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Previous Article | Next Article 
Infection and Immunity, February 2002, p. 665-671, Vol. 70, No. 2
0019-9567/01/$04.00+0 DOI: 70.2.665-671.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Abteilung Molekulare Biologie, Max-Planck-Institut für Infektionsbiologie, D-10117 Berlin, Germany
Received 13 August 2001/ Returned for modification 16 October 2001/ Accepted 8 November 2001
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ABSTRACT |
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INTRODUCTION |
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Several in vitro and in vivo studies have demonstrated that H. pylori induces host cell vacuolation (17, 33), cytoskeletal rearrangements (5, 11, 36), and the secretion of a range of inflammatory mediators, including cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor alpha and chemokines IL-8, Gro-
, RANTES, ENA-78, MCP-1, and Mip-1 (8, 9, 25, 41). Of these factors, IL-8 is a potent neutrophil and T-cell chemoattractant and activator, which is believed to play a key role in the pathogenesis of H. pylori-induced tissue damage (31, 34, 42). H. pylori is thought to stimulate IL-8 gene expression by activating the transcription factor nuclear factor 
B (19, 37).
Several H. pylori virulence factors have been determined. First, bacterial adhesion is essential for host-pathogen interaction. The Lewis b-binding adhesin BabA (21) and two outer-membrane proteins, designated AlpA and AlpB (30), have been identified. Second, patients with gastritis, ulcers, or malignancies are often infected with H. pylori type I strains. Although type I and type II strains do not constitute two distinct lineages, some virulence factors have been reported to be prevalent in type I strains (14). These strains frequently express the adhesin BabA, cytotoxic alleles of the vacuolating cytotoxin (VacA), and the immunodominant cytotoxin-associated antigen (CagA). While VacA induces the formation of vacuoles in infected cells (33), CagA has been associated with rearrangements of the actin cytoskeleton resulting in phenotypic changes called the scattering or "hummingbird" phenotype (5, 36).
Genetically, type I strains are characterized by the presence of a 40-kb DNA segment (containing up to 31 open reading frames [ORFs]) termed the cag pathogenicity island (PAI) that is often divided into the cag I and cag II loci (9, 14). While CagA is encoded within the cag PAI, VacA is encoded outside the pathogenicity island. Some of the cag PAI ORFs show significant homology to the virulence (vir) genes virB4, virB7, virB8, virB9, virB10, virB11, and virD4 of the so-called VirB/D complex of type IV secretion systems known from Agrobacterium tumefaciens and Bordetella pertussis (10, 14). Type IV systems can be regarded as molecular syringes that inject their specific substrate into the cytosol of target cells. They are involved in conjugative DNA transfer of prokaryotes and in the delivery of bacterial virulence factors into the cells of their eukaryotic hosts.
Recent studies have revealed compelling evidence that the cag PAI of H. pylori encodes a functional type IV machinery that translocates the CagA protein into the cytosol of gastric epithelial cells (3, 5, 7, 29, 36, 38, 43). Translocated CagA is tyrosine-phosphorylated by an unidentified host kinase and undergoes processing into p100CagA and p35CagA fragments that have been observed in both gastric epithelial cells (6) and phagocytic cells (26, 28). However, their importance is not understood. Conspicuously, the IL-8-inducing activity of some H. pylori strains mutated in a series of cag PAI genes was strongly reduced (1, 9, 23, 40). These data led to the speculation that H. pylori induces IL-8 secretion by an unidentified factor that is translocated through the type IV secretion system. However, there are also conflicting reports in which inactivation of virD4, virB10, and virB11 enhanced IL-8 secretion (16, 18). Another group has reported that water-soluble extracts of H. pylori containing nonprotein low-molecular-weight compounds can induce IL-8 secretion (22). These phenomena are not understood.
Thus, there are still open questions concerning the function of the type IV secretion system. A systematic functional analysis using genes throughout the cag I and cag II loci and different read-out systems (for the function of the type IV secretion system) in parallel has not yet been carried out. Moreover, in almost all recent studies, possible polar effects of cag PAI mutagenesis on expression of neighboring genes were not investigated. No studies using genetic complementation of individual cag PAI genes have been performed so far.
In this study, we have generated a large set of isogenic cag PAI mutants, including all virB/D genes, and analyzed their ability to induce IL-8 secretion, CagA phosphorylation, and phenotypic changes. To rule out the possibility of polar effects in our mutants, we complemented two of our knockout strains with wild-type genes in trans. Our results demonstrate that CagA translocation and induction of IL-8 secretion depend on all vir homologs and other cag PAI genes. While phenotypic changes were found to be strictly correlated with CagA translocation, IL-8 release was independent of CagA. VirD4 played a special role, as it was essential for CagA translocation but dispensable for the induction of IL-8 secretion, indicating important differences between these processes.
