Assembly and molecular mode of action of theHelicobacter pylori Cag type IV secretion apparatus Wolfgang Fischer Max von Pettenkofer-Institut, Ludwig-Maximilians-Universita¨t, Mu¨nchen,
Trang 1Assembly and molecular mode of action of the
Helicobacter pylori Cag type IV secretion apparatus
Wolfgang Fischer
Max von Pettenkofer-Institut, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany
Introduction
Type IV secretion systems (T4SS) represent a family of
macromolecule transporters that is widely distributed
in prokaryotes, and individual members of this family
have adapted to their cellular background and to a
variety of substrates as DNA import and export
sys-tems, conjugation systems or effector protein
translo-cation systems [1] Several pathogenic bacteria have
adopted T4SS for the secretion of virulence-associated
proteins to the extracellular milieu or for their
injec-tion into different host cells, mediating host cell
manipulation in different ways and thereby facilitating
mucosa-associated or intracellular lifestyles The
human gastric pathogen Helicobacter pylori, which is
the principal cause of chronic active gastritis and
pep-tic ulcer disease, and also is involved in the
develop-ment of mucosa-associated lymphoid tissue lymphoma
and gastric cancer [2], uses different T4SS for horizon-tal gene transfer, and the cytotoxin-associated gene (Cag) T4SS for interactions with various host cells [3,4] The Cag-T4SS is encoded on the cytotoxin-asso-ciated gene-pathogenicity island (cagPAI), a 37 kb genomic island representing one of the major variable genome regions of H pylori that is clearly associated with an enhanced risk of developing peptic ulcers or adenocarcinoma The percentage of cagPAI-positive strains varies considerably between geographically dis-tinct groups, ranging from universal presence in East Asian isolates to a complete absence in certain African populations [5] Strains carrying the cagPAI are often equipped with a vacuolating cytotoxin (vacA) s1⁄ m1 genotype, suggesting a common selective pressure for these two major virulence factors, and have been
Keywords
CagA; Helicobacter pylori; pathogenicity
island; protein translocation; secretion
apparatus; type IV secretion
Correspondence
W Fischer, Max von Pettenkofer-Institut,
Ludwig-Maximilians-Universita¨t,
Pettenkoferstrasse 9a, 80336 Mu¨nchen,
Germany
Fax: +49 89 51605223
Tel: +49 89 51605277
E-mail: fischer@mvp.uni-muenchen.de
(Received 15 November 2010, accepted
10 January 2011)
doi:10.1111/j.1742-4658.2011.08036.x
Bacterial type IV secretion systems (T4SS) form supramolecular protein complexes that are capable of transporting DNA or protein substrates across the bacterial cell envelope and, in many cases, also across eukaryotic target cell membranes Because of these characteristics, they are often used
by pathogenic bacteria for the injection of host cell-modulating virulence factors One example is the human pathogen Helicobacter pylori, which uses the Cag-T4SS to induce a pro-inflammatory response and multiple cytoskeletal and gene regulatory effects in gastric epithelial cells Work in recent years has shown that the Cag-T4SS exhibits marked differences in relation to other systems, both with respect to the composition of its secre-tion apparatus and the molecular details of its secresecre-tion mechanisms This review describes the molecular properties of the Cag-T4SS and compares these with prototypical systems of this family of protein transporters
Abbreviations
cagPAI, cytotoxin-associated gene-pathogenicity island; IL, interleukin; T4SS, type IV secretion system.
