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

MINIREVIEW Structural and functional aspects of unique type IV secretory components in the Helicobacter pylori cag-pathogenicity island Laura Cendron 1 and Giuseppe Zanotti 2 1 Department of Biological Chemistry, University of Padua, Italy 2 Venetian Institute of Molecular Medicine (VIMM), Padua, Italy Introduction Cytotoxin-associated gene-pathogenicity island (cagPAI) characterizes the type I strains of Helicobacter pylori (i.e. the virulent strains) responsible for most gastroduo- denal diseases, including active chronic gastritis, peptic ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma [1–3]. CagA, a major anti- genic factor of the bacterium, is the main signature of the cagPAI-positive strains. Indeed, cagPAI confers H. pylori the capability to express and translocate the CagA protein inside the host cell through a secretion machinery, which is coded by the components of the PAI; see the accompanying review by Fischer [4]. Once translocated, CagA associates with the inner side of the membrane and is phosphorylated at EPIYA motifs by Keywords 3D structure; Cag proteins; gastric cancer; Helicobacter pylori; type IV secretion system Correspondence G. Zanotti, Department of Biological Chemistry, University of Padua, Viale G. Colombo 3, 35121 Padua, Italy Fax: +39 0498073310 Tel: +39 0498276409 E-mail: giuseppe.zanotti@unipd.it (Received 16 November 2010, revised 10 January 2011, accepted 27 January 2011) doi:10.1111/j.1742-4658.2011.08038.x Helicobacter pylori cytotoxin-associated gene-pathogenicity island (cagPAI) is responsible for the secretion of the CagA effector through a type IV secretion system (T4SS) apparatus, as well as of peptidoglycan and possibly other not yet identified factors. Twenty-nine different polypeptide chains are encoded by this cluster of genes, although only some of them show a significant similarity with the constitutive elements of well characterized secretion systems from other bacteria. The other cagPAI components repre- sent almost unique proteins in this scenario. The majority of the T4SS include approximately fifteen components, taking into account either the transmembrane complex subunits, ATPases or substrate factors. The com- position of the cagPAI is very complex: it includes proteins most likely involved at different levels in the pilus assembly, stabilization and process- ing of secreted substrate, as well as regulatory particles possibly involved in the control of the entire apparatus. Despite recent findings with respect to components that play a role in the interaction with the host cell, the func- tion of several cagPAI proteins remains unclear or unknown. This is partic- ularly true for those that represent unique members with no clear similarity to those of other T4SS and no obvious evidence of involvement in the secretion of CagA or induction of pro-inflammatory responses. We summarize what is known about these accessory components, both from a molecular and structural point of view, as well as their putative physiological role. Abbreviations cagPAI, cytotoxin-associated gene-pathogenicity island; IL, interleukin; T4SS, type IV secretion system. FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1223 host tyrosine kinases. The phosphorylation triggers a series of interactions between CagA and human proteins that interfere with the signalling cascades at multiple levels, resulting in a dramatic change of cellular mor- phology, known as the ‘hummingbird phenotype’, and a remarkable enhancement of cellular motility, causing cell scattering [5]; see the accompanying review by Tegtmeyer et al. [6]. The entire cagPAI region is 37 kb long, including approximately 29 genes [7], which encode for the com- ponents of a type IV secretion system (T4SS), homolo- gous to the VirB ⁄ D4 machinery of Agrobacterium tumefaciens, the best characterized T4SS that is regarded as the prototype among that family members [8]. T4SS are multicomponent membrane-spanning transport systems ancestrally related to the conjugation processes, which can be responsible for diverse pro- cesses such as DNA transfer, DNA uptake and release, and translocation of proteins that have an effector role in the target cell. Eleven out of the 29 Cag proteins can be ascribed to the secretion machinery itself or have been proposed to represent functional homologues of VirB proteins [9–11]. For a more detailed description of the correspondence; see the accompanying review by Fischer [4], who describes the relationships between cag and virB ⁄ D4 genes in detail. A second major effect on the host cells, which is elicited only by H. pylori strains harbouring a func- tional cagPAI, is the activation of nuclear transcription factor-jB and the induction of pro-inflammatory pro- duction of cytokines, mainly interleukin (IL)-8 [12]. The activation