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MATERIALS AND METHODS |
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We constructed isogenic cagA, cagH, and vir gene knockout mutants in strain P1 by insertion of a chloramphenicol resistance gene cassette (Cmr, 1-kb BamHI/BglII fragment of plasmid pTnMax1) using a protocol described previously (7, 26). 
cagF, cagI, and cagM knockout mutants were created in strain G27 by transposon-mediated mutagenesis and were provided by Antonello Covacci (9). The mutant lacking the complete cag PAI (PAI) was derived from strain P12 by insertion of a kanamycin resistance cassette as described previously (28). The P12vacA and P1alpA/B double mutants were kindly provided by Rainer Haas (30, 35).
H. pylori strains were cultivated on horse serum agar plates supplemented with vancomycin (10 µg/ml), nystatin (1 µg/ml), and trimethoprim (5 µg/ml). Depending on the specific strain, chloramphenicol (4 to 6 µg/ml) and/or kanamycin (20 µg/ml) was added. Bacteria were cultivated for 2 days at 37°C in an anaerobic jar containing a Campygen gas mix of 5% O2, 10% CO2, and 85% N2 (Oxoid, Wesel, Germany).
Complementation of CagA and VirD4. For construction of complementation vector constructs, cagA and virD4 were amplified from strain 26695 by PCR (NCBI database accession numbers AAD07614 and D64585, respectively). For cagA, the primers 5'GTCGACCATCTTTAGCGTTGCATTTGATTT (forward) and 5'GTCGACCAACACAAGTAGCCCCTAAAACTT (reverse) were used. These primers contained unique AccI sites that were used to clone the PCR product into the ClaI site of the Escherichia coli/H. pylori shuttle vector pHel2 containing the oriT of RP4 and a kanamycin resistance gene cassette (aph-A3) as a selectable marker (20).
virD4 was amplified using the primers 5' TCCATATGGAAGACTTTTTGTATAACAC (forward) and 5' CGGGATCCGACACACTTCTACTATCTTATG (reverse). The forward primer contained an NdeI site, and the reverse primer contained a BamHI site for cloning in pHel2. virD4 was expressed under the constitutive H. pylori flaA promoter, which was cloned as described (20). The cagA and virD4 complementation constructs were transferred into H. pylori by conjugation of E. coli XL1-Blue with P1cagA and P1virD4, respectively, using E. coli GC7[pRK2013] as the mobilizer (20). Plasmids were reisolated from transformed H. pylori strains, and restriction fragment length polymorphism patterns were confirmed in order to exclude integration of the constructs into the bacterial chromosome.
Synchronized infection assays. AGS cells (ATCC CRL 1739; a human gastric adenocarcinoma epithelial cell line) were cultivated in six-well tissue culture dishes in 2 ml of RPMI 1640 medium (Gibco-BRL, Eggenstein, Germany) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL) per well for 2 days to reach monolayers of about 70% confluence. H. pylori cells were suspended in phosphate-buffered saline (PBS) and added to AGS cells at a multiplicity of infection (MOI) of 100. In order to synchronize the infection, bacteria were centrifuged onto the cells for 5 min at 600 x g. The alpA/B mutant, which is defective for adherence, was added to the cells without centrifugation.
After incubation in a 5% CO2-95% air incubator for 5 h, the phenotype was analyzed by phase contrast microscopy. When more than 60% of the cells were elongated (see Fig. 3), the hummingbird phenotype was considered to have been induced. After 6 h, the cell culture medium was collected for the IL-8 assay and stored at -20°C. Cells were washed once with ice-cold PBS containing 1 mM Na3VO4 (Sigma-Aldrich, Deisenhofen, Germany), harvested with a rubber policeman in the same buffer, and pelleted together with attached bacteria at 600 x g and 4°C.
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Tyrosine-phosphorylated proteins were detected with the monoclonal phosphotyrosine-specific antibody PY99 (Santa Cruz Biotechnology, Santa Cruz, Calif.), in a 1:1,000 dilution in TBS-T for 1 h at room temperature. CagA was detected with a polyclonal rabbit anti-CagA antibody, a gift of S. Censini and A. Covacci (12). Blots were washed three times in TBS-T and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin secondary antibodies (Amersham Pharmacia Biotech) for 1 h. After three more washing steps, the bound antibodies were detected with the Renaissance Western blot kit system for enhanced chemiluminescence immunostaining (ICN Biochemicals, Eschwege, Germany). The same blot was used for both antiphosphotyrosine and anti-CagA immunoblotting. Between the blotting procedures, the membranes were stripped for 30 min at 50°C in stripping buffer (62.5 mM Tris-HCl [pH 6.7], 100 mM 2-mercaptoethanol, 2% SDS).
