Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori
Cell Communication and Signaling
Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori
Steffen Backert 0
Marguerite Clyne 1
Nicole Tegtmeyer 0
0 University College Dublin, School of Biomolecular and Biomedical Sciences, Science Center West , Belfield Campus, Dublin-4 , Ireland
1 University College Dublin, School of Medicine and Medical Science, Health Science Center , Belfield Campus, Dublin-4 , Ireland
Helicobacter pylori is a highly successful pathogen uniquely adapted to colonize humans. Gastric infections with this bacterium can induce pathology ranging from chronic gastritis and peptic ulcers to gastric cancer. More virulent H. pylori isolates harbour numerous well-known adhesins (BabA/B, SabA, AlpA/B, OipA and HopZ) and the cag (cytotoxin-associated genes) pathogenicity island encoding a type IV secretion system (T4SS). The adhesins establish tight bacterial contact with host target cells and the T4SS represents a needle-like pilus device for the delivery of effector proteins into host target cells such as CagA. BabA and SabA bind to blood group antigen and sialylated proteins respectively, and a series of T4SS components including CagI, CagL, CagY and CagA have been shown to target the integrin b1 receptor followed by injection of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine may also play a role in the delivery process. While substantial progress has been made in our current understanding of many of the above factors, the host cell receptors for OipA, HopZ and AlpA/B during infection are still unknown. Here we review the recent progress in characterizing the interactions of the various adhesins and structural T4SS proteins with host cell factors. The contribution of these interactions to H. pylori colonization and pathogenesis is discussed.
Helicobacter pylori; adherence; adhesin; integrin; receptor; signalling; type IV secretion
H. pylori colonises the stomach of about half of the
human world population, which is associated with
chronic, often asymptomatic gastritis in all infected
individuals. Depending on various criteria, more severe
gastric diseases including peptic ulcer disease can occur in
up to 10-15% of infected persons [1-3]. H. pylori
infections are commonly diagnosed with a strong
inflammatory response, but the bacteria evolved numerous
mechanisms during evolution to avoid recognition and
clearance by the host defence machineries, and if not
treated with antibiotics, they can persist for life. H.
pylori-induced gastritis is the strongest singular risk
factor for developing cancers of the stomach; however,
only a small proportion of infected individuals develop
malignancy such as mucosa-associated lymphoid tissue
(MALT) lymphoma and even gastric adenocarcinoma
[1-3]. Gastric adenocarcinoma constitutes the second
leading cause of cancer-associated death worldwide, and
about 700,000 people die from this malignancy annually
. The clinical outcome of infection with H. pylori is
dependent on a very complex scenario of host-pathogen
crosstalk. Disease progression is determined by various
factors including the genetic predisposition of the host,
the bacterial genotype and environmental parameters
[1-3]. The cellular and molecular mechanisms developed
by H. pylori to undermine host defence strategies have
been under intense investigation worldwide.
Clinical H. pylori strains are highly diverse both in their
genetic information and potential to induce pathogenicity.
Myriads of bacterial factors have been reported to
influence the pathogenesis of H. pylori infections. There are
two classical virulence determinants expressed by
H. pylori, the CagA protein encoded by the
cytotoxinassociated genes pathogenicity island (cagPAI) and the
vacuolating cytotoxin (VacA). Secreted VacA can trigger
various responses including pore formation in the host
cell membrane, modification of endo-lysosomal
trafficking, cellular vacuolation, immune cell inhibition and
apoptosis. VacAs activities are highlighted in several
review articles [1-4] and will not be discussed here. In
the mid nineties, the cagPAI was entirely sequenced from
various H. pylori strains and found to represent a 40-kb
DNA insertion element in the chromosome, which is
flanked by 31-bp direct repeats and carrying up to 32
genes [5,6]. Large scientific interest concentrates on the
CagA protein which is present in more virulent isolates,
but is typically absent in less virulent H. pylori strains.
