Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both?
Cell Communication and Signaling
Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both?
Steffen Backert 0
Manja Boehm 0
Nicole Tegtmeyer 0
0 Department of Biology, Institute for Microbiology, Friedrich Alexander University Erlangen/Nuremberg , Staudtstr. 5, D-91058, Erlangen , Germany
Intact intercellular junctions and cellular matrix contacts are crucial structural components for the formation and maintenance of epithelial barrier functions in humans to control the commensal flora and protect against intruding microbes. Campylobacter jejuni is one of the most important zoonotic pathogens causing food-borne gastroenteritis and potentially more severe diseases such as reactive arthritis or Guillain-Barr syndrome. Crossing the intestinal epithelial barrier and host cell invasion by C. jejuni are considered to represent the primary reasons of gut tissue damage in humans and various animal model systems including monkeys, piglets, rabbits, hamsters and ferrets. C. jejuni is also able to invade underlying tissues such as the lamina propria, can enter the bloodstream, and possibly reach distinct organs such as spleen, liver or mesenteric lymph nodes. However, the molecular mechanisms as well as major bacterial and host cell factors involved in these activities are poorly understood. Various models exist by which the pathogen can trigger its own transmigration across polarized intestinal epithelial cells in vitro, the paracellular and/or transcellular mechanism. Recent studies suggest that bacterial factors such as flagellum, serine protease HtrA and lipooligosaccharide LOS may play an active role in bacterial transmigration. Here we review our knowledge on transmigration of C. jejuni as well as some other Campylobacter species, and discuss the pros and cons for the route(s) taken to travel across polarized epithelial cell monolayers. These studies provide fresh insights into the infection strategies employed by this important pathogen.
Adherens junctions; Cell polarity; E-cadherin; Fibronectin; HtrA; Integrins; Invasion; Molecular pathogenesis; Cellular invasion; Signaling; TER; Tight junctions; Transmigration; Virulence
Campylobacter jejuni is a wide-spread Gram-negative
bacterium living as commensal in the gut of most birds
and domestic animals. However, C. jejuni is infectious for
humans and consumption of contaminated food products
is a major cause of human bacterial gastroenteritis, which
may be responsible for as many as 400500 million cases
annually . The clinical outcome of C. jejuni infection
varies from mild, non-inflammatory, self-limiting diarrhoea
to severe, inflammatory, bloody diarrhoea that can
continue for few weeks [2-5]. In some cases, C. jejuni
infections can be also associated with the development
of reactive arthritis and peripheral neuropathies, known
as MillerFisher and GuillainBarr syndromes [6,7].
Despite the significant health burden caused by C. jejuni
infections, our present knowledge about the interplay
between C. jejuni and its various hosts is still very limited.
The availability of complete genome sequences from
various C. jejuni isolates has started to improve our
understanding in genetics, physiology, pathogenesis and
immunity of C. jejuni infections in recent years. C. jejuni
is the first bacterium reported to encode for both O- and
N-linked glycosylation systems, a property that is likely
influencing the host-pathogen crosstalk and disease
outcome. In addition, a multitude of infection studies in
various animal and in vitro cell model systems revealed the
importance of C. jejuni motility and chemotaxis as critical
features important for establishing successful infections
[2,8-10]. In particular, the high motility (Mot+) permits
C. jejuni to effectively move to its favored colonization
niche at the inner mucus layer of the human intestine.
Various in vivo and in vitro studies have shown that
this pathogen encodes numerous virulence determinants
involved in important disease-associated processes such as
bacterial adhesion to, transmigration across, invasion into
and intracellular survival within infected intestinal
epithelial cells . In the present article we review our
current knowledge including various recent developments
in C. jejuni research on how this bacterium can breach
the gut epithelial barrier and transmigrate across polarised
cell layers. In particular, we focus on the two major known
routes that could be taken, the transcellular and
paracellular ways of C. jejuni transmigration. Better molecular
understanding of these pathways and the identification of
involved bacterial and host factors is crucial for the future
development of effective treatment regimes.
The intestinal mucosa is a first barrier against microbial
The intestinal mucosal epithelium in humans is an
important cell layer that controls not only digestive,
absorptive and secretory functions, but also forms the
first barrier against pathogenic microbes . The intact
structure of healthy intestinal epithelial cells is maintained
by the integrity of the apical-basal polarity, forming
microvilli structures with a well-defined brush border, a highly
organized actin-cytoskeleton and proper junctional
complexes [13,14]. Importantly, well-established junctions
are built up on the lateral cell-to-cell contacts including
tight junctions (TJs) and E-cadherin-based adherens
junctions (AJs) as well as basally located integrin-mediated
cell-matrix contacts such as focal adhesions (FAs) and
hemidesmosomes (HDs). While FAs are present both
in cultured polarised and non-polarised cells, TJs, AJs,
and HDs are only established in polarised and absent
in non-polarised epithelial cells (Figure 1A,B). A model
for the overall protein composition of these junction
complexes is shown in Figure 2.