IL-8 ELISA. The amount of IL-8 secreted into the cell culture medium after 6 h of infection was determined by sandwich enzyme-linked immunosorbent assay (ELISA) using the CytoSets system (BioSource International, Camarillo, Calif.) according to the manufacturer's instructions. All samples were measured in triplicate from at least three independent experiments.
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RESULTS |
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Infection of AGS cells with type I H. pylori wild-type strains P1, P12, and G27 induced IL-8 secretion (Fig. 2A) and translocation and phosphorylation of CagA (Fig. 2B and C). Cells infected with these wild-type strains were significantly elongated (25 to 70 µm) and had a spindle-like morphology in more than 60% of the infected cells (Fig. 3) that has been referred to as the scattering or hummingbird phenotype (5, 36). An exception was wild-type strain J99, which induced IL-8 secretion but no tyrosine phosphorylation of CagA or phenotypic changes in host cells. This might be due to the lack of tyrosine phosphorylation motifs (5, 29). An alpA/B mutant failed to induce any of these host responses. None of the investigated host responses were induced by H. pylori culture supernatant (Fig. 2). This indicated that no bacterial factor secreted into the culture medium is responsible for IL-8 secretion, translocation and phosphorylation of CagA, and induction of the hummingbird phenotype.
In agreement with these results, we found that all virB gene knockout strains (virB4, virB7, virB8, virB9, virB10, and virB11) of the contact-dependent type IV transporter complex and a mutant lacking the entire cag PAI were defective for IL-8 secretion (Fig. 2A), translocation and phosphorylation of CagA (Fig. 2B and C), and induction of the hummingbird phenotype (Fig. 3, Table 1), demonstrating their essential function in each of these host responses. Moreover, several other genes of the cag PAI (cagF, cagH, cagI, and cagM) having no significant homology to other known genes or gene products were also needed for these processes. Hence, the ability of the bacteria to stimulate IL-8 production largely correlated with their ability to actively translocate CagA (Table 1).
We observed two interesting exceptions. First, the cagA knockout strain stimulated a 5.1-fold increase in IL-8 release by infected AGS cells compared to uninfected controls. This IL-8 level is similar to levels obtained during infection with wild-type H. pylori, indicating that CagA as a translocated effector protein does not play a role in IL-8 induction. The second exception was the virD4 knockout strain. Although the intact virD4 gene was essential for translocation and phosphorylation of CagA (Fig. 2B and C) and induction of the hummingbird phenotype (Fig. 3), inactivation of the virD4 gene did not abolish IL-8 secretion compared to all other cag PAI mutant strains but reduced the IL-8 level by about 60% (Fig. 2A). As the IL-8 release stimulated by this mutant strain was significantly above background levels, virD4 can be considered nonessential for induction of IL-8 release.
In order to rule out polar effects of our insertional mutagenesis, we complemented P1virD4 and P1cagA mutants with intact virD4 and cagA, respectively, expressed from the E. coli-H. pylori shuttle vector pHel2. The complemented strains, P1cagA/cagA and P1virD4/virD4, respectively, induced translocation and phosphorylation of CagA (Fig. 2B) and induction of the hummingbird phenotype (Fig. 3). This demonstrated that in our cagA and virD4 knockout mutants, the expression of flanking genes was not affected, and therefore, these mutations are not polar. Thus, both cagA and virD4 and possibly all other cag PAI genes investigated in this study played an essential role in translocation and phosphorylation of CagA and induction of the hummingbird phenotype, whereas cagA and virD4 were not essential for induction of IL-8 secretion.
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DISCUSSION |
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Our data showed that stimulation of IL-8 release, CagA translocation, and induction of phenotypic changes were dependent on many genes within the cag PAI. Specifically, all strains carrying knockout mutations in the virB gene homologs were unable to elicit these host responses. This result strongly supports the view that H. pylori possesses a functional type IV secretion system that plays a crucial role in the host-pathogen interaction. As bacterial culture supernatants and bacteria defective in adherence (alpA/B mutant) did not induce these effects, the type IV-mediated transport of H. pylori effector molecule(s) into the host cell cytoplasm is obviously a direct process that does not proceed through secretion into the extracellular medium.