Thus, CagA serves as a virulence marker for the cagPAI
[7,8]. Work in the last ten years has shown that the
cagPAI encodes type-IV secretion system (T4SS) which
injects CagA into target cells where it interferes with
multiple host cell signaling cascades [9,10]. Other
welldescribed pathogenicity-associated mechanisms include
flagella-driven bacterial motility, urease-mediated
neutralization of pH, HtrA-mediated cleavage of E-cadherin,
modification of host cell cholesterol, shedding of
outermembrane vesicles and peptidoglycan-dependent immune
responses [1-3,11-13]. In addition, H. pylori carries
several classical surface adhesins permitting tight adherence
of the bacteria to gastric epithelial cells. Here we review
the various molecular adhesion strategies of H. pylori to
gastric epithelial target cells which facilitate bacterial
binding. We also discuss the structure and function of
the T4SS, and how it makes contact with host cell
surface factors to inject the CagA effector protein.
Role of the classical H. pylori adhesins
Intensive research in recent years has demonstrated
that H. pylori encodes a broad set of various adhesion
factors, for some of which the corresponding host cell
receptor(s) have been identified (Table 1). The H.
pylori genomes from various strains contain more
than 30 genes which encode outer membrane proteins
(OMPs) that have been divided into Hop
(Helicobacter outer membrane porins) and Hor (hop-related)
subgroups. The Hop family of proteins includes
several well described adhesins of H. pylori such as
BabA, SabA, AlpA/B, HopZ and OipA. However,
among clinical strains of H. pylori considerable
diversity in the expression of OMPs exists. This is thought
to reflect a selective pressure for bacterial adhesion
which may differ both across and within infected
individuals over time. It has been shown that some of
the classical adhesion molecules discussed below act
in conjunction with factors from the cagPAI in order
to highjack several host cell processes including
altered transcription, cytoskeletal rearrangements,
opening of cell-to-cell junctions, onset of
inflammation and others as summarized in a simplified model
Table 1 Characteristics of cagPAI-independent and cagPAI-dependent host cell adhesion factorsa
AGS, GD25 vs. GD25b1,
HeLa, mouse fibroblasts
RIA and Scatchard analysis
Glycoconjugate array binding studies
Bacterial adherence assays
Flow cytometry, Biacore binding studies,
surface adherence assay
AGS cell adhesion assays
Y2H, PD with magnetic beads, FACS
Biacore binding studies, Peptide competition, [61,81]
cell adhesion assays, AB blocking, CF
Y2H, PD with magnetic beads, FACS
a Abbreviations: AB (antibody); CF (cellular fractionation); FACS (Fluorescence-activated cell sorting); IF (co-localization by immunofluorescence); PD (pull-down
experiments); RIA (Radioimmuno assay); Y2H (yeast two hybrid screen).
The OMP member BabA was the first H. pylori adhesin
discovered. BabA mediates binding of the bacteria to
Lewis B antigens, Leb  and related terminal fucose
residues found on blood group O (H antigen), A and B
antigens  that are expressed on the gastric mucosa.
Multiple chromosomal loci and alleles for BabA have been
reported to exist and Leb binding activity was shown to be
facilitated by the BabA2 allele . However, more recent
work suggests that BabA1 alleles occur only very rarely
and are difficult to detect  Substantial amino acid
polymorphisms exist among BabA proteins expressed by
different strains . Diversity also appears with respect to
the binding of strains to Leb and BabA expression, and
binding affinity for A, B and O antigens correlates with
blood group antigen expression of the host [15,18]. A
closely related gene babB, encodes for a translation product
which has significant N- and C-terminal similarity to
BabA. BabA and babB are nearly identical in their 5 and
3 regions but there is sequence divergence in their mid
region  indicating that the central variable regions
likely encode unique functions. Many Leb non-binding
strains express silent babA gene sequences which may
ASHlpaoAbpA/ZB,,, 1 Apical colonization
become activated by recombination into the babB locus
forming chimeric babB/A genes . BabA expression in
vivo, however, appears to be highly dynamic. Experimental
infection of Rhesus Macaques, mice and Mongolian gerbils
resulted in loss of BabA expression and Leb binding
[21,22]. Post-infection strains isolated from gerbils
contained a BabA2 protein that was modified by six amino
acids from the strain used for inoculation.