of a pro-inflammatory signalling cascade has been confirmed to be Nod1 cytoplasmic receptor dependent and a model has been proposed [13] according to which bacterial peptidoglycan is the key effector of such a response. Indeed, the peptidogly- can muropeptides could be transferred to and internal- ized into the gastric epithelial cells only if a functional cagPAI is present. However, it is yet to be established whether this kind of stimuli as a result of muropep- tides secretion is promoted by a syringe-like mecha- nism, analogous to CagA, or whether the only intimate contact with the host cell surface of the com- plex coded by the cagPAI could induce a facilitated internalization of H. pylori peptydoglycan fragments. CagA deficient mutants do not affect that response to wide extent, whereas, in the absence of a functional cagPAI, peptidoglycan release may still occur but with much lower efficiency. Electron microscopy studies indicate that, upon attachment of H. pylori to gastric epithelial cells, pilus- like structures are formed between the bacteria and host cells. Partial characterizations of the secretion organelle allow a description of some of the interac- tions occurring in the H. pylori T4SS, mainly those concerning components that belong to the external pilus, protruding from the bacterial surface toward the extracellular milieu. At least five of the VirB func- tional ⁄ structural homologues of this syringe-like com- plex have been localized in this way: CagC (VirB2), CagL (VirB5), CagT (VirB7), CagX (VirB9) and CagY (VirB10) [9,14,15]. At the same time, yeast two-hybrid system approaches combined with immunoprecipita- tion studies allow a description of the interactions that were assumed to involve Cag proteins [16,17]. These findings, combined with previous analysis on single components, have allowed the proposal of preliminary models of the H. pylori T4SS. Systematic studies have established that some Cag proteins are essential or important for CagA transloca- tion, whereas others are involved in IL-8 secretion, or both [18,19]. A few are apparently unnecessary for any of these effects. Despite the fact that many of the cagPAI proteins have been demonstrated to be involved in the CagA translocation and⁄ or IL-8 induction ⁄ peptidoglycan release, very little is known about their specific function, and this is particularly true for those components that are unique to the Cag apparatus. In this minireview, we concentrate on this last class of cagPAI proteins, and summarize what is known about them both from a molecular and structural point of view, as well as their putative physiological roles. The unique members of the Cag-T4SS Cagf (cag1/HP0520), Cage (cag2/HP0521) Very little is known about the proteins encoded by these two genes. Both were found not to be necessary for either translocation of CagA or for IL-8 induction [18]. cagf (cag1⁄ HP0520) was found to be among the most highly expressed genes in an H. pylori transcript profile analysis from infected human gastric mucosa, together with cagC (cag25 ⁄ HP0546), whereas Cage was not detected at all. Interestingly, the expression levels of cagC and cagf reached values analogous to genes important for bacterial survival and homeostasis, such as catalase, urease and NapA; by contrast, the transcript abundance of other cag genes appeared to be much lower, indicating that cagPAI consists of mul- tiple operons tightly controlled by different promoter regions [20]. The presence of the cagf gene was also found to be related to specific ethnic groups, although an association with virulence and disease has not yet been demonstrated [21]. Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti 1224 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS Cagd (cag3/HP0522) Cagd is a 55 kDa protein essential both for the secre- tion of CagA and the induction of IL-8. The corre- sponding sequence is characterized by a clear N-terminal signal sequence for export into the peri- plasmic space, although no transmembrane helices can be predicted by the most common software. Sequence alignments with a nonredundant database allow the identification of some weak but interesting similarities to proteins associated with adhesion and interaction with eukaryotic cells, such as the surface-exposed Streptococcus pneumoniae choline-binding protein A. When H. pylori bacteria were investigated in the absence of host cell contact, Cagd was found to enrich in the membrane fractions, even if a minor contribu- tion was also present in the soluble pool. Moreover, it was shown to co-purify with other Cag proteins, mainly CagT, Cagb, CagD and Cagf, which represent the most specific interaction partners. Other Cag-T4SS members were coimmunoprecipitated with Cagd, even if with a lower abundance: CagM, CagX, CagC, CagE and Caga [22]. Previous yeast two-hybrid experiments