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http://europepmc.org/articles/PMC3733401;jsessionid=TTuOpoJQmfWbFgaULPVS.3
The bacterial pathogen Helicobacter pylori has co-evolved with humans and colonizes roughly one half of the human population, but only causes overt gastric disease in a subset of infected hosts. In this Review, we discuss the pathogenesis of this bacterium and the mechanisms it uses to promote persistent colonization of the gastric mucosa, with a focus on recent insights into the role of the virulence factors VacA, CagA and CagL. We also describe the immunobiology of H. pylori infection and highlight how this bacterium manipulates the innate and adaptive immune systems of the host to promote its own persistence.
Helicobacter pylori is a highly successful human pathogen that colonizes roughly one half of the world‘s population. It is typically transmitted orally within families during early childhood and can persist for decades in its preferred niche, the gastric mucosa, despite triggering vigorous innate and adaptive immune responses. H. pylori infection causes chronic gastritis, which is asymptomatic in the majority of carriers but is considered a major risk factor for the development of gastric and duodenal ulcers and the two gastric malignancies, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma1. In addition to its tight association with cancer, H. pylori stands out among other Gram-negative bacterial pathogens in its ability to persist and establish chronic infection.
Contrary to long-held dogma, the stomach is not a sterile organ and is estimated to support a community composed of up to 200 different species2. However, when H. pylori is present it tends to be numerically dominant and readily visible in gastric biopsy tissue sections as helical rod-shaped organisms covering the gastric epithelial cells and surrounding mucus. Initial colonization depends on bacterial urease activity and cell-shape modulation to penetrate the gastric mucus. Constitutive DNA and protein repair pathways, combined with bacterial genome diversification and attenuation of host cell chemical radical production are now recogized as essential for persistence of the bacterium in this niche. The two known H. pylori toxins, VacA and CagA, have been the focus of much work aimed at understanding H. pylori virulence. While work on VacA has been reviewed recently3, we highlight new insights on functional interactions between VacA and CagA and the modulation of immune responses by VacA and another secreted virulence factor, the γ-glutamyl transpeptidase (GGT).
Besides its aresenal of virulence factors, persistence of H. pylori is strongly influenced by the ability of the bacterium to evade, subvert and manipulate the host‘s immune system. This bacterium can evade detection by several innate immune receptors through target modification and it can also subvert other innate recognition pathways through the suppression of downstream signal transduction, while evasion of adaptive immunity is achieved by the modulation of T-cell effector functions.
In this review we discuss the remarkable ability of H. pylori to colonize and persist in the hostile environment of the human stomach, through the interplay of several secreted virulence factors and sophisticated manipulation of innate and adaptive immune responses. We also highlight progress on understanding the consequences of persistence for both the bacterium and the host.
The stomach is a particularly challenging niche for bacterial habitation. In the lower bowel, which has a neutral or slightly alkaline pH, bacterial density is highest in the lumen; by contrast, the production of gastric acid in the stomach, which results in a pH of 1-2, severely limits luminal colonization. Indeed, H. pylori can only survive for minutes in the stomach lumen and must quickly migrate to the gastric epithelial surface4. Similar to the intestine, the mucus layer in the stomach forms a physical barrier to bacterial penetration and likely a scaffold for the binding of antimicrobial compounds of the host5. Bacterial urease production is required for acid resistance through the localized production of ammonium ions, and flagellar-based motility allows penetration of the mucus (Figure 1)6. Furthermore, urease activity facilitates flagellar motility through the mucus layer by changing the viscoelasticity properties of gastric mucins. At low pH, gastric mucins form a gel that effectively traps the bacteria, but urease-catalyzed production of ammonium ions raises the pH to near neutral and the mucus gel transitions to a viscoelastic solution through which H. pylori can swim7, 8. Regulators of motility, including chemotaxis9-12 and cell shape13-15 have been probed to discover additional colonization factors and to better define H. pylori‘s optimal niche. Helical cell shape is thought to enhance motility through viscous media by a corkscrew mechanism and cell shape mutants that have lost helical twist and/or curvature show attenuated colonization13-15. Mutants in several chemotaxis components show altered localization including lower numbers of bacteria in close association with gastric epithelial cells16 or deeply penetrating the gastric glands9. In addition to promoting clearance, the altered localization of chemotaxis mutants correlates with lower inflammation, impaired recruitment of CD4+ T-cells and the absence of a T-helper 17 response10, 16. Thus, the intimate association with the gastric epithelium promotes stable infection while simultaneously provoking more inflammation. Higher inflammation correlates with lower bacterial loads17, which suggests that H. pylori must actively manage its interaction with the host epithelium to avoid clearance and persist at this site (Figure 1).