Complementation experiments confirmed these six amino acid residues
are critical for binding to fucosylated blood group antigens
. In a recent study, experimental infection of
Mongolian gerbils resulted in complete absence of expression of
BabA at six months post-infection. Loss of BabA
expression was attributable to nucleotide changes within the
babA gene that resulted in a truncated BabA translation
product . BabA-mediated adherence of H. pylori to
Leb on the surface of epithelial cells has been shown in
vitro, using Leb transfected MDCK cells, and in vivo, using
infection of Mongolian gerbils, to augment
cagPAI-dependent H. pylori pathogenicity by triggering the production
of proinflammatory cytokines and precancer-related
factors  (Figure 1A). Thus, the expression of the BabA
adhesin seems tightly connected to the onset of
T4SSFigure 1 H. pylori interactions with epithelial cells highlighting the roles of adhesins and the type-IV secretion system (T4SS) encoded
by cagPAI. (A) (1) H. pylori adhesins mediate apical binding to known and unknown receptors on gastric epithelium and probably also direct
signal transduction as indicated. (2) Upregulation of transcription factors such as NF- B leads to production of pro-inflammatory cytokines and
chemokines. (3) Secretion of mediators at basolateral surfaces attracts immune cells to the site of infection. (4) Upon host cell contact, H. pylori
assembles T4SS pili at their surface enabling delivery of molecules, CagA and peptidoglycan, from bacterial cytoplasm into host cells. cagPAI
proteins (CagA, CagI, CagL and CagY) interact with integrin receptors. Interactions with phosphatidylserine (PS) and cholesterol in lipid rafts are also
involved in T4SS processes. T4SS and CagA are involved in numerous cellular effects including disruption of cell-to-cell junctions (5), cytoskeletal
rearrangements (6) and nuclear signalling (7). (B) Two models for the assembled T4SS machinery in H. pylori are proposed. Model-1 assumes
VirB111 proteins, the coupling factor VirD4 and accessory factors such as CagF (a proposed chaperone of CagA) assemble in a similar fashion to that
proposed for A. tumefaciens T4SS . Model-2 assumes that the T4SS requires the same VirB/D proteins as model-1 with two major differences.
The T4SS pilus surface is covered with CagY (VirB10) molecules and VirB5 is excluded . H. pylori VirB10 is a very large protein (~250 kDa) carrying
two transmembrane domains to form a hairpin-loop structure in the pilus as depicted . Immunogold labelling of the loop region in CagY
indicated that this is exposed to the extracellular space and is transported to the pilus surface by an unknown mechanism . Abbreviations: AJ
(adherens junction); HtrA (High temperature requirement A protease); Leb (Lewis B antigens); MF (macrophage); NTP (nucleotide triphosphate);
NDP (nucleotide diphosphate); P (phosphate group); SDL (sialyl-dimeric-Lewis glycosphingolipid); TJ (tight junction).
related host cell responses in vivo. The presence of babA is
associated with cagA and vacA s1 alleles, and strains that
possess all three of these genes incur the highest risk of
gastric cancer development .
for human IL-8) and IL-6 . In contrast to these
results, in another recent study alpA and alpB gene
mutants of H. pylori SS1 induced more severe
inflammation than the parental strain in infected gerbils .
Expression of sialyl-dimeric-Lewis glycosphingolipid is
upregulated upon infection with H. pylori and
inflammation. This molecule also acts as a receptor for the
pathogen and binding is mediated through the bacterial OMP
member, SabA . No binding to gangliosides was
obtained with a SabA-negative mutant strain using a thin
layer chromatography overlay assay . Infection of the
gastric epithelial cancer cell line MKN45 with H. pylori
upregulated expression of the gene encoding 3 GlcNAc
T5, a GlcNAc transferase essential for the biosynthesis of
Lewis antigens. Overexpression of this gene in both the
MKN45 and AGS gastric adenocarcinoma cell lines lead
to expression of the SabA ligand, sialyl Lex, suggesting
that H. pylori can modulate receptor expression .