demonstrated that Cagd associates both in homotypic oligomers a nd heterotypic complexes with CagV (VirB8), CagT (Vi rB7), CagM and CagG [16]. In particular, the interactionwithCagT,acorecomponentinvolvedin the Cag-T4SS outer membrane sub-complex, was identi- fied by mu ltiple i ndependen t t echniques and more accurately elucidated. Size exclusion chromatography analysis allowed the isolation of both Cagd-in depend ent oligomers and large Cagd-CagT complexes, reminiscent of what occurs in vivo. Finally, pulse-chase assays demonstrated a mutual correlation between expression levels and the stability of Cagd and CagT proteins [22]. Taken together, these results suggest that Cagd represents a unique and essential core component of the Cag-T4SS. CagZ (cag6/HP0526) CagZ protein, a 23 kDa soluble protein, was found to be absolutely essential for the translocation of CagA but not for the induction of IL-8 [18]. The crystal structure [23] shows that it consists of a single compact L-shaped domain, composed of seven a-helices, includ- ing approximately 70% of the total residues (Fig. 1A). The protein fold can be described as an up-and-down bundle: four long, twisted stretches run antiparallel to each other. A twist in each stretch produces an L-shaped molecule: its longest arm has dimensions of approximately 15 · 25 · 60 A ˚ , whereas the shortest is 15 · 14 · 30 A ˚ . The side chains located at the protein interior are all hydrophobic, with the exception of three residues; thus, packing of the entire bundle is dri- ven by hydrophobic forces. By contrast, the molecular surface is heavily charged: 26 negative- and 21 posi- tively-charged side chains, over a total of 199 residues. The presence of a flexible C-terminal tail and the heav- ily-charged surface suggest that CagZ may participate in the interaction of effector proteins with one or more components of the H. pylori T4SS on the cytoplasmic side of the inner membrane. One or more clusters of surface exposed amino acids have been suggested to represent structural motifs of some relevance for pro- tein activity (NEST prediction, ProFunc server; http:// www.ebi.ac.uk/thornton-srv/databases/profunc/). An exhaustive search of structural similarities in the Protein Data Bank did not provide any remarkable information about the function of the protein. The Fig. 1. (A) Cartoon representation of CagZ protein fold. The L-shape is clearly visible. (B) Two views of the electrostatic potential surface of CagZ. In the overall, the surface is strongly hydrophilic, with patches of positive and negative charges. L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1225 only weak and partial similarity found by the most common servers involve Rab GTPases, comprising proteins that regulate the maturation and transport of endoplasmic-reticulum-derived vesicles in eukaryotic cells (ProFunc server analysis: http://www.ebi.ac.uk/ thornton-srv/databases/profunc/). CagZ has been detected by 2D differential in-gel electrophoresis from H. pylori cultures in vitro [16], demonstrating that it is expressed at a relatively high abundance compared to other Cag proteins. No pro- cessing of eventual N-terminal signal peptide was observed, which is in agreement with predictions based on the sequence only. This supports the idea that its surface and charge distribution make it prone to be involved in protein assemblies, most likely from the cytoplasmic side. Finally, CagZ has been proposed to interact with multiple Cag components, not only non- VirB homologues, such as CagF, CagM, CagG and CagI, but also with some T4SS core components, such as CagV, CagY and the ATPase CagE [16]. CagS (cag13/HP0534) The CagS gene is located immediately after the cluster of cagPAI genes whose putative products show homol- ogies with the VirB proteins that define the structural core of T4SS. Experimental evidence showed that, similar to CagZ, CagS is expressed at a reasonable abundance in H. pylori cultures in vitro [16]. Primary sequence analyses do not show any strong similarity of CagS with proteins of known function, except for some weak similarities with components involved in the peptidoglycan biosynthesis, which belong to the FemABX family of enzymes [24]. The crystal structure of CagS has been determined at a res- olution of 2.3 A ˚ [25]. The protein is a single compact domain, with an all-a structure (Fig. 2). Ten a-helices, labelled from A to J, can be distinguished. Helices B, E, F and H, arranged in an up-and-down topology, form the structural core, whereas the short helices C, D and G on one side, and A, I and J on the other, represent types of appendices, conferring a ‘peanut shape’ to the overall structure. The ten helices that form the protein are held together mainly by hydro- phobic forces, with few hydrophilic interhelical inter- actions. The model lacks 20 amino acids at the