TJs are based on junction adhesion molecules (JAMs),
claudins, occludin and other proteins, which represent
important structural elements in establishing epithelial cell
polarity. They are crucial for the tight sealing of the cellular
sheets, thus controlling paracellular ion flux and therefore
maintaining tissue homeostasis. The tight apposition of the
membranes at TJs, which are localized at the apical end
of the lateral membrane, also blocks lateral mobility of
membrane proteins and lipids allowing the segregation
of membrane components in an apical and basolateral
compartment (Figures 1B and 2A). The AJs are
positioned basal to TJs and form a network of membrane
proteins and associated molecules, which are responsible
for the mechanical adhesion between neighboring cells
(Figures 1B and 2B). AJs assemble via homophilic,
calciumdependent interactions between the extracellular domains
of E-cadherin on the surface of two adjacent epithelial
cells. E-cadherin does not only act as an adhesive protein,
but also has important functions as a regulator of cell
proliferation. By modulating the availability of -catenin, which
binds to the intracellular domain of E-cadherin and helps
to connect AJs with the actin cytoskeleton,
E-cadherinbased AJs are involved in cell signaling and transcriptional
regulation. Therefore, disturbed E-cadherin signaling is also
associated with tumorigenesis . The FAs comprise the
third group of cell adhesion structures and consist of
integrin heterodimers (composed of and chains), which are
transmembrane receptors that link the extracellular matrix
to intracellular FA proteins (Figures 1B and 2C). FAs
modulate multiple signaling cascades to regulate cell
attachment, proliferation, migration, differentiation and
gene expression events. These processes are controlled by
classical outside in and inside out signal transduction
pathways [16,17]. The extracellular domain of a given
integrin can directly bind to extracellular matrix proteins
such as fibronectin, while the cytoplasmic tail is linked to
the actin-cytoskeleton via a large number of adapter
proteins, including vinculin, paxillin or talin, and signaling
enzymes such as focal adhesion kinase (FAK) or Src kinase
(Figure 2C). These protein complexes continually assemble
and disassemble, and this turnover process must be
differentially controlled at the leading edge versus the
trailing edge of a migrating cell. In addition, HDs
constitute adhesive protein complexes that mediate stable
attachment of basal epithelial cells to the underlying
tissues . Similar to FAs, the organization of HDs relies
on a complex network of protein-protein interactions, but
in HDs integrin 64, laminin and plectin play essential
roles (Figures 1B and 2D). Interestingly, many microbial
pathogens including C. jejuni have adapted mechanisms
during evolution to exploit TJs, AJs, FAs and/or HDs in
infected cells in order to proliferate, survive and sometimes
persist within the host [12,19-21].
Detection of C. jejuni in the intestinal mucus, lamina
propria, blood and other organs during infection in vivo
A major goal of current C. jejuni research is to define the
exact role of bacterial adhesion, invasion and
transmigration across enterocytes for the induction or absence of
pathogenesis in different hosts. Several in vivo studies of
human biopsies and infected animal models reported on
observations of C. jejuni entering gut epithelial cells and
underlying subepithelial tissues during infection (Table 1).
For example, electron microscopic studies of biopsies from
patients with campylobacteriosis have shown that C. jejuni
can closely associate to the surface or within the intestinal
epithelium, especially in Goblet cells, and was focally
present in the lamina propria . The majority of patients
exhibited the histological picture of acute infectious colitis
associated with massive infiltration of immune cells and
Figure 1 A schematic presentation of non-polarised and polarized intestinal cell epithelial cells under non-infective conditions or
during infection with C. jejuni. (A) Cultured non-polarised intestinal epithelial cells such as INT-407 do not express typical cell-to cell junctions.
Thus, basolateral receptors such as focal adhesion structures are accessible and not protected by tight or adherens junctions. (B) Polarised
intestinal epithelial cells such as mucin-producing HT29-MTX-E12 cells express the different types of intercellular junctions including the tight
junctions (orange), adherens junctions (light blue), focal adhesions (dark blue) and hemidesmosomes (green) which exhibit specific localization in
the lateral or basal membranes as indicated. GAP junctions and desmosomes are other examples which are not discussed in this review article.
(C,D) C. jejuni is able to infect both cell variants in vitro. This pathogen encodes numerous described pathogenicity-associated factors involved in
important processes including bacterial adhesion to, transmigration across, invasion into and intracellular survival within intestinal epithelial cells.
For more details see text.
marked distortion of crypt architecture. Penetration of C.
jejuni into the intestinal tissue is also supported by the
presence of blood and leukocytes in stool samples. Similar
observations were obtained during C. jejuni infection
experiments in monkeys , hamsters , piglets ,
rabbits  and ferrets . In addition, live C. jejuni were
recovered from other organs in infected animals such as
the spleen [28-30], liver [27,29,30], mesenteric lymph nodes
 and blood . This suggests that C. jejuni exhibits the
capability not only to adhere to and enter into enterocytes,
but can also travel within the host, pass the intestinal
epithelial barrier, enter the lamina propria and even access
other organs of various infected hosts. Interestingly, a
mixed set of results were obtained from infection
experiments in chicken and mice. In multiple studies C. jejuni
was regularly seen attached to or within colonic epithelial
cells, lamina propria and other organs of chicken and mice
[28-31]. However, C. jejuni infection resulted either in
no obvious pathology or in less pathology as compared to
humans or the above discussed other animal model systems
(Table 1). In contrast, in some other reports C. jejuni
was not seen attached to or inside intestinal epithelial
cells, although high loads of bacteria were noted in the
corresponding extracellular mucus layer [32,33]. These
studies suggest that adhesion of C. jejuni to and invasion
into intestinal epithelial tissues occurs in vivo, but may vary
substantially in various chicken and mouse model systems,
probably depending on the C. jejuni strain, variant and age
of animals and other infection parameters (Table 1).
