Báo cáo khoa học: Structural and functional aspects of unique type IV secretory components in the Helicobacter pylori cag-pathogenicity island potx

MINIREVIEW
Structural and functional aspects of unique type IV
secretory components in the Helicobacter pylori
cag-pathogenicity island
Laura Cendron
1
and Giuseppe Zanotti
2
1 Department of Biological Chemistry, University of Padua, Italy
2 Venetian Institute of Molecular Medicine (VIMM), Padua, Italy
Introduction
Cytotoxin-associated gene-pathogenicity island (cagPAI)
characterizes the type I strains of Helicobacter pylori
(i.e. the virulent strains) responsible for most gastroduo-
denal diseases, including active chronic gastritis, peptic
ulcers, gastric adenocarcinoma and mucosa-associated
lymphoid tissue lymphoma [1–3]. CagA, a major anti-
genic factor of the bacterium, is the main signature of
the cagPAI-positive strains. Indeed, cagPAI confers
H. pylori the capability to express and translocate the
CagA protein inside the host cell through a secretion
machinery, which is coded by the components of the
PAI; see the accompanying review by Fischer [4]. Once
translocated, CagA associates with the inner side of the
membrane and is phosphorylated at EPIYA motifs by
Keywords
3D structure; Cag proteins; gastric cancer;
Helicobacter pylori; type IV secretion
system
Correspondence
G. Zanotti, Department of Biological

Chemistry, University of Padua, Viale G.
Colombo 3, 35121 Padua, Italy
Fax: +39 0498073310
Tel: +39 0498276409
E-mail:
(Received 16 November 2010, revised 10
January 2011, accepted 27 January 2011)
doi:10.1111/j.1742-4658.2011.08038.x
Helicobacter pylori cytotoxin-associated gene-pathogenicity island (cagPAI)
is responsible for the secretion of the CagA effector through a type IV
secretion system (T4SS) apparatus, as well as of peptidoglycan and possibly
other not yet identified factors. Twenty-nine different polypeptide chains
are encoded by this cluster of genes, although only some of them show a
significant similarity with the constitutive elements of well characterized
secretion systems from other bacteria. The other cagPAI components repre-
sent almost unique proteins in this scenario. The majority of the T4SS
include approximately fifteen components, taking into account either the
transmembrane complex subunits, ATPases or substrate factors. The com-
position of the cagPAI is very complex: it includes proteins most likely
involved at different levels in the pilus assembly, stabilization and process-
ing of secreted substrate, as well as regulatory particles possibly involved in
the control of the entire apparatus. Despite recent findings with respect to
components that play a role in the interaction with the host cell, the func-
tion of several cagPAI proteins remains unclear or unknown. This is partic-
ularly true for those that represent unique members with no clear similarity
to those of other T4SS and no obvious evidence of involvement in the
secretion of CagA or induction of pro-inflammatory responses. We summarize
what is known about these accessory components, both from a molecular
and structural point of view, as well as their putative physiological role.
Abbreviations

cagPAI, cytotoxin-associated gene-pathogenicity island; IL, interleukin; T4SS, type IV secretion system.
FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1223
host tyrosine kinases. The phosphorylation triggers a
series of interactions between CagA and human proteins
that interfere with the signalling cascades at multiple
levels, resulting in a dramatic change of cellular mor-
phology, known as the ‘hummingbird phenotype’, and a
remarkable enhancement of cellular motility, causing
cell scattering [5]; see the accompanying review by
Tegtmeyer et al. [6].
The entire cagPAI region is 37 kb long, including
approximately 29 genes [7], which encode for the com-
ponents of a type IV secretion system (T4SS), homolo-
gous to the VirB ⁄ D4 machinery of Agrobacterium
tumefaciens, the best characterized T4SS that is
regarded as the prototype among that family members
[8]. T4SS are multicomponent membrane-spanning
transport systems ancestrally related to the conjugation
processes, which can be responsible for diverse pro-
cesses such as DNA transfer, DNA uptake and release,
and translocation of proteins that have an effector role
in the target cell. Eleven out of the 29 Cag proteins can
be ascribed to the secretion machinery itself or have
been proposed to represent functional homologues of
VirB proteins [9–11]. For a more detailed description of
the correspondence; see the accompanying review by
Fischer [4], who describes the relationships between cag
and virB ⁄ D4 genes in detail.
A second major effect on the host cells, which is
elicited only by H. pylori strains harbouring a func-