H. pylori uses diverse strategies to promote its survival in the face of robust immune responses. All H. pylori strains encode proteins important for detoxifying reactive oxygen species (for example, catalase and superoxide dismutase) and H. pylori arginase limits NO production by macrophage-, neutrophil- and epithelial cell-derived nitric oxide synthase18, 19. Moreover, multiple DNA repair pathways contribute to efficient colonization20 even while the surrounding host tissue accumulates DNA lesions21, 22. Recent work has shown that H. pylori strains constitutively express DNA repair proteins such as RecA and thus lack a classic SOS response to DNA damage23, 24. Upon DNA damage, H. pylori instead upregulates natural competence which promotes chronic persistence, likely through enhanced genetic diversification23, 25.
The H. pylori genome contains multiple intragenic and extragenic repeat sequences26. CagY, which is expressed on the cell surface and is required for the T4SS-mediated translocation of the effector CagA (see below), can undergo recombination between internal repeat motifs that generally preserve the reading frame27. During experimental infection of mice and rhesus macaques, CagY variants accumulate that have gained or lost T4SS activity. These results suggest that CagA translocation and the associated biological responses, including inflammation, may have both beneficial and detrimental effects on bacterial persistence leading to selection for both retention and loss of T4SS activity28.
Among the 60 predicted outer membrane proteins (OMPs), the HOP family shares highly similar or identical sequences at their amino and carboxy termini and includes sevearl known or predicted H. pylori adhesins that promote binding to the gastric epithelium29. These shared sequences could promote intra- or intergenomic recombination. Sequencing of H. pylori HOP loci from human clinical strain collections has revealed likely gene conversion of the Lewis B binding adhesin gene babA with babB or babC30-33 and of sabB with the sialyl-Lewis binding adhesin sabA or omp2734-36. During experimental rhesus macaque or mouse infection, replacement of babA with babB results in strains that have lost the ability to bind immobilized Lewis B antigens32, 37. Additionally, replacement of sabA with sabB leads to strains expressing two copies of sabA, which results in increased binding to sialyl-Lewis antigens on murine gastric tissues36. Some alleles of babA and sabA can undergo phase variation by slipped strand mispairing at dinucleotide sequences in the coding sequences or homopolymeric tracts in their promoters, again leading to either loss or elevated gene expression38-40. The carbohydrate antigens bound by these adhesins can be expressed on the cell surface and/or on secreted glycoproteins such as mucin. Furthermore some of these antigens, such as sialyl-Lewis antigens, are induced during inflammation. Phase variation by gene conversion and slipped strand mispairing allows for development of subpopulations with variable adherence properties that could allow the pathogen to evade immune responses or resist shedding. This ability to generate diverse subpopulations may also impact transmission to new hosts.
H. pylori strains actively manipulate host tissues and promote their own persistence through the activity of a number of secreted toxins, some of which are discussed below. Vacuolating cytotoxin A (VacA), which was recently reviewed3, is a pore-forming toxin that disrupts cell polarity, promotes apoptosis of epithelial cells and inhibits T-cell proliferation and effector functions3. The vacA gene is carried by all H. pylori strains, and sequence variation in several domains of this protein is linked to varying expression levels and cell type specific toxicity, as well as disease severity3. Another important toxin is cytotoxin-associated gene A (CagA). Originally characterized as an immunodominant antigen from patients infected with highly virulent vacA alleles41, 42, CagA is translocated into host cells by the Cag type IV secretion system (T4SS) encoded on the cag pathogenicity island (PAI)1, 43. Strains expressing CagA are associated with an increased cancer risk and transgenic expression of CagA in mice induces gastric carcinoma and other malignancies, which has lead to its designation as a bacterial oncoprotein44.
To act as an oncoprotein, CagA must persist in cells or act in a hit and run manner. CagA has not been readily detected in gastric cancer tissues45 and thus was suggested to have an early causative role in cancer progression. It has now been elegantly shown that translocated CagA is degraded by autophagy during infection by H. pylori strains carrying the m1 allele of VacA via the ability of this VacA isoform to bind the cell surface receptor low-density lipoprotein receptor-related protein-1 (LRP1)46. VacA binding of LPR1 leads to loss of reduced glutathione (GSH) in the cell and increased production of reactive oxygen species (ROS). This in turn activates Akt kinase-dependent degradation of the tumour suppressor p53 and induction of autophagy that degrades CagA. Interestingly, autophagy is not activated in cells expressing a variant form of the CD44 adhesion molecule46. These cells have increased intracellular levels of GSH due to activation of xCT, a glutamate-cysteine transporter47 and therefore do not induce ROS or autophagy upon VacA binding. CD44 is a cell surface marker associated with epithelial cancer stem cells and CagA could be detected in variant-CD44 expressing cells from gastric cancer patients46. Paradoxically, tissue changes associated with H. pylori-induced gastric carcinogenesis, including development of intestinal metaplasia, were thought to render the stomach less hospitable for H. pylori colonization leading to lower colonization loads. However, H. pylori was shown to intimately interact with gastric progenitor cells in a mouse infection model48. The ability of H. pylori to colonize cells having stem cell-like properties and persistance of CagA protein in these cells due to the activiation of xCT and thus impaired autophagy dependent CagA degradation could provide a mechanism for a sustained role of H. pylori colonization and CagA in oncogenesis.