What is the advantage for C. jejuni to cross the epithelial
barrier and infect underlying tissues?
The degree by which a given pathogen can translocate
through an epithelial cell barrier and the bacteriums fate
beyond the local environment differs substantially between
different known microbes. For example, Salmonella typhi
rapidly translocates across a polarized monolayer, causing
cellular disruption leading to a complete loss of cell
monolayer integrity. In contrast, the transcytosis of Salmonella
typhimurium across polarized cells results in minimal
damage to the epithelial monolayer [34,35]. Presumably
these observations reflect disease manifestation in vivo,
Figure 2 Composition of major intercellular junctions in the polarized intestinal epithelium. Schematic presentation of specific junctional
complexes and associated signaling pathways. (A) Tight junctions (TJs) contain at least four major groups of transmembrane proteins: the JAMs,
claudins, occludin and a number of cytoplasmic peripheral proteins. While the transmembrane proteins mediate cell-to-cell adhesion, the
cytosolic TJ complex connects to different factors (e.g. ZO-1/-2/-3, MUPP1 or cingulin) that link the involved transmembrane proteins to the
actin-cytoskeleton. The integrity of TJs is maintained by a regulatory complex including atypical PKC (aPKC), Rac1, Cdc42, Par6 and Par3. (B) The
calcium-dependent integrity of adherens junctions (AJs) is stabilized by binding of E-cadherin to the intracellular catenins. The carboxy-terminal
domain of E-cadherin binds to the cytoplasmic protein -catenin. p120-catenin binds to the juxtamembrane part of E-cadherin and stabilizes the
AJ complex. The E-cadherin--catenin structure is connected to the actin-cytoskeleton via binding to -catenin and Eplin. When the E-cadherin
complex is disrupted, -catenin can translocate into the nucleus and activate Tcf/LEF transcription factors. (C) Focal adhesions (FAs) are structural
complexes that link the extracellular matrix (ECM) to the intracellular actin-cytoskeleton. They contain various integrin heterodimers which are
transmembrane receptors composed of and chains. The extracellular integrin tail directly binds to ECM proteins such as fibronectin, while the
cytoplasmic domain is linked to the actin-cytoskeleton via a large number of indicated adapter/signaling proteins to transmit signaling. (D)
Hemidesmosomes are also located at the basal side of epithelial cells where they link laminins to the intracellular intermediate filament network.
Thus, hemidesmosomes provide stable adhesion of epithelial cell layers to the basement tissue. They consist of integrin 64, CD151 and BP180
which are transmembrane proteins, while plectin and BPAG1e are located in the cytoplasm. Plectin mediates linkage of hemidesmosomes to the
cytokeratin network and not to F-actin filaments.
where infections with S. typhi are commonly septic in
patients whereas infections with S. typhimurium are
commonly restricted to the intestinal mucosa. By
comparison to Salmonella, infections with C. jejuni are
usually less acute. Advantages for C. jejuni reaching
the underlying tissues and submucosa include that the
bacteria are no longer subject to peristaltic forces in the
intestine and they may gain pronounced access to certain
nutrients such as iron. In addition, invasive C. jejuni can
achieve contact with a set of basal host cell receptors such
as fibronectin, which are normally not present at apical
surfaces. Another advantage could be that the intracellular
environment is better protected to antibiotics as compared
to the gut lumen. Finally, by causing inflammatory diarrhea
in the intestine, C. jejuni can improve its own spread to find
a new host. This is in agreement with observations that
stools from patients are diarrheal and remain C.
jejunipositive for several weeks [2-5].
Use of non-polarised and polarised cells to study C. jejuni
infection in vitro
Among others, the C. jejuni isolates 81176, NCTC11168,
F38011 and 81116 are the most commonly used strains in
laboratories for infection studies in vitro (Table 2). Infection
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experiments of cultured cell lines with C. jejuni have shown
that the bacteria can bind to (Bind+), invade into (Inv+)
and survive inside a defined intracellular compartment
(Surv+), called the Campylobacter-containing vacuole.
These phenotypes, have been reported for both C. jejuni
infection of non-polarised and polarised epithelial cells
(Figure 1C,D). Studies of the translocation capabilities of C.
jejuni strains across an intestinal epithelium in vitro require
tight polarized cell monolayers. Typical chosen cell lines
expressing TJs, AJs and FAs include Caco-2 [36-40], T84
[41-43], MDCK-I  or MKN-28 [44,45]. In addition,
some of the polarised epithelial cell lines such as
HT29MTX-E12 have been shown to produce a mucus layer and
thus maybe also very useful as they could better mimic the
natural environment in the intestine [46,47]. It has been
described that while C. jejuni can adhere to different cell lines
with similar extend, the bacterial invasion and
transmigration capacities can vary considerably between the different
cell lines [43,48-50]. It was proposed that C. jejuni can enter
cultured epithelial cell lines of human origin with higher
efficacy as compared to non-human cells, suggesting
that the pathogen is particularly specialised for
diseasetriggering infection of the human host .