SabA has been identified as a haemagglutinin that binds
to sialylated structures found on the surface of red blood
cells and there is a good correlation among strains
between sialic acid dependent haemagglutination and
sialyl Lex binding . Like observations with BabA, a high
level of polymorphism was reported in sialyl binding
properties among clinical H. pylori isolates, which
suggests that SabA adapts to its host depending on the
mucosal sialylation pattern of the infected individual
. SabA has also been shown to mediate binding of H.
pylori to sialylated moieties on the extracellular matrix
protein laminin .
Two strongly homologous genes termed alpA and alpB
were also characterized and shown to encode for
integral OMPs. Adhesion experiments indicated that they
are also involved in adherence of H. pylori to human
gastric tissue biopsies . OMP expression profiling of
200 strains from Germany revealed that virtually all
clinical isolates produced the AlpA and AlpB proteins in
contrast to many other OMPs that were produced at
very variable rates . Recently both AlpA and AlpB
proteins have been shown to bind to mouse laminin in
vitro and plasmid-borne alpA conferred laminin-binding
ability on E. coli . No other binding partners or
receptors for AlpA and AlpB have yet been identified.
The alpA/B locus has also been shown to influence host
cell signaling and cytokine production upon infection.
Deletion of alpA/B genes reduced IL-8 induction during
infection with East Asian but not with Western H. pylori
strains . The alpA/B mutants poorly colonized the
stomachs of C57BL/6 mice and were associated with
lower mucosal levels of induced KC (the mouse name
OipA (outer inflammatory protein A), encoded by the
hopH gene, was initially identified as a surface protein
that promoted IL-8 production in a T4SS-independent
fashion . OipA expression by H. pylori was shown to
be significantly associated with the presence of duodenal
ulcers and gastric cancer, high H. pylori density, and
severe neutrophil infiltration . Later studies
identified that hopH knockout mutant strains adhered
significantly less to gastric cancer cell lines, AGS and Kato-III,
than wild-type strains, and complementation of the
hopH gene restored the adherence properties of the
hopH mutant . The presence of oipA has also been
shown to clearly enhance production of IL-8 in vitro but
only in the presence of the cagPAI . Further insights
came from infection studies for up to 52 weeks in the
Mongolian gerbil model system. All infected gerbils
developed gastritis; however, inflammation was
significantly attenuated in animals infected with the cagA,
but not the single vacA or oipA strains .
However, inactivation of oipA decreased nuclear localization
of b-catenin, a factor involved in transcriptional
up-regulation of genes implicated in carcinogenesis, and
reduced the incidence of cancer in the gerbils. OipA
expression was detected significantly more frequently
among H. pylori strains isolated from human subjects
with gastric cancer precursor lesions versus persons
with gastritis alone . The host receptor for OipA,
however, remains unknown.
The hopZ gene encodes a protein which was shown by
immunofluoresence to be located on the surface of the
bacteria. A knockout mutant strain showed significantly
reduced binding to the AGS cell line, compared to the
corresponding wild-type strain . Lack of production
of HopZ did not affect the ability of the bacterium to
colonize the stomachs of guinea pigs . However, a
role for HopZ in colonization in vivo has recently been
proposed as deletion of hopZ reduced the ability of H.
pylori to survive in a germ-free transgenic mouse model
of chronic atrophic gastritis . In addition, one of the
few differences identified in H. pylori strains isolated
from infected volunteers, was an OFF/ON switch in the
phase-variable hopZ gene suggesting strong in vivo
selection for HopZ during colonization . Similar to
OipA, the host receptor for HopZ has not yet been
identified and will be a major challenging aim for future
Role of the cagPAI in cell adhesion and T4SS
Composition of the H. pylori T4SS apparatus
The T4SS in the cagPAI belongs to a large group of
transmembrane transporters that are ancestrally related
to plasmid DNA conjugation systems of Gram-negative
bacteria and have been found in many pathogenic and
non-pathogenic organisms [9,43,44]. Although
evolutionary conserved, T4SSs are functionally heterogenous
in respect to both the delivered substrate (DNA-protein
complexes or proteins) and the involved recipients.