Trang 2induction of a pronounced pro-inflammatory response
in vitro and in vivo, and the translocation and
subse-quent tyrosine phosphorylation of its effector protein
CagA into various host cells is a hallmark of
Cag-T4SS activity Despite the molecular characterization
of a number of effects in host cells, the exact function
of CagA translocation during infection is still not fully
clear However, this process significantly increases the
risk of gastric cancer in the Mongolian gerbil model
[6], and CagA has been shown to act as a bacterial
oncoprotein capable of malignant cell transformation
[7] CagA is the only protein that has been described
so far as an effector protein of the Cag-T4SS, although
it has been suggested that the secretion apparatus
transports peptidoglycan fragments into the host cell
cytoplasm as well, thereby inducing a
pro-inflamma-tory response via activation of the pattern-recognition
molecule Nod1 [8] However, the mechanistic details of
type IV secretion-dependent transport of peptidoglycan
fragments are not known
Although the Cag system is evolutionarily related to
other T4SS, it contains only few proteins with high
sequence similarities to components of other T4SS,
and many essential components are unique for the Cag
system These pronounced differences are likely to be
reflected in details of secretion apparatus assembly, as
well as in the molecular mechanisms of effector protein
secretion This minireview describes the composition of
the cagPAI and the properties of both conserved and
unique components of the Cag-T4SS, together with
potential implications for the current understanding of
its mode of action
The cagPAI and the Cag type IV
secretion apparatus
Gene arrangement and variants of the cagPAI
The cagPAI, which was originally identified by
sequencing the genome region upstream of the cagA
gene or DNA found only in CagA-positive strains, was
shown to encode proteins with sequence similarity to
Agrobacterium tumefaciensVir proteins, and it was
fur-ther demonstrated that these proteins are necessary for
inducing secretion of the chemokine interleukin (IL)-8
from infected epithelial cells [9,10] In the
correspond-ing strain NCTC11638, the cagPAI is not a contiguous
genome island but was split as a result of integration of
an IS605 insertion element and an associated genome
genes encoding a Sel1 repeat-containing protein and glutamate racemase, respectively, and it is flanked by a
31 bp sequence duplication (Fig 1A) The cagPAI has
an overall size of 37 kb and harbours 30 genes The amount of sequence diversity among these genes
in isolates from different geographic groups has recently been taken as an indication that the cagPAI was acquired only once in the history of
H pylori[13]
Although the gene order on the cagPAI is con-served, recent genome sequencing projects have revealed certain variations of the general gene arrange-ment For example, some strains isolated from Ameri-can Indians have a duplication of cagA (in a nonfunctional form) and cagB inserted into the inter-genic locus between cagP and cagQ [14] (Fig 1B) Additionally, these islands have an inversion of the cagQ gene, which is also frequently found in East Asian strains [12,13] A more complex rearrangement was identified in a strain colonizing Mongolian gerbils [15] It includes an inversion of all cagPAI genes except cagAin conjunction with several flanking genes, and a second inversion comprising most of these flanking genes (hp0511–hp0518) Similar rearrangements would also account for earlier observations that the cagA gene is not adjacent to cagB in some strains [11,16] Interestingly, the corresponding gene locus of Helicob-acter acinonychis Sheeba, which is the closest relative
to H pylori known, but probably diverged before acquisition of the cagPAI [17], contains a similar inver-sion of the same flanking genes and also fragments of
a helicase gene that is frequently found downstream of cagA (Fig 1B) Because the cagA downstream region
is highly polymorphic and contains remnants of an IS606 insertion element [13], this observation suggests that these genes or gene fragments were not originally part of the cagPAI but were inserted at a later time point after cagPAI acquisition
Components of the Cag-T4SS Prototypical T4SS, such as the T-DNA transfer system
of A tumefaciens, usually contain 11 essential compo-nents (VirB1–VirB11) of the secretion apparatus and a coupling protein (VirD4) that mediates substrate rec-ognition [1] By contrast to other T4SS found in
H pylori, the Cag-T4SS is only distantly related to T4SS found in other species [4], and only a few cag genes encode proteins with clear sequence similarities
Trang 3to known T4SS proteins Obvious similarities exist
only for CagE (to VirB4), CagX (to VirB9), CagY (to
VirB10), Caga (to VirB11) and Cagb (to VirD4),
although even these proteins, particularly CagX and
CagY, are remarkably different from their
counter-parts in prototypical systems Nevertheless, protein
topology predictions and determinations, localization
studies and functional studies suggested that Cagc
(VirB1), CagC (VirB2), CagL (VirB5), CagW (VirB6),
CagT (VirB7) and CagV (VirB8) are further VirB
homologues [18–22] (Fig 1A and Table 1)
Early systematic studies with isogenic mutants in
each cag gene [23,24] identified 14–15 genes that are
essential for inducing IL-8 secretion and for CagA
translocation, suggesting that these genes encode
com-ponents of the secretion apparatus (Table 1) These
essential secretion apparatus components include all
VirB-like proteins mentioned above and several further components that are unique to the Cag system Three further gene products are not absolutely necessary, although their absence results in a reduced efficiency
of both phenotypes, and these proteins (supporting components) thus appear to be involved in assembling the secretion apparatus as well An additional group
of genes was shown to be required for CagA transloca-tion but not for IL-8 inductransloca-tion [23], and the encoded gene products were accordingly termed CagA translo-cation factors
Finally, several cagPAI gene products do not appear
to have a function for the type IV secretion-related phenotypes examined They might have other as yet unknown functions or even be further effector pro-teins, or they might simply be unrelated to the T4SS Interestingly, however, one of these genes (cagf) was
A
B
Fig 1 Gene arrangement and variants of the cag pathogenicity island (A) Integration of the cagPAI at a chromosomal locus flanked by gene hp0519, with numbering according to the genome sequence of strain 26695 [51], encoding a Sel1 repeat-containing protein and gene hp0549 encoding glutamate racemase Gene designations and putative homologies to components of the A tumefaciens T-DNA transfer system are indicated The left (LJ) and right junctions (RJ) of the cagPAI represent a 31 bp direct repeat (B) Rearrangements of the cagPAI found in complete H pylori genome sequences Apart from complete deletions in cag-negative strains, rearrangements include an inversion
of cagQ, a duplication of cagA and cagB associated with cagA degeneration, and a more complex rearrangement comprising all cag genes except cagA and a second inversion of several flanking genes (green) Examples of strains containing the depicted arrangements are given.