Bacterial and host factors with proposed roles in C. jejuni
adhesion and invasion of intestinal cells
Although the exact molecular mechanisms triggering
adhesion of C. jejuni to intestinal epithelial cells are
still not fully understood, several studies have provided
evidence in recent years that this is a multifactorial process
requiring the concerted activity of various C. jejuni factors,
which are under much debate [2,11,51]. Application of the
gentamycin protection assay (GPA) and other approaches
led to reports of more than 20 bacterial gene products
potentially mediating the interaction of C. jejuni with
various host cell lines. These factors comprise numerous
genes of the flagellar apparatus [52-56], pseA modifying
flagellin with the acetamidino form of pseudaminic acid
, lipooligosaccharide (LOS) biosynthesis gene galE ,
sialyltransferase cst-II gene , N-glycosylation genes pglB
and pglE , capsule biosynthesis genes kpsM and kpsE
[61,62], autotransporter CapA , Peb1-4 membrane
proteins [64-67], serine protease HtrA [45,68,69] and
the fibronectin-binding proteins CadF and FlpA [70-73]. In
addition, the proposed lipoprotein Cj0497 , cytochrome
c oxidoreductase SOR encoded by the genes cj0004c and
cj0005c , CJIE1 prophage homologs , components
of a type VI secretion system (T6SS)  and
surfaceexposed lipoprotein JlpA  have also been shown to
influence C. jejuni host cell adhesion. However, it is
not yet clear if all above factors contribute directly or
indirectly to C. jejuni-mediated host cell binding. Another
handicap is that some described adherence factors cannot
be confirmed by other groups . For example, JlpA is
dispensable for the binding of C. jejuni to chicken-derived
hepatocellular epithelial carcinoma cells . However,
mutagenesis of most of these adhesion-related factors also
exhibited distinct defects in invasion of C. jejuni suggesting
a possible positive correlation between bacterial adhesion
and host entry events. One interesting exception is again
the jlpA mutant. This mutant exhibits a reduced adherence
phenotype by ~20% of wild-type level , but was not
defective for host cell entry [73,79,80]. Taken together, while a
considerable number of putative bacterial binding factors
have been described for C. jejuni, there is a large gap in our
knowledge on the corresponding host cell receptors.
C. jejuni transmigration across polarised epithelial cells in
transwell chambers: role of protein biosynthesis and
Different well-known intestinal pathogens such as Listeria,
Shigella, Salmonella or Yersinia have the capability to
transmigrate across the gut epithelial barrier (Trans+ strains),
gain access to deeper tissues, trigger cell damage and cause
disease in humans. There are two general mechanisms how
bacterial pathogens can overcome the epithelial barrier,
described as the paracellular and the transcellular
migration routes [34,35]. Pathogens utilising the
paracellular mechanism break the TJ and AJ complexes and cross
the epithelial barrier by passage between neighboring
epithelial cells . In contrast, some other pathogens
specialised on the transcellular mechanism and invade
epithelial or specialised M cells at the apical surface
followed by intracellular trafficking and exit these cells
at the basolateral membrane [81,82]. Studies on the
translocation capabilities of C. jejuni across an intestinal
epithelium layer in vitro have been performed with
multiple strains and polarized cell lines grown in transwell
chambers (Table 2). Migration of various Trans+ C. jejuni
strains from the apical compartment of transwells through
polarized cells was confirmed by determination of colony
forming units (CFU) obtained from the lower chamber,
GPA and other functional assays. Application of
chloramphenicol, a well-known inhibitor of bacterial protein
biosynthesis, reduced the transmigration potential of
C. jejuni significantly . The failure of chloramphenicol
to completely abolish translocation may indicate that
some of the bacteria possess the factors necessary to
facilitate penetration while others may have to synthesize
such components de novo . C. jejuni adherence,
penetration and transmigration activities were also inhibited at
lower temperatures when investigated at 20C and 4C as
compared to 37C . These data suggest that adhesion,
internalization and translocation of C. jejuni require
active bacterial and host cell processes at optimal
temperature. The current, common opinion is that C. jejuni
can effectively transmigrate in vivo and in vitro, but the
involved mechanisms (paracellular and/or transcellular)
are controversial in the literature (Table 2) and will be
Role of the chosen polarised cell line and variability
among wild-type C. jejuni strains
Most of the polarised cell lines applied to study C. jejuni
transmigration were of human origin (Caco-2, HCA-7, T84
and MKN-28). The majority of utilised C. jejuni strains can
markedly interact with these cells and actively transmigrate
in large quantities within 1-6 h, while a negative control
(non-pathogenic Escherichia coli) did not (Table 2). One of
the most popular and well-investigated cell lines is Caco-2,
and invasive wild-type C. jejuni strains such as 81176,
NCTC11168, F38011 and 81116 also revealed pronounced
capabilities to transmigrate across polarised Caco-2 cells and
represent typical Inv+/Trans+ isolates (Table 2). Based on
these criteria, however, it is not possible to decide if the
strains transmigrate either by the paracellular or transcellular
pathway, respectively. Inv+/Trans+ strains could take either
route while transmigrating non-invasive C. jejuni strains
(Inv-/Trans+) would be limited to the paracellular pathway.
