 1.8 million deaths . Various Campylobacter species have been identified as the leading enteric bacterial infection worldwide [2, 3]. Campylobacter jejuni is considered as a classical zoonotic pathogen, as it is found in the normal intestinal flora in many birds and mammals. Since C. jejuni colonizes various food animals, it can contaminate food products during processing . After ingestion by a human host, these bacteria use their flagella-driven motility to colonize the epithelial cells of the ileum and colon. Here, they can interfere with normal functions in the intestinal tract, leading to diseases associated with fever, malaise, abdominal pain and watery diarrhoea [2, 3]. In addition, a minority of infected individuals may develop late complications such as Reiter’s reactive arthritis or Guillain-Barrè and Miller-Fisher syndromes . There is increasing evidence showing that C. jejuni disturbs the normal absorptive capacity of the human intestine by damaging epithelial cell functions, either by cell invasion, the production of pathogenicity-associated factors or indirectly by triggering inflammatory responses [3, 6–8].
It has been proposed that transmigration across and invasion into intestinal epithelial cells during infection is a major reason of C. jejuni-triggered tissue damage [2–4]. Investigation of gut biopsies obtained from infected patients and in vitro infection experiments of intestinal epithelial cells indicated that C. jejuni can enter human host cells [9–11]. Campylobacter jejuni expresses various adhesins in the outer-membrane including CadF, FlpA, JlpA and PEB1 [12–15]. For example, in vitro CadF is a well-known bacterial factor that binds to fibronectin, an important extracellular matrix (ECM) protein and bridging factor to integrin receptors [13, 16, 17]. Maximal bacterial adherence and invasion of INT-407 intestinal epithelial cells is dependent on CadF and is associated with tyrosine phosphorylation of paxillin, a focal adhesion-based scaffolding factor . The expression of CadF also seems to be required for the stimulation of the small Rho GTPases Rac1 and Cdc42 via fibronectin and integrin member β1, that are required for C. jejuni host cell entry. The signalling pathways involved in the latter process have been described in detail [19–21]. However, fibronectin and integrin β1 are basolateral receptor molecules and not commonly exposed at apical surfaces in the intestine. It is therefore unclear how C. jejuni gains access to these receptors during infection.
To access deeper tissues and cause short- or long-term infections in the human body, various pathogenic bacteria, including Salmonella, Shigella Listeria or Neisseria, must overcome the epithelial barrier [22, 23]. These important bacterial pathogens are able to cross polarised intestinal epithelial cells by different mechanisms, known as the paracellular and the transcellular routes. Bacteria using the transcellular route enter host cells at apical surfaces followed by intracellular trafficking and leave these cells at the basolateral surface. In contrast, bacteria specialised on the paracellular route cross the epithelial barrier by passage between neighbouring epithelial cells and overcome the tight junctions and adherens junctions . In the case of C. jejuni, the literature is highly controversial. While some groups reported the paracellular route, others described the transcellular model or a mix of both [25–30]. In general, the host factors and bacterial factors involved in the transmigration process of C. jejuni are still unclear .
We have recently shown that a closely related pathogen, Helicobacter pylori, secretes a novel bacterial virulence determinant into the culture supernatant, the serine protease HtrA [32–34], which is also present in C. jejuni[35–37]. HtrA proteins constitute a group of heat shock induced serine proteases that influence the adhesion and invasion properties of different bacterial pathogens. HtrA proteins typically consist of a signal peptide, a trypsin-like serine protease domain and one or two protein interaction (PDZ) domains. In addition, by binding of the PDZ domain in one HtrA molecule to that in other HtrA molecules, HtrA can build-up to highly proteolytic active oligomers that also function as a chaperone . The HtrA protease domain consists of an active site, called the catalytic triad, which is formed by the conserved amino acid residues histidine, aspartatic acid and serine . Many bacterial HtrA proteins are suggested to be localized in the periplasm and to be involved in quality control of envelope proteins by degradation of misfolded proteins as well as prevention of formation of aggregates . Thus, it was surprising to find that HtrA exhibits the capability of extracelluar transport in H. pylori[34, 41], where it could cleave host surface molecules. We identified that H. pylori HtrA directly cleaves the junctional protein and tumor suppressor E-cadherin and fibronectin on the surface of gastric epithelial host cells. HtrA-mediated cleavage of E-cadherin facilitated the loss of the adherence junction complex leading to the disruption of the epithelial barrier function in response to H. pylori infection  and may also apply to C. jejuni HtrA . Here, we present the results from a detailed investigation to determine if C. jejuni HtrA can cleave both E-cadherin and fibronectin, and whether HtrA protease activity is required for transmigration across polarised epithelial cells. Our findings show that C. jejuni can effectively cross polarised epithelial cells in an HtrA-protease dependent fashion without affecting TER.