tional cagPAI, is the activation of nuclear transcription
factor-jB and the induction of pro-inflammatory pro-
duction of cytokines, mainly interleukin (IL)-8 [12].
The activation of a pro-inflammatory signalling
cascade has been confirmed to be Nod1 cytoplasmic
receptor dependent and a model has been proposed
[13] according to which bacterial peptidoglycan is the
key effector of such a response. Indeed, the peptidogly-
can muropeptides could be transferred to and internal-
ized into the gastric epithelial cells only if a functional
cagPAI is present. However, it is yet to be established
whether this kind of stimuli as a result of muropep-
tides secretion is promoted by a syringe-like mecha-
nism, analogous to CagA, or whether the only
intimate contact with the host cell surface of the com-
plex coded by the cagPAI could induce a facilitated
internalization of H. pylori peptydoglycan fragments.
CagA deficient mutants do not affect that response to
wide extent, whereas, in the absence of a functional
cagPAI, peptidoglycan release may still occur but with
much lower efficiency.
Electron microscopy studies indicate that, upon
attachment of H. pylori to gastric epithelial cells, pilus-
like structures are formed between the bacteria and
host cells. Partial characterizations of the secretion
organelle allow a description of some of the interac-
tions occurring in the H. pylori T4SS, mainly those
concerning components that belong to the external
pilus, protruding from the bacterial surface toward the
extracellular milieu. At least five of the VirB func-

tional ⁄ structural homologues of this syringe-like com-
plex have been localized in this way: CagC (VirB2),
CagL (VirB5), CagT (VirB7), CagX (VirB9) and CagY
(VirB10) [9,14,15]. At the same time, yeast two-hybrid
system approaches combined with immunoprecipita-
tion studies allow a description of the interactions that
were assumed to involve Cag proteins [16,17]. These
findings, combined with previous analysis on single
components, have allowed the proposal of preliminary
models of the H. pylori T4SS.
Systematic studies have established that some Cag
proteins are essential or important for CagA transloca-
tion, whereas others are involved in IL-8 secretion, or
both [18,19]. A few are apparently unnecessary for any
of these effects. Despite the fact that many of the
cagPAI proteins have been demonstrated to be involved
in the CagA translocation and⁄ or IL-8 induction ⁄
peptidoglycan release, very little is known about their
specific function, and this is particularly true for those
components that are unique to the Cag apparatus.
In this minireview, we concentrate on this last class
of cagPAI proteins, and summarize what is known
about them both from a molecular and structural
point of view, as well as their putative physiological
roles.
The unique members of the Cag-T4SS
Cagf (cag1/HP0520), Cage (cag2/HP0521)
Very little is known about the proteins encoded by
these two genes. Both were found not to be necessary
for either translocation of CagA or for IL-8 induction

[18]. cagf (cag1⁄ HP0520) was found to be among the
most highly expressed genes in an H. pylori transcript
profile analysis from infected human gastric mucosa,
together with cagC (cag25 ⁄ HP0546), whereas Cage
was not detected at all. Interestingly, the expression
levels of cagC and cagf reached values analogous to
genes important for bacterial survival and homeostasis,
such as catalase, urease and NapA; by contrast, the
transcript abundance of other cag genes appeared to
be much lower, indicating that cagPAI consists of mul-
tiple operons tightly controlled by different promoter
regions [20]. The presence of the cagf gene was also
found to be related to specific ethnic groups, although
an association with virulence and disease has not yet
been demonstrated [21].
Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti
1224 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS
Cagd (cag3/HP0522)
Cagd is a 55 kDa protein essential both for the secre-
tion of CagA and the induction of IL-8. The corre-
sponding sequence is characterized by a clear
N-terminal signal sequence for export into the peri-
plasmic space, although no transmembrane helices can
be predicted by the most common software. Sequence
alignments with a nonredundant database allow the
identification of some weak but interesting similarities
to proteins associated with adhesion and interaction
with eukaryotic cells, such as the surface-exposed
Streptococcus pneumoniae choline-binding protein A.
When H. pylori bacteria were investigated in the

absence of host cell contact, Cagd was found to enrich
in the membrane fractions, even if a minor contribu-
tion was also present in the soluble pool. Moreover, it
was shown to co-purify with other Cag proteins,
mainly CagT, Cagb, CagD and Cagf, which represent
the most specific interaction partners. Other Cag-T4SS
members were coimmunoprecipitated with Cagd, even
if with a lower abundance: CagM, CagX, CagC, CagE
and Caga [22]. Previous yeast two-hybrid experiments
demonstrated that Cagd associates both in homotypic
oligomers a nd heterotypic complexes with CagV (VirB8),
CagT (Vi rB7), CagM and CagG [16]. In particular, the
interactionwithCagT,acorecomponentinvolvedin
the Cag-T4SS outer membrane sub-complex, was identi-
fied by mu ltiple i ndependen t t echniques and more
accurately elucidated. Size exclusion chromatography
analysis allowed the isolation of both Cagd-in depend ent
oligomers and large Cagd-CagT complexes, reminiscent
of what occurs in vivo. Finally, pulse-chase assays
demonstrated a mutual correlation between expression
levels and the stability of Cagd and CagT proteins
[22]. Taken together, these results suggest that Cagd
represents a unique and essential core component of
the Cag-T4SS.
CagZ (cag6/HP0526)
CagZ protein, a 23 kDa soluble protein, was found to
be absolutely essential for the translocation of CagA
but not for the induction of IL-8 [18]. The crystal
structure [23] shows that it consists of a single compact
L-shaped domain, composed of seven a-helices, includ-

ing approximately 70% of the total residues (Fig. 1A).
The protein fold can be described as an up-and-down
bundle: four long, twisted stretches run antiparallel to
each other. A twist in each stretch produces an
L-shaped molecule: its longest arm has dimensions of
approximately 15 · 25 · 60 A
˚
, whereas the shortest is
15 · 14 · 30 A
˚
. The side chains located at the protein
interior are all hydrophobic, with the exception of
three residues; thus, packing of the entire bundle is dri-
ven by hydrophobic forces. By contrast, the molecular
surface is heavily charged: 26 negative- and 21 posi-
tively-charged side chains, over a total of 199 residues.
The presence of a flexible C-terminal tail and the heav-
ily-charged surface suggest that CagZ may participate
in the interaction of effector proteins with one or more
components of the H. pylori T4SS on the cytoplasmic
side of the inner membrane. One or more clusters of
surface exposed amino acids have been suggested to
represent structural motifs of some relevance for pro-
tein activity (NEST prediction, ProFunc server; http://
www.ebi.ac.uk/thornton-srv/databases/profunc/).
An exhaustive search of structural similarities in the
Protein Data Bank did not provide any remarkable
information about the function of the protein. The
Fig. 1. (A) Cartoon representation of CagZ
protein fold. The L-shape is clearly visible.

(B) Two views of the electrostatic potential
surface of CagZ. In the overall, the surface
is strongly hydrophilic, with patches of
positive and negative charges.
L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI
FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1225
only weak and partial similarity found by the most
common servers involve Rab GTPases, comprising
proteins that regulate the maturation and transport of
endoplasmic-reticulum-derived vesicles in eukaryotic
cells (ProFunc server analysis: />thornton-srv/databases/profunc/).
CagZ has been detected by 2D differential in-gel
electrophoresis from H. pylori cultures in vitro [16],
demonstrating that it is expressed at a relatively high
abundance compared to other Cag proteins. No pro-
cessing of eventual N-terminal signal peptide was
observed, which is in agreement with predictions based
on the sequence only. This supports the idea that its
surface and charge distribution make it prone to be
involved in protein assemblies, most likely from the
cytoplasmic side. Finally, CagZ has been proposed to
interact with multiple Cag components, not only non-
VirB homologues, such as CagF, CagM, CagG and
CagI, but also with some T4SS core components, such
as CagV, CagY and the ATPase CagE [16].
CagS (cag13/HP0534)
The CagS gene is located immediately after the cluster
of cagPAI genes whose putative products show homol-
ogies with the VirB proteins that define the structural
core of T4SS. Experimental evidence showed that,

similar to CagZ, CagS is expressed at a reasonable
abundance in H. pylori cultures in vitro [16].
Primary sequence analyses do not show any strong
similarity of CagS with proteins of known function,
except for some weak similarities with components
involved in the peptidoglycan biosynthesis, which
belong to the FemABX family of enzymes [24]. The
crystal structure of CagS has been determined at a res-
olution of 2.3 A
˚
[25]. The protein is a single compact
domain, with an all-a structure (Fig. 2). Ten a-helices,
labelled from A to J, can be distinguished. Helices B,
E, F and H, arranged in an up-and-down topology,
form the structural core, whereas the short helices C,
D and G on one side, and A, I and J on the other,
represent types of appendices, conferring a ‘peanut
shape’ to the overall structure. The ten helices that
form the protein are held together mainly by hydro-
phobic forces, with few hydrophilic interhelical inter-
actions. The model lacks 20 amino acids at the
N-terminus and 25 at the C-terminus, which are flexi-
ble or disordered. Some confuse electron density is
present in correspondence to the N-terminus, although
it was not possible to trace the polypeptide chain, with
the exception of few helical turns.
The model shows a highly charged surface, with 24
positively- and 24 negatively-charged residues. In par-
ticular, the tertiary structure defines a negatively-
charged region including several glutamate and aspar-

tate residues confined to the portion of the molecule
involving a-helix A, the nearby loop, the first and last
turns of helices E and F, and the C-terminus helices I
and J. In addition, there is a lysine-rich N- and C-ter-
minus, in accordance with the basic isoelectric point of
CagS. However, these lysine rich unmodelled N- and
C-terminal appendages might define some positively-
charged brunches playing a potential role in Cag pro-
teins interactions. As mentioned in the case of CagZ,
even if to a minor extent, some putative interactions
with the other cagPAI components have been detected
by a yeast two-hybrid system, involving mainly CagZ
and CagM proteins. Another peculiar feature of the
molecule is the presence of fourteen methionine resi-
dues over a sequence of 199 amino acids, which is an
unusually high content compared to other proteins.
Four of them, M69, M130, M133, M138, define a clus-
ter in the 3D structure, approximately located in the
internal side of the peanut. The results of the crystallo-
graphic model of CagS do not show any clear evidence
of architectural similarity to other known structures,
with the exception of a weak structural homology with
the phosphotransfer domain (HPt) of CheA, a histi-
dine protein kinase that controls chemotaxis response
in bacteria [26]. This homology is too weak to be con-
sidered as providing any clues with respect to protein
function. Even a primary sequence alignment with a
nonredundant database shows very limited similarities;
the one with the best score being that with phosphoch-
oline cytidylyltransferases, comprising rate-limiting

enzymes for surfactant phospholipid synthesis.
Fig. 2. (A) Cartoon view of CagS protein. Methionine residues are
represented by ball and sticks. A cluster of four methionine resi-
dues is visible in the lower part. (B) van der Waals representation
which shows the ‘peanut’ shape of the protein.
Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti
1226 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS
CagQ (cag14/HP0535), CagP (cag15/HP0536)
The products of these two short genes correspond to
putative proteins of 126 and 114 amino acids, respec-
tively. They are both predicted to be membrane pro-
teins [27] and they were found not to be necessary for
either translocation of CagA or for IL-8 induction in
H. pylori 26695 strain [18]. CagP appears to play some
role in H. pylori adherence to gastric epithelial cells, in
addition to classical adhesins, because mutations in its
gene may affect bacterium pathogenicity by reducing
either the ability of the bacteria to attach to gastric
epithelial cells or the intensity of bacteria–host cell
interactions [28].
CagM (cag16/HP0537)
CagM protein is a 43.7 kDa protein, characterized by
a putative N-terminal signal sequence, and at least
three transmembrane helices can be predicted from the
sequence pattern. It has been detected in the mem-
brane-bound fractions isolated from both in vivo (from
gastric patients isolates) and in vitro H. pylori cultures
by 2D-electrophoresis and MS studies [16,29]. In par-
ticular, the results of the 2D differential in-gel electro-
phoresis analysis performed by Busler et al. [16] is

suggestive of an N-terminal processing as hypothesized
by the predictions.
Systematic mutagenesis analysis clearly showed that
DcagM mutants are neither able to produce an efficient
CagA translocation, nor to release peptidoglycan degra-
dation fragments [13,18,30], thus suggesting an essential
role for the cagM gene product. By using a reporter
assay in human gastric cancer cells, CagM (along with
cagPAI coded protein, CagL) has also been demon-
strated to promote the activation of nuclear factor-jB.
More recent experiments with DcagE, DcagM and
DcagA isogenic mutant strains of H. pylori provided
preliminary evidence that these genes could be
involved in the repression of the gene coding for the
catalytic subunit of a human gastric H ⁄ K-ATPase
{{ 770 Saha,A. 2008;}}. Generally, this effect might be
stimulated by a functional Cag-T4SS, allowing
H. pylori to inhibit acid secretion by gastric cells and
induce episodes of transient hypochlorhydria that facil-
itate bacterial colonization. Evidence for protein–pro-
tein interactions involving CagM has been observed by
yeast two-hybrid analysis. In such experiments, CagM
was found to form complexes with many other Cag
proteins both belonging to the core apparatus, includ-
ing CagX, CagY, CagT, CagV and Cagd, as well as
other Cag components such as the ATPase CagE,
CagF, CagG, CagZ and CagS [16]. In a different study
employing a similar approach, interactions with CagX
and partial interactions with CagT were confirmed,
whereas those with CagF and CagY were not [17].

Furthermore, both studies identified a clear tendency
for CagM to associate, forming homotypic oligomers.
An analogous behaviour was observed when we puri-
fied a recombinant CagM construct expressed in
E. coli for structural studies. Oligomers composed of
five to six subunits were isolated and partially charac-
terized by gel filtration and preliminary electron
microscopy analysis (L. Cendron, unpublished results).
In any case, the proposed rich network of interac-
tions of this protein agrees with its functional rele-
vance and localization studies, where it was found to
enrich both in the inner and partially in the outer
membrane fractions. A model has been proposed
according to which CagM, together with CagX and
CagT, associates in the outer membrane basal body of
the Cag-T4SS, and the results obtained are in good
agreement with the main studies in this respect.
CagN (cag17/HP0538)
Full-length CagN protein (306 amino acids, 35 kDa)
has been demonstrated to be processed at the C-termi-
nus, giving rise to a product of approximately 24 kDa
(CagN
1–216
), most likely by a mechanism that is not
dependent on other cagPAI proteins [31]. Interestingly,
the first 24 amino acids are intact in the endogenous
protein, despite it shows a clear N-terminus hydro-
phobic pattern and a putative cleavage site that can be
easily predicted. The entire primary sequence is pre-
dicted to be largely unfolded (foldindex, http://bip.

weizmann.ac.il/fldbin/findex). CagN localization studies
demonstrated that it is not delivered into the host cell
together with CagA but, in contrast, it remains local-
ized at the bacterial membrane, most likely anchored
by a N-terminal hydrophobic helix [31]. cagN gene
deletion appears not to abolish directly the main conse-
quences of a functional cagPAI (i.e. CagA translocation
and IL-8 induction), even if a variable efficiency of both
processes has been observed [18].
Recombinant CagN deleted forms have been pro-
duced (His6-CagN
25–306
, CagN
25–216
-His
6
) and par-
tially characterized in our laboratory. Although the
DN-terminal hydrophobic construct was strongly
prone to aggregation, the one also truncated at the
C-terminal resulted in a soluble protein that behaves
similar to a monomer in solution, showing a secondary
structure content composed of 13% b-sheet, 30%
a-helix, with a certain fraction not being ascribed
to any well characterized secondary structure motifs
(L. Cendron, unpublished results).
L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI
FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1227
CagI (cag19/HP0540), CagH (cag20/HP0541)
Very little is known about CagI (41.5 kDa) and

CagH (39 kDa), despite the fact that knockout stud-
ies demonstrated they are essential for CagA translo-
cation and tyrosine phosphorylation. CagH was also
shown to be involved in IL-8 induction in epithelial
cells, whereas a cagI deletion mutant does not affect
that ability [18]. CagH is also predicted to be secreted
out of the inner membrane as a result of an N-termi-
nal signal sequence, whereas only one of three differ-
ent algorithms tested suggests the presence of a
putative hydrophobic helix in the mature protein.
CagI most likely is a nonsecretory protein, anchored
to the inner membrane toward the periplasmic space
as a result of a N-terminal hydrophobic helix, span-
ning residues 26–51, thus supporting the idea that it
might be involved in the translocation as a putative
effector protein rather than being a component of the
T4SS apparatus. Finally, interaction studies indicated
that CagI might interact with CagZ and CagG, and
weak evidence of interaction with Cagb was also
observed [16].
CagG (cag21/HP0542)
The product of this gene is a 16 kDa protein with a
very acidic isoelectric point and a predicted N-terminal
signal peptide with a putative cleavage site between
residues 27 and 28. A weak homology with the flagel-
lar motor switch protein or toxin co-regulated pilus
biosynthesis protein D has been detected [32]. Yeast
two-hybrid screens, as noted elsewhere, indicate that
CagG is involved in multiple protein–protein interac-
tions with CagM, Cagd, CagF and CagZ and, to a

minor extent, with CagT and the VirD4 homologous
Cagb [16].
Analogous to cagI, cagG deletion mutants are inca-
pable of delivering CagA into gastric epithelial cells,
although they retain the capacity to induce IL-8 pro-
duction, pointing toward a potential effector role for
the protein. Other studies provided different evidence,
showing a marked reduction of IL-8 production from
gastric epithelial cells, as well as a reduced capacity to
adhere to epithelial cells in vitro and to colonize Mon-
golian gerbils in vivo [33]. Similar results were found
in an experiment with cagG-deleted strains tested on
cultivated KATOIII cells [34].
CagF (cag22/HP0543)
This 268 amino acids protein was demonstrated to
interact with CagA, presumably at the inner bacterial
membrane, and this interaction is essential for CagA
translocation in the host. These data were used to sug-
gest that CagF might play a chaperone function in the
early steps of CagA recruitment and delivery into the
T4SS channel [35,36]. Subsequently CagF was shown
to interact with the 100 amino acids region adjacent to
the C-terminal secretion signal of CagA [37]. Weak
interactions involving three other Cag proteins (CagZ,
CagT and CagM) were also detected. Localization
studies indicated that it is both present in the mem-
brane fractions and in the cytoplasm. A His6-tagged
construct in our hands behaves as a soluble protein,
even if it has a clear tendency to form oligomers of dif-
ferent sizes, coexisting with a major fraction approxi-

mately corresponding to a monomer. Detergent
treatments appeared to reduce the contribution of very
large unspecific oligomers and favour the presence of
dimers and ⁄ or monomers.
CagD (cag24/HP0545)
The cagD locus is present in a majority of clinical iso-
lates, although little is known about its role. Its amino
acid sequence contains a predicted signal sequence for
secretion in the periplasmic space.
The crystal structure of CagD, solved in two differ-
ent crystal forms at medium resolution (2.2 A
˚
and
2.75 A
˚
for the monoclinic and the hexagonal forms,
respectively), shows that, in both cases, CagD is a
homodimer, where the two monomers are covalently
linked by a disulfide bridge (Fig. 3) [19]. In both crys-
tal forms, the N-terminal domain is not visible in one
monomer and absent in the other, as a result of prote-
olysis. Consequently, the model is available only for
residues 47–176. The visible part of each monomer
folds as a single domain, characterized by a b-sheet
flanked by a-helices. Five b-strands, labelled from A to
E, run all contiguous. The N-terminus of the monomer
includes strand A, an a-helix and a long stretch, and
the C-terminal portion includes two relatively long
a-helices and a final b-strand, F, which protrudes from
the core of the monomer and runs anti-parallel to the

same strand of a second monomer, allowing for the
formation of the dimer. The surface of interaction
between monomers also involves portion of chains D
and E of the two monomers, which are held together
not only by the S-S bridge between two Cys172, but
also by hydrogen bonds. A second intramolecular
disulfide bridge, between Cys120 and Cys133, helps to
stabilize the 3D structure. The dimer presents a large
crevice inbetween the two monomers, and the 46
N-term amino acids of one monomer could partially
fill in this cavity.
Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti
1228 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS
The CagD overall fold is relatively common: the
most relevant among proteins that present a similar
fold is the SycT chaperone of Yersinia enterocolitica
type III secretion system [38]. In particular, SycT
shares the same topology in the region including the
N-terminal helix and the b-sheet element, whereas it
displays a remarkable difference in the orientation of
the a-helices located at the C-terminus. Analogous to
Yersinia SycT chaperone, CagD presents all the main
a-helical motifs grouped on just one side of the
b-strands, whereas all the other type III secretion sys-
tem chaperones display a third a-helix on the opposite
side, and this last is widely involved in the dimeriza-
tion process. However, the dimeric arrangement of
CagD is quite different from SycT as well as that of
other members of this family.
Finally, when crystallized in the presence of Cu(II),

the protein shows the presence of the ion coordinated
in a small cavity of the surface at the polar opposites
of each monomer, close to another dimer present in
the crystal packing and partially involving it in the
coordination. It is likely that the presence of Cu(II) is
an artefact of the crystallization, although the possibil-
ity that the protein can physiologically bind cations
cannot be ruled out completely.
Disruption of the cagD gene was first reported to
have an intermediate and variable effect on CagA deliv-
ery and IL-8 induction phenotype [18]. Subsequently,
CagA tyrosine phosphorylation and IL-8 assays have
demonstrated that CagD is involved in CagA transloca-
tion into the host epithelial cells, although it is not an
absolute requirement for T4SS pilus assembly [19].
As suggested by the presence of a secretion signal at
the protein N-terminus, CagD is mainly found in the
periplasmic space, partially associated with the inner
membrane. Interestingly, it was found to be secreted
in the culture supernatant and this result was found
not to be a result of generic bacterial lysis. Moreover,
in a H. pylori infection experiment with AGS cells, sig-
nificant amounts of CagD were found to be associated
with the host cell membranes, and this interaction
appeared to be independent of CagA translocation or
the components of the T4SS, such as CagF. Because
this localization was independent of the various tested
cag mutants, these findings may indicate that CagD
is released into the supernatant during host cell infec-
tion by an unknown independent mechanism and then

binds to the host cell surface or is incorporated in the
pilus structure.
Taken together, these results suggest that CagD may
serve as a multifunctional component of the T4SS,
which is involved in CagA secretion at the inner mem-
brane and may localize outside the bacteria to promote
additional effects on the host cell; however, whether
these effects are required for CagA translocation or
trigger CagA-independent virulence functions remains
unclear.
Conclusions
Despite several studies carried out during the last
15 years on cagPAI, several questions about its compo-
nents still remain unanswered. Those members that are
not strictly structural are, in this sense, particularly
puzzling because the role they play in the process of
CagA secretion or IL-8 induction is still unknown or
uncertain. However, partial maps of the H. pylori trans-
membrane core apparatus and external pilus have been
defined as a result of recent localization and interaction
studies. Together with the VirB ⁄ D homologues CagV,
Fig. 3. (A, B) Two different views of CagD dimer. Disulfide bridges are shown in yellow. It is possible to see the two b-strands, one per
each monomer, that favour dimerization of the protein. (C) The electrostatic potential surface of CagD dimer.
L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI
FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1229
CagX, CagY, CagT, Caga, Cagb and CagE, the pro-
teins Cagd, CagM and CagZ have been identified as
part of a wide network of interactions, with the first two
most likely as unique oligomeric core components.
CagF has been recognized to act as a chaperone of the

major effector CagA, with CagL as a component play-
ing a role in pilus adhesion to gastric epithelial cells.
Finally, for a few other Cag proteins, localization in the
bacterial compartments has been characterized. As
described in this minireview, the 3D structure of only
three of these unique components (CagZ, CagS and
CagD) is now available, along with Caga ATPase.
The lack of a clear picture of the biological func-
tion and organization of some cagPAI components is
also a major obstacle to structural studies because
most of these gene products possibly do not act as
single proteins, but perhaps as subunits of larger
complexes, or they are made to act in concert with
other partners. For these reasons, further accurate
studies on the interactions among cagPAI compo-
nents will be relevant not only to clarify the function
of these proteins, but also for future structural inves-
tigations.
Acknowledgements
We acknowledge all the PhD students and post-doctoral
students that have contributed to the structural studies
on the cag proteins over the years. This work was sup-
ported by the Ministero dell’Istruzione, dell’Universita
`
e
della Ricerca, MIUR (PRIN 2007LHN9JL) and by the
University of Padua, Italy.
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