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 comple
Trang 1Architecture of the Helicobacter pylori Cag-type IV
secretion system
Laurent Terradot1and Gabriel Waksman2
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: laurent.terradot@ibcp.fr 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: g.waksman@bbk.ac.uk or
g.waksman@ucl.ac.uk
(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.
Trang 2macromolecules, 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 retrans-ferred
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 pyloriComB 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
Trang 3Cag 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.
Trang 4Each 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.
Trang 5T4SS, 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].
Trang 6(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 platplat-form
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
Trang 7the 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.
Trang 8recently 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
References
1 Durand E, Verger D, Rego AT, Chandran V, Meng G,
Fronzes R & Waksman G (2009) Structural biology of
bacterial secretion systems in gram-negative
patho-gens – potential for new drug targets Infect Disord
Drug Targets 9, 518–547
2 Fronzes R, Christie PJ & Waksman G (2009) The
struc-tural biology of type IV secretion systems Nat Rev
Microbiol 7, 703–714
3 Alvarez-Martinez CE & Christie PJ (2009) Biological
diversity of prokaryotic type IV secretion systems
Microbiol Mol Biol Rev 73, 775–808
4 Dreiseikelmann B (1994) Translocation of DNA across
bacterial membranes Microbiol Rev 58, 293–316
5 Wallden K, Rivera-Calzada A & Waksman G (2010)
Type IV secretion systems: versatility and diversity in
function Cell Microbiol 12, 1203–1212
6 Christie PJ (2004) Type IV secretion: the Agrobacterium
VirB⁄ D4 and related conjugation systems Biochim
Biophys Acta 1694, 219–234
7 Hooykaas PJ & Schilperoort RA (1992) Agrobacterium and plant genetic engineering Plant Mol Biol 19, 15–38
8 Hamilton HL, Dominguez NM, Schwartz KJ, Hackett
KT & Dillard JP (2005) Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion sys-tem Mol Microbiol 55, 1704–1721
9 Hofreuter D, Odenbreit S & Haas R (2001) Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secre-tion system Mol Microbiol 41, 379–391
10 Kersulyte D, Velapatino B, Mukhopadhyay AK, Cah-uayme L, Bussalleu A, Combe J, Gilman RH & Berg
DE (2003) Cluster of type IV secretion genes in Helicob-acter pylori’s plasticity zone J BHelicob-acteriol 185, 3764–3772
11 Fischer W, Windhager L, Rohrer S, Zeiller M, Karnholz
A, Hoffmann R, Zimmer R & Haas R (2010) Strain-spe-cific genes of Helicobacter pylori: genome evolution dri-ven by a novel type IV secretion system and genomic island transfer Nucleic Acids Res 38, 6089–6101
12 Kersulyte D, Lee W, Subramaniam D, Anant S,
Herre-ra P, CabreHerre-ra L, Balqui J, BaHerre-rabas O, Kalia A, Gilman
RH et al (2009) Helicobacter pylori’s plasticity zones are novel transposable elements PLoS ONE 4, e6859
13 Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W
& Haas R (2000) Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion Science 287, 1497–1500
14 Tegtmeyer N, Wessler S & Backert S (2011) Role of the cagpathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis FEBS J 278, 1190–1202
15 Hatakeyama M & Higashi H (2005) Helicobacter pylori CagA: a new paradigm for bacterial carcinogenesis Cancer Sci 96, 835–843
16 Backert S, Tegtmeyer N & Selbach M (2010) The versa-tility of Helicobacter pylori CagA effector protein func-tions: the master key hypothesis Helicobacter 15, 163–176
17 Hatakeyama M (2008) SagA of CagA in Helicobacter pyloripathogenesis Curr Opin Microbiol 11, 30–37
18 Backert S, Moese S, Selbach M, Brinkmann V & Meyer
TF (2001) Phosphorylation of tyrosine 972 of the Helicobacter pyloriCagA protein is essential for induc-tion of a scattering phenotype in gastric epithelial cells Mol Microbiol 42, 631–644