N-terminus and 25 at the C-terminus, which are flexi- ble or disordered. Some confuse electron density is present in correspondence to the N-terminus, although it was not possible to trace the polypeptide chain, with the exception of few helical turns. The model shows a highly charged surface, with 24 positively- and 24 negatively-charged residues. In par- ticular, the tertiary structure defines a negatively- charged region including several glutamate and aspar- tate residues confined to the portion of the molecule involving a-helix A, the nearby loop, the first and last turns of helices E and F, and the C-terminus helices I and J. In addition, there is a lysine-rich N- and C-ter- minus, in accordance with the basic isoelectric point of CagS. However, these lysine rich unmodelled N- and C-terminal appendages might define some positively- charged brunches playing a potential role in Cag pro- teins interactions. As mentioned in the case of CagZ, even if to a minor extent, some putative interactions with the other cagPAI components have been detected by a yeast two-hybrid system, involving mainly CagZ and CagM proteins. Another peculiar feature of the molecule is the presence of fourteen methionine resi- dues over a sequence of 199 amino acids, which is an unusually high content compared to other proteins. Four of them, M69, M130, M133, M138, define a clus- ter in the 3D structure, approximately located in the internal side of the peanut. The results of the crystallo- graphic model of CagS do not show any clear evidence of architectural similarity to other known structures, with the exception of a weak structural homology with the phosphotransfer domain (HPt) of CheA, a histi- dine protein kinase that controls chemotaxis response in bacteria [26]. This homology is too weak to be con- sidered as providing any clues with respect to protein function. Even a primary sequence alignment with a nonredundant database shows very limited similarities; the one with the best score being that with phosphoch- oline cytidylyltransferases, comprising rate-limiting enzymes for surfactant phospholipid synthesis. Fig. 2. (A) Cartoon view of CagS protein. Methionine residues are represented by ball and sticks. A cluster of four methionine resi- dues is visible in the lower part. (B) van der Waals representation which shows the ‘peanut’ shape of the protein. Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti 1226 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS CagQ (cag14/HP0535), CagP (cag15/HP0536) The products of these two short genes correspond to putative proteins of 126 and 114 amino acids, respec- tively. They are both predicted to be membrane pro- teins [27] and they were found not to be necessary for either translocation of CagA or for IL-8 induction in H. pylori 26695 strain [18]. CagP appears to play some role in H. pylori adherence to gastric epithelial cells, in addition to classical adhesins, because mutations in its gene may affect bacterium pathogenicity by reducing either the ability of the bacteria to attach to gastric epithelial cells or the intensity of bacteria–host cell interactions [28]. CagM (cag16/HP0537) CagM protein is a 43.7 kDa protein, characterized by a putative N-terminal signal sequence, and at least three transmembrane helices can be predicted from the sequence pattern. It has been detected in the mem- brane-bound fractions isolated from both in vivo (from gastric patients isolates) and in vitro H. pylori cultures by 2D-electrophoresis and MS studies [16,29]. In par- ticular, the results of the 2D differential in-gel electro- phoresis analysis performed by Busler et al. [16] is suggestive of an N-terminal processing as hypothesized by the predictions. Systematic mutagenesis analysis clearly showed that DcagM mutants are neither able to produce an efficient CagA translocation, nor to release peptidoglycan degra- dation fragments [13,18,30], thus suggesting an essential role for the cagM gene product. By using a reporter assay in human gastric cancer cells, CagM (along with cagPAI coded protein, CagL) has also been demon- strated to promote the activation of nuclear factor-jB. More recent experiments with DcagE, DcagM and DcagA isogenic mutant strains of H. pylori provided preliminary evidence that these genes could be involved in the repression of the gene coding for the catalytic subunit of a human gastric H ⁄ K-ATPase {{ 770 Saha,A. 2008;}}. Generally, this effect might be stimulated by a functional Cag-T4SS, allowing H. pylori to inhibit acid secretion by gastric cells and induce episodes of transient hypochlorhydria that facil- itate bacterial colonization. Evidence for protein–pro- tein interactions involving CagM has been observed by yeast two-hybrid analysis. In such experiments, CagM was found to form complexes with many other Cag proteins both belonging to the core