Bras and Ketley  described 6 C. jejuni isolates exhibiting
various phenotypes in the Caco-2 infection model including
Inv+/Trans,+ Inv+/Trans- and Inv-/Trans+ strains,
respectively. This study demonstrates that at least some Inv-/Trans+
C. jejuni strains are to abound, which should travel
exclusively by the paracellular route. In addition, the study shows
that Inv+/Trans- strains exist. This means that invasive C.
jejuni strains do not necessarily exit polarised epithelial cells
at the basal membrane to complete the transmigration step,
but obviously stay within the intracellular environment. In
another study, transcytosis across polarized Caco-2
monolayers was seen in 18 of the 21 colitis-associated C. jejuni
strains, compared with only 11 of the 23 isolates from
noninflammatory diarrhoea . Interestingly, 6 strains from the
latter group had the Inv-/Trans+ phenotype; again in such
cases transcytosis can be unambiguously attributed to a
paracellular passage . Harvey and co-workers 
compared 10 C. jejuni wild-type isolates and showed that they
differ by at least 10-fold in invasiveness and transmigration
across Caco-2 cells. C. jejuni transmigration did not
quantitatively correlate with the intracellular invasiveness of these
isolates and a similar repertoire of strains including Inv+/
Trans,+ Inv+/Trans- and Inv-/Trans+ isolates were found as
described above. Taken together, these data suggest that
different phenotypic wild-type C. jejuni isolates exist in nature
and that bacterial transmigration capabilities may correlate
with colitis disease outcome. However, more studies are
certainly necessary to substantiate this hypothesis.
Transepithelial electrical resistance during C. jejuni
transmigration: changed or unchanged?
A well-established technique to confirm the presence of
tight cell monolayers with proper TJs and to monitor
changes in cell permeability during infection is
measuring the transepithelial electrical resistance (TER)
with electrodes . For this purpose, polarized cell
lines are commonly seeded and differentiated for up to
23 weeks in transwell chambers and TER values are
followed over time and during the course of infection.
However, in a few studies the cells were grown just for
about 1 week or the growth period and TER values were
not provided (Table 2). A common opinion is that bacterial
transmigration by the paracellular route or toxic effects by
the bacteria lead to disruption of TJ and AJ junctional
complexes, consequently TER should drop, thus increasing cell
monolayer permeability. Interestingly, while infection
with Listeria, Shigella, Neisseria and Salmonella reduced
TER substantially over time, infection with the C. jejuni
Inv+/Trans+ strains 81176, F38011 und NCTC11168 did
not influence TER significantly [44,45]. Many other
transmigration studies determined and followed TER during
the course of C. jejuni infection [38,40,43,49,50,84,85]. In
most of these reports, C. jejuni traversed the polarised
cell monolayers without any apparent damage to the host
cells. Commonly, within 48 h almost no difference was
observed in the morphologic appearance of host cells and
TER values of C. jejuni-infected versus non-infected
monolayers [37,38,40,44,50,84,85]. Interestingly, both C. jejuni
Inv+/Trans+ and Inv-/Trans+ strains traversed the Caco-2
cell monolayer without causing apparent cell damage as
judged by TER . This inability to measure TJ disruption
led many researchers to hypothesize that C. jejuni cannot
pass the epithelial barrier by the paracellular route, at least
not at early times of infection [38,43,49,84,85]. In prolonged
studies, some groups observed no TER changes even after
48 h of infection with C. jejuni , while others saw TER
remaining unchanged until 24 h followed by a drop of TER
either after 24 h  or 48 h . The drop of TER in vitro
indicates that opening of TJs and AJs by C. jejuni associated
with massive cell monolayer disruption can occur at late
times of infection. Immunofluorescence microscopy of
polarized T84 monolayers infected for 24 h with C. jejuni
revealed a redistribution of the TJ transmembrane protein
occludin from an intercellular to an intracellular location
associated with a change in phosphorylation . Host
cell degeneration is consistent with the tissue damage and
inflammation found in many cases of campylobacteriosis
in vivo (Table 1).
Electron microscopy reveals C. jejuni within and between
neighboring epithelial cells
Proper monolayers and junction formation in polarized
cells when grown in transwells have been confirmed by
scanning electron microscopy (SEM) or transmission
electron microscopy (TEM), which also illustrated the
presence of microvilli and well-defined brush borders at the
apical cell surface [39,40]. In addition, immunofluorescence
microscopy staining for JAM (a TJ marker) and E-cadherin
(an AJ marker) was used to verify intact cell-to-cell
junctions . SEM and TEM studies were then applied
to investigate the interaction of C. jejuni with polarized
Caco-2 cells. The efficiency of C. jejuni invasion of Caco-2
cells was 2- to 3-fold less as compared to non-polarised
INT-407 cells . Interestingly, only 11-17% of
differentiated Caco-2 cells were found to contain bound or
internalized C. jejuni, and even smaller percentage of Caco-2
cells contained 520 internalized bacteria per cell after
2 h of infection . Furthermore, SEM and TEM
demonstrated that C. jejuni were present extracellularly between
two neighboring Caco-2 cells as well as intracellularly
[39,40]. Similar TEM observations have been obtained by
some other researchers showing intracellular C. jejuni,
which remain tightly surrounded by a host-derived
membrane (the Campylobacter vacuole) after invasion into
epithelial cells in vitro and in vivo [22,25,39,40,43,50,86]. It
was therefore suggested that C. jejuni could translocate
across polarized cell monolayers by passing through
single cells (Figure 3A) and/or between two neighboring
cells (Figure 3B).