19 Viala J, Chaput C, Boneca IG, Cardona A, Girardin
SE, Moran AP, Athman R, Memet S, Huerre MR, Coyle AJ et al (2004) Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island Nat Immunol 5, 1166–1174
20 Stingl K, Muller S, Scheidgen-Kleyboldt G, Clausen M
& Maier B (2010) Composite system mediates two-step DNA uptake into Helicobacter pylori Proc Natl Acad Sci USA 107, 1184–1189
Trang 921 Fischer W (2011) Assembly and molecular mode of
action of the Helicobacter pylori Cag type IV secretion
apparatus FEBS J 278, 1203–1212
22 Cendron L & Zanotti G (2011) Structural and
func-tional aspects of unique type IV secretory components
in the Helicobacter pylori cag pathogenicity island
FEBS J 278, 1223–1231
23 Fischer W, Puls J, Buhrdorf R, Gebert B, Odenbreit S
& Haas R (2001) Systematic mutagenesis of the
Heli-cobacter pyloricag pathogenicity island: essential genes
for CagA translocation in host cells and induction of
interleukin-8 Mol Microbiol 42, 1337–1348
24 Christie PJ & Cascales E (2005) Structural and dynamic
properties of bacterial type IV secretion systems
(review) Mol Membr Biol 22, 51–61
25 Gomis-Ruth FX, Moncalian G, Perez-Luque R,
Gonzalez A, Cabezon E, de la Cruz F & Coll M
(2001) The bacterial conjugation protein TrwB
resembles ring helicases and F1-ATPase Nature 409,
637–641
26 Jurik A, Hausser E, Kutter S, Pattis I, Prassl S, Weiss
E & Fischer W (2010) The coupling protein Cag{beta}
and its interaction partner CagZ are required for type
IV secretion of the Helicobacter pylori CagA protein
Infect Immun 78, 5244–5251
27 Schroder G, Krause S, Zechner EL, Traxler B, Yeo HJ,
Lurz R, Waksman G & Lanka E (2002) TraG-like
proteins of DNA transfer systems and of the
Helicob-acter pyloritype IV secretion system: inner membrane
gate for exported substrates? J Bacteriol 184, 2767–
2779
28 Yeo HJ, Savvides SN, Herr AB, Lanka E & Waksman
G (2000) Crystal structure of the hexameric traffic
ATPase of the Helicobacter pylori type IV secretion
system Mol Cell 6, 1461–1472
29 Savvides SN, Yeo HJ, Beck MR, Blaesing F, Lurz R,
Lanka E, Buhrdorf R, Fischer W, Haas R & Waksman
G (2003) VirB11 ATPases are dynamic hexameric
assemblies: new insights into bacterial type IV secretion
EMBO J 22, 1969–1980
30 Christie PJ, Atmakuri K, Krishnamoorthy V,
Jakubow-ski S & Cascales E (2005) Biogenesis, architecture, and
function of bacterial type IV secretion systems Annu
Rev Microbiol 59, 451–485
31 Rain JC, Selig L, De Reuse H, Battaglia V, Reverdy C,
Simon S, Lenzen G, Petel F, Wojcik J, Schachter V
et al.(2001) The protein–protein interaction map of
Helicobacter pylori Nature 409, 211–215
32 Terradot L, Durnell N, Li M, Li M, Ory J, Labigne A,
Legrain P, Colland F & Waksman G (2004)
Biochemi-cal characterization of protein complexes from the
Heli-cobacter pyloriprotein interaction map: strategies for
complex formation and evidence for novel interactions
within type IV secretion systems Mol Cell Proteomics
3, 809–819
33 Hare S, Fischer W, Williams R, Terradot L, Bayliss R, Haas R & Waksman G (2007) Identification, structure and mode of action of a new regulator of the
Helicobacter pyloriHP0525 ATPase EMBO J 26, 4926–4934
34 Kutter S, Buhrdorf R, Haas J, Schneider-Brachert W, Haas R & Fischer W (2008) Protein subassemblies of the Helicobacter pylori Cag type IV secretion system revealed by localization and interaction studies J Bacte-riol 190, 2161–2171
35 Terradot L, Bayliss R, Oomen C, Leonard GA, Baron
C & Waksman G (2005) Structures of two core subunits
of the bacterial type IV secretion system, VirB8 from Brucella suisand ComB10 from Helicobacter pylori Proc Natl Acad Sci USA 102, 4596–4601
36 Bayliss R, Harris R, Coutte L, Monier A, Fronzes R, Christie PJ, Driscoll PC & Waksman G (2007) NMR structure of a complex between the VirB9⁄ VirB7 inter-action domains of the pKM101 type IV secretion sys-tem Proc Natl Acad Sci USA 104, 1673–1678