apparatus, includ- ing CagX, CagY, CagT, CagV and Cagd, as well as other Cag components such as the ATPase CagE, CagF, CagG, CagZ and CagS [16]. In a different study employing a similar approach, interactions with CagX and partial interactions with CagT were confirmed, whereas those with CagF and CagY were not [17]. Furthermore, both studies identified a clear tendency for CagM to associate, forming homotypic oligomers. An analogous behaviour was observed when we puri- fied a recombinant CagM construct expressed in E. coli for structural studies. Oligomers composed of five to six subunits were isolated and partially charac- terized by gel filtration and preliminary electron microscopy analysis (L. Cendron, unpublished results). In any case, the proposed rich network of interac- tions of this protein agrees with its functional rele- vance and localization studies, where it was found to enrich both in the inner and partially in the outer membrane fractions. A model has been proposed according to which CagM, together with CagX and CagT, associates in the outer membrane basal body of the Cag-T4SS, and the results obtained are in good agreement with the main studies in this respect. CagN (cag17/HP0538) Full-length CagN protein (306 amino acids, 35 kDa) has been demonstrated to be processed at the C-termi- nus, giving rise to a product of approximately 24 kDa (CagN 1–216 ), most likely by a mechanism that is not dependent on other cagPAI proteins [31]. Interestingly, the first 24 amino acids are intact in the endogenous protein, despite it shows a clear N-terminus hydro- phobic pattern and a putative cleavage site that can be easily predicted. The entire primary sequence is pre- dicted to be largely unfolded (foldindex, http://bip. weizmann.ac.il/fldbin/findex). CagN localization studies demonstrated that it is not delivered into the host cell together with CagA but, in contrast, it remains local- ized at the bacterial membrane, most likely anchored by a N-terminal hydrophobic helix [31]. cagN gene deletion appears not to abolish directly the main conse- quences of a functional cagPAI (i.e. CagA translocation and IL-8 induction), even if a variable efficiency of both processes has been observed [18]. Recombinant CagN deleted forms have been pro- duced (His6-CagN 25–306 , CagN 25–216 -His 6 ) and par- tially characterized in our laboratory. Although the DN-terminal hydrophobic construct was strongly prone to aggregation, the one also truncated at the C-terminal resulted in a soluble protein that behaves similar to a monomer in solution, showing a secondary structure content composed of 13% b-sheet, 30% a-helix, with a certain fraction not being ascribed to any well characterized secondary structure motifs (L. Cendron, unpublished results). L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1227 CagI (cag19/HP0540), CagH (cag20/HP0541) Very little is known about CagI (41.5 kDa) and CagH (39 kDa), despite the fact that knockout stud- ies demonstrated they are essential for CagA translo- cation and tyrosine phosphorylation. CagH was also shown to be involved in IL-8 induction in epithelial cells, whereas a cagI deletion mutant does not affect that ability [18]. CagH is also predicted to be secreted out of the inner membrane as a result of an N-termi- nal signal sequence, whereas only one of three differ- ent algorithms tested suggests the presence of a putative hydrophobic helix in the mature protein. CagI most likely is a nonsecretory protein, anchored to the inner membrane toward the periplasmic space as a result of a N-terminal hydrophobic helix, span- ning residues 26–51, thus supporting the idea that it might be involved in the translocation as a putative effector protein rather than being a component of the T4SS apparatus. Finally, interaction studies indicated that CagI might interact with CagZ and CagG, and weak evidence of interaction with Cagb was also observed [16]. CagG (cag21/HP0542) The product of this gene is a 16 kDa protein with a very acidic isoelectric point and a predicted N-terminal signal peptide with a putative cleavage site between residues 27 and 28. A weak homology with the flagel- lar motor switch protein or toxin co-regulated pilus biosynthesis protein D has been detected [32]. Yeast two-hybrid screens, as noted elsewhere, indicate that CagG is involved in multiple protein–protein interac- tions with CagM, Cagd, CagF and CagZ and, to a minor extent, with CagT and the VirD4 homologous Cagb [16]. Analogous to cagI, cagG deletion mutants are inca- pable of delivering CagA into gastric epithelial cells, although they retain the capacity to induce IL-8 pro- duction, pointing toward a potential effector role for the protein. Other studies provided different evidence, showing a marked reduction of IL-8 production from gastric epithelial cells, as well as a reduced capacity to adhere to