Bacterial and host factors involved in C. jejuni
transmigration across polarised cells
The process of C. jejuni transmigration across polarised
intestinal cells is not fully understood because only a
handful putative bacterial and host factors have been
reported yet. The application of pharmacological inhibitors
has indicated that the activity of phosphoinositid-3-kinase
is necessary for C. jejuni transcytosis . The role of
membrane lipid rafts was assessed by pharmacological
depletion of cholesterol and caveolin co-localization
using immunofluorescence microscopy . In addition, it
was shown that C. jejuni transmigration was enhanced
by adding interferon-gamma, probably because of its
TER-reducing capabilities during inflammation . Many
other studies have shown that inactivation of flagellar genes
in C. jejuni resulted in a colonization-negative phenotype in
various animal models [2,8,9]. Early studies using polarized
Caco-2 cells and various flagellar and motility mutants
indicated that either C. jejuni motility or the flagellin gene
products or both appear to be essential for translocation
across the polarized monolayer in vitro . For example,
C. jejuni mutants GRK5 and GRK7 (FlaA FlaB Mot-) and
GRK17 (FlaA FlaB+ Mot-) were unable to cross the Caco-2
cellular barrier as compared to Mot+ control strains .
Another flagellin knockout (flaA/B) and flgF mutants
with Mot- phenotype were also diminished in passing
polarised T84 or MKN-28 cells, respectively [43,44,85]. In
addition, the wild-type strain NCTC12189, a C. jejuni
variant that has reduced motility (Mot/+) despite retaining
intact flagella, was unable to colonise the intestinal tract of
infant mice  and elicited no histological changes in the
rabbit intestinal mucosa nor C. jejuni-positive blood culture
. It was therefore proposed that flagella and associated
motility are the driving forces for colonization, invasion
and transmigration properties of C. jejuni.
The flagellum does not only have a distinct function
in bacterial motility and cell binding, but also acts as a
type III secretion system (T3SS) for the delivery of Cia
(Campylobacter invasion antigens) proteins into the
extracellular space or into the host cell [89-93]. The
first described Cia protein member is CiaB . The
CiaB protein was reported to be translocated into the
cytoplasm of host cells, suggesting that it is a T3SS effector
molecule facilitating invasion . CiaB expression was also
shown to be crucial for the secretion of at least eight other
Cia proteins, ranging in size from 12.8 to 108 kDa, that
were induced upon host cell contact or by the presence of
calf serum . However, the exact function of CiaB is not
yet clear. Interestingly, the invasion-defective ciaB mutant
was able to transmigrate across polarised T84 cells like
wild-type bacteria suggesting that apical cell invasion is
not necessary for C. jejuni transmigration, thus favoring
the paracellular route . Further arguments for the
paracellular route came from competition experiments
with soluble fibronectin and observations that the cadF
mutant (deficient in fibronectin-binding and invasion) also
transmigrated as effectively as wild-type C. jejuni strain
F38011 . This report is counteracted by another
publication showing that a LOS-deficient cstII mutant in
C. jejuni strain GB11 exhibited a strong deficiency of
invasion and transmigration as determined by GPA,
immunofluorescence microscopy and transwell assays, thus favoring
a transcellular route . Unfortunately, in the latter two
studies two different cell systems were used and TER was
not followed over time (Table 2). Thus, differences in these
observations are not yet clear, but could be explained by
Role of serine protease HtrA and E-cadherin cleavage in
C. jejuni transmigration
Recently, another factor was identified to be a novel
virulence determinant in C. jejuni, the serine protease
HtrA (high temperature resistant protein A) . Deletion
of the htrA gene in two strains resulted in strong deficiency
of C. jejuni to travel across polarised MKN-28 cells [44,45].
This mutant was not affected with regard to motility and
flagella production, suggesting that bacterial motility per
se is not sufficient for C. jejuni transmigration , but
htrA-mediated cell binding maybe involved . Another
important new discovery was that HtrA can be secreted
into the cell culture supernatant by C. jejuni, although this
class of proteases has well-known functions as chaperone
and protein quality controllers in the periplasm of E. coli
[44,45]. Secretion of HtrA by C. jejuni was enhanced during
host cell contact or in the presence of calf serum, but was
Figure 3 Models for transepithelial migration across polarised epithelial cells by C. jejuni. Simplified schematic diagram depicting cell
junctions and two considered routes of bacterial travel across a polarized epithelium. The apical surface of the epithelial monolayer faces the
external environment to the gut and forms the first barrier for C. jejuni invasion. Cell junctions important for the structural stability of a polarized
epithelium include the tight junctions, adherens junctions, and matrix receptors as indicated. Various routes for C. jejuni transmigration have been
proposed. (A) The transcellular route is characterized by pathogens crossing the epithelial barrier through entering the cells at the apical surface
and exiting the cells at the basal membrane. (B) The paracellular route is taken by the bacteria entering the epithelium between two
neighboring cells, thus crossing cells through the tight and adherens junctions. Opening of the cell-to-cell junctions maybe a temporal process
and potentially close again after C. jejuni have passed. Basal exiting C. jejuni express the adhesin CadF which can bind to the fibronectinintegrin
complex utilized for invasion from the bottom of epithelial cells.
independent of the flagellar T3SS . Infection studies
and protease assays showed that HtrA cleaves the major AJ
protein E-cadherin on epithelial cells and the recombinant
protein in vitro [44,45]. Interestingly, E-cadherin cleavage
has also been found for the HtrA proteins of other enteric
pathogens including Shigella, Helicobacter and EPEC,
but not for the urogenital pathogen Neisseria .