Once translocated into host cells, CagA can be tyrosine phosphorylated on EPIYA motifs49 by Src- and Abl family kinases. These two types of kinases are activated sequentially and in a tightly regulated manner, with Src kinases mediating the initial, preferential phosphorylation of EPIYA-C (and –D) motifs and Abl kinases phosphorylating any EPIYA motif at later time points post-infection50. Phosphorylated CagA interacts with SHP-2 tyrosine phosphatase and Csk kinase while unphosphorylated CagA is known to interact with Crk adaptor, c-Met, Grb2, PAR1/MARK and E-cadherin43. Collectively, these interactions lead to altered cell signalling and changes in cell polarity, extrusion, motility, proliferation and pro-inflammatory cytokine secretion1, 43 As discussed below, many of these phenotypes have now been linked to acquistion of nutrients by the bacterium to promote persistance and/or host pathology.
Under standard conditions, CagA expression is not required for stomach colonization, but promotes inflammation in the Mongolian gerbil model51. Cag T4SS activity is often lost during murine infection, which complicates efforts to elucidate the pathophysiological roles of CagA during chronic H. pylori infection28, 52, 53. However, in a polarized cell culture model, CagA promotes increased basolateral uptake and transcytosis of transferrin while VacA drives mislocalization of the transferrin receptor to sites of bacterial attachment to facilitate iron acquisition by the bacterium54. In cagA mutants, the formation of microcolonies on the apical surface of the cell requires iron supplementation, whereas this is not a requirement for wild-type bacteria, suggesting that CagA- and VacA-dependent cell polarity perturbations confer a nutritional benefit. Consistent with this hypothesis, CagA is required for efficient colonization of Mongolian gerbils under iron-limiting conditions54. Thus CagA and VacA collaborate to promote efficient colonization in the iron-limited environment of the stomach and to moderate pathologic effects of CagA.
Given the importance of CagA in persistance and pathology, there has been much interest in the mechanisms governing CagA delivery into host cells. Translocation of CagA from the bacterium to the host cell cytosol is mediated by the Cag T4SS. This is a contact-dependent secretion system that forms a large complex spanning the inner and outer membrane, containing a pilus and several ATPases that promote T4SS assembly, pilus formation and/or CagA translocation55. The H. pylori cag PAI encodes homologues or paralogues of the prototypical Agrobacterium tumefaciens Vir T4SS56, including the putative VirB7 (CagT), B9 (CagX) and B10 (CagY) inner and outer membrane spanning channel subunits57, the major (VirB2, CagC) and minor (VirB5, CagL) pilus subunits and several additional H. pylori specific cag proteins that are required for CagA translocation (for example, CagH and CagI)58, 59. Many Cag T4SS components have distinct domain structures from their Vir counterparts. For example, the VirB10 homologue, CagY, is considerably larger (~220 kDa) and contains additional domains composed of repeat regions27. Additionally, the three core cell envelope spanning channel subunit homologues, CagY, CagT and CagX, have been suggested to localize to the pilus surface or to the base of the pilus using transmission electron microscopy (TEM)60, 61. A later study localized CagL and CagA to the tip of the pilus62. CagL was suggested to act as a tip adhesin which binds to α5β1 integrin (the host cell receptor for CagL) through an RGD motif and neighbouring sequences (see insert Figure 1)62, 63. CagL binding and α5β1 integrin signalling were found to be required for both pilus extension and CagA translocation. Soluble RGD peptide could partially rescue the CagA translocation defect of a cagLRGA mutant, but not a ΔcagL deletion strain suggesting a two-step model, where surface exposed CagL binds and activates α5β1 integrin, partially activating focal adhesion kinase (Fak) and Src kinase, promoting pilus extension. In a second step, pilus associated CagL further stimulates α5β1 integrin, in addition to stimulating the activities of Fak and Src, thereby inducing CagA translocation and ensuring its rapid tyrosine phosphorylation by Src.