37 Chandran V, Fronzes R, Duquerroy S, Cronin N, Navaza J & Waksman G (2009) Structure of the outer membrane complex of a type IV secretion system Nature 462, 1011–1015
38 Fronzes R, Schafer E, Wang L, Saibil HR, Orlova EV
& Waksman G (2009) Structure of a type IV secretion system core complex Science 323, 266–268
39 Pinto-Santini DM & Salama NR (2009) Cag3 is a novel essential component of the Helicobacter pylori Cag type
IV secretion system outer membrane subcomplex
J Bacteriol 191, 7343–7352
40 Jones AL, Lai EM, Shirasu K & Kado CI (1996) VirB2
is a processed pilin-like protein encoded by the Agro-bacterium tumefaciensTi plasmid J Bacteriol 178, 5706–5711
41 Eisenbrandt R, Kalkum M, Lai EM, Lurz R, Kado CI
& Lanka E (1999) Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits J Biol Chem 274, 22548–22555
42 Kalkum M, Eisenbrandt R, Lurz R & Lanka E (2002) Tying rings for sex Trends Microbiol 10, 382–387
43 Andrzejewska J, Lee SK, Olbermann P, Lotzing N, Katzowitsch E, Linz B, Achtman M, Kado CI, Suer-baum S & Josenhans C (2006) Characterization of the pilin ortholog of the Helicobacter pylori type IV cag pathogenicity apparatus, a surface-associated protein expressed during infection J Bacteriol 188, 5865–5877
44 Yeo HJ, Yuan Q, Beck MR, Baron C & Waksman G (2003) Structural and functional characterization of the VirB5 protein from the type IV secretion system encoded by the conjugative plasmid pKM101 Proc Natl Acad Sci USA 100, 15947–15952
45 Backert S, Fronzes R & Waksman G (2008) VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems? Trends Microbiol 16, 409–413
Trang 1046 Rohde M, Puls J, Buhrdorf R, Fischer W & Haas R
(2003) A novel sheathed surface organelle of the
Helicobacter pyloricag type IV secretion system Mol
Microbiol 49, 219–234
47 Tanaka J, Suzuki T, Mimuro H & Sasakawa C (2003)
Structural definition on the surface of Helicobacter pylori
type IV secretion apparatus Cell Microbiol 5, 395–404
48 Jimenez-Soto LF, Kutter S, Sewald X, Ertl C, Weiss E,
Kapp U, Rohde M, Pirch T, Jung K, Retta SF et al
(2009) Helicobacter pylori type IV secretion apparatus
exploits beta1 integrin in a novel RGD-independent
manner PLoS Pathog 5, e1000684
49 Kwok T, Zabler D, Urman S, Rohde M, Hartig R,
Wessler S, Misselwitz R, Berger J, Sewald N, Konig W
et al.(2007) Helicobacter exploits integrin for type IV
secretion and kinase activation Nature 449, 862–866
50 Zhong Q, Shao S, Mu R, Wang H, Huang S, Han J,
Huang H & Tian S (2010) Characterization of
peptido-glycan hydrolase in Cag pathogenicity island of
Helicobacter pylori Mol Biol Rep 38, 503–539
51 Angelini A, Tosi T, Mas P, Acajjaoui S, Zanotti G,
Terradot L & Hart DJ (2009) Expression of
Helicobact-er pyloriCagA domains by library-based construct
screening Febs J 276, 816–824
52 Nesic D, Miller MC, Quinkert ZT, Stein M, Chait BT
& Stebbins CE (2010) Helicobacter pylori CagA inhibits
PAR1-MARK family kinases by mimicking host sub-strates Nat Struct Mol Biol 17, 130–132
53 Stebbins CE & Galan JE (2001) Maintenance of an unfolded polypeptide by a cognate chaperone in bacte-rial type III secretion Nature 414, 77–81
54 Cendron L, Couturier M, Angelini A, Barison N, Stein
M & Zanotti G (2009) The Helicobacter pylori CagD (HP0545, Cag24) protein is essential for CagA translo-cation and maximal induction of interleukin-8 secretion
J Mol Biol 386, 204–217
55 Cendron L, Tasca E, Seraglio T, Seydel A, Angelini A, Battistutta R, Montecucco C & Zanotti G (2007) The crystal structure of CagS from the Helicobacter pylori pathogenicity island Proteins 69, 440–443
56 Cendron L, Seydel A, Angelini A, Battistutta R & Zan-otti G (2004) Crystal structure of CagZ, a protein from the Helicobacter pylori pathogenicity island that encodes for a type IV secretion system J Mol Biol 340,
881–889
57 Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton
GG, Fleischmann RD, Ketchum KA, Klenk HP, Gill
S, Dougherty BA et al (1997) The complete genome sequence of the gastric pathogen Helicobacter pylori Nature 388, 539–547