HtrA-mediated E-cadherin cleavage led to the disruption of
AJs allowing H. pylori or C. jejuni to enter the intercellular
space. In further studies, the HtrA ortholog in H. pylori has
also been shown to cleave another protein, fibronectin,
but C. jejuni HtrA has obviously lost this activity over
fibronectin during evolution [44,96]. The molecular
reason for this observation is unknown, however, it is in
agreement with the concept that fibronectin is a major
basolateral host factor necessary for C. jejuni binding to
integrins and host cell invasion, at least during infection
of non-polarised cells [42,71,72,97,98].
Deletion of htrA or substitution of htrA with a
proteasedeficient S197A point mutant in the bacteria resulted
in severe defects for E-cadherin cleavage and C. jejuni
transcytosis through MKN-28 cells [44,45]. Thus, cleavage
of host junctional proteins like E-cadherin (and probably
other yet unidentified host factors) by secreted HtrA could
explain how C. jejuni may transmigrate intercellularly
between neighboring cells using the paracellular route.
Interestingly, in a time course of C. jejuni infection the
total amount of cell-associated E-cadherin dropped to
some extent, but did not lead to a complete cleavage,
not even in 8h infections . It was therefore proposed
that cleavage of E-cadherin by HtrA could be strictly
controlled, in a temporal and spatial manner, during infection.
Such a localized action of the bacterial protease could
possibly be achieved by restricting secretion of HtrA to the
time point, when the bacteria attach to cell-cell adhesion
sites . As the host cell translation machinery
continuously produces large amounts of E-cadherin, the host cells
can quickly substitute cleaved proteins. This hypothesis
could also explain why no significant reduction in TER
was observed during infection with C. jejuni and suggests
that these bacteria could somehow open and close the
door between two neighboring cells . Such a
mechanism could be analogous to transendothelial
migration of neutrophils, which transmigrate effectively from the
bloodstream to the site of infection and do not cause any
damage to the endothelial cells . If our hypothesis turns
out to be true, it may represent a clever novel infection
strategy for transmigration of pathogens such as C. jejuni
across polarised host epithelial cells.
Translocation studies of other Campylobacter species
Studies on the epithelial transmigration of Campylobacters
other than C. jejuni are rare in the literature, with only a
few reports on C. fetus, C. rectus and C. coli. In a first study,
the ability to translocate across epithelial barriers has
been investigated during infection with C. fetus, a
recognized pathogen of cattle, sheep and humans . Using
cultured Caco-2 cells, C. fetus was found to translocate
efficiently within 24h without altering TER, similar to
C. jejuni as discussed above. C. fetus was also observed
to invade and subsequently egress from Caco-2 cells as
shown in a modified GPA procedure and this occurred
independently of C. fetus S layer expression . SEM
and TEM studies revealed the presence of C. fetus both
at apical and basal surfaces as well as in intracellular
locations, but not in the paracellular space.
Pharmacological inhibitor studies demonstrated the requirement
of a functional tubulin cytoskeleton, and together with the
TEM data support a transcellular mechanism for C. fetus
transmigration across Caco-2 monolayers . Thus, the
ability to invade and subsequently egress may contribute
to establishing C. fetus infections in various hosts and
can explain bacterial recovery from extraintestinal sites
. In a second report, C. rectus, a periodontal pathogen
associated with human fetal exposure and adverse
pregnancy outcomes including preterm delivery, was
investigated . Infection experiments in pregnant
BALB/c mice have demonstrated that C. rectus can
translocate from a distant site of infection to the placenta
where it can induce fetal growth restriction and impairs
placental development . Infection with C. rectus was
detected in 63% of placentas after two weeks and
significantly decreased fetoplacental weight. In invasion
assays, C. rectus was able to effectively invade human
trophoblasts in vitro (but not trophoblasts of murine origin),
and showed a trend for higher invasiveness as compared
to C. jejuni. Interestingly, C. rectus infection significantly
upregulated IL-6 and TNF- levels in a dose-dependent
manner in human trophoblasts, but not in murine cells,
suggesting a correlation between invasion and cytokine
activation . It was proposed that the invasive trait of
C. rectus in human trophoblasts may play a role in
facilitating bacterial translocation and placental inflammation
during early gestation . A third report investigated
C. coli, which is the dominant Campylobacter species
commonly found in pigs . An experimental trial
was conducted to evaluate the colonisation and
translocation ability of the porcine C. coli strain 5981 in weaned
pigs over 28 days. Excretion of C. coli 5981 was seen for
all piglets 7 days after inoculation and highest counts were
detectable on day 10 . Post-mortem, of luminal C. coli
was observed for gut tissues of the small intestine and
for the gut associated lymphatic tissues, such as jejunal
mesenteric lymph nodes and tonsils as well as for spleen
and gall bladder. In conclusion, this trial indicates that
C. coli exhibit translocation and invasion capabilities in pigs
making it a useful model system to study colonisation and
pathogenicity of this pathogen .