Recipients can be either bacteria of the different or
same species, or species from other kingdoms including
plants, fungi and mammalian cells. Besides H. pylori,
T4SSs have also been found in Agrobacterium,
Legionella, Bartonella, Bordetella and other pathogens, and
typically consist of a distinct set of VirB/D proteins. The
latter include the VirB1-VirB11 components and the
socalled coupling factor, the NTPase VirD4. The
agrobacterial T-DNA system is the prototype of a T4SS
transporter and its VirB proteins have been classified into
three groups: (i) the putative core or channel subunits
(VirB6-10), (ii) the energetic components (the NTPases
VirB4 and VirB11) and (iii) the pilus-associated proteins
(VirB2, and possibly VirB3 and VirB5). VirB1 is a
proposed transglycosylase for limited lysis of the murein
layer at the T4SS assembly site in the membrane
[45,46]. In case of the H. pylori T4SS, all orthologs of
the 11 VirB proteins and VirD4 as well as some
accessory factors have been identified to be encoded by the
cagPAI [10,47,48]. Mutagenesis of all individual cagPAI
genes revealed at least 14 essential and two accessory
components while some other genes are not required
for injecting CagA [9,49,50]. The function of many
accessory T4SS factors is not yet clear, however, the
role of CagF and CagD was recently elucidated. CagD
appears to serve as a potential multifunctional
component of the T4SS which may be involved in CagA
injection at the inner membrane and may localize outside
the bacteria to promote other responses in host cells
. In addition, CagF is a chaperone-like protein for
CagA that binds close to the carboxy-terminal secretion
motif of the effector protein, which is important for its
translocation by the T4SS [52,53]. Further studies using
yeast two-hybrid technology, fractionation and
immunoprecipitation approaches identified selective interactions
of numerous cagPAI proteins which are likely to have
an important role in early T4SS assembly steps [50,54].
Crystal structure of the T4SS core complex and several
A major contribution to our current knowledge about
T4SS nanomachineries in bacteria came from resolution
of crystal structures of the T4SS-core from plasmid
pKM101 [45,55]. Three proteins (VirB7, VirB9 and
VirB10) assemble into a ~1 megadalton structure
spanning the inner and outer membranes. This core
structure consists of 14 copies of each of the proteins and
forms two layers, inserting in the inner and outer
membrane, respectively . The crystal structure of a ~0.6
megadalton outer-membrane complex containing the
entire O layer was solved at higher resolution .
Comparison of the structures points to conformational
changes regulating T4SS channel opening and closing,
which could be involved in the transport of effector
molecules [45,55]. In addition to these major findings,
the crystal structures of four individual structural
cagPAI proteins have been reported. The structure of
VirB11 revealed a hexameric ring complexed with the
regulatory protein HP1451, which functions as a gating
factor in the inner membrane, proposed to cycle
through closed and open conformations as triggered by
ATP-binding and ATP-hydrolysis, respectively [56-58].
The crystal structures of CagS, a 23-kDa protein coded
by a well-conserved cagPAI gene whose exact function
remains elusive, and CagZ, a 23-kDa protein involved in
the translocation of CagA, have also been solved [59,60].
Moreover, the structural characterization of CagD
indicated that it exists as a covalent dimer in which each
monomer folds as a single domain that is composed of
three a-helices and five b-strands . In addition, the
structure of CagL has been modelled based on the
crystal structure available from TraC of pKM101, another
VirB5 ortholog . CagL seems to form a three
a-helical bundle structure with an exposed domain, which is
in agreement with its published circular dichroism (CD)
spectrum that revealed ~65% helical sequences .
Finally, the 2.2- crystal structure of a
carboxyterminal part of CagA in complex with one of its
cellular targets, the human kinase Par1b/MARK2, was
recently solved . The CagA peptide interacted
with the kinase as an extended coil. The visible
14amino acid peptide sequence spanned the
FPLKRHDKVDDLSK motif, a sequence occurring
twice in the crystallized CagA construct. This CagA
peptide was named MKI (for MARK2 kinase inhibitor)
in analogy to PKI, a well-described peptide inhibitor
of protein kinase A. Interestingly, the manner in
which the MKI sequence of CagA binds in the
substrate-binding cleft of Par1b/MARK2 is reminiscent of
the manner by which PKI binds to and inhibits PKA.
