Chronic infection of the human gastric mucosa with Helicobacter pylori is a major cause of gastroduodenal pathologies, including peptic ulcerations, mucosa-associated lymphoid tissue (MALT) lymphoma and adenocarcinoma. Helicobacter pylori strains carrying the cag pathogenicity island, which encodes an active type IV protein secretion system (cag+ or type I strains), are preferentially associated with strong gastric inflammation and severe disease. We show here that cag+H. pylori strains use the type IV secretion system to inject the bacterial protein CagA into various types of professional phagocytes, including human polymorphonuclear leucocytes (PMNs) and the human and murine macrophage cell lines THP-1 and J774A.1 CagA is rapidly tyrosine phosphorylated and proteolytically processed to generate a stable 35–45 kDa C-terminally tyrosine-phosphorylated protein fragment. H. pylori was efficiently ingested by the different types of phagocytic cells. A chromosomal deletion of the complete pathogenicity island had no significant effect on the rate of ingestion. Furthermore, the survival rate of H. pylori in the phagosome was unchanged between the wild type and a deletion mutant lacking the type IV secretion system. Thus, the type IV secretion system seems to be involved neither in active phagocytosis resistance nor in prolonged survival of the bacteria in phagocytic cells.
Helicobacter pylori is a microaerophilic Gram-negative bacterium which colonizes the gastric epithelium of about 50% of the human population. The bacteria induce a gastric inflammation, chronic gastritis, and play a causative role in the development of peptic ulceration, mucosa-associated lymphoid tissue (MALT) lymphoma and adenocarcinoma of the stomach ( Blaser, 1996). Despite a vigorous host immune response, if untreated the infection usually persists for years and even decades.
To date, H. pylori strains have been divided into two groups, type I and type II strains. Type I strains are characterized by the production of the vacuolating cytotoxin (VacA) and the cytotoxin-associated antigen CagA, an immunodominant antigen of H. pylori that has no known function. They possess the cag pathogenicity island (cag-PAI), a 40 kb chromosomal DNA sequence harbouring the cagA gene at its border ( Covacci and Rappuoli, 1998; Covacci et al., 1999 ). Type I strains are associated with strong gastric inflammation and more severe pathologies than type II strains, which secrete only little VacA and are devoid of the cag-PAI. The H. pylori infection induces the secretion of proinflammatory cytokines, such as interleukin 6 (IL-6), IL-8, granulocyte–macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor α (TNF-α), that results in the recruitment of neutrophils, mononuclear phagocytes and lymphocytes to the gastric mucosa. For IL-8 induction, several genes in the cag-PAI seem to be essential, as has been shown by defined knockout mutants ( Münzenmaier et al., 1997; Covacci and Rappuoli, 1998).
Several proteins encoded by the cag-PAI reveal significant homologies to so-called type IV secretion system components in other bacteria, especially the VirB/D system of Agrobacterium tumefaciens ( Fullner et al., 1996 ) and proteins involved in DNA transfer during bacterial conjugation of several broad host range plasmids ( Winans et al., 1996 ). Furthermore, Bordetella pertussis uses a similar secretion system for the export of its major proteinaceous virulence factor, the pertussis toxin ( Winans et al., 1996 ). Recent work in several laboratories provided clear evidence that the cag-PAI actually encodes a functional type IV secretion system of type I H. pylori strains and CagA was identified as the first protein translocated into epithelial cells by the secretion system (effector protein). After translocation, CagA is tyrosine phosphorylated by a eukaryotic kinase activity and converted into CagAP–Tyr ( Asahi et al., 2000 ; Backert et al., 2000; Odenbreit et al., 2000 ; Stein et al., 2000 ).
The function of CagAP–Tyr in epithelial cells is unclear. Our data from H. pylori infection experiments using the AGS cell line suggested that CagAP–Tyr might be involved in dephosphorylation of proteins in the 80 kDa and the 120–130 kDa range in epithelial cells ( Odenbreit et al., 2000 ). This dephosphorylation of host cell proteins is reminiscent of pathogenic Yersinia strains, which translocate the phosphatase YopH into professional phagocytes by a type III secretion system to avoid their phagocytosis (anti-phagocytosis). In this context, we sought to determine whether H. pylori might use the type IV secretion system to induce a similar anti-phagocytosis mechanism. That H. pylori encounters professional phagocytes in its niche in vivo has been shown in an elegant electron microscopic study by Steer (1975) before H. pylori was recognized as a human pathogen. Steer (1975) showed that bacteria, now known as H. pylori, are associated with and phagocytosed by polymorphonuclear leucocytes in the gastric mucosa of patients with gastritis. Alternatively, H. pylori might tolerate phagocytosis but modulate the phagosome to survive within phagocytes, as is the case for other Gram-negative bacterial pathogens, including Mycobacterium tuberculosis, Legionella pneumophila or Salmonella typhimurium ( Buchmeier et al., 2000 ).