A helicase gene (hel), fragments of which are often located close to the cagPAI right junction (orange), is also present in H acinonychis strain Sheeba or in some H pylori strains as part of a strain-specific segment integrated next to a lipoprotein gene (blue) and has therefore probably not originally been part of the cagPAI.
Trang 4found to be among the most highly transcribed among
all cag genes in vitro and in vivo, and transcripts of
cagS, cagQ and cagP were also found [25] Moreover,
a recent determination of the complete H pylori
tran-scriptome identified a number of transcriptional start
sites on the cagPAI, suggesting transcription of the
cagBgene, transcription of an operon comprising cagf,
cage, cagd and cagc, transcription of an operon
com-prising cagQ, cagS and possibly the small ORF hp0533
(which is not present in many strains) and, finally,
transcription of the cagP gene, probably together with
a small noncoding RNA upstream of cagP [26] These
observations suggest that all non-essential genes are
expressed, and their organization in operons indicates
a functional relationship with the T4SS For a
discus-sion of these non-essential components, see the
accom-panying review by Cendron and Zanotti [27]
The putative type IV secretion apparatus core complex
The assembly of different type IV secretion machines and functions of their essential components have been studied in detail [1], although the structural details of
a type IV secretion apparatus have emerged only recently from the structural determination of a core complex of the pKM101 conjugation system [28,29]; see also the accompanying review by Terradot and Waksman [30] This core complex consists of 14 mono-mers each of the VirB7, VirB9 and VirB10 homologues and does not require other secretion system compo-nents for assembly It is reasonable to assume that the Cag system forms a similar core complex composed of CagT, CagX and CagY (Fig 2) However, it should be noted that all these proteins are considerably different
NE, non-essential for both phenotypes References report protein structures, functions, localizations or interactions.
Gene Protein Size (aa) Localization Classification (Putative) function(s) or homologies Reference
a
Replaced by hp0521B in several strains.bConflicting data with respect to requirement for IL-8 induction [9,23,24]. cConflicting data with respect to requirement for CagA translocation [23,56].
Trang 5from their counterparts in the pKM101 or other T4SS.