epithelial cells in vitro and to colonize Mon- golian gerbils in vivo [33]. Similar results were found in an experiment with cagG-deleted strains tested on cultivated KATOIII cells [34]. CagF (cag22/HP0543) This 268 amino acids protein was demonstrated to interact with CagA, presumably at the inner bacterial membrane, and this interaction is essential for CagA translocation in the host. These data were used to sug- gest that CagF might play a chaperone function in the early steps of CagA recruitment and delivery into the T4SS channel [35,36]. Subsequently CagF was shown to interact with the 100 amino acids region adjacent to the C-terminal secretion signal of CagA [37]. Weak interactions involving three other Cag proteins (CagZ, CagT and CagM) were also detected. Localization studies indicated that it is both present in the mem- brane fractions and in the cytoplasm. A His6-tagged construct in our hands behaves as a soluble protein, even if it has a clear tendency to form oligomers of dif- ferent sizes, coexisting with a major fraction approxi- mately corresponding to a monomer. Detergent treatments appeared to reduce the contribution of very large unspecific oligomers and favour the presence of dimers and ⁄ or monomers. CagD (cag24/HP0545) The cagD locus is present in a majority of clinical iso- lates, although little is known about its role. Its amino acid sequence contains a predicted signal sequence for secretion in the periplasmic space. The crystal structure of CagD, solved in two differ- ent crystal forms at medium resolution (2.2 A ˚ and 2.75 A ˚ for the monoclinic and the hexagonal forms, respectively), shows that, in both cases, CagD is a homodimer, where the two monomers are covalently linked by a disulfide bridge (Fig. 3) [19]. In both crys- tal forms, the N-terminal domain is not visible in one monomer and absent in the other, as a result of prote- olysis. Consequently, the model is available only for residues 47–176. The visible part of each monomer folds as a single domain, characterized by a b-sheet flanked by a-helices. Five b-strands, labelled from A to E, run all contiguous. The N-terminus of the monomer includes strand A, an a-helix and a long stretch, and the C-terminal portion includes two relatively long a-helices and a final b-strand, F, which protrudes from the core of the monomer and runs anti-parallel to the same strand of a second monomer, allowing for the formation of the dimer. The surface of interaction between monomers also involves portion of chains D and E of the two monomers, which are held together not only by the S-S bridge between two Cys172, but also by hydrogen bonds. A second intramolecular disulfide bridge, between Cys120 and Cys133, helps to stabilize the 3D structure. The dimer presents a large crevice inbetween the two monomers, and the 46 N-term amino acids of one monomer could partially fill in this cavity. Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti 1228 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS The CagD overall fold is relatively common: the most relevant among proteins that present a similar fold is the SycT chaperone of Yersinia enterocolitica type III secretion system [38]. In particular, SycT shares the same topology in the region including the N-terminal helix and the b-sheet element, whereas it displays a remarkable difference in the orientation of the a-helices located at the C-terminus. Analogous to Yersinia SycT chaperone, CagD presents all the main a-helical motifs grouped on just one side of the b-strands, whereas all the other type III secretion sys- tem chaperones display a third a-helix on the opposite side, and this last is widely involved in the dimeriza- tion process. However, the dimeric arrangement of CagD is quite different from SycT as well as that of other members of this family. Finally, when crystallized in the presence of Cu(II), the protein shows the presence of the ion coordinated in a small cavity of the surface at the polar opposites of each monomer, close to another dimer present in the crystal packing and partially involving it in the coordination. It is likely that the presence of Cu(II) is an artefact of the crystallization, although the possibil- ity that the protein can physiologically bind cations cannot be ruled out completely. Disruption of the cagD gene was first reported to have an intermediate and variable effect on CagA deliv- ery and IL-8 induction phenotype [18]. Subsequently, CagA tyrosine phosphorylation and IL-8 assays have demonstrated that CagD is involved in CagA transloca- tion into the host epithelial cells, although it is not an absolute requirement for T4SS pilus assembly [19]. As suggested by the presence of a secretion signal at the protein N-terminus, CagD is mainly found in the periplasmic space, partially associated with the inner membrane. Interestingly, it was found