The relationship between pilus formation and CagA secretion was further explored by field emission scanning EM (FESEM), which readily detects Cag T4SS dependent pili64. This technique confirmed the requirement of CagL for pilus formation and also revealed a hyperpiliated phenotype for cagH mutants which, like cagL mutants, fail to translocate CagA64. A ΔcagY mutant produces pili28, which is surprising because CagL was found to be unstable in a ΔcagY mutant in another study59. At present, the mechanism by which CagL (or CagA, T, X and Y) becomes surface exposed or incorporated into pili has not been explored. Collectively these data suggest that pilus formation is not sufficient for CagA translocation, that pilus formation can proceed in the absence of at least one core T4SS component, and that there may be CagL-independent mechanisms of integrin activation, of pilus assembly and CagA translocation in some strains. In fact, CagA, CagI and CagY were shown to bind α5β1 integrin in vitro and using yeast two hybrid studies65. CagA, in particular, shows much higher integrin binding affinity in vitro than CagL; CagA binding is not inhibited by the Yersinia entercolitica RGD-containing invasin whereas CagL binding is inhibited, which would indicate that CagA and CagL use different integrin interaction surfaces. Antibodies that prevent integrin switching between a bent and open configuration block CagA translocation65 and the α5β1 integrin interaction domain of CagA was shown to inhibit CagA translocation when provided as a soluble peptide66 indicating a complex series of molecular interactions are required for integrin activation and CagA secretion. Further insights into the precise nature of the interactions between CagA, CagL and host interaction partners are beggining to be revealed by structural and molecular evolution studies (see text box 1). .
While the CagA translocation defect of cagL mutants suggested that CagL has a structural role as part of the T4SS, a number of studies suggest additional functions67-70. Studies with purified recombinant CagL revealed that the protein can induce cell spreading and focal adhesion formation in a similar manner to the host extracellular matrix RGD-containing protein fibronectin69. CagL activates epidermal growth factor receptor (EGFR) more efficiently than fibronectin, and this was shown to result from RGD-dependent displacement of ADAM17 (a disintegrin and metalloprotease) from α5β1 integrin, activating ADAM17 protease activity68. ADAM17 cleaves and releases surface bound heparin-binding EGF-like growth factor. The resulting activation of EGFR in gastric epithelial cells represses H,K-ATPase activity (and thus diminshes acid secretion) via a repressive NF-κB binding site in the H,K-ATPase promoter. CagL can also bind αvβ5 integrin independently of its RGD motif, which mediates the induction of gastrin70. Gastrin is a potent inducer of acid secretion, thus, simultaneous activation of gastrin and repression of the H,K-ATPase could explain the observed hypergastrinaemia and hypochlorhydria during chronic H. pylori infection. Finally, CagL RGD-dependent activation of α5β1 integrin activates the pro-inflammatory cytokine interleukin-8 (IL-8) independently of CagA translocation and Nod1 signalling67, indicating that CagL induces inflammation. Increased risk cancer and ulcer which is associated with the carriage of the Cag PAI, has largely been attributed to CagA but these studies indicate that CagL may be an equally important effector. Furthermore, studies on the evolution of the Cag PAI suggest that additional Cag proteins may directly interact with host proteins via exposure on the cell surface or as novel effectors (see text box 1).
In addition to the multiple virulence factors that H. pylori uses to manipulate the host and ensure its persistence, the pathogen has evolved elaborate strategies to evade and subvert host immune defences, which are key to the success of this pathogen. The first defence barrier against H. pylori is formed by mucus-producing epithelial cells lining the gastric mucosa and by innate immune cells that either reside in the gastric lamina propria under steady state conditions or are recruited there during infection. The detection of conserved pathogen-derived molecular structures (pathogen-associated molecular patterns, PAMPs) by epithelial cells and innate immune cells occurs via four distinct classes of innate immune receptors (so-called pattern recognition receptors, PRRs) that differ in their subcellular localization, coupling to downstream signalling pathways and specificity. H. pylori has evolved to avoid detection by several types of PRRs that are crucial for the recognition of other Gram-negative enteropathogens.
The best-defined among the four classes of PRRs are the Toll-like receptors (TLRs). TLRs are either surface-exposed on the plasma membrane or localize to endosomes and bind diverse classes of microbially derived PAMPs. Among these are the ligands for TLR4 (LPS), TLR2 (lipoteichoic acid and lipoproteins), TLR3 (dsRNA and polyinosinic:polycytidylic acid), TLR5 (flagellin) and TLR9 (unmethylated CpG). H. pylori largely avoids recognition by TLRs, with evasion of TLR4 detection of LPS being the best understood example. H. pylori LPS is predominantly tetra-acylated and is 1000-fold less bioactive than the hexa-acylated LPS of E. coli 71. Furthermore, the relative inactivity of H. pylori LPS was recently shown to result from the removal of phosphate groups from the 1‘- and 4‘- positions of the lipid A backbone, which generates LPS that exhibits less negative charge, resists binding by antimicrobial peptides such as polymyxin B and escapes detection by TLRs72. The phosphatases responsible for lipid A modification in H. pylori have been identified and the respective gene deletion mutants indeed fail to colonize experimentally infected mice72. The TLR(s) involved in the residual detection of H. pylori LPS remain a matter of debate; whereas several studies utilizing purified LPS have implicated the classical LPS sensor TLR473, 74, others suggest that TLR2 is the main sensor of H. pylori LPS75, 76 (Figure 2). A clear interpretation of the published studies is complicated by the fact that they rely on models in which the respective TLR is ectopically expressed, often in the absence of its co-receptor, and the fact that both TLR4 and TLR2 participate in the detection of other, non-LPS-related PAMPs of H. pylori77, 78, which may contaminate LPS preparations.