Taken together, the first CagA substructure revealed
that it mimics host cell kinase substrates, using a
short MKI peptide to attach to the substrate binding
site of Par1b/MARK2 . However, injected CagA
also interacts with many other host cell proteins,
involved in multiple signalling cascades (Figure 1A),
which are discussed elsewhere [7,8].
Structure of the T4SS apparatus in live H. pylori
Electron microcopy of infecting H. pylori has
demonstrated that assembly of the T4SS is induced after host
cell contact and represents a needle-like structure
extending from the bacterial outer membrane, also
called T4SS-pili [61,63,64]. These pili are proposed to
consist of CagC, which was identified as the major
VirB2 pilin subunit ortholog , however, direct
labeling of the pili with a-VirB2 antibodies was not yet
demonstrated. Studies using antibody-labelling with
immunogold have shown that the bacterial protrusions
are decorated by VirB10 (CagY)  and VirB5 (CagL)
. CagY proteins are about 250-kDa in size and can
differ enormously in size between strains and changes
size during colonization of a given host. In-frame
deletions or duplications rearrangements in VirB10 can
result in reduced host antibody recognition which may
allow immune evasion . The T4SS-needle base can
be stained with antibodies against VirB7 (CagT) and
VirB9 (CagW) proteins [63,64]. In addition,
immunogold-staining indicated the presence of CagA at the tip
of the appendages, providing the first direct evidence
that CagA may be indeed delivered through the pilus,
an observation not yet reported for T4SS substrates in
other bacteria . However, transport of CagA through
the T4SS is proposed to occur by an energy-dependent
mechanism stimulated by the concerted action of
NTPases VirB4, VirB11 and VirD4 [46,56,67].
There are two T4SS-pilus assembly models proposed
for H. pylori. As outlined above, all orthologs of the 11
VirB proteins and VirD4 have been identified to be
encoded in the cagPAI as well as some accessory
proteins , leading to a T4SS model similar to that of
the agrobacterial T4SS (Figure 1B, left). In line with
these conclusions, immunogold-labelling studies
indicated that the tips of the T4SS pilus are decorated with
CagL/VirB5 , which exhibited a similar distribution
of VirB5 orthologs on the T4SS pilus in Agrobacterium
. In a second model (Figure 1B, right), it has been
proposed that the appendages in H. pylori are covered
locally or completely by CagY  and the T4SS
includes all VirB components, except VirB5 .
Remarkably, CagY is a very large protein containing two
transmembrane segments with the mid region (also
called the repeat domain) exposed to the extracellular
side like a hairpin-like structure . As described
above, VirB10 forms the inner core in a common T4SS
model [45,55], but H. pylori CagY/VirB10 clearly differs
from their counterparts in other T4SSs . Thus,
further studies are necessary to investigate if the T4SS
pilus in H. pylori is more similar to that in
Agrobacterium (mainly composed of VirB2 and VirB5 subunits) or
if it is made-up of CagY as major pilus protein, or if it
is a mix of both (Figure 1B).
The function of the T4SS depends on the used cell
Although H. pylori is a stomach-specific pathogen in
humans, infection studies in vitro have shown that
CagA can be injected into many different cell types. A
summary of human cell types with reported
susceptibility for the uptake of T4SS-delivered CagA in vitro is
shown in Table 2. The major criterium for successful
translocation in a given cell line is that CagA undergoes
tyrosine phosphorylation (CagAPY) by host kinases of
the Src and Abl family [70-73], which is commonly
monitored in cell lysates or immunoprecipiates (IPs)
using monoclonal phosphotyrosine-specific antibodies
(Table 2). Interestingly, various studies noted significant
cell type-specific differences in CagAPY levels during
infection of human cell lines. In addition, injected CagAPY was
reported for some cell types from mice and monkeys
(Table 3), while selected other cell lines from humans,
hamster or dog appeared to be resistant for detection of
CagAPY (Table 4). As controls, in vitro phosphorylation
experiments of CagA with various cell lysates indicated
that the kinases are active and strongly phosphorylated
CagA . Thus, the variation in CagAPY levels during
infection evidently resulted from different levels of CagA
translocation [74,75]. There are several scenarios that may
explain the observed host cell specificity. One potential
explanation is that specific host cell factors might be
required to activate the T4SS. This activation could
operate at the level of protein expression. For example, this is
the case for the type-III secretion apparatus in Yersinia
species . However, CagA is one of the most abundant
proteins in the proteome of H. pylori even in the absence
of host cell contact  indicating that the translocation
process is repressed, rather than a CagA expression effect.
Indeed, despite its abundant presence, CagA is not
secreted into the supernatant . This represents a clever
resource-saving strategy reminiscent to Shigella species,
where effector proteins are stored in the bacterial
cytoplasm before contacting host cells. In the latter case,
translocation is triggered by a variety of factors, such as
extracellular matrix proteins, bile salts or Congo red .
It was therefore proposed that the H. pylori T4SS might
be similarly activated by specific factors exposed on the
surface of specific target cells .
The T4SS receptor hypothesis: role of host cell
Despite the above reports, it was assumed for many
years that CagA can be randomly injected into gastric
epithelial cells. However, this is obviously not the case
because more recent studies showed that numerous host
cell surface molecules are required for T4SS function,
suggesting a sophisticated control mechanism through
which H. pylori injects CagA . The first identified
Table 2 Reported phosphorylation/injection of CagA in human cell linesa
Cell line Origin H. pylori strains used
host receptor for the T4SS was integrin b1 . Based
on a series of experiments including the use of integrin
b1 knockout cell lines (GD25 and GD25b1), gene
silencing RNAs, function-blocking antibodies and
competition experiments with a well-known integrin b1
bacterial adhesin (InvA from Yersinia), compelling
evidence was provided that integrin b1 plays a crucial role
for injection of CagA during infection of several
nonTable 4 Cell lines with reported resistance for phosphorylation/injection of CagA a
H. pylori strains used
P1, P12, 26695, P310
a Abbreviations: anti-PY WB (Westernblotting of infected cell lysates using several pan-phospho-tyrosine antibodies); IPA (in vitro phosphorylation assays of CagA
with cell lysates to ensure that active kinases are present in the cells of interest); SI (synchronised infection assays: bacteria were centrifuged onto Hek293 cells to
ensure proper contact of bacteria with these host cells).
polarised AGS and mouse cell lines . In line with
these observations, various structural T4SS proteins
have been demonstrated to bind to integrin b1 in vitro,
including CagL, CagA, CagI and CagY (Table 1).
However, while very little is known about interactions of
CagA and CagI with integrin, CagL has been
investigated intensively. Like the human extracellular matrix
protein fibronectin, CagL carries a RGD-motif shown to
be important for interaction with integrin b1 on host
cells, as well as downstream signaling to activate
tyrosine kinases including EGFR, FAK and Src .
However, mutation of the RGD-motif in CagL revealed no
reduction of injected CagAPY during infection in
another study . Another unsolved question is the
structure of CagY with respect to which domain is
exposed to the extracellular space. While the repeat
domain in the middle of CagY on bacteria has been
shown to be accessible to recognition by antibodies ,
in vitro binding studies and yeast-two hybrid screens
revealed that the very carboxy-terminus interacted with
integrin b1 . However, although it seems clear that
each of the above factors exhibits an important
functional role for injecting CagA, their interaction
capabilities with integrin b1 during infection are unknown, and
need to be investigated in future studies.
Role of phosphatidylserine and cholesterol for
injection of CagA
Another factor interacting with T4SS functions emerged
from a recent study indicating that CagA binds directly
to phosphatidylserine (PS) of the host cell and that this
interaction is involved in CagA delivery into AGS cells
based on saponin-fractionation experiments . In
vitro, the full-length protein binds to PS and this
required a K-Xn-R-X-R motif present in the central
region of CagA . During H. pylori infection, the
PSbinding protein annexinV and anti-PS antibodies both
reduced CagA injection levels , suggesting that
bacterial contact with PS is important for CagA delivery
across the host membrane. Mutagenesis experiments
showed that two arginine residues (R619 and R621) in
the above motif are crucial for binding of purified CagA
to PS and ensure membrane localization of transfected
CagA in polarized MDCK cells . Murata-Kamiya
and co-workers proposed that the reported
CagL-b1integrin interaction may stabilize the CagA-PS
interaction and may contribute to internalization of PS-bound
CagA into host cells through activation of integrin
signaling . However, the injection mechanism of CagA
into polarized MDCK cells is not completely clear
because many other studies performed CagA
transfection or biochemical fractionation experiments, but in
most cases did not investigate phosphorylation of CagA
[83-87]. The situation becomes even more puzzling
because another group reported that MDCK cells are
resistant to injection/phosphorylation of CagA upon
infection . Thus, the exact mechanism of CagA
injection into polarized host cells requires further
elucidation. Nevertheless, these findings point to the lipid
bilayer in the host cell membrane as a second platform
for T4SS-host cell interplay, rather then a piercing"-like
injection mechanism by the T4SS-pilus. Interestingly,
recent findings from other groups indicated that
cholesterol in lipid rafts, another component of the lipid
bilayer, is also required for CagA translocation and
proinflammatory signaling [88,89]. Taken together, these
studies indicate that there are at least three host cell
factors being involved in proper T4SS functions of H.
H. pylori is one of the most successful human
pathogens. Studies of host-bacterial interactions using their
fundamental adhesins and the virulence factors CagA
and T4SS have provided us with many detailed insights
in processes ultimately connected to H. pylori
colonisation and pathogenesis. The current opinion implies a
model in which the major adhesins BabA, SabA and
others make the initial and sustained host cell contact
important for bacterial colonisation. Once intimate
contact is established, the T4SS further interacts with
specific host cell surface molecules including integrins and
PS to facilitate injection of CagA, probably in
cholesterol-rich microdomains on host cells, the lipid rafts.
The above discussed studies indicate that at least three
known host factors are involved in CagA injection
(integrin, PS and cholesterol). However, it also seems
clear that some cell lines are resistant to injection of
CagA indicating that none of the reported host factors
alone can fascililtate the injection process. We therefore
assume that the T4SS injection mechanism is much
more complicated than originally proposed and probably
requires even more host factors, probably acting
cooperatively. It should also be noted that almost all of the
functional T4SS studies have been made in vitro using
cultured cell lines, which are indeed very helpful.
However, we are aware of only one in vivo study, in which
phosphorylated CagA was isolated from biopsies of
atrophic gastritis and in noncancerous tissues from
H. pylori-positive patients using immunoprecipitation
and Western blotting approaches . Thus, more
studies are clearly necessary to investigate under which
circumstances and how CagA is injected during infection
in vivo. In particular, it remains to be investigated if
CagA injection occurs at the apical, basolateral and/or
other sites of the gastric epithelium. In addition, it has
been convincingly shown that CagA can be very efficiently
translocated into certain immune cells in vitro (Table 2).
Thus, future studies are necessary to investigate the
importance of these findings in vivo. Finally, the
evolutionary advantage of the T4SS for H. pylori is also not yet clear
and needs to be investigated more thoroughly. For
example, recent studies indicated that injected CagA enables
H. pylori to grow as microcolonies adhered to the host cell
surface even in conditions that do not support growth of
free-swimming bacteria . Thus, it appears that the
H. pylori T4SS will continue to be a fascinating and
rewarding research topic in future studies.
The work of S.B. is supported through grants from the Deutsche
Forschungsgemeinschaft DFG grant (Ba1671/8-1), Science Foundation Ireland
(UCD 09/IN.1/B2609) and from the National Institute of Diabetes and
Digestive and Kidney Diseases (R56DK064371). The work of MC is supported
through grants from Science foundation Ireland (UCD 08SRCB1393).
Received: 3 July 2011 Accepted: 1 November 2011
Published: 1 November 2011
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