to be secreted in the culture supernatant and this result was found not to be a result of generic bacterial lysis. Moreover, in a H. pylori infection experiment with AGS cells, sig- nificant amounts of CagD were found to be associated with the host cell membranes, and this interaction appeared to be independent of CagA translocation or the components of the T4SS, such as CagF. Because this localization was independent of the various tested cag mutants, these findings may indicate that CagD is released into the supernatant during host cell infec- tion by an unknown independent mechanism and then binds to the host cell surface or is incorporated in the pilus structure. Taken together, these results suggest that CagD may serve as a multifunctional component of the T4SS, which is involved in CagA secretion at the inner mem- brane and may localize outside the bacteria to promote additional effects on the host cell; however, whether these effects are required for CagA translocation or trigger CagA-independent virulence functions remains unclear. Conclusions Despite several studies carried out during the last 15 years on cagPAI, several questions about its compo- nents still remain unanswered. Those members that are not strictly structural are, in this sense, particularly puzzling because the role they play in the process of CagA secretion or IL-8 induction is still unknown or uncertain. However, partial maps of the H. pylori trans- membrane core apparatus and external pilus have been defined as a result of recent localization and interaction studies. Together with the VirB ⁄ D homologues CagV, Fig. 3. (A, B) Two different views of CagD dimer. Disulfide bridges are shown in yellow. It is possible to see the two b-strands, one per each monomer, that favour dimerization of the protein. (C) The electrostatic potential surface of CagD dimer. L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1229 CagX, CagY, CagT, Caga, Cagb and CagE, the pro- teins Cagd, CagM and CagZ have been identified as part of a wide network of interactions, with the first two most likely as unique oligomeric core components. CagF has been recognized to act as a chaperone of the major effector CagA, with CagL as a component play- ing a role in pilus adhesion to gastric epithelial cells. Finally, for a few other Cag proteins, localization in the bacterial compartments has been characterized. As described in this minireview, the 3D structure of only three of these unique components (CagZ, CagS and CagD) is now available, along with Caga ATPase. The lack of a clear picture of the biological func- tion and organization of some cagPAI components is also a major obstacle to structural studies because most of these gene products possibly do not act as single proteins, but perhaps as subunits of larger complexes, or they are made to act in concert with other partners. For these reasons, further accurate studies on the interactions among cagPAI compo- nents will be relevant not only to clarify the function of these proteins, but also for future structural inves- tigations. Acknowledgements We acknowledge all the PhD students and post-doctoral students that have contributed to the structural studies on the cag proteins over the years. This work was sup- ported by the Ministero dell’Istruzione, dell’Universita ` e della Ricerca, MIUR (PRIN 2007LHN9JL) and by the University of Padua, Italy. References 1 Parsonnet J (1994) Gastric adenocarcinoma and Helicobacter pylori infection. West J Med 161, 60. 2 Goodwin CS (1997) Helicobacter pylori gastritis, peptic ulcer, and gastric cancer: clinical and molecular aspects. Clin Infect Dis 25, 1017–1019. 3 Covacci A & Rappuoli R (2000) Tyrosine-phosphory- lated bacterial proteins: Trojan horses for the host cell. J Exp Med 191, 587–592. 4 Fischer W (2011) Assembly and molecular mode of action of the Helicobacter pylori Cag type IV secretion apparatus. FEBS J 278, 1203–1212. 5 Hatakeyama M (2006) The role of Helicobacter pylori CagA in gastric carcinogenesis. Int J Hematol 84, 301–308. 6 Tegtmeyer N, Wessler S & Backert S (2011) Role of the cag pathogenicity island encoded type IV secretion system in Helicobacter pylori pathogenesis. FEBS J 278, 1190–1202. 7 Covacci A, Telford JL, Del Giudice G, Parsonnet J & Rappuoli R (1999) Helicobacter pylori virulence and genetic geography. Science 284, 1328–1333. 8 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 pathogenicity island of Helicobacter pylori. Mol Microbiol 28 , 37–53. 9 Andrzejewska J, Lee SK, Olbermann P, Lotzing N, Katzowitsch E, Linz B, Achtman M, Kado CI, Suerbaum 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. 10 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. 11 Zhong Q, Shao S, Mu R, Wang H, Huang S, Han J, Huang H & Tian S. (2011). Characterization of pepti- doglycan hydrolase in Cag pathogenicity island of Helicobacter pylori. Mol Biol Rep 38, 503–509. 