Another putative H. pylori PAMP, flagellin, escapes recognition by TLR5 due to modifications in the N-terminal TLR5 recognition domain79 (Figure 2). Mutating residues 89-96 of Salmonella flagellin to the corresponding flaA sequence of H. pylori abolishes its recognition by TLR580. Experiments using dendritic cells (DCs) lacking TLRs 2, 4, 7 and 9, or combinations thereof, revealed that the innate immune system recognizes H. pylori nucleic acids77. Intracellular delivery of H. pylori DNA to DCs by lipofection efficiently activates endosomally localized TLR977, however the net effect of this activation is anti- rather than pro-inflammatory81-83 (Figure 2). TLR9 signalling has anti-inflammatory consequences in the early stages of infection in a mouse model81 and H. pylori DNA can even be used therapeutically to treat experimentally induced inflammatory bowel disease in mice82, 83. The bioactivity of H. pylori DNA may account for the inverse correlation between H. pylori colonization and the risk of developing inflammatory bowel diseases84, which has been attributed to a specific “immuno-regulatory“ sequence (TTTAGGG) that appears to be unique to the H. pylori genome82, 83. H. pylori RNA sensing by DCs has been suggested to be mediated by endosomally localized TLR877, as well as a cytoplasmic nucleic acid sensor belonging to the RIG-like helicase receptor family (RLRs), RIG-I. RIG-I appears to be required for the detection of 5‘ triphosphorylated H. pylori RNA and the ensuing IRF3/7-dependent induction of type I interferons (IFNs) by DCs77 (Figure 2). Whether the activation of RIG-I and the H. pylori-induced production of type I IFNs has predominantly pro- or anti-inflammatory effects remains to be determined.
The detection of H. pylori non-LPS ligands by TLR2 represents another example of how H. pylori exploits the immune system for the induction of anti-inflammatory responses. Activation of TLR2 triggers the MyD88-dependent expression of a number of anti-inflammatory genes, most notably interleukin-1077 (Figure 2). Furthermore, TLR2−/− mice infected with Helicobacter felis, a close relative of H. pylori, control experimental infections better than wild type mice and develop stronger T-cell responses and T-cell-driven immunopathology78. The effects of TLR2 gene deletion are phenocopied by MyD88−/− mice, indicating that the absence of anti-inflammatory signals induced by Helicobacter is phenotypically dominant over the simultaneous lack of MyD88-dependent pro-inflammatory signals induced by other TLRs78.
In addition to its TLR and RLR ligands, H. pylori also harbors ligands for a third class of PRRs, the C-type lectin receptors (CLRs). The best characterized of these are fucosylated ligands that bind to the CLR family member DC-SIGN85. In contrast to pathogens such as M. tuberculosis and HIV, which express mannosylated DC-SIGN ligands and activate pro-inflammatory downstream signalling pathways, the fucose residues of H. pylori‘s DC-SIGN ligands actively dissociate the signalling complex downstream of DC-SIGN (consisting of the scaffold proteins LSP1, KSR1 and CNK and the kinase Raf-1) and suppress pro-inflammatory signalling85 (Figure 2). The differential biological effects of mannosylated and fucosylated DC-SIGN ligands are in line with the proposed role of this PRR in tailoring and fine-tuning adaptive immunity to specific pathogens via the DC-SIGN/Raf-1-mediated acetylation of TLR-activated nuclear NF-κB86. Acetylation of the NF-κB subunit p65both prolongs and increases IL-10 transcription to enhance anti-inflammatory cytokine responses86.
In summary, most of the available data support the conclusion that H. pylori avoids the induction of a strong pro-inflammatory response, as well as subsequent adaptive immunity and clearance, through two main mechanisms: the evasion of innate immune detection by pro-inflammatory TLRs and the preferential activation and manipulation of anti-inflammatory TLRs and CLRs. Together, these strategies promote persistence of the organism.
The heterogeneous cytoplasmic family of Nod-like receptors (NLRs) comprise the fourth and final family of PRRs. NLRs detect a wide range of PAMPs and are also essential for sensing host-derived, damage-associated molecular patterns (DAMPs) that are released upon perturbations of tissue homeostasis87. Broadly speaking, NLRs fall into two categories: Nod1 and Nod2 recognize peptidoglycan metabolites and activate the transcription factor NF-κB to induce innate and adaptive immune response genes88, while most other NLRs promote the assembly of multiprotein complexes called inflammasomes, which activate the cysteine protease caspase-189.
