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

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 re

Structural and functional aspects of unique type IV

secretory components in the Helicobacter pylori

cag-pathogenicity island

Laura Cendron1and Giuseppe Zanotti2

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.

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 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 descripimmunoprecipita-tion of the interacimmunoprecipita-tions 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 secretransloca-tion, 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]

Cagd (cag3/HP0522)

Cagd is a 55 kDa protein essential both for the

secre-tion of CagA and the inducsecre-tion 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 pneumoniaecholine-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 and heterotypic complexes with CagV (VirB8),

CagT (VirB7), CagM and CagG [16] In particular, the

interaction with CagT, a core component involved in

the Cag-T4SS outer membrane sub-complex, was

identi-fied by multiple independent techniques and more

accurately elucidated Size exclusion chromatography

analysis allowed the isolation of both Cagd-independent

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.

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.

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 pylori26695 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 (CagN1–216), 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-CagN25–306, CagN25–216-His6) 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)

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 sigN-termi-nal 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

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.

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 organizafunc-tion 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

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