MINIREVIEW Assembly 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 secretion 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. FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1203 termed type I strains. By contrast, type II H. pylori strains have vacA m2 genotypes, lack the cagPAI and are less virulent. The Cag secretion system is responsible for the induction 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 tumefaciens Vir 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 rearrangement. However, comparative sequence analy- sis of multiple cag islands [11–13] suggested that this situation is rather an exception. In its most common gene arrangement, the cagPAI is inserted between the 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 cagA in 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 Cag protein functions W. Fischer 1204 FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS to 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 induction [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. W. Fischer Cag protein functions FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1205 found 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 cagB gene, 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 Table 1. Overview of characteristics and functions of cagPAI-encoded proteins. Gene designations and protein sizes are in accordance with a previous study [51]; aa, amino acids; NA, not annotated. Localization according to sequence prediction or fractionation studies; C, cytoplas- mic; IM, inner membrane; PP, periplasmic; OM, outer membrane; S, surface-exposed or supernatant. Functional classification according to requirement for IL-8 induction and ⁄ or CagA translocation. SA, secretion apparatus component required for IL-8 and CagA translocation; TF, translocation factor required for CagA translocation, but not IL-8 induction; SC, supportive, but not essential component for both phenotypes; 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 hp0520 Cagf ⁄ Cag1 116 IM NE hp0521 a Cage ⁄ Cag2 80 C NE hp0522 Cagd ⁄ Cag3 481 OM SA OM complex 33,38 hp0523 Cagc ⁄ Cag4 169 PP SA PG hydrolase, VirB1 18 hp0524 Cagb ⁄ Cag5 748 IM TF Coupling protein, VirD4 49,52 hp0525 Caga ⁄ VirB11 330 C, IM SA ATPase, VirB11 40,41 hp0526 CagZ 199 C, IM TF Cagb stabilization 38,49,53 hp0527 CagY 1927 IM, OM, S SA Core complex, integrin binding, VirB10 22,34,36 hp0528 CagX 522 IM, OM, S SA Core complex, VirB9 22 hp0529 CagW 535 IM SA IM channel, VirB6 hp0530 CagV 252 IM SA Core complex, VirB8 21 hp0531 CagU 218 IM SA hp0532 CagT 280 OM, S SA Core complex, OM lipoprotein, VirB7 22,33,35 hp0534 CagS 196 C NE 54 hp0535 CagQ 126 IM NE hp0536 CagP 114 IM NE hp0537 CagM 376 OM SA OM complex 22 hp0538 CagN 306 PP, IM SC 55 hp0539 CagL 237 PP, S SA Integrin binding, VirB5 42 hp0540 CagI 381 PP, S TF ⁄ SA b 36 hp0541 CagH 370 IM SA hp0542 CagG 142 PP SC ⁄ TF hp0543 CagF 268 C, IM TF Secretion chaperone 47,48 hp0544 CagE 983 IM SA ATPase, VirB3 ⁄ B4 hp0545 CagD 207 IM, PP, S SC ⁄ TF c 56 hp0546 CagC 115 IM, OM, S SA Pilus subunit, VirB2 19,22 NA CagB 75 C Unknown hp0547 CagA 1186 C, S Effector protein 47–49,57 a Replaced by hp0521B in several strains. b Conflicting data with respect to requirement for IL-8 induction [9,23,24]. c Conflicting data with respect to requirement for CagA translocation [23,56]. Cag protein functions W. Fischer 1206 FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS from 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. W. Fischer Cag protein functions FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1207 and without a positional preference. In the A. tumefac- iens Vir system, spatial targeting of the secretion appa- ratus has been suggested to be determined by VirB8 as a nucleating factor [37], possibly together with the pep- tidoglycan-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 to b1 integrin subunits, suggesting a VirB5-like adhesin function for CagL [42], although conflicting results were obtained with respect to the requirement of this motif during CagA translocation [36,42]. On the other 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 information 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 Cag protein functions W. Fischer 1208 FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS residues 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 secre- tion signal(s) or protein domains containing the secre- tion information 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 recognition 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 secretion 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-terminal 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 binding 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. W. Fischer Cag protein functions FEBS Journal 278 (2011) 1203–1212 ª 2011 The Author Journal compilation ª 2011 FEBS 1209 Conclusions Although T4SS have emerged as a common theme in microbial interactions with eukaryotic cells, they have also 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 ¨ tMu ¨ 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. 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MINIREVIEW Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus Wolfgang Fischer Max von Pettenkofer-Institut,. Cendron and Zanotti [27]. The putative type IV secretion apparatus core complex The assembly of different type IV secretion machines and functions of their