Nod1-mediated detection of H. pylori peptidoglycan was one of the first PRR-mediated innate immune pathways found to become activated upon H. pylori infection90. Although initial work indicated that only T4SS-proficient H. pylori strains (harboring a functional Cag T4SS) could deliver peptidoglycan and its active metabolite (meso-diaminopimelate-containing N-acetylglucosamine-N-acetylmuramic acid) into the cytoplasm of host epithelial cells90, it is now clear that outer membrane vesicles (OMVs) from Cag PAI-negative strains of H. pylori can also target peptidoglycan to Nod191 (Figure 3). Intragastric delivery of OMVs in mice induces innate and adaptive immune responses through a NOD1-dependent but TLR-independent mechanism91. The delivery of peptidoglycan by both OMVs and the T4SS occurs at cholesterol-rich lipid rafts91, 92 (Figure 3). In addition to the initially reported Nod1 signalling pathway resulting in NF-κB translocation to the nucleus90, Nod1 also activates the transcription factor AP-1 via the ERK and p38-dependent pathways93. A direct consequence of Nod1 signalling is efficient killing of H. pylori by beta-defensin 2, an antimicrobial peptide produced by Nod1-activated gastric epithelial cells94. The idea that H. pylori-induced activation of NF-κB depends on Nod1 has recently been challenged by a report showing that introduction of an siRNA specific for Nod1 does not alter the nuclear translocation of the NF-κB subunit p6595. This study rather provides evidence for an alternative Nod1-dependent signalling pathway, which activates the IRF3/7 transcription factor to induce the production of type I IFNs that are required for H. pylori-specific cytokine and chemokine responses and infection control95 (Figure 3).
H. pylori harbors one or more ligands that trigger activation of the inflammasome and of caspase-1, a cysteine protease that controls the processing and secretion of two cytokine precursors, pro-IL-1β and pro-IL-1887. Like other caspases, caspase-1 is synthesized as an inactive precursor, which becomes auto-proteolytically activated only upon inflammasome assembly. Inflammasome assembly in turn is regulated by ligand binding and subsequent hetero-oligomerization of inflammasome sensors in conjunction with an adaptor molecule (apoptosis-associated speck-like protein containing a CARD, ASC) and pro-caspase-187, 89. Whereas the inflammasome ligands and NLR sensors involved in H. pylori detection remain obscure, in vitro and in vivo studies have demonstrated that caspase-1 becomes activated in DCs upon co-culture with H. pylori and that IL-18 and IL1-β are processed and released into the infected gastric mucosa96, 97 (Figure 3).
There is no evidence to suggest that H. pylori actively avoids inflammasome or caspase-1 activation. In fact, mice lacking caspase-1 due to targeted deletion of the corresponding gene clear an experimental infection with Helicobacter felis or H. pylori more efficiently than wild-type animals, and exhibit more pronounced pathogen-specific T-cell responses and T-cell-driven immunopathology96. The explanation for this unexpected observation was provided by mouse strains lacking either IL-18 or its receptor, IL-18R: these mice phenocopy the effects of caspase-1 gene deletion, i.e. they clear the infection better than wild-type mice due to enhanced T-cell responses, and as a consequence, develop more severe immunopathology.96, 98 Further analysis revealed that IL-18 is crucial for inducing regulatory T-cell (Treg) responses to H. pylori (Figure 3), which in turn restrict excessive effector T-cell activation and promote persistence98. Interestingly, IL-1β (the other caspase-1 cytokine substrate) apparently opposes IL-18 function. IL-1R−/− animals lacking the receptor for IL-1β fail to launch H. pylori-specific Th1 and Th17 responses, and cannot control an experimental infection (even when vaccinated against H. pylori before challenge) and, as a consequence, are protected against even the mildest forms of infection-associated immunopathology96. These data corroborate an earlier report showing that stomach-specific expression of human IL-1β is sufficient to induce gastric inflammation and gastric cancer in transgenic mice99, and also explain why promoter polymorphisms associated with increased steady-state levels of IL-1β predispose carriers to a high gastric cancer risk100. Furthermore, the effects of IL-1R gene deletion appear to be phenocopied by H. pylori-infected mice lacking the inflammasome adaptor ASC101. In conclusion, detection of H. pylori via NLRs and subsequent activation of the inflammasome and downstream signalling pathways is crucial for efficient infection control (in the case of Nod1 signalling and inflammasome-mediated IL-1β secretion) and at the same time ensures the restriction of excessive T-cell responses and immunopathological tissue damage (via inflammasome-mediated IL-18 secretion).
Experimental infection studies have shed light on the elements of the innate and adaptive immune system required for control of H. pylori infections, and in particular for the generation of vaccine-induced protective immunity17, 102-107. Whereas B-cells and antibodies are dispensible for H. pylori control103, 104, 107 (at least for the sub-optimal, non-sterilizing reduction in colonization by 1-2 orders of magnitude that is considered the gold standard in the H. pylori vaccinology field, see text box 2), it is now clear that CD4+ effector T-cells (not to be confused with the CD4+ Tregs mentioned above), and in particular Th1 and Th17-polarized T-effector cell subsets and their signature cytokines, are critical for proper control of this infection17, 102, 106. The same T-cell subtypes have been implicated in promoting the immunopathological changes of the chronically infected gastric mucosa that manifest histologically as atrophic gastritis, compensatory epithelial hyperplasia and intestinal metaplasia in experimentally infected animals and symptomatic human carriers108, 109.