We show here that CagA is translocated into professional phagocytes. In contrast to epithelial cells, a dephosphorylation of host cell proteins is not observed, but CagA is rapidly processed in phagocytic cells. Furthermore, H. pylori strains are efficiently internalized by phagocytes in vitro.
Helicobacter pylori CagA is translocated, tyrosine phosphorylated and proteolytically processed in macrophage cell lines and human PMNs
The interaction of H. pylori with epithelial cells results in translocation of CagA and its tyrosine phosphorylation ( Odenbreit et al., 2000 ). We were interested to determine whether type I H. pylori strains are able to translocate CagA into professional phagocytes, which are important in terms of controlling the H. pylori infection in the host. Therefore, a P12 strain carrying a precise deletion of the complete cag-PAI (P12ΔPAI) was constructed ( Fig. 2A) which served as a negative control to determine CagA protein translocation. First, a murine macrophage cell line, J774A.1, was used. As a control for successful translocation of H. pylori protein(s) into phagocytic cells, we took advantage of the tyrosine phosphorylation of CagA by a yet undefined eukaryotic kinase, which occurs only when CagA enters the cytoplasm of eukaryotic cells ( Odenbreit et al., 2000 ). Independent H. pylori strains (P1, P12, ATCC43526 and J99) produce CagA of variable size, as seen in the CagA-specific immunoblot ( Fig. 1A). The same H. pylori strains were incubated with phagocytic cells for 4 h.
The tyrosine-specific immunoblot showed a tyrosine-phosphorylated protein between 35 and 45 kDa for H. pylori wild-type (wt) strains interacting with J774A.1 cells, but not in the cell line alone or upon infection with the P12ΔPAI mutant strain ( Fig. 1B). The same bands were detected with the anti-CagA antiserum AK257 ( Fig. 1C), demonstrating that CagA is translocated into the cytosol of the phagocyte where it is tyrosine phosphorylated and cleaved. The size variation of CagAP–Tyr between 35 and 45 kDa seen between independent wt strains reflects the difference in the overall molecular weight of CagA expressed by the corresponding strains, as seen in the immunoblot of the bacterial lysates without J774A.1 cells ( Fig. 1A). The size-variable region of CagA is in the C-terminal part of the protein ( Yamaoka et al., 1998 ), indicating that the tyrosine-phosphorylated CagA domain corresponds to a C-terminal fragment. This is in agreement with the identification of two putative tyrosine phosphorylation motifs in the C-terminal region of CagA proteins ( Odenbreit et al., 2000 ). As already seen with AGS epithelial cells, CagA of H. pylori strain J99 is also not tyrosine phosphorylated in the J774A.1 macrophage cells, which can be explained by the absence of all putative tyrosine phosphorylation sites in the sequence of the J99 CagA protein. Interestingly, the J99 CagA protein is either not proteolytically processed or the corresponding C-terminal fragment is unstable in the non-phosphorylated form because no fragment between 35 and 45 kDa is detected in the CagA-specific immunoblot ( Fig. 1C).
Processing of CagAP–Tyr in the mouse J774A.1 cell line was not seen in human epithelial cells ( Odenbreit et al., 2000 ). To verify and to extend the observation to other phagocytic cells, a human macrophage cell line, THP-1, as well as freshly isolated human PMNs were used. In these different types of phagocytic cells, the translocation of CagA, its tyrosine phosphorylation and efficient processing could be reproduced ( Fig. 1D–G). The next obvious question was whether or not a contact between H. pylori and phagocytic cells is required for translocation of CagA and whether an intact type IV secretion apparatus, as demonstrated in the AGS model, would be necessary.
