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

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 ⁄ 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

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-

(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,

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

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].

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

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-

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),

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

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