For example, CagT is a lipoprotein similar to TraN of
pKM101 but has a size of 280 amino acids compared
to 48 amino acids for TraN or 55 amino acids for
VirB7, suggesting additional functions for CagT
Lipo-proteins of 150–300 amino acids are also found to be
essential components of conjugation systems such as
those of plasmids RP4 and F [31], and of the less
related type IVB secretion systems, where the structure
of a domain of the DotD lipoprotein was shown to be
similar to secretin domains from type II or type III
secretion systems [32]
Consistent with the assumption that CagT is a
VirB7 homologue is the fact that it takes part in an
outer membrane-associated subcomplex of the Cag
sys-tem, which also contains CagX [22] By contrast to
other T4SS, however, this subcomplex appears to
har-bour two additional components, CagM and Cagd
Both proteins have N-terminal signal sequences and
were shown to be associated with the outer membrane
and to interact with CagT, with CagX, and with each
other [22,33] Moreover, both CagM and Cagd were
found to interact with themselves, suggesting that they
might contribute to oligomerization of the outer
mem-brane subcomplex [22,33] Interestingly, the interaction
between CagT and CagX was lost in a cagM mutant,
indicating that it is either an indirect interaction via
CagM, or that only a ternary complex comprising all three proteins is stable [22] In support of this view,
a cagM mutant produces significantly reduced levels of CagT Furthermore, Cagd was found to stabilize the lipoprotein CagT, and vice versa [33] Taken together, these observations suggest that the Cag system elabo-rates a more complex core structure than other T4SS Both CagT and CagX were also found exposed at the bacterial surface [34,35]
The most divergent core protein is CagY, a huge protein with a peculiar middle region containing exten-sive sequence repeats CagY was shown to interact with the outer membrane-associated subcomplex, although this interaction was only detected in the presence of CagX [22], suggesting that CagY does not interact directly with CagM or CagT Apart from its putative membrane localization spanning both bacterial mem-branes, CagY was also detected on type IV secretion pilus-like surface appendages [34] Interestingly, CagY was identified as one of several bacterial interaction partners of b1 integrins, which represent the secretion apparatus receptors on target cells [36] (see below)
Assembly of the secretion apparatus Although T4SS core complexes are able to form autonomously, they are unlikely to do so constitutively
Fig 2 Assembly and interaction model of
the Cag type IV secretion apparatus Cag
proteins are depicted in their most likely
localizations according to sequence
predic-tion or experimental data and designated by
their last letters (e.g ‘A’ for CagA) Essential
or supportive secretion apparatus
compo-nents are indicated in green, translocation
factors in orange, and the effector protein
CagA in red Overlapping boxes indicate
probable protein–protein interactions.
Integrin heterodimers are indicated as
receptors on the target cell surface (a, b1).
CagA, CagL and CagY are also shown on
the pilus as a result of their integrin-binding
capacities Note that not all interactions are
depicted, and that CagD, CagG and CagI, as
well as non-essential components, are not
shown IM, inner (bacterial) membrane;
PG, peptidoglycan layer; OM, outer
(bacterial) membrane; CM, cytoplasmic
membrane of a eukaryotic target cell.
Trang 6tidoglycan-degrading lytic transglycosylase VirB1 [1].
In accordance with this, both the lytic transglycosylase
of the Cag system, Cagc [18], and CagV, a bitopic
inner membrane protein with features similar to VirB8
[21], are essential secretion apparatus components The
lytic transglycosylase activity of Cagc would require a
periplasmic localization (Fig 2), although it is unclear
how Cagc is exported because it does not have an
N-terminal signal sequence Interactions of CagV with
Cagd, CagM and CagT were identified in a yeast
two-hybrid screen and partly confirmed by pulldown
exper-iments [38], supporting the idea that CagV might act
as a nucleator by forming contacts with other core
complex proteins A further step in the assembly of the
secretion apparatus would be the recruitment of
com-ponents forming the inner membrane pore of the
secre-tion apparatus The Cag system contains three
essential proteins that might constitute a cytoplasmic
membrane pore CagW is a polytopic inner membrane
protein with features that are common among
VirB6-like proteins [22], CagU is a second polytopic inner
membrane protein with three predicted transmembrane
helices that has no counterpart in other systems, and
CagH is an essential bitopic inner membrane protein,