Two virulence factors have been specifically implicated in the manipulation and inhibition of human T-cells (Figure 4). VacA inhibits T-cell proliferation by interfering with the T-cell receptor/interleukin-2 signalling pathway at the level of the Ca2+/calmodulin-dependent phosphatase calcineurin110, 111. VacA prevents nuclear translocation of the T-cell transcription factor NF-AT and its subsequent transactivation of T-cell-specific immune response genes110, 111 (Figure 4). Follow up studies have since identified β2 integrin (CD18) as the receptor for VacA on human T-cells112; β2 integrin associates with CD11a on T-cells to form the heterodimeric transmembrane receptor LFA-1 (lymphocyte function-associated antigen-1). H. pylori exploits the recycling of LFA-1 to facilitate VacA uptake112 in a manner that depends on protein kinase C-mediated serine/threonine phosphorylation of the β2 integrin cytoplasmic tail113. The other H. pylori virulence determinant implicated in T-cell inhibition is γ-glutamyl-transpeptidase GGT114, 115. Similar to VacA, GGT is a secreted factor that blocks proliferation of T-cells through a mechanism that involves the inhibition of cyclin-dependent kinase activity in the G1 phase of the cell cycle by the disruption of the Ras signalling pathway114, 115 (Figure 4).
Both VacA and GGT also affect T-cell activity in an indirect manner, by promoting the preferential differentiation of naive T-cells into Tregs116. Treg differentiation in response to H. pylori infection requires the direct interaction of naive T-cellswith ‘tolerogenic‘ DCs that have been exposed to H. pylori, either in the gastric mucosa or in the stomach-draining (gastric or mesenteric) lymph nodes98, 117, 118H. pylori-exposed DCs fail to induce effector T-cell responses of the Th1 and Th17 type in vitro and in vivo; instead, such DCs preferentially induce the expression of the Treg-specific transcription factor FoxP3, the surface marker CD25 and the anti-inflammatory cytokine IL-10 in naive T-cells 98, 107, 117 (Figure 4). Such peripherally induced Tregs affect the control of H. pylori profoundly, as shown in chronically infected patients119-123 and by animal experiments in which Tregs were systemically depleted in infected hosts52, 107. Tregs accumulate in the H. pylori-infected human gastric mucosa119, 121, especially in children123 and in asymptomatic carriers122, and effectively suppress H. pylori-specific memory T-cell responses120.
Experimental depletion of Tregs facilitates clearance of H. pylori in infected animals52 and enhances vaccine-induced protective immunity in vaccinated mice107. The Treg-facilitated persistence of H. pylori requires T-cell-specific expression of IL-10; in fact, IL-10−/− mice and a strain lacking IL-10 expression in the CD4+ T-cell compartment are capable of spontaneously controlling experimental infections52, 78, 124. The efficient control or even clearance of H. pylori in animals invariably comes at the price of enhanced gastric immunopathology (gastritis and epithelial changes such as atrophy and intestinal metaplasia). Interestingly, an analogous observation has been reported for human carriers, which either accumulate large numbers of IL-10-producing, H. pylori-specific Tregs and are colonized heavily (asymptomatic carriers), or develop gastric ulcers because their Treg response is inadequate122. The H. pylori-specific ‘tolerization‘ of DCs, which appears to be a prerequisite for the skewing of T-cell responses (at least in experimental models98, 117), requires the activity of both VacA and GGT 116 (Figure 4). While the exact mechanism of VacA- and GGT-specific DC tolerization remains to be elucidated, the newly assigned function of both factors in Treg induction and persistence is consistent with previous reports showing that gene deletion mutants lacking VacA or GGT have colonization defects relative to their parental, VacA/GGT-proficient wild-type isolates125, 126.
The active inhibition and manipulation of adaptive, T-cell-driven immune responses by H. pylori has a variety of consequences for the host. The persistence mechanisms of H. pylori are dominant enough to override the protective effects conferred by H. pylori-specific vaccination; a challenge infection can only be cleared (or at least strongly reduced) by vaccinated mice if Tregs or DCs are depleted107. These observations may partly explain the difficulties and obstacles faced in H. pylori vaccine development (see text box 2). An interesting side effect of H. pylori-specific immunomodulation and manipulation is evident in Western societes from which H. pylori is gradually disappearing due to reduced transmission rates, the frequent use of antibiotics in childhood and generally improved sanitation conditions127. In these populations, the incidence of allergic asthma, other allergic disease manifestations and chronic inflammatory diseases is steadily increasing and an inverse association with H. pylori colonization has been documented for allergic asthma128-132 and inflammatory bowel diseases84 (see text box 3). While the exact mechanisms underlying this inverse association remain to be elucidated, the idea that H. pylori-induced immune regulation and manipulation are causally linked to protection from such immune disorders is compelling (see text box 3). The fact that Tregs isolated from H. pylori-infected mice are sufficient to protect naive recipients against allergen-induced asthma in adoptive transfer models argues in favor of Treg-mediated cross-protection against allergen-specific immune responses98, 133. Further work in this area is urgently needed to shed more light on the intricate interactions of this extraordinarily well-adapted, persistent pathogen with the host adaptive immune system.