CagA tyrosine phosphorylation and processing in phagocytic cells is dependent on a functional type IV secretion system
To verify whether tyrosine phosphorylation and processing of CagA in phagocytes is dependent on an intact type IV secretion system of H. pylori type I strains, J774A.1 cells were infected with H. pylori P12 and two congenic mutants in genes of the cag-PAI. H. pylori P12(hp523) and P12(hp544) carry transposon insertions in the putative open reading frames (ORFs) hp523 and hp544 respectively ( Tomb et al., 1997 ; Odenbreit et al., 2000 ). In both mutant strains, the function of the secretion system is disrupted, but expression of the cagA gene is not affected; the cagA insertion mutant P12(cagA) ( Odenbreit et al., 2000 ) contains an intact secretion system, but no cagA gene ( Fig. 2A).
Without contact between bacteria and phagocytes, wt and mutant H. pylori strains produce a full-size CagA ( Fig. 2C), but no tyrosine-phosphorylated CagA is detectable by the tyrosine-specific monoclonal antibody PY99 ( Fig. 2B). In the infection experiment, however, CagA of the wt strain P12 is tyrosine phosphorylated and proteolytically processed, yet the mutant strains P12(hp523), P12(hp544) and P12(cagA) were unable to translocate and convert CagA into CagAP–Tyr ( Fig. 2B and C). This indicates that translocation of CagA into phagocytes, and/or its tyrosine phosphorylation, might be a prerequisite for the proteolytic processing of CagA, which probably occurs by a protease in the phagocytic cells. In conclusion, the experiments show that CagA is translocated into professional phagocytes by type IV secretion, which indicates that phagocytic cells might be natural targets for CagA and the processed CagAP–Tyr might have a biological function in these cells.
Kinetics of CagA translocation, tyrosine phosphorylation and processing
To study the kinetics of CagA translocation into phagocytic cells, J774A.1 cells were infected with H. pylori P12 for various time periods between 0 and 240 min. The lysates were analysed by immunoblotting with CagA- and tyrosine phosphate-specific antisera ( Fig. 3A and B). Tyrosine phosphorylation of CagA accumulated continuously up to 4 h. Completely processed CagAP–Tyr is seen first after 20 min of infection, and processed CagA is detected by AK257 after 40 min. The increase in CagA and CagAP–Tyr associated with the phagocytic cells might be explained by continuous phagocytosis over time.
Thus, translocation of CagA, tyrosine phosphorylation and proteolytic processing is a rapid process in phagocytes. Our data also suggest that the processing takes place after translocation of CagA into the cytoplasm of the eukaryotic cell, which might occur when H. pylori attaches to phagocytes, which means from an extracellular location or after phagocytosis from inside the phagosome. Translocation-defective mutants are rapidly ingested by the phagocytes (data not shown), but they are unable to proteolytically process CagA ( Fig. 2A–C).
Function of the cag-PAI in phagocytosis of H. pylori by human PMNs
We initially observed a dephosphorylation of host cell proteins after translocation of CagA into epithelial cells ( Odenbreit et al., 2000 ). The successful translocation of CagA into professional phagocytes prompted us to test whether type I H. pylori strains are able to block phagocytosis, similar to the function of the phosphatase YopH of pathogenic Yersinia species ( Persson et al., 1997 ). We therefore compared the ingestion of wt and the congenic mutant in the cag-PAI. As positive and negative controls, the Yersinia enterocolitica strains WA-P and WA-C were chosen, which either contain or not the virulence plasmid pYV respectively. Strain WA-P translocates YopH by its active type III secretion system into phagocytes and is resistant against phagocytosis, but strain WA-C, which is cured from the virulence plasmid ( Heesemann and Laufs, 1983), is efficiently ingested by human PMNs ( Fig. 4A and B). This demonstrated that the granulocytes were active and the conditions for phagocytosis were appropriate (see Experimental procedures).
Under these conditions, H. pylori P12 and H. pylori P12ΔPAI were incubated with PMNs for 1 h at a multiplicity of infection (MOI) of 10 and the ratio among extracellular, adherent and intracellular phagocytosed H. pylori was determined by fluorescence microscopy with the anti-H. pylori antiserum AK175 (see Experimental procedures). A typical experiment revealed an extensive phagocytosis of both wt and deletion mutants ( Fig. 4C and D), suggesting that the wt strain as well as the defined mutant strain without the cag-dependent type IV secretion system are internalized by PMNs. The same experiments were performed with opsonized and non-opsonized H. pylori strains, but opsonization with human serum did not result in major differences in the phagocytosis behaviour of H. pylori. Thus, the cag-PAI does not seem to be involved in an anti-phagocytosis mechanism of H. pylori towards human granulocytes under the in vitro conditions used here.