12 Rieder G, Hatz RA, Moran AP, Walz A, Stolte M & Enders G (1997) Role of adherence in interleukin-8 induction in Helicobacter pylori-associated gastritis. Infect Immun 65, 3622–3630. 13 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. 14 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. 15 Rohde M, Puls J, Buhrdorf R, Fischer W & Haas R (2003) A novel sheathed surface organelle of the Helicobacter pylori cag type IV secretion system. Mol Microbiol 49, 219–234. 16 Busler VJ, Torres VJ, McClain MS, Tirado O, Fried- man DB & Cover TL (2006) Protein-protein interactions among Helicobacter pylori cag proteins. J Bacteriol 188, 4787–4800. 17 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. 18 Fischer W, Puls J, Buhrdorf R, Gebert B, Odenbreit S & Haas R (2001) Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8. Mol Microbiol 42, 1337–1348. 19 Cendron L, Couturier M, Angelini A, Barison N, Stein M & Zanotti G (2009) The Helicobacter pylori CagD Unique type IV components of H. pylori cag PAI L. Cendron and G. Zanotti 1230 FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS (HP0545, Cag24) protein is essential for CagA translo- cation and maximal induction of interleukin-8 secretion. J Mol Biol 386, 204–217. 20 Boonjakuakul JK, Syvanen M, Suryaprasad A, Bowlus CL & Solnick JV (2004) Transcription profile of Helicobacter pylori in the human stomach reflects its physiology in vivo. J Infect Dis 190, 946–956. 21 Schmidt HM, Andres S, Nilsson C, Kovach Z, Kaako- ush NO, Engstrand L, Goh KL, Fock KM, Forman D & Mitchell H (2010) The cag PAI is intact and func- tional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore. Eur J Clin Microbiol Infect Dis 29, 439–451. 22 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. 23 Cendron L, Seydel A, Angelini A, Battistutta R & 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. 24 Hegde SS & Shrader TE (2001) FemABX family members are novel nonribosomal peptidyltransferases and important pathogen-specific drug targets. J Biol Chem 276, 6998–7003. 25 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. 26 Mourey L, Da Re S, Pedelacq JD, Tolstykh T, Faurie C, Guillet V, Stock JB & Samama JP (2001) Crystal structure of the CheA histidine phosphotransfer domain that mediates response regulator phosphorylation in bacterial chemotaxis. J Biol Chem 276, 31074–31082. 27 Tusnady GE & Simon I (1998) Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283, 489–506. 28 Zhang ZW, Dorrell N, Wren BW & Farthingt MJ (2002) Helicobacter pylori adherence to gastric epithelial cells: a role for non-adhesin virulence genes. J Med Microbiol 51, 495–502. 29 Backert S, Kwok T, Schmid M, Selbach M, Moese S, Peek RM Jr, Konig W, Meyer TF & Jungblut PR (2005) Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimen- sional gel electrophoresis and mass spectrometry. Proteomics 5, 1331–1345. 30 Segal ED, Lange C, Covacci A, Tompkins LS & Falkow S (1997) Induction of host signal transduction pathways by Helicobacter pylori. Proc Natl Acad Sci USA 94, 7595–7599. 31 Bourzac KM, Satkamp LA & Guillemin K (2006) The Helicobacter pylori cag pathogenicity island protein CagN is a bacterial membrane-associated protein that is processed at its C terminus. Infect Immun 74, 2537–2543. 32 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. 33 Saito H, Yamaoka Y, Ishizone S, Maruta F, Sugiyama A, Graham DY, Yamauchi K, Ota H & Miyagawa S (2005) Roles of virD4 and cagG genes in the cag patho- genicity island of Helicobacter pylori using a Mongolian gerbil model. Gut 54, 584–590. 34 Mizushima T, Sugiyama T, Kobayashi T, Komatsu Y, Ishizuka J, Kato M & Asaka M (2002) Decreased adherence of cagG-deleted Helicobacter pylori to gastric epithelial cells in Japanese clinical isolates. Helicobacter 7, 22–29. 35 Fischer W, Pu ¨ ls J, Buhrdorf R, Gebert B, Odenbreit S & Haas R (2003) Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8. Mol Microbiol 47, 1759. 36 Couturier MR, Tasca E, Montecucco C & Stein M (2006) Interaction with CagF is required for trans- location of CagA into the host via the Helicobacter pylori type IV secretion system. Infect Immun 74, 273–281. 37 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. 38 Buttner CR, Cornelis GR, Heinz DW & Niemann HH (2005) Crystal structure of Yersinia enterocolitica type III secretion chaperone SycT. Protein Sci 14, 1993–2002. L. Cendron and G. Zanotti Unique type IV components of H. pylori cag PAI FEBS Journal 278 (2011) 1223–1231 ª 2011 The Authors Journal compilation ª 2011 FEBS 1231 . MINIREVIEW Structural and functional aspects of unique type IV secretory components in the Helicobacter pylori cag-pathogenicity island Laura. portion of the molecule involving a-helix A, the nearby loop, the first and last turns of helices E and F, and the C-terminus helices I and J. In addition, there