The work summarized in this Review outlines how H. pylori uses a combination of virulence factors and immune subversion and manipulation mechanisms to colonize and persist in the challenging environment of the gastric mucosa. Experimental work in recent years has elucidated exciting details on the structure and function of the type IV secretion system, the pleiotropic effects of CagA delivery, the CagA-independent effects of the secretion system and the newly discovered function of the extracellular effector CagL. The role of cell shape and chemotaxis in persistent colonization is now well documented with respect to the genes involved. Progress in other areas, in particular H. pylori-specific vaccine development, has suffered from setbacks in phase I clinical trials and the lack of continuous industry support. The necessity of overriding the bacteria‘s persistence strategies has been identified as a major challenge in H. pylori-specific vaccine development. More and more interest in the field has shifted to gaining a better understanding of the benefits, suggested from epidemiological studies, that the infection may bestow on the large majority of asymptomatic carriers, and experimental evidence has been forthcoming to support such claims. In particular, it is now becoming increasingly clear that the virulence factors deployed by H. pylori and the mechanisms that are exploited to override T-cell-driven immunity and to ensure persistent infection have systemic immunomodulatory effects that likely explain the benefits of the infection to asymptomatic carriers. The molecular mechanisms allowing H. pylori to suppress T-cell activation via production of VacA and GGT, and to skew T-cell responses towards regulatory T-cells, are increasingly well understood.
Other aspects of the Helicobacter-host interaction have received surprisingly little, if any, attention; these include the specifics of inflammasome activation by H. pylori and of innate immune activation by H. pylori in general, the molecular basis of its host species specificity, and the relative (or perhaps additive) contributions of its direct and indirect (inflammation-mediated) carcinogenic properties to gastric cancer development. Another important aspect of H. pylori biology that has been largely ignored relates to its transmission. While several independent, mostly older, studies indicate that mothers serve as the predominant source of their children‘s H. pylori infection, the transmission route remains unclear. Also, very little is currently known about the vast differences in gastric cancer risk of (often closely related, and physically close) human populations, which is likely to be influenced by human genetic predisposition, population ecology and behaviour. In summary, many of the peculiarities that set H. pylori apart from other Gram-negative enteropathogens remain underexplored and deserve further work.
Work in the laboratories of NRS and AM is supported by grants AI054423 and AI094839 from the US National Institutes of Health to NRS and grants from the Swiss and Zurich Cantonal Cancer Leagues, the Gebert-Rüf Foundation and the Swiss National Science Foundation to AM. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of these funding agencies.
Anne Müller
Anne Müller studies the pathogenesis of Helicobacter-induced gastric cancer and gastric lymphoma. Additional research interests include H. pylori virulence factors, persistence mechanisms and innate immune recognition. She received her Ph.D. in 2000 at the Max Planck Institute of Infection Biology, Berlin, Germany, and then moved to the laboratory of Stanley Falkow at Stanford University, California, to pursue postdoctoral research on Helicobacter-host interactions. She has been an independent researcher and Faculty member at the Institute of Molecular Cancer Research, University of Zürich, Switzerland, since 2006.
Mara L. Hartung
Mara L. Hartung studied biology at the University of Tübingen, Germany. She is currently pursuing her doctoral studies at the Institute of Molecular Cancer Research, University of Zürich, Switzerland. Her research focuses on investigating the mechanism of DNA damage inflicted on gastric epithelial cells by H. pylori and the implications of this process for gastric carcinogenesis.
Nina Salama
Nina Salama investigates H. pylori pathogenesis with a focus on bacterial determinants that promote persistent colonization. Current insterests include generation and consequeces of helical cell morphology, genetic variation during chronic colonization, mechanisms of transmission and H. pylori interactions with other bacteria and tissues in the upper gastrointestinal tract. She received her Ph.D. in 1995 at the University of California, Berkely, and then moved to the laboratory of Stanley Falkow at Stanford University, California, to pursue postdoctoral research on Helicobacter pylori bacterial genetics. She has been an independent research and Faculty member at the Fred Hutchinson Cancer Research Center and Affiliate Professor of Microbiology at the University of Washington in Seattle Washington, since 2001.
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