Báo cáo khoa học: Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus doc

MINIREVIEW
Assembly and molecular mode of action of the
Helicobacter pylori Cag type IV secretion apparatus
Wolfgang Fischer
Max von Pettenkofer-Institut, Ludwig-Maximilians-Universita
¨
t, Mu
¨
nchen, Germany
Introduction
Type IV secretion systems (T4SS) represent a family of
macromolecule transporters that is widely distributed
in prokaryotes, and individual members of this family
have adapted to their cellular background and to a
variety of substrates as DNA import and export sys-
tems, conjugation systems or effector protein translo-
cation systems [1]. Several pathogenic bacteria have
adopted T4SS for the secretion of virulence-associated
proteins to the extracellular milieu or for their injec-
tion into different host cells, mediating host cell
manipulation in different ways and thereby facilitating
mucosa-associated or intracellular lifestyles. The
human gastric pathogen Helicobacter pylori, which is
the principal cause of chronic active gastritis and pep-
tic ulcer disease, and also is involved in the develop-
ment of mucosa-associated lymphoid tissue lymphoma
and gastric cancer [2], uses different T4SS for horizon-
tal gene transfer, and the cytotoxin-associated gene
(Cag) T4SS for interactions with various host cells
[3,4]. The Cag-T4SS is encoded on the cytotoxin-asso-
ciated gene-pathogenicity island (cagPAI), a 37 kb

genomic island representing one of the major variable
genome regions of H. pylori that is clearly associated
with an enhanced risk of developing peptic ulcers or
adenocarcinoma. The percentage of cagPAI-positive
strains varies considerably between geographically dis-
tinct groups, ranging from universal presence in East
Asian isolates to a complete absence in certain African
populations [5]. Strains carrying the cagPAI are often
equipped with a vacuolating cytotoxin (vacA)s1⁄ m1
genotype, suggesting a common selective pressure for
these two major virulence factors, and have been
Keywords
CagA; Helicobacter pylori; pathogenicity
island; protein translocation; secretion
apparatus; type IV secretion
Correspondence
W. Fischer, Max von Pettenkofer-Institut,
Ludwig-Maximilians-Universita
¨
t,
Pettenkoferstrasse 9a, 80336 Mu
¨
nchen,
Germany
Fax: +49 89 51605223
Tel: +49 89 51605277
E-mail: fi
(Received 15 November 2010, accepted
10 January 2011)
doi:10.1111/j.1742-4658.2011.08036.x

Bacterial type IV secretion systems (T4SS) form supramolecular protein
complexes that are capable of transporting DNA or protein substrates
across the bacterial cell envelope and, in many cases, also across eukaryotic
target cell membranes. Because of these characteristics, they are often used
by pathogenic bacteria for the injection of host cell-modulating virulence
factors. One example is the human pathogen Helicobacter pylori, which
uses the Cag-T4SS to induce a pro-inflammatory response and multiple
cytoskeletal and gene regulatory effects in gastric epithelial cells. Work in
recent years has shown that the Cag-T4SS exhibits marked differences in
relation to other systems, both with respect to the composition of its secre-
tion apparatus and the molecular details of its secretion mechanisms. This
review describes the molecular properties of the Cag-T4SS and compares
these with prototypical systems of this family of protein transporters.
Abbreviations
cagPAI, cytotoxin-associated gene-pathogenicity island; IL, interleukin; T4SS, type IV secretion system.
FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1203
termed type I strains. By contrast, type II H. pylori
strains have vacA m2 genotypes, lack the cagPAI and
are less virulent.
The Cag secretion system is responsible for the
induction of a pronounced pro-inflammatory response
in vitro and in vivo, and the translocation and subse-
quent tyrosine phosphorylation of its effector protein
CagA into various host cells is a hallmark of Cag-
T4SS activity. Despite the molecular characterization
of a number of effects in host cells, the exact function
of CagA translocation during infection is still not fully
clear. However, this process significantly increases the
risk of gastric cancer in the Mongolian gerbil model
[6], and CagA has been shown to act as a bacterial

oncoprotein capable of malignant cell transformation
[7]. CagA is the only protein that has been described
so far as an effector protein of the Cag-T4SS, although
it has been suggested that the secretion apparatus
transports peptidoglycan fragments into the host cell
cytoplasm as well, thereby inducing a pro-inflamma-
tory response via activation of the pattern-recognition
molecule Nod1 [8]. However, the mechanistic details of
type IV secretion-dependent transport of peptidoglycan
fragments are not known.
Although the Cag system is evolutionarily related to
other T4SS, it contains only few proteins with high
sequence similarities to components of other T4SS,
and many essential components are unique for the Cag
system. These pronounced differences are likely to be
reflected in details of secretion apparatus assembly, as
well as in the molecular mechanisms of effector protein
secretion. This minireview describes the composition of
the cagPAI and the properties of both conserved and
unique components of the Cag-T4SS, together with
potential implications for the current understanding of
its mode of action.
The cagPAI and the Cag type IV
secretion apparatus
Gene arrangement and variants of the cagPAI
The cagPAI, which was originally identified by
sequencing the genome region upstream of the cagA
gene or DNA found only in CagA-positive strains, was
shown to encode proteins with sequence similarity to
Agrobacterium tumefaciens Vir proteins, and it was fur-

ther demonstrated that these proteins are necessary for
inducing secretion of the chemokine interleukin (IL)-8
from infected epithelial cells [9,10]. In the correspond-
ing strain NCTC11638, the cagPAI is not a contiguous
genome island but was split as a result of integration of
an IS605 insertion element and an associated genome
rearrangement. However, comparative sequence analy-
sis of multiple cag islands [11–13] suggested that this
situation is rather an exception. In its most common
gene arrangement, the cagPAI is inserted between the
genes encoding a Sel1 repeat-containing protein and
glutamate racemase, respectively, and it is flanked by a
31 bp sequence duplication (Fig. 1A). The cagPAI has
an overall size of  37 kb and harbours  30 genes.
The amount of sequence diversity among these genes
in isolates from different geographic groups has
recently been taken as an indication that the
cagPAI was acquired only once in the history of
H. pylori [13].
Although the gene order on the cagPAI is con-
served, recent genome sequencing projects have
revealed certain variations of the general gene arrange-
ment. For example, some strains isolated from Ameri-
can Indians have a duplication of cagA (in a
nonfunctional form) and cagB inserted into the inter-
genic locus between cagP and cagQ [14] (Fig. 1B).
Additionally, these islands have an inversion of the
cagQ gene, which is also frequently found in East
Asian strains [12,13]. A more complex rearrangement
was identified in a strain colonizing Mongolian gerbils

[15]. It includes an inversion of all cagPAI genes except
cagA in conjunction with several flanking genes, and a
second inversion comprising most of these flanking
genes (hp0511–hp0518). Similar rearrangements would
also account for earlier observations that the cagA
gene is not adjacent to cagB in some strains [11,16].
Interestingly, the corresponding gene locus of Helicob-
acter acinonychis Sheeba, which is the closest relative
to H. pylori known, but probably diverged before
acquisition of the cagPAI [17], contains a similar inver-
sion of the same flanking genes and also fragments of
a helicase gene that is frequently found downstream of
cagA (Fig. 1B). Because the cagA downstream region
is highly polymorphic and contains remnants of an
IS606 insertion element [13], this observation suggests
that these genes or gene fragments were not originally
part of the cagPAI but were inserted at a later time
point after cagPAI acquisition.
Components of the Cag-T4SS
Prototypical T4SS, such as the T-DNA transfer system
of A. tumefaciens, usually contain 11 essential compo-
nents (VirB1–VirB11) of the secretion apparatus and a
coupling protein (VirD4) that mediates substrate rec-
ognition [1]. By contrast to other T4SS found in
H. pylori, the Cag-T4SS is only distantly related to
T4SS found in other species [4], and only a few cag
genes encode proteins with clear sequence similarities
Cag protein functions W. Fischer
1204 FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS
to known T4SS proteins. Obvious similarities exist

only for CagE (to VirB4), CagX (to VirB9), CagY (to
VirB10), Caga (to VirB11) and Cagb (to VirD4),
although even these proteins, particularly CagX and
CagY, are remarkably different from their counter-
parts in prototypical systems. Nevertheless, protein
topology predictions and determinations, localization
studies and functional studies suggested that Cagc
(VirB1), CagC (VirB2), CagL (VirB5), CagW (VirB6),
CagT (VirB7) and CagV (VirB8) are further VirB
homologues [18–22] (Fig. 1A and Table 1).
Early systematic studies with isogenic mutants in
each cag gene [23,24] identified 14–15 genes that are
essential for inducing IL-8 secretion and for CagA
translocation, suggesting that these genes encode com-
ponents of the secretion apparatus (Table 1). These
essential secretion apparatus components include all
VirB-like proteins mentioned above and several further
components that are unique to the Cag system. Three
further gene products are not absolutely necessary,
although their absence results in a reduced efficiency
of both phenotypes, and these proteins (supporting
components) thus appear to be involved in assembling
the secretion apparatus as well. An additional group
of genes was shown to be required for CagA transloca-
tion but not for IL-8 induction [23], and the encoded
gene products were accordingly termed CagA translo-
cation factors.
Finally, several cagPAI gene products do not appear
to have a function for the type IV secretion-related
phenotypes examined. They might have other as yet

unknown functions or even be further effector pro-
teins, or they might simply be unrelated to the T4SS.
Interestingly, however, one of these genes (cagf) was
A
B
Fig. 1. Gene arrangement and variants of the cag pathogenicity island. (A) Integration of the cagPAI at a chromosomal locus flanked by gene
hp0519, with numbering according to the genome sequence of strain 26695 [51], encoding a Sel1 repeat-containing protein and gene
hp0549 encoding glutamate racemase. Gene designations and putative homologies to components of the A. tumefaciens T-DNA transfer
system are indicated. The left (LJ) and right junctions (RJ) of the cagPAI represent a 31 bp direct repeat. (B) Rearrangements of the cagPAI
found in complete H. pylori genome sequences. Apart from complete deletions in cag-negative strains, rearrangements include an inversion
of cagQ, a duplication of cagA and cagB associated with cagA degeneration, and a more complex rearrangement comprising all cag genes
except cagA and a second inversion of several flanking genes (green). Examples of strains containing the depicted arrangements are given.
A helicase gene (hel), fragments of which are often located close to the cagPAI right junction (orange), is also present in H. acinonychis
strain Sheeba or in some H. pylori strains as part of a strain-specific segment integrated next to a lipoprotein gene (blue) and has therefore
probably not originally been part of the cagPAI.
W. Fischer Cag protein functions
FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1205
found to be among the most highly transcribed among
all cag genes in vitro and in vivo, and transcripts of
cagS, cagQ and cagP were also found [25]. Moreover,
a recent determination of the complete H. pylori tran-
scriptome identified a number of transcriptional start
sites on the cagPAI, suggesting transcription of the
cagB gene, transcription of an operon comprising cagf,
cage, cagd and cagc, transcription of an operon com-
prising cagQ, cagS and possibly the small ORF hp0533
(which is not present in many strains) and, finally,
transcription of the cagP gene, probably together with
a small noncoding RNA upstream of cagP [26]. These
observations suggest that all non-essential genes are

expressed, and their organization in operons indicates
a functional relationship with the T4SS. For a discus-
sion of these non-essential components, see the accom-
panying review by Cendron and Zanotti [27].
The putative type IV secretion apparatus core
complex
The assembly of different type IV secretion machines
and functions of their essential components have been
studied in detail [1], although the structural details of
a type IV secretion apparatus have emerged only
recently from the structural determination of a core
complex of the pKM101 conjugation system [28,29];
see also the accompanying review by Terradot and
Waksman [30]. This core complex consists of 14 mono-
mers each of the VirB7, VirB9 and VirB10 homologues
and does not require other secretion system compo-
nents for assembly. It is reasonable to assume that the
Cag system forms a similar core complex composed of
CagT, CagX and CagY (Fig. 2). However, it should be
noted that all these proteins are considerably different
Table 1. Overview of characteristics and functions of cagPAI-encoded proteins. Gene designations and protein sizes are in accordance with
a previous study [51]; aa, amino acids; NA, not annotated. Localization according to sequence prediction or fractionation studies; C, cytoplas-
mic; IM, inner membrane; PP, periplasmic; OM, outer membrane; S, surface-exposed or supernatant. Functional classification according to
requirement for IL-8 induction and ⁄ or CagA translocation. SA, secretion apparatus component required for IL-8 and CagA translocation; TF,
translocation factor required for CagA translocation, but not IL-8 induction; SC, supportive, but not essential component for both phenotypes;
NE, non-essential for both phenotypes. References report protein structures, functions, localizations or interactions.
Gene Protein Size (aa) Localization Classification (Putative) function(s) or homologies Reference
hp0520 Cagf ⁄ Cag1 116 IM NE
hp0521
a