Until now, the CagA protein has been the only known substrate for the H. pylori type IV secretion system. We observed that translocation of CagA was always correlated with cytoskeletal rearrangements, resulting in a scattering phenotype, suggesting that CagA triggers this host response (5; this study). The cagA and the virD4 gene knockout mutants did not induce changes in the actin cytoskeleton. However, knockout procedures by insertional mutagenesis can interfere with the expression of neighboring genes. Here we were able to exclude such polar effects by genetic complementation of our cagA and virD4 mutants, which indeed restored wild-type function.
VirD4 is a key component in the intensively studied type IV secretion system of the plant pathogen Agrobacterium tumefaciens. The agrobacterial VirD4 protein is located in the inner membrane (32) and is, in contrast to other Vir proteins, absolutely required for VirD2/T-DNA transfer into a wide range of host plants (24). VirD4 contains a Walker A nucleotide triphosphate binding motif that is necessary for function. Consequently, VirD4 is thought to mediate introduction of the nucleoprotein complex into the transporter by an energy-dependent mechanism (44). As the H. pylori virD4 gene shows significant homology to its counterpart in A. tumefaciens, the function of these proteins is probably similar. Thus, the H. pylori VirD4 protein presumably acts as an adapter protein for the transfer of CagA from the bacterial cell into the transfer channel formed by proteins encoded by the other vir genes.
Apart from inducing host cytoskeletal rearrangements, the H. pylori type IV secretion system has been speculated to play a role in the induction of proinflammatory host responses. However, until now there has been no systematic approach analyzing all vir genes. We used four well-characterized H. pylori strains and demonstrated that virB4, virB7, virB8, virB9, virB10, virB11, cagI, cagF, cagH, and cagM are involved in proinflammatory responses, suggesting that the integrity of the type IV secretion system is essential. Yet the only bacterial factor known to be translocated through the type IV machinery, CagA, does not induce proinflammatory signaling, as the cagA knockout mutant induced wild-type levels of IL-8 secretion. These observations strongly suggest that H. pylori induces IL-8 secretion by the transfer of another, as yet unknown effector molecule.
In this context it is interesting that our virD4 gene knockout mutant induced intermediate levels of IL-8 secretion. This observation contrasts with the finding that a virD4 knockout mutant of the TIGR strain 26695 shows 3.6-fold increased IL-8 secretion (16). The reason for this discrepancy is not clear. The possibility that our mutant is polar, however, can be ruled out. Perhaps the different findings are the result of differences between the two wild-type strains used in the study of Crabtree and coworkers and our study (26695 and P1, respectively). Those authors suggested that VirD4 is a bacterial downregulator (modulator) of IL-8 synthesis. An important common result from both studies, however, is that VirD4 plays a role in H. pylori-induced host responses that is different from those of the other vir genes. In particular, inactivation of virD4 prevented CagA translocation and phosphorylation but did not block proinflammatory responses.
Collectively, our results suggest that both CagA translocation and induction of IL-8 release are dependent on the type IV secretion system. However, the mechanism is apparently not identical, as the role of virD4 in these processes is different. If IL-8 secretion is induced by another translocated bacterial factor, then this unidentified factor does not depend on VirD4 in order to be transferred to the type IV secretion channel. Together with preliminary biochemical evidence (22; unpublished data), these observations suggest that the factor may not even be a protein. Alternatively, Covacci and Rappuoli suggested that proinflammatory responses could be induced by the type IV secretion channel itself, which could perturb membranes (13), or bind to and activate a relevant cell surface receptor(s), known as the receptor hypothesis.
The model presented in Fig. 4 summarizes the results obtained in this study. The H. pylori type IV secretion system spans both bacterial membranes. This needle-like structure is thought to inject effector molecules into the cytoplasm of host cells. However, a typical pilus protein is unknown, as a virB2 homologue is absent from the H. pylori genome (2, 39). We suggest that VirD4 is located at the cytoplasmic face of the inner membrane (similar to its agrobacterial counterpart) and guides CagA to the translocation channel.
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We favor a model in which H. pylori translocates an unknown effector molecule into the cytoplasm of host cells. Yet the transfer mechanism is substantially different from CagA transfer and may under some circumstances even be cag PAI independent. Purification and identification of this unknown factor will shed new light on the pathogenic mechanisms of this important human pathogen.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the Fonds der Chemischen Industrie to T.F.M.
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FOOTNOTES |
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) and induces actin cytoskeletal rearrangements associated with the scattering phenotype. cagA and virD4 mutants are unable to translocate CagA, and hence, rearrangement of the host cell cytoskeleton failed. Stimulation of IL-8 secretion, however, is VirD4-CagA independent. It is induced either by the translocation of an as yet unidentified factor or by binding of the transporter itself to relevant cell surface receptors (receptor hypothesis). virB mutants do not assemble a functional transporter and therefore are unable to elicit any of these host responses. For more details, see the text.