also without counterparts in other systems Functional
studies with all these components are lacking so far
On the cytoplasmic face of the secretion apparatus,
two ATPases provide the energy for secretion
appara-tus assembly and⁄ or substrate transport CagE
proba-bly has two transmembrane helices at its N-terminus
and a large region with sequence similarity to the VirB4
ATPase It has been speculated that the N-terminal
extension represents a VirB3-like domain fused to the
VirB4-like component [22], which is consistent with the
recent observation that a fusion protein of A
tumefac-iens VirB3 and VirB4 retains both functions [39] The
VirB11-like ATPase Caga has been shown to form
hexamers in solution and to undergo conformational
changes upon binding of ADP or ATP, suggesting a
dynamic cycling process [40,41]; for details, see the
accompanying review by Terradot and Waksman [30]
Finally, the Cag-T4SS elaborates sheathed surface
appendages that are dissimilar to the pili commonly
found in DNA-transporting T4SS Nevertheless, these
appendages are considered to be composed of the
VirB2-like pilin subunit CagC [19] but, in addition,
they can be stained with immunogold labels directed
against CagY, CagT, CagX and CagL [34,35,42] It
was shown that purified CagL binds via its RGD motif
hand, CagY, CagA and CagI were also identified as Cag proteins binding to b1 integrins [36] In any case, interaction of secretion system components with b1 integrins is an important prerequisite for T4SS function
Mechanisms of CagA recognition and transfer
CagA as a type IV secretion substrate
By contrast to most other virulence-associated T4SS, the Cag system transports only CagA as a protein sub-strate, although CagA is an effector protein with mul-tiple functions in target cells; see the accompanying review by Tegtmeyer et al [43] The different functions are partly dependent on, and partly independent of, CagA tyrosine phosphorylation, and the phosphoryla-tion motifs located in the C-terminal region of CagA (EPIYA motifs) are thus essential for some pheno-types A second conserved motif found to be required for phosphorylation-independent effects is located adjacent to the EPIYA motifs and has been termed microtubule affinity-regulating kinase inhibitor motif because of its binding to this kinase [44] Translocation reporter assays using the phosphorylatable glycogen synthase kinase epitope tag showed that these motifs are not required for translocation of CagA (I Pattis
& W Fischer, unpublished results) Translocation of CagA critically depends on its C-terminal 20 amino acids [45] This is consistent with the situation for T4SS substrates in other bacteria, which are generally considered to use C-terminal secretion signals [1] Although the CagA C-terminus contains a number of positively charged amino acids and positive charges are important for some type IV effector proteins, site-specific mutation of the CagA C-terminus has not resulted in a clear picture concerning the nature of the secretion signal [45] However, domain-swapping experiments using the C-terminal regions of other type
IV substrates indicated that this part contains a secre-tion informasecre-tion that is common among different T4SS By contrast to most other type IV effector pro-teins analyzed so far, the CagA C-terminus is not suffi-cient for translocation Possible explanations for this are that binding of an N-terminal domain of CagA to b1 integrins [36] might be required for transport into the target cell, or that CagA translocation might depend on an interaction with phosphatidylserine in the target cell membrane, for which two arginine
Trang 7residues in the middle CagA region were shown to be
necessary [46]
Substrate recognition and the role of CagA
translocation factors
Before entering the translocation channel, CagA
tion signal(s) or protein domains containing the
secre-tion informasecre-tion have to be recognized by a signal
recognition protein Because the three essential CagA
translocation factors CagF, CagZ and Cagb are all
pre-dicted to be localized in the bacterial cytoplasm or at
the inner membrane, each of them could fulfill a
func-tion as signal recognifunc-tion factor CagA
immunoprecipi-tation experiments from bacterial lysates resulted in the
identification of CagF as major component interacting
with CagA [47,48] This interaction is independent of
other secretion apparatus components and probably
takes place at the inner face of the cytoplasmic
mem-brane The CagF-binding region comprises 100
amino acids in the C-terminal region of CagA but does
not contain the putative C-terminal secretion signal,
suggesting that CagF binding is not a signal
recogni-tion event [48] However, a fusion of GFP to the
C-terminal 195 amino acids of CagA (containing the