A further parameter to test was the influence of the multiplicity of infection (MOI) on the phagocytosis behaviour. Freshly isolated human PMNs were compared in phagocytosis experiments with H. pylori at a MOI of 10 and 75. The number of adherent and internalized Hp was counted (100 PMNs per experiment) and analysed statistically. As seen in Fig. 4E and F, there was no significant difference in adherence between P12 and P12ΔPAI to PMNs, but at the MOI of 75 a slightly lower number of wt P12 bacteria were found within the PMNs compared with P12ΔPAI. The difference was, however, not statistically significant because of the low numbers of bacteria per cell and the relatively high standard deviation.
Phagocytosis of H. pylori by J774A.1 macrophages and quantification of adherence and ingestion
We then extended our analysis to other phagocytic cells, including macrophages, which might be involved in the natural defence against H. pylori during chronic active gastritis. The immunofluorescence data showed again, as already seen with PMNs, that P12 was internalized by the macrophage cell line J774A.1 as efficiently as the P12ΔPAI mutant strain ( Fig. 5A and B).
To quantify the internalization process, the J774A.1 cell line was used for phagocytosis experiments as isolated phagocytes were less homogeneous and might vary in their phagocytosis activity among independent blood donors. The number of intracellular (internalized) and extracellular (adherent) bacteria was counted from 100 J774A.1 cells in several infection experiments with a MOI of 10. The quantitative analysis did not prove a statistical difference in the number of bacteria adherent to or phagocytosed by J774A.1 cells between strain P12 and P12ΔPAI ( Fig. 5C and D).
To extend the data obtained with one H. pylori strain, H. pylori P12, three genetically well-characterized H. pylori type I wt strains (P1, ATCC43526 and CCUG17875) as well as 11 fresh patient isolates without previous laboratory passage were tested in phagocytosis experiments with human macrophages. These isolates behaved very similarly to P12 in phagocytosis by human macrophages (data not shown). In conclusion, phagocytosis of H. pylori by professional phagocytes seems to be common and very efficient and the type IV secretion system does not affect the phagocytosis of H. pylori in vitro.
The type IV secretion system does not influence the survival of H. pylori within phagocytes
As we could not observe a significant contribution of the cag-PAI on phagocytosis resistance, the question arose whether ingested H. pylori with an active type IV secretion system might have an advantage in survival within phagocytes. In a recent paper, Allen et al. (2000) showed that H. pylori type I strains are efficiently ingested by the J774A.1 macrophage cell line and that they survive for a prolonged time compared with type II strains, which are rapidly destroyed in macrophages. To test the role of the type IV secretion system in this process, we used the P12 and the P12ΔPAI mutant strains. After infection of J774A.1 cells with both strains, extracellular H. pylori were killed by gentamicin and intracellular bacteria were rescued by lysis of the phagocytes and plating on serum plates after 2 and 20 h (gentamicin killing assay). To monitor the efficiency of ingestion and extracellular killing of H. pylori by gentamicin, a cytochalasin D control was used. Cytochalasin D blocks the phagocytic uptake of H. pylori, as can be monitored by fluorescence microscopy. Cytochalasin D treatment of the phagocytes resulted in a more than 100-fold reduction of surviving H. pylori after the gentamicin-killing assay ( Fig. 6A and B). Without cytochalasin D, however, both P12 and the P12ΔPAI mutant could be recovered efficiently after gentamicin treatment, suggesting that the bacteria survived within the phagocytes. Exposure of H. pylori without phagocytic cells resulted in a strong reduction of the living bacteria after 1 h gentamicin treatment (> 4 logs) ( Fig. 6C). The P12 wt strain and the P12ΔPAI mutant showed, however, no significant difference in survival after 2 h or 20 h within phagocytes ( Fig. 6A and B), indicating that the cag-PAI is not directly involved in a modulation of the phagosome to allow prolonged survival of H. pylori. Thus, our experimental data do not support a role of the cag-PