Báo cáo khoa học: Architecture of the Helicobacter pylori Cag-type IV secretion system pdf

MINIREVIEW
Architecture of the Helicobacter pylori Cag-type IV
secretion system
Laurent Terradot
1
and Gabriel Waksman
2
1 Institut de Biologie et Chimie des Prote
´
ines, Biologie Structurale des Complexes Macromole
´
culaires Bacte
´
riens, UMR 5086
CNRS Universite
´
de Lyon, France
2 Institute of Structural and Molecular Biology, UCL and Birkbeck, London, UK
Introduction
Secretion systems are widespread in bacteria for which
they provide means of exchange between the extra-
and intracellular milieus. Seven different systems have
been described in Gram-negative bacteria [1]. Amongst
them, the type IV secretion systems (T4SS) have raised
considerable attention during the past 10 years because
of their role in bacterial pathogenesis. T4SS are macro-
molecular devices that bacteria use to transport various
Keywords
bacterial pathogenesis; CagA; secretion
system; stomach cancer; translocation
Correspondence

L. Terradot, Institut de Biologie et Chimie
des Prote
´
ines, Biologie Structurale des
Complexes Macromole
´
culaires Bacte
´
riens,
UMR 5086 CNRS Universite
´
de Lyon,
IFR128, 7 Passage du Vercors, F-69367
Lyon Cedex 07, France
Fax: +33 472 722604
Tel: +33 472 722652
E-mail: and
G. Waksman, Institute of Structural and
Molecular Biology, UCL and Birkbeck, Malet
Street, London, WC1E 7HX, UK
Fax: +44 (0)207 631 6803
Tel: +44 (0)207 631 6833
E-mail: or

(Received 15 November 2010, revised 18
January 2011, accepted 27 January 2011)
doi:10.1111/j.1742-4658.2011.08037.x
Type IV secretion systems (T4SS) are macromolecular assemblies used by
bacteria to transport material across their membranes. T4SS are generally
composed of a set of twelve proteins (VirB1–11 and VirD4). This repre-

sents a dynamic machine powered by three ATPases. T4SS are widespread
in pathogenic bacteria where they are often used to deliver effectors into
host cells. For example, the human pathogen Helicobacter pylori encodes a
T4SS, the Cag-T4SS, which mediates the injection of the toxin CagA. We
review the progress made in the past decade in our understanding of T4SS
architecture. We translate this new knowledge to derive an understanding
of the structure of the H. pylori Cag system, and use recent protein–protein
interaction data to refine this model.
Abbreviations
cagPAI, cytotoxin-associated gene-pathogenicity island; CTD, C-terminal domain; EM, electron microscopy; NTD, N-terminal domain; T4SS,
type IV secretion system.
FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS 1213
macromolecules, including protein, DNA or nucleic
acid ⁄ protein complexes, across the cell envelope [2,3].
These systems are remarkably versatile and have been
classified into three different groups according to their
function [3]. T4SS in the first group are used to trans-
fer DNA from one cell to another in a process referred
to as conjugation [4]. Incorporating new DNA
sequences is a major selective advantage for these
organisms, which can rapidly acquire new genetic fea-
tures and adapt to changes in the environment. Such
mechanisms are involved in the spread of antibiotic
resistance genes among pathogenic bacteria [5]. One of
the most studied T4SS of this group is encoded by the
Agrobacterium tumefaciens VirB ⁄ D system, which is
able to deliver nucleoprotein complexes into plant cells
leading to crown gall disease [6]. The artificial utiliza-
tion of a modified VirB ⁄ D system has proved instru-
mental in the production of genetically modified plants

[7]. The second group of T4SS, exemplified by the
ComB system of H. pylori and the GGI system of Nei-
sseria gonorrhoeae, is used for DNA uptake and release
from and to the extracellular milieu [8]. The third
group consists of T4SS that transfer protein effectors
and are used by numerous pathogenic bacteria, includ-
ing Bordetella pertusis, Legionella pneumophila, Bru-
cella spp., H. pylori and Bartonella spp. In these
species, T4SS can be described as molecular pumping
devices that facilitate host–pathogen interaction and ⁄ or
inject toxins into the host cell [5].
In the human pathogen H. pylori, T4SS of the three
different groups have been identified. The most con-
served one is the so-called ComB system (group 2),
which is considered to mediate the import and integra-
tion of environmental DNA fragments into its genome
[9]. Another one (named Tfs3, group 1) is less con-
served and appears to play important roles in deter-
mining the remarkable plasticity of the H. pylori
genome [10–12]. The last T4SS (group 3) is present
only in the most virulent H. pylori strains that produce
a major toxin, the CagA protein [13]. This toxin and
the T4SS apparatus responsible for its transfer to the
eukaryotic cell are encoded by the so-called cytotoxin-
associated gene-pathogenicity island (cagPAI), a
40 kbp DNA fragment that is transmitted horizontally;
see the accompanying review by Tegtmeyer et al. [14 ].
CagA is considered as a paradigm for bacterial carci-
nogenesis [15]. Briefly, once injected into the host cell,
CagA is phosphorylated and interacts with more than

20 different human proteins involved in signal trans-
duction [16]. As a consequence of CagA action, epithe-
lial cells will have some of their major functions
disturbed, such as cell–cell adhesion, signalling, adher-
ence and proliferation [17]. CagA translocation and
phosphorylation are required to trigger the so-called
‘hummingbird phenotype’, a form of cell scattering
[18]. The cagPAI system also delivers bacterial pepti-
doglycan that triggers the Nod1-response and induc-
tion of the nuclear factor-jB pathway [19].
How these effectors are injected into the host cell is
still poorly understood. However, the last decade has
seen a number of spectacular advances in the struc-
tural definition of individual components of the T4SS
and, more recently, on how they associate into large
macromolecular complexes. We review these advances,
as well as how, when integrated within the larger con-
text of the numerous functional studies on the Cag
proteins, a clearer picture of the architecture and func-
tion of the Cag-encoded T4SS can be derived.
Architecture of T4SS
The T4SS apparatus generally consists of twelve pro-
teins named VirB1–11 and VirD4 based on the nomen-
clature used for A. tumefaciens T4SS (Fig. 1). They
assemble to form three interlinked subparts: a cyto-
plasmic ⁄ inner membrane complex, a double mem-
brane-spanning channel and an external pilus (Fig. 1).
Variations exist among the different types of T4SS but
the composition of the compartments is generally con-
served. The cytoplasmic ⁄ inner membrane complex is

composed of three NTPases (VirB4, VirB11 and
VirD4), VirB6 and VirB8; the trans-membranes pore
complex (VirB7, 9, 10; also termed ‘the core complex’)
forms a channel from the inner to the outer mem-
brane; and the external pilus generally consists of the
VirB2 and VirB5 proteins. Other components are
essential for the formation of the T4SS complex: VirB1
allows for the insertion of the system in the periplasm
and VirB3, the function of which is unknown, is often
associated with VirB4. In certain bacteria, some of
these components are absent. This is the case for the
H. pylori ComB system where the apparatus is used to
import DNA at the outer membrane and relies on the
other competence system ComEC to transport DNA
into the cytoplasm [20].
The conserved components of the cagPAI encoded
T4SS (Cag-T4SS) have been identified first by sequence
comparison with those of the VirB ⁄ D system; see the
accompanying review by Fischer [21]. However,
although the archetypal VirB ⁄ D system has 12 compo-
nents, the cagPAI encodes for 27 proteins. Note that
several nomenclatures exist for cagPAI proteins, which
are listed in the review by Fischer [21]. Except for the
VirB ⁄ D system homologues, these proteins are unique
to H. pylori; see the accompanying reviews by Fischer
[21] and Cendron and Zanotti [22]. The role of most
Structure of the Cag-T4SS L. Terradot and G. Waksman
1214 FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS
Cag proteins in the T4SS apparatus is not clear,
although several of them are essential for the secretion

of CagA or the induction of interleukin-8 [23].
The NTPases battery of the Cag-T4SS
The Cag-T4SS powering machinery is composed of
three cytoplasmic NTPases, HP0525 (VirB11), HP0524
(VirD4) and HP0544 (VirB4 putative homologue),
which supply the energy necessary to assemble the
apparatus and secrete CagA. Structural information is
available for the VirD4 homologue TrwB from Escher-
ichia coli conjugative plasmid R388 and for HP0525
but not for VirB4. These NTPases have the canonical
walker A and B motifs, and couple NTP hydrolysis to
conformational changes. These might in turn be cou-
pled to unfolding or transfer of the substrate [24].
Coupling protein VirD4 (HP0524)
VirD4 proteins are also named ‘coupling’ proteins
because they can recruit substrates to the T4SS appa-
ratus, although this function is absent in some systems.
The most studied VirD4 protein is probably TrwB,
which is required for the translocation of DNA by the
E. coli R388 conjugation system. The crystal structure
of TrwB showed that the protein is a globular hexamer
with an orange-like shape, with each subunit forming
an orange segment (Fig. 2A) [25]. The protein is com-
posed of a 70 residues N-terminal trans-membrane seg-
ment (absent in the crystal structure) and two
domains, a nucleotide binding domain and an all-a-
domain. TrwB binds to ATP at the interface between
subunits and hydrolysis is stimulated by DNA. In the
Cag-T4SS, VirD4 is encoded by the hp0524 and the
resulting protein is much larger (748 residues). HP0524

is essential for CagA translocation but not for interleu-
kin-8 induction [23]. Evidence has recently been pro-
vided that HP0524 interact with CagA, suggesting that
HP0524 might also act as a coupling protein in the
Cag-T4SS, as in other systems [26,27].
VirB11 (HP0525) and its regulation
HP0525 is essential for CagA secretion [23]. The struc-
ture of HP0525 showed that the protein also forms
hexamers, with each subunit consisting of two
domains, an N-terminal domain (NTD) and a RecA-
like C-terminal domain (CTD) containing the motifs
found in all members of the traffic ATPases family [2].
Fig. 1. (A) Schematic view of the cagPAI encoded by the H. pylori strain 26695. Numbers correspond to the HP0XXX number of the ORF
[57] represented by arrows; see also the accompanying review by Fischer [21]. (B) Schematic representation of the prototypal T4SS VirB ⁄ D
from A. tumefaciens (left) and comparison with components of the Cag-T4SS (right). Cytoplasmic NTPases are coloured in blue, proteins
forming the core trans-membrane complex are indicated in various shades of green, and pilus components in yellow ⁄ orange. Integral trans-
membrane segments or proteins are depicted as squares. Note the presence of additional components (coloured in pink) that have been
shown to participate in the Cag-T4SS complex. In addition, the effector CagA (coloured in black) has been located at the tip of the pilus.
L. Terradot and G. Waksman Structure of the Cag-T4SS
FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS 1215
Each domain of HP0525 forms an hexameric ring that
surrounds a central chamber. The CTD ring has a
grapple-like shape, with two helices from each of the
monomers pointing into the centre of the ring to form
the claws of the grapple [28]. VirB11 proteins are
dynamic assemblies, the conformations of which
depend on their nucleotide binding state [29]. Indeed,
in HP0525 crystal structures, the nucleotides bind at
the NTD–CTD interface and stabilize their interaction.
By contrast, in the absence of nucleotide, the NTDs

become disordered and point outwards from the centre
of the hexamer, leaving an open NTD ring. When nu-
cleotides are bound, the NTDs are ordered and point
inwards in a closed ring conformation. Such important
structural changes generated by nucleotide binding,
together with functional analysis of other T4SS com-
ponents, suggest that VirB11 proteins play a role in
substrate translocation by participating in the local
unfolding of effector proteins during translocation [30].
Nucleotides are not solely responsible for structural
changes in HP0525. A protein, HP1451, was originally
identified to interact with HP0525 in a high through-
put yeast two-hybrid experiment and the interaction
was confirmed biochemically [31,32]. The structure of
the complex between HP0525 and a large portion of
HP1451 revealed that two molecules of the latter inter-
acted with the hexamer of HP0525 [33] (Fig. 2C). The
HP1451 monomer structure consists of two consecutive
KH domains that are used by the protein to interact
with several parts of the HP0525 NTDs. The two
HP1451 molecules lock the HP0525 NTDs in the
closed state and obstruct the chamber. From this
structure, HP1451 was suggested to play an inhibitor
role for HP0525 ATPase activity and CagA transfer, a
hypothesis supported by ATPase assays and in vivo
observation of complex formation. It is therefore likely
that HP1451 acts as a negative regulator for toxin
secretion [33], thereby controlling part of the secretion
process.
HP0544 (VirB3 ⁄ B4)

Little is known about the HP0544 (also named CagE).
On the basis of sequence signature, HP0544 contains
motif conserved in both VirB3 and VirB4 proteins
[34]. This is reminiscent of the Campylobacter jejuni
Fig. 2. Structures of cytoplasmic NTPases. Side and top views of the crystal structures of T4SS NTPases shown as ribbon. (A) Structure of
TrwB, the VirD4 homologue from E. coli conjugative plasmid R388 [25]. (B) Structure of HP0525 [28], the VirB11 homologue from H. pylori
Cag-T4SS with the NTDs and the CTDs coloured in light and dark blue, respectively. (C) Structure of the HP0525 ⁄ HP1451 [33]. HP0525 pro-
tomers are coloured in light blue and are in the same orientation as in (B). The two molecules of HP1451 are coloured in pink and magenta.
Structure of the Cag-T4SS L. Terradot and G. Waksman
1216 FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS
T4SS, where VirB3 and VirB4 appear to be fused into
a single protein. Thus, it is possible that CagE may
also combine VirB3 and VirB4 functions. In other
T4SS, VirB4 is known to make numerous interactions
with other T4SS components, including VirB3, VirB8,
VirB10, VirB11 and VirD4 [2]. Yet, in A. tumefaciens,
it does not interact with the substrate DNA.VirB4
activity is required for T4SS function and might also
have a structural role at the inner membrane.
The translocation pore of the Cag-T4SS
Until recently, the periplasmic core of the T4SS was
considered to be composed of VirB8, VirB7, VirB9
and VirB10. The structures of these proteins or parts
of these proteins have been solved individually (VirB8,
VirB9 [35]) or in a complex (VirB9:VirB7 [36]; VirB7-
VirB9-VirB10 [37,38]) and have provided important
information on the architecture of the periplasmic core
(Fig. 3A). Compared to these structures, the homo-
logues from the cagPAI are significantly different.
Indeed, similarities between VirB10 and HP0527 and

VirB9 and HP0528 reside only in the C-terminal por-
tion (Fig. 3C). This discrepancy is particularly appar-
ent for HP0527 that consists of almost 2000 residues,
of which only 400 at the C-terminus correspond to
VirB10 (approximately 400 residues in all T4SS). The
remaining 1600 residues have a unique composition
with a number of tandem repeats regions; see the
accompanying review by Fischer [21]. HP0532 and
HP0530 are considered as putative homologues of
VirB7 and VirB8, respectively, although the similarities
are very poor and it can be anticipated that their prop-
erties might also be different (Fig. 3B).
Structure of the translocation pore
Recently, two major advances have provided the
molecular details of the translocation pore of a T4SS.
First, the cryo-electron microscopy (EM) structure of a
VirB7-VirB9-VirB10 complex, from the plasmid
pKM101 T4SS, was determined at 15 A
˚
resolution
Fig. 3. Periplasmic core complex. (A) Ribbon representation of the crystal structures of VirB8 (Brucella suis) and ComB10 (VirB10 homo-
logue from H. pylori) and NMR structure of the TraO ⁄ TraN complex (VirB7 ⁄ 9 homologues from the pKM101 plasmid) [35,36]. (B) Cryo-EM
structure of the T4SS core complex at 15 A
˚
resolution composed of TraO, TraN and TraF (VirB7 ⁄ 9 ⁄ 10). The 1.05 MDa complex spans the
entire periplasmic space and forms channels in the inner and outer membranes. It is subdivided into two layers: the I layer inserting into the
inner membrane and the O layer inserting into the outer membrane [38]. (C) Graphic representation of the VirB homologues from the Cag-
T4SS: HP0527 (VirB10), HP0528 (VirB9). The coloured circles represent the homologous regions with VirB ⁄ D systems protein structures. (D)
Crystal structure of the O-layer with the individual components coloured as in (A) [37].
L. Terradot and G. Waksman Structure of the Cag-T4SS

FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS 1217
(Fig. 3C). The complex forms a large approximately
1 MDa complex spanning both the inner and the outer
membranes and containing 14 copies of each of the
three proteins [38]. The structure is cylindrical (length
185 A
˚
, diameter 185 A
˚
) with two distinct layers termed
‘I’ and ‘O’ connected by narrow linkers and with a
central channel spanning the entire structure (Fig. 3C).
The channel is open on the cytoplasmic side (55 A
˚
opening) but constricted on the outer-membrane side
(10 A
˚
). Two chambers, one in each of the layers, are
clearly visible.
The I and O layer connect, respectively, the inner
and outer membranes and each display double-walled
architecture but have different composition and struc-
tures [38]. The I layer consists of the N-terminal part
of VirB9 and VirB10 and is inserted into the inner
membrane. The I ring has a large central chamber
with a narrower 55 A
˚
base that forms a cup. The O
layer has two different parts, a main body and a cap
that is inserted in the outer membrane, and is formed

by the CTDs of VirB9 and VirB10 and the full-length
VirB7.
The second advance has been the crystal structure of
the O-layer [37]. This structure demonstrates a number
of surprising features. First, VirB10 forms the outer
membrane channel. Indeed, the inside of the O-layer is
lined by VirB10, and VirB10 contributes the part
crossing the outer membrane. Because VirB10 is also
known to insert into the inner membrane, this endows
VirB10 with the remarkable and unique property of
spanning both membranes. Second, the structure span-
ning the outer membrane is helical, instead of
b-stranded as are the vast majority of proteins forming
pores in the outer membrane. Indeed, VirB10 projects
a helical segment through the outer membrane, and 14
of them form the outer membrane channel. Although,
in the EM structure, this channel was con-
stricted ⁄ closed (only a 10 A
˚
hole was observed; see
above), in the X-ray crystal structure, the channel is
open. Third, VirB9 interacts closely with VirB7 and 14
VirB7 ⁄ VirB9 complexes form an outer ring stabilizing
the VirB10 tetradecameric channel. Finally, the CTD
of VirB10 exhibits an extended approximately 30 resi-
dues sequence at its N-terminus, termed the ‘lever
arm’, which embraces three consecutive VirB10 subun-
its in the tetradecameric structure and forms a plat-
form inside the O-layer. Interestingly, this platform
locates at a different level in the crystal and EM struc-

tures. This observation, coupled with the fact that, in
the EM structure, the outer membrane channel is
closed, whereas, in the crystal structure, this channel is
open, has led to the suggestion that the lever arms
might regulate the open ⁄ closed state of the channel.
Although the VirB7-VirB9-VirB10 (HP0532-
HP0528-HP0527) complex appears to be conserved,
additional Cag-T4SS specific proteins participate in the
periplasmic complex (Fig. 1); for details, see the
accompanying review by Fischer [21]. For example,
HP0532 (VirB7) does not interact directly with
HP0528 (VirB9) but requires HP0537 (CagM) that sta-
bilizes the translocation pore HP0528, HP0527 and
HP0532 complex [34]. Moreover, other protein–protein
interactions occur between the core complex and the
periplasmic proteins HP0538, HP0522, HP0530 and
HP0537. In particular, HP0522 was found to be part
of a large complex involving several Cag proteins,
including cytoplasmic, periplasmic and pilus compo-
nents, and therefore might be an important part of the
outer membrane complex of the T4SS [39].
The T4SS pilus
T4SS pili are generally composed of two proteins,
VirB2 and VirB5. VirB2 is considered the major pilin
subunit and VirB5, although less abundant, decorates
the external part of the appendage formed by VirB2.
In the VirB ⁄ D system, VirB2 is a small protein pro-
cessed into a 7.2 kDa T-pilin that is cyclized before
pilus formation [40,41]. HP0546 was proposed to be a
functional homologue of VirB2 based on sequence sim-

ilarity [34,42,43]. HP0546 is present in membrane frac-
tions and at the bacterial surface but is only a minor
component of the Cag-T4SS specific pilus (see below)
[43].
The structure of TraC, the VirB5 homologue from
the pKM101 T4SS, showed that the protein consists of
a helix bundle capped by a globular domain [44]
(Fig. 4). Mutational studies of TraC have suggested
that VirB5 proteins play a role in adhesion, mediating
cell–cell interaction during conjugation [44]. There is
no obvious homologue of VirB5 in the cagPAI. How-
ever, a detailed analysis of the HP0539 (also named
CagL) sequence suggested that it could be a structural
homologue of VirB5, which is consistent with its inter-
action with host-cell receptors and its location at the
Cag-T4SS specific pilus (see below) [34,45].
Cag-T4SS specific pilus
The Cag-T4SS pilus is remarkably unusual. Its compo-
sition appears more complex than the prototypal
VirB2 ⁄ B5 pilus produced by other T4SS. The Cag-
T4SS pilus involves not only HP0546 and HP0539 pro-
teins, but also HP0527, HP0528, HP0532, as well as
CagA. By contrast with other T4SS, there is no evi-
dence suggesting that the VirB2 homologue HP0546 is
Structure of the Cag-T4SS L. Terradot and G. Waksman
1218 FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS
the main component of the needle-like structure
described in two studies [46,47]. Perhaps the most sur-
prising finding is that part of the translocation core
complex is also involved in the pilus external structure.

Indeed, HP0527 (VirB10) and HP0532 (VirB7) associ-
ate with the pilus surface and were detected by immu-
nogold labelling [46,47]. HP0527 is able to make
intramolecular interactions with itself [34]. The central
region of HP0527 interacts with the C-terminal portion
and this interaction could provide a means of oligo-
merization to form a super-structure in direct prolon-
gation of the translocation pore (Fig. 4). Pilus
formation might be coupled with receptor binding
because Cag-T4SS assembly first requires a contact
with epithelial cells [46]. This receptor is likely to be
the a5b1 integrin. Indeed, several Cag proteins, includ-
ing HP0527, HP0539, HP0540 and CagA itself, were
shown to bind to different domains of the integrin
a5b1 [48,49]. This suggests that HP0540 might also be
exposed at the surface of the Cag-T4SS pilus. Some of
these results are conflicting (see the accompanying
review by Fischer [21]) and a general consensus has yet
to emerge. However, all studies emphasize the role of
the Cag-T4SS pilus in mediating interactions with
a5b1.
Concluding remarks
How the Cag-T4SS is assembled is still poorly under-
stood. Very recently, HP0523 was proposed to act as a
lytic transglycosylase, suggesting that HP0523 might
be the H. pylori homologue of VirB1 [50]. VirB1 pro-
teins are important with respect to piercing the pepti-
doglycan layer in the periplasm. Formation of the
double-membrane spanning core complex formed by
the HP0532-HP0528-HP0527 (VirB7-VirB9-VirB10)

proteins is likely to occur next because these proteins
assemble spontaneously in other T4SS [38]. Because
HP0527 is a major component of the Cag-T4SS pilus,
core complex formation might be coupled with pilus
formation. Subsequent steps might include recruitment
of the cytoplasmic ⁄ inner membrane ATPase complex.
Once assembled, the Cag-T4SS delivers two types of
effectors: the CagA protein and peptidoglycan frag-
ments. These have different effects on the cell and it is
unclear whether they are secreted together. Little struc-
tural information is available on the main effector
CagA, which cannot be produced as a recombinant
protein under standard conditions [51]. However, a
crystal structure of a C-terminal fragment of CagA in
complex with mitogen-activated protein kinase was
Fig. 4. A model of Cag-T4SS pilus assembly upon contact with the cellular receptor integrin a5b1. Protein directly interacting with the a5b1
integrins are HP0539 [49], HP0527, HP0540 and CagA [48]. A possible function of these interactions would be to trigger a signal for oligo-
merization of HP0527, assembly of the Cag-T4SS injection machinery, and locking of the a5b1 receptor, as suggested by [48]. The pilus sub-
structure is composed of the VirB2 functional homologue HP0546 (indicated by the yellow colour) of the pilus that is initially present only at
some areas of the cell surface [43]. The protein HP0539 (CagL) could be a homologue of VirB5 and binds to a5b1 via a RGD motif. The
structure of the VirB5 homologue TraC [44] is shown in ribbon representation (left). After sequential binding of CagA, HP0527 and possibly
HP540 to the receptor, the assembly of the injection apparatus would take place and effectors (CagA and peptidoglycan fragments) could be
translocated.
L. Terradot and G. Waksman Structure of the Cag-T4SS
FEBS Journal 278 (2011) 1213–1222 ª 2011 The Authors Journal compilation ª 2011 FEBS 1219
recently determined [52]. This structure was sufficient
to reveal 12 residues of CagA bound to the kinase
active site, demonstrating that the toxin inhibits the
enzyme by mimicking its natural substrate [52]. How-
ever, only 12 of 120 residues bound to the kinase were

visible, suggesting that the remaining part of the poly-
peptide was unfolded in the crystal. This is somehow
reminiscent of bacterial effectors delivered by other
systems that are unfolded during translocation [53]. It
is therefore possible that a large part of CagA is
unfolded during and after translocation, although
more studies are necessary to decipher the structural
details of this process. Although structural data are
accumulating on T4SS, a number of specific questions
remain unanswered concerning the Cag-T4SS machin-
ery. This is illustrated by the structural studies of ‘not-
T4SS’ Cag proteins, which have revealed that these
proteins do not resemble known structures [54–56]; see
the accompanying review by Cendron and Zanotti
[22]. Therefore, although evolutionary related to other
T4SS, the Cag-T4SS displays numerous specific fea-
tures, and more studies will be necessary to obtain a
more complete understanding of this fascinating
machinery, which is involved in one of the main steps
of H. pylori infection.
Acknowledgements
This work was funded by grant 082227 from the Well-
come Trust to G.W. and by an ATIP-Avenir and
Ligue contre le cancer grant to L.T.
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