Cage ⁄ Cag2 80 C NE
hp0522 Cagd ⁄ Cag3 481 OM SA OM complex 33,38
hp0523 Cagc ⁄ Cag4 169 PP SA PG hydrolase, VirB1 18
hp0524 Cagb ⁄ Cag5 748 IM TF Coupling protein, VirD4 49,52
hp0525 Caga ⁄ VirB11 330 C, IM SA ATPase, VirB11 40,41
hp0526 CagZ 199 C, IM TF Cagb stabilization 38,49,53
hp0527 CagY 1927 IM, OM, S SA Core complex, integrin binding, VirB10 22,34,36
hp0528 CagX 522 IM, OM, S SA Core complex, VirB9 22
hp0529 CagW 535 IM SA IM channel, VirB6
hp0530 CagV 252 IM SA Core complex, VirB8 21
hp0531 CagU 218 IM SA
hp0532 CagT 280 OM, S SA Core complex, OM lipoprotein, VirB7 22,33,35
hp0534 CagS 196 C NE 54
hp0535 CagQ 126 IM NE
hp0536 CagP 114 IM NE
hp0537 CagM 376 OM SA OM complex 22
hp0538 CagN 306 PP, IM SC 55
hp0539 CagL 237 PP, S SA Integrin binding, VirB5 42
hp0540 CagI 381 PP, S TF ⁄ SA
b
36
hp0541 CagH 370 IM SA
hp0542 CagG 142 PP SC ⁄ TF
hp0543 CagF 268 C, IM TF Secretion chaperone 47,48
hp0544 CagE 983 IM SA ATPase, VirB3 ⁄ B4
hp0545 CagD 207 IM, PP, S SC ⁄ TF
c
56
hp0546 CagC 115 IM, OM, S SA Pilus subunit, VirB2 19,22
NA CagB 75 C Unknown

hp0547 CagA 1186 C, S Effector protein 47–49,57
a
Replaced by hp0521B in several strains.
b
Conflicting data with respect to requirement for IL-8 induction [9,23,24].
c
Conflicting data with
respect to requirement for CagA translocation [23,56].
Cag protein functions W. Fischer
1206 FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS
from their counterparts in the pKM101 or other T4SS.
For example, CagT is a lipoprotein similar to TraN of
pKM101 but has a size of 280 amino acids compared
to 48 amino acids for TraN or 55 amino acids for
VirB7, suggesting additional functions for CagT. Lipo-
proteins of 150–300 amino acids are also found to be
essential components of conjugation systems such as
those of plasmids RP4 and F [31], and of the less
related type IVB secretion systems, where the structure
of a domain of the DotD lipoprotein was shown to be
similar to secretin domains from type II or type III
secretion systems [32].
Consistent with the assumption that CagT is a
VirB7 homologue is the fact that it takes part in an
outer membrane-associated subcomplex of the Cag sys-
tem, which also contains CagX [22]. By contrast to
other T4SS, however, this subcomplex appears to har-
bour two additional components, CagM and Cagd.
Both proteins have N-terminal signal sequences and
were shown to be associated with the outer membrane

and to interact with CagT, with CagX, and with each
other [22,33]. Moreover, both CagM and Cagd were
found to interact with themselves, suggesting that they
might contribute to oligomerization of the outer mem-
brane subcomplex [22,33]. Interestingly, the interaction
between CagT and CagX was lost in a cagM mutant,
indicating that it is either an indirect interaction via
CagM, or that only a ternary complex comprising all
three proteins is stable [22]. In support of this view,
a cagM mutant produces significantly reduced levels of
CagT. Furthermore, Cagd was found to stabilize the
lipoprotein CagT, and vice versa [33]. Taken together,
these observations suggest that the Cag system elabo-
rates a more complex core structure than other T4SS.
Both CagT and CagX were also found exposed at the
bacterial surface [34,35].
The most divergent core protein is CagY, a huge
protein with a peculiar middle region containing exten-
sive sequence repeats. CagY was shown to interact with
the outer membrane-associated subcomplex, although
this interaction was only detected in the presence of
CagX [22], suggesting that CagY does not interact
directly with CagM or CagT. Apart from its putative
membrane localization spanning both bacterial mem-
branes, CagY was also detected on type IV secretion
pilus-like surface appendages [34]. Interestingly, CagY
was identified as one of several bacterial interaction
partners of b1 integrins, which represent the secretion
apparatus receptors on target cells [36] (see below).
Assembly of the secretion apparatus

Although T4SS core complexes are able to form
autonomously, they are unlikely to do so constitutively
Fig. 2. Assembly and interaction model of
the Cag type IV secretion apparatus. Cag
proteins are depicted in their most likely
localizations according to sequence predic-
tion or experimental data and designated by
their last letters (e.g. ‘A’ for CagA). Essential
or supportive secretion apparatus compo-
nents are indicated in green, translocation
factors in orange, and the effector protein
CagA in red. Overlapping boxes indicate
probable protein–protein interactions.
Integrin heterodimers are indicated as
receptors on the target cell surface (a, b1).
CagA, CagL and CagY are also shown on
the pilus as a result of their integrin-binding
capacities. Note that not all interactions are
depicted, and that CagD, CagG and CagI, as
well as non-essential components, are not
shown. IM, inner (bacterial) membrane;
PG, peptidoglycan layer; OM, outer
(bacterial) membrane; CM, cytoplasmic
membrane of a eukaryotic target cell.
W. Fischer Cag protein functions
FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1207
and without a positional preference. In the A. tumefac-
iens Vir system, spatial targeting of the secretion appa-
ratus has been suggested to be determined by VirB8 as
a nucleating factor [37], possibly together with the pep-

tidoglycan-degrading lytic transglycosylase VirB1 [1].
In accordance with this, both the lytic transglycosylase
of the Cag system, Cagc [18], and CagV, a bitopic
inner membrane protein with features similar to VirB8
[21], are essential secretion apparatus components. The
lytic transglycosylase activity of Cagc would require a
periplasmic localization (Fig. 2), although it is unclear
how Cagc is exported because it does not have an
N-terminal signal sequence. Interactions of CagV with
Cagd, CagM and CagT were identified in a yeast two-
hybrid screen and partly confirmed by pulldown exper-
iments [38], supporting the idea that CagV might act
as a nucleator by forming contacts with other core
complex proteins. A further step in the assembly of the
secretion apparatus would be the recruitment of com-
ponents forming the inner membrane pore of the secre-
tion apparatus. The Cag system contains three
essential proteins that might constitute a cytoplasmic
membrane pore. CagW is a polytopic inner membrane
protein with features that are common among VirB6-
like proteins [22], CagU is a second polytopic inner
membrane protein with three predicted transmembrane
helices that has no counterpart in other systems, and
CagH is an essential bitopic inner membrane protein,
also without counterparts in other systems. Functional
studies with all these components are lacking so far.
On the cytoplasmic face of the secretion apparatus,
two ATPases provide the energy for secretion appara-
tus assembly and ⁄ or substrate transport. CagE proba-
bly has two transmembrane helices at its N-terminus

and a large region with sequence similarity to the VirB4
ATPase. It has been speculated that the N-terminal
extension represents a VirB3-like domain fused to the
VirB4-like component [22], which is consistent with the
recent observation that a fusion protein of A. tumefac-
iens VirB3 and VirB4 retains both functions [39]. The
VirB11-like ATPase Caga has been shown to form
hexamers in solution and to undergo conformational
changes upon binding of ADP or ATP, suggesting a
dynamic cycling process [40,41]; for details, see the
accompanying review by Terradot and Waksman [30].
Finally, the Cag-T4SS elaborates sheathed surface
appendages that are dissimilar to the pili commonly
found in DNA-transporting T4SS. Nevertheless, these
appendages are considered to be composed of the
VirB2-like pilin subunit CagC [19] but, in addition,
they can be stained with immunogold labels directed
against CagY, CagT, CagX and CagL [34,35,42]. It
was shown that purified CagL binds via its RGD motif
to b1 integrin subunits, suggesting a VirB5-like adhesin
function for CagL [42], although conflicting results
were obtained with respect to the requirement of this
motif during CagA translocation [36,42]. On the other
hand, CagY, CagA and CagI were also identified as
Cag proteins binding to b1 integrins [36]. In any case,
interaction of secretion system components with b1
integrins is an important prerequisite for T4SS function.
Mechanisms of CagA recognition and
transfer
CagA as a type IV secretion substrate

By contrast to most other virulence-associated T4SS,
the Cag system transports only CagA as a protein sub-
strate, although CagA is an effector protein with mul-
tiple functions in target cells; see the accompanying
review by Tegtmeyer et al. [43]. The different functions
are partly dependent on, and partly independent of,
CagA tyrosine phosphorylation, and the phosphoryla-
tion motifs located in the C-terminal region of CagA
(EPIYA motifs) are thus essential for some pheno-
types. A second conserved motif found to be required
for phosphorylation-independent effects is located
adjacent to the EPIYA motifs and has been termed
microtubule affinity-regulating kinase inhibitor motif
because of its binding to this kinase [44]. Translocation
reporter assays using the phosphorylatable glycogen
synthase kinase epitope tag showed that these motifs
are not required for translocation of CagA (I. Pattis
& W. Fischer, unpublished results). Translocation of
CagA critically depends on its C-terminal 20 amino
acids [45]. This is consistent with the situation for
T4SS substrates in other bacteria, which are generally
considered to use C-terminal secretion signals [1].
Although the CagA C-terminus contains a number of
positively charged amino acids and positive charges
are important for some type IV effector proteins, site-
specific mutation of the CagA C-terminus has not
resulted in a clear picture concerning the nature of the
secretion signal [45]. However, domain-swapping
experiments using the C-terminal regions of other type
IV substrates indicated that this part contains a secre-

tion information that is common among different
T4SS. By contrast to most other type IV effector pro-
teins analyzed so far, the CagA C-terminus is not suffi-
cient for translocation. Possible explanations for this
are that binding of an N-terminal domain of CagA to
b1 integrins [36] might be required for transport into
the target cell, or that CagA translocation might
depend on an interaction with phosphatidylserine in
the target cell membrane, for which two arginine
Cag protein functions W. Fischer
1208 FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS
residues in the middle CagA region were shown to be
necessary [46].
Substrate recognition and the role of CagA
translocation factors
Before entering the translocation channel, CagA secre-
tion signal(s) or protein domains containing the secre-
tion information have to be recognized by a signal
recognition protein. Because the three essential CagA
translocation factors CagF, CagZ and Cagb are all pre-
dicted to be localized in the bacterial cytoplasm or at
the inner membrane, each of them could fulfill a func-
tion as signal recognition factor. CagA immunoprecipi-
tation experiments from bacterial lysates resulted in the
identification of CagF as major component interacting
with CagA [47,48]. This interaction is independent of
other secretion apparatus components and probably
takes place at the inner face of the cytoplasmic mem-
brane. The CagF-binding region comprises  100
amino acids in the C-terminal region of CagA but does

not contain the putative C-terminal secretion signal,
suggesting that CagF binding is not a signal recogni-
tion event [48]. However, a fusion of GFP to the C-
terminal 195 amino acids of CagA (containing the
CagF-binding domain together with the C-terminal
signal region) exerted a dominant-negative effect on
translocation of full-length CagA. This indicated that
the corresponding region is sufficient for recruitment to
the secretion apparatus and that CagF binding plays a
role in this recruitment, similar to the function of secre-
tion chaperones in type III secretion systems.
In almost all conjugation systems and also in many
effector protein translocation systems, coupling pro-
teins are essential for the secretion process [1]. Cou-
pling proteins are ATPases interacting both with
substrates and with secretion apparatus components
that determine substrate specificity of a given T4SS. In
the Cag system, the coupling protein homologue Cagb
is dispensable for IL-8 induction but essential for
CagA translocation [23,24], which is consistent with its
putative role as a type IV substrate receptor. Similar
to other coupling proteins, Cagb is predicted to con-
tain two or three transmembrane helices in its N-termi-
nal region, with the major C-terminal part of the
protein being located in the cytoplasm [22]. Recently,
it was shown that this cytoplasmic part of Cagb is able
to interact with CagA [49], although it is not clear
whether this binding involves the putative C-terminal
secretion signal. Furthermore, a robust interaction
between Cagb and the third translocation factor CagZ

was identified in yeast two-hybrid screens and con-
firmed biochemically in H. pylori [38,49]. Deletion of
the cagZ gene resulted not only in CagA translocation
deficiency, but also in a strong reduction of Cagb pro-
tein levels, and both defects could be restored by com-
plementation of the mutant with a myc-tagged cagZ
gene [49]; for structural details of CagZ, see the
accompanying review by Cendron and Zanotti [27].
Taken together, these data suggest that Cagb and
CagZ form a stable complex at the bacterial cytoplas-
mic membrane that might constitute the functional
CagA signal recognition receptor.
Mechanisms of CagA translocation
The molecular mechanisms of CagA translocation
through the secretion apparatus are only poorly under-
stood. For the A. tumefaciens VirB system, subsequent
contacts of the secreted nucleoprotein complex with
VirD4, VirB11, VirB6 ⁄ VirB8 and VirB2 ⁄ VirB9 have
been defined [50]. An analogous secretion route might
be taken by CagA but, as a result of the high variabil-
ity among T4SS [1], considerable differences are also
possible. Although putative interactions between CagA
and different secretion apparatus components have
been identified in a yeast two-hybrid approach [22,49],
they have not been confirmed so far in H. pylori cells.
As for other T4SS, it is currently also unclear whether
the extracellular pilus-like appendages are used as con-
duits for protein transport or rather represent struc-
tures required for T4SS-dependent cell contact. Several
studies have shown that CagA is located at the bacte-

rial surface, particularly at the pilus tip [36,42,46],
although it has not been examined whether surface- or
pilus-associated CagA represents a translocation inter-
mediate. Such a scenario is suggested by a study show-
ing that CagA binding to phosphatidylserine at the
outer leaflet of the host cell cytoplasmic membrane
induces its uptake into the cell [46]. However, it has
also been established that translocation of CagA
depends on the presence of b1 integrins as receptors
for the Cag secretion apparatus at the target cell sur-
face [36,42]. Irrespective of which components of the
secretion apparatus bind to b1 integrin extracellular
domains, inhibitory effects of different integrin anti-
bodies on CagA translocation, as well as the observa-
tion that CagA itself binds strongly to b1 integrin [36],
suggest that pilus-associated CagA has an important
function for translocation. The uptake process into the
host cell cytoplasm is not understood. Incubation of
target cells with different inhibitors interferes with
CagA tyrosine phosphorylation [36,46], although it
remains unclear whether CagA uptake involves pore
formation in the host cell cytoplasmic membrane or
other cellular processes.
W. Fischer Cag protein functions
FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1209
Conclusions
Although T4SS have emerged as a common theme in
microbial interactions with eukaryotic cells, they have
also been evolutionarily adapted to various needs, and
major deviations from ancestral systems might accord-

ingly be expected with respect to their structure and
function. This is well-reflected in the H. pylori Cag sys-
tem, which includes a number of unique essential com-
ponents and probably relies on a specific translocation
mechanism. Given that this system also poses a major
health problem by enhancing the risk of cancer devel-
opment, it is important to understand its molecular
principles in detail. Defining the molecular mechanisms
of CagA transport to the bacterial surface and across
the target cell membrane will thus be of particular
interest for future research.
Acknowledgements
The author is grateful to Rainer Haas for continuous
support, and to Claudia Ertl and Rainer Haas for
critically reading the manuscript. This work was
supported by a FoeFoLe research grant from the
Ludwig-Maximilians-Universita
¨
tMu
¨
nchen.
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