CagF-binding domain together with the C-terminal
signal region) exerted a dominant-negative effect on
translocation of full-length CagA This indicated that
the corresponding region is sufficient for recruitment to
the secretion apparatus and that CagF binding plays a
role in this recruitment, similar to the function of
secre-tion chaperones in type III secresecre-tion systems
In almost all conjugation systems and also in many
effector protein translocation systems, coupling
pro-teins are essential for the secretion process [1]
Cou-pling proteins are ATPases interacting both with
substrates and with secretion apparatus components
that determine substrate specificity of a given T4SS In
the Cag system, the coupling protein homologue Cagb
is dispensable for IL-8 induction but essential for
CagA translocation [23,24], which is consistent with its
putative role as a type IV substrate receptor Similar
to other coupling proteins, Cagb is predicted to
con-tain two or three transmembrane helices in its
N-termi-nal region, with the major C-termiN-termi-nal part of the
protein being located in the cytoplasm [22] Recently,
it was shown that this cytoplasmic part of Cagb is able
to interact with CagA [49], although it is not clear
whether this binding involves the putative C-terminal
secretion signal Furthermore, a robust interaction
between Cagb and the third translocation factor CagZ
was identified in yeast two-hybrid screens and
con-firmed biochemically in H pylori [38,49] Deletion of
the cagZ gene resulted not only in CagA translocation deficiency, but also in a strong reduction of Cagb pro-tein levels, and both defects could be restored by com-plementation of the mutant with a myc-tagged cagZ gene [49]; for structural details of CagZ, see the accompanying review by Cendron and Zanotti [27] Taken together, these data suggest that Cagb and CagZ form a stable complex at the bacterial cytoplas-mic membrane that might constitute the functional CagA signal recognition receptor
Mechanisms of CagA translocation The molecular mechanisms of CagA translocation through the secretion apparatus are only poorly under-stood For the A tumefaciens VirB system, subsequent contacts of the secreted nucleoprotein complex with VirD4, VirB11, VirB6⁄ VirB8 and VirB2 ⁄ VirB9 have been defined [50] An analogous secretion route might
be taken by CagA but, as a result of the high variabil-ity among T4SS [1], considerable differences are also possible Although putative interactions between CagA and different secretion apparatus components have been identified in a yeast two-hybrid approach [22,49], they have not been confirmed so far in H pylori cells
As for other T4SS, it is currently also unclear whether the extracellular pilus-like appendages are used as con-duits for protein transport or rather represent struc-tures required for T4SS-dependent cell contact Several studies have shown that CagA is located at the bacte-rial surface, particularly at the pilus tip [36,42,46], although it has not been examined whether surface- or pilus-associated CagA represents a translocation inter-mediate Such a scenario is suggested by a study show-ing that CagA bindshow-ing to phosphatidylserine at the outer leaflet of the host cell cytoplasmic membrane induces its uptake into the cell [46] However, it has also been established that translocation of CagA depends on the presence of b1 integrins as receptors for the Cag secretion apparatus at the target cell sur-face [36,42] Irrespective of which components of the secretion apparatus bind to b1 integrin extracellular domains, inhibitory effects of different integrin anti-bodies on CagA translocation, as well as the observa-tion that CagA itself binds strongly to b1 integrin [36], suggest that pilus-associated CagA has an important function for translocation The uptake process into the host cell cytoplasm is not understood Incubation of target cells with different inhibitors interferes with CagA tyrosine phosphorylation [36,46], although it remains unclear whether CagA uptake involves pore formation in the host cell cytoplasmic membrane or other cellular processes
Trang 8also been evolutionarily adapted to various needs, and
major deviations from ancestral systems might
accord-ingly be expected with respect to their structure and
function This is well-reflected in the H pylori Cag
sys-tem, which includes a number of unique essential
com-ponents and probably relies on a specific translocation
mechanism Given that this system also poses a major
health problem by enhancing the risk of cancer
devel-opment, it is important to understand its molecular
principles in detail Defining the molecular mechanisms
of CagA transport to the bacterial surface and across
the target cell membrane will thus be of particular
interest for future research
Acknowledgements
The author is grateful to Rainer Haas for continuous
support, and to Claudia Ertl and Rainer Haas for
critically reading the manuscript This work was
supported by a FoeFoLe research grant from the
Ludwig-Maximilians-Universita¨t Mu¨nchen
References
1 Alvarez-Martinez CE & Christie PJ (2009) Biological
diversity of prokaryotic type IV secretion systems
Microbiol Mol Biol Rev 73, 775–808
2 Suerbaum S & Michetti P (2002) Helicobacter pylori
infection N Engl J Med 347, 1175–1186
3 Fischer W, Karnholz A, Jime´nez-Soto LF & Haas R
(2008) Type IV secretion systems in Helicobacter pylori
In Helicobacter pylori Molecular Genetics and Cellular
Biology (Yamaoka Y ed.), pp 115–136 Caister
Academic Press, Norfolk, UK
4 Fischer W, Windhager L, Rohrer S, Zeiller M,
Karnholz A, Hoffmann R, Zimmer R & Haas R (2010)
Strain-specific genes of Helicobacter pylori: genome
evolution driven by a novel type IV secretion system
and genomic island transfer Nucleic Acids Res 38,
6089–6101
5 Gressmann H, Linz B, Ghai R, Pleissner KP,
Schlap-bach R, Yamaoka Y, Kraft C, Suerbaum S, Meyer TF
& Achtman M (2005) Gain and loss of multiple genes
during the evolution of Helicobacter pylori PLoS Genet
1, e43
6 Amieva MR & El-Omar EM (2008) Host-bacterial
interactions in Helicobacter pylori infection
Gastroen-terology 134, 306–323
7 Hatakeyama M (2008) SagA of CagA in Helicobacter
pyloripathogenesis Curr Opin Microbiol 11, 30–37
9 Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R & Covacci A (1996) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors Proc Natl Acad Sci USA 93, 14648–14653
10 Akopyants NS, Clifton SW, Kersulyte D, Crabtree JE, Youree BE, Reece CA, Bukanov NO, Drazek ES, Roe
BA & Berg DE (1998) Analyses of the cag pathogenic-ity island of Helicobacter pylori Mol Microbiol 28, 37–53
11 Blomstergren A, Lundin A, Nilsson C, Engstrand L & Lundeberg J (2004) Comparative analysis of the com-plete cag pathogenicity island sequence in four Helicob-acter pyloriisolates Gene 328, 85–93
12 Azuma T, Yamakawa A, Yamazaki S, Ohtani M, Ito Y, Muramatsu A, Suto H, Yamazaki Y, Keida Y, Higashi H et al (2004) Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains
in Japan J Clin Microbiol 42, 2508–2517
13 Olbermann P, Josenhans C, Moodley Y, Uhr M, Sta-mer C, Vauterin M, Suerbaum S, Achtman M & Linz
B (2010) A global overview of the genetic and func-tional diversity in the Helicobacter pylori cag pathoge-nicity island PLoS Genet 6, e1001069
14 Mane SP, Dominguez-Bello MG, Blaser MJ, Sobral
BW, Hontecillas R, Skoneczka J, Mohapatra SK, Crasta OR, Evans C, Modise T et al (2010) Host-interactive genes in Amerindian Helicobacter pylori diverge from their Old World homologs and mediate inflammatory responses J Bacteriol 192, 3078–3092
15 Farnbacher M, Jahns T, Willrodt D, Daniel R, Haas R, Goesmann A, Kurtz S & Rieder G (2010) Sequencing, annotation, and comparative genome analysis of the gerbil-adapted Helicobacter pylori strain B8 BMC Genomics 11, 335
16 McGee DJ, May CA, Garner RM, Himpsl JM & Mobley
HL (1999) Isolation of Helicobacter pylori genes that modulate urease activity J Bacteriol 181, 2477–2484
17 Eppinger M, Baar C, Linz B, Raddatz G, Lanz C, Keller H, Morelli G, Gressmann H, Achtman M & Schuster SC (2006) Who ate whom? Adaptive Helicob-acter genomic changes that accompanied a host jump from early humans to large felines PLoS Genet 2, e120
18 Zahrl D, Wagner M, Bischof K, Bayer M, Zavecz B, Beranek A, Ruckenstuhl C, Zarfel GE & Koraimann G (2005) Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems Microbiology 151, 3455–3467
19 Andrzejewska J, Lee SK, Olbermann P, Lotzing N, Katzowitsch E, Linz B, Achtman M, Kado CI,
Trang 9Suerbaum 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
20 Backert S, Fronzes R & Waksman G (2008) VirB2 and
VirB5 proteins: specialized adhesins in bacterial type-IV
secretion systems? Trends Microbiol 16, 409–413
21 Buhrdorf R, Fo¨rster C, Haas R & Fischer W (2003)
Topological analysis of a putative virB8 homologue
essential for the cag type IV secretion system in
Heli-cobacter pylori Int J Med Microbiol 293, 213–217
22 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 Bacteriol 190, 2161–2171
23 Fischer W, Pu¨ls J, Buhrdorf R, Gebert B, Odenbreit S
& Haas R (2001) Systematic mutagenesis of the
Heli-cobacter pylori cagpathogenicity island: essential genes
for CagA translocation in host cells and induction of
interleukin-8 Mol Microbiol 42, 1337–1348 Erratum
in: Mol Microbiol 47, 1759 (2003)
24 Selbach M, Moese S, Meyer TF & Backert S (2002)
Functional analysis of the Helicobacter pylori cag
path-ogenicity island reveals both VirD4-CagA-dependent
and VirD4-CagA-independent mechanisms Infect
Immun 70, 665–671
25 Boonjakuakul JK, Canfield DR & Solnick JV (2005)
Comparison of Helicobacter pylori virulence gene
expression in vitro and in the Rhesus macaque Infect
Immun 73, 4895–4904
26 Sharma CM, Hoffmann S, Darfeuille F, Reignier J,
Findeiss S, Sittka A, Chabas S, Reiche K, Hackermu¨ller
J, Reinhardt R et al (2010) The primary transcriptome
of the major human pathogen Helicobacter pylori
Nature 464, 250–255
27 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
28 Fronzes R, Scha¨fer E, Wang L, Saibil HR, Orlova EV
& Waksman G (2009) Structure of a type IV secretion
system core complex Science 323, 266–268
29 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
30 Terradot L & Waksman G (2011) Architecture of the
Helicobacter pyloriCag- Type IV secretion system
FEBS J 278, 1213–1222
31 Lawley TD, Klimke WA, Gubbins MJ & Frost LS
(2003) F factor conjugation is a true type IV secretion
system FEMS Microbiol Lett 224, 1–15
32 Nakano N, Kubori T, Kinoshita M, Imada K & Nagai
H (2010) Crystal structure of Legionella DotD: insights
into the relationship between type IVB and typeII⁄ III secretion systems PLoS Pathog 6, e1001129
33 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
34 Rohde M, Pu¨ls J, Buhrdorf R, Fischer W & Haas R (2003) A novel sheathed surface organelle of the Helicobacter pylori cagtype IV secretion system Mol Microbiol 49, 219–234
35 Tanaka J, Suzuki T, Mimuro H & Sasakawa C (2003) Structural definition on the surface of Helicobacter pyloritype IV secretion apparatus Cell Microbiol 5, 395–404
36 Jime´nez-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 b1 integrin in a novel RGD-independent man-ner PLoS Pathog 5, e1000684
37 Judd PK, Kumar RB & Das A (2005) Spatial location and requirements for the assembly of the Agrobacterium tumefacienstype IV secretion apparatus Proc Natl Acad Sci USA 102, 11498–11503
38 Busler VJ, Torres VJ, McClain MS, Tirado O, Friedman DB & Cover TL (2006) Protein–Protein interactions among Helicobacter pylori Cag proteins
J Bacteriol 188, 4787–4800
39 Mossey P, Hudacek A & Das A (2010) Agrobacterium tumefacienstype IV secretion protein VirB3 is an inner membrane protein and requires VirB4, VirB7, and VirB8 for stabilization J Bacteriol 192, 2830–2838
40 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
41 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
42 Kwok T, Zabler D, Urman S, Rohde M, Hartig R, Wessler S, Misselwitz R, Berger J, Sewald N, Ko¨nig W
et al.(2007) Helicobacter exploits integrin for type IV secretion and kinase activation Nature 449, 862–866
43 Tegtmeyer N, Wessler S & Backert S (2011) Role of the cagpathogenicity island encoded type IV secretion sys-tem in Helicobacter pylori pathogenesis FEBS J 278, 1190–1202
44 Nesic D, Miller MC, Quinkert ZT, Stein M, Chait BT
& Stebbins CE (2010) Helicobacter pylori CagA inhibits PAR1-MARK family kinases by mimicking host substrates Nat Struct Mol Biol 17, 130–132
45 Hohlfeld S, Pattis I, Pu¨ls J, Plano GV, Haas R & Fischer W (2006) A C-terminal secretion signal is necessary, but not sufficient for type IV secretion
Trang 10tion, and pathophysiological action of the CagA
onco-protein Cell Host Microbe 7, 399–411
47 Couturier MR, Tasca E, Montecucco C & Stein M
(2006) Interaction with CagF is required for
transloca-tion of CagA into the host via the Helicobacter pylori
type IV secretion system Infect Immun 74, 273–281
48 Pattis I, Weiss E, Laugks R, Haas R & Fischer W
(2007) The Helicobacter pylori CagF protein is a type
IV secretion chaperone-like molecule that binds close
to the C-terminal secretion signal of the CagA effector
protein Microbiology 153, 2896–2909
49 Jurik A, Hausser E, Kutter S, Pattis I, Prassl S, Weiss
E & Fischer W (2010) The coupling protein Cagb and
its interaction partner CagZ are required for type IV
secretion of the Helicobacter pylori CagA protein Infect
Immun, doi: 10.1128/IAI.00796-10
50 Cascales E & Christie PJ (2004) Definition of a bacterial
type IV secretion pathway for a DNA substrate
Science 304, 1170–1173
51 Tomb J-F, 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
52 Schro¨der G, Krause S, Zechner EL, Traxler B, Yeo HJ,
Lurz R, Waksman G & Lanka E (2002) TraG-like
Zanotti 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
54 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
55 Bourzac KM, Satkamp LA & Guillemin K (2006) The Helicobacter pylori cagpathogenicity island protein CagN is a bacterial membrane-associated protein that
is processed at its C terminus Infect Immun 74, 2537– 2543
56 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
57 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 Helicobacter pyloriprotein interaction map: strategies for complex formation and evidence for novel interactions within type IV secretion systems
Mol Cell Proteomics 3, 809–819