Different mechanisms are used by various enteropathogens
to transmigrate across the host intestinal epithelium,
including transcytosis through specialized M cells,
phagocytosis by interepithelial leukocytes or transcytosis of
enterocytes [35,81,82]. C. jejuni is a predominant zoonotic
pathogen causing enterocolitis in humans worldwide.
However, despite the high prevalence of C. jejuni induced
disease and research progress made in recent years, our
knowledge is still relatively limited as compared to other
invasive pathogens such as Salmonella, Listeria or Shigella.
A series of studies on human biopsies and animal infection
experiments have demonstrated that C. jejuni is able to
cross the intestinal epithelial barrier and enter underlying
tissues, bloodstream and even other organs (Table 1).
However, the mechanism of C. jejuni translocation is
controverse and not well understood. There is only one
report that M cells in the Peyer's patches may facilitate
transport of C. jejuni from the intestine in rabbits .
From in vitro studies it seems clear that C. jejuni
transmigration across polarised cultured cells requires de
novo protein synthesis and depends on functional flagella.
Unfortunately, there is no consensus on the transepithelial
route followed by C. jejuni, both the transcellular and
paracellular routes have been described (Table 2). If apical
binding of C. jejuni to epithelial cells is a prerequisite for
subsequent invasion and transcellular migration is also
unclear. There is rapid increase in reports on putative
bacterial adhesion factors we have now a list of more
than 20 bacterial factors with proposed role in binding
and subsequent invasion . In contrast, there is a
large gap in our knowledge on corresponding host cell
receptors. Thus, there is an urgent need for identifying
and characterizing host receptors which can be attributed
to certain bacterial factors. The only receptor pathway
intensively studied and verified by various independent
research groups is the CadFfibronectinintegrin
signaling cascade [42,71,72,97,98,104]. These studies have
presented high resolution SEM pictures of various invading
C. jejuni strains (showing details of the invagination process)
in multiple non-polarised cell types [71,97,98], but
corresponding qualitative and quantitative SEM data for a
set of C. jejuni strains invading polarised cells from apical
or basal membranes are currently not available. Alternative
possibilities include the involvement of ganglioside-like
LOS in apical invasion, thus favoring a transcellular route
, but this model is in contrast to the paracellular model
for HtrA-mediated opening of AJs and basal invasion as
triggered by the CadFfibronectinintegrin complex
[44,45]. How C. jejuni can open the TJs after longer
coincubation times is yet unclear. Several studies exist that
could support the apical invasion model, but can C. jejuni
also enter host cells from basal surfaces? Basal engulfment
and entry of C. jejuni into non-polarised Chang or polarised
Caco-2 cells has been demonstrated by TEM and
immunofluorescence microscopy, and this process has been called
subvasion [79,105]. However, if paracellular transmigration
is a prerequisite for subvasion in polarised cells is not
yet clear. Furthermore, it is also unclear how the
T3SSdependent injection of certain Cia proteins fits in any
of the above models. Thus, more studies are clearly
required to unravel the sequence of events that allow
C. jejuni strains to travel across polarised intestinal
epithelial cells, either by a transcellular or paracellular
pathway or a mix of both. It should be also considered
that individual C. jejuni strains might switch from one
to the other mode under specific culturing or infection
conditions. Finally, besides the commonly applied transwell
system, a vertical diffusion chamber model system has been
recently described, which creates microaerobic
conditions at the apical surface and aerobic conditions at the
basolateral surface of cultured intestinal epithelial cells,
thus producing an in vitro system that probably closely
mimics in vivo conditions of the human intestine .
The use of this vertical diffusion chamber for studying
the interactions of C. jejuni with intestinal epithelial cells
demonstrated the importance of performing such
experiments under conditions that converge to the in vivo
situation and will allow novel insights into C. jejuni
pathogenic mechanisms . In addition, it should be noted
that most of the cell lines used for in vitro studies are
already transformed because they originate from cancer
patients. A possible alternative option came from recent
studies demonstrating that single Lgr5 (the receptor for the
Wnt-agonistic R-spondins)-positive stem cells isolated from
the intestine can grow in culture into complex epithelial
organoid structures, called miniguts, which retain their
original organ identity [107-109]. This important example of
self-organization could be used for stem cell research and
regenerative medicine, but also disease modeling of
infections with enteric pathogens including Campylobacter. It
therefore appears that transmigration of C. jejuni and that
of many other Campylobacter species will continue to be a
fascinating and rewarding research subject in the future.
The authors declare that they have no competing interests.
SB performed the data collection and wrote the text. MB designed Table 1
and SW designed Table 2. NT was drawing the figures and wrote the figure
legends. All authors discussed, read and approved the text including the
The work of SB is supported through a DFG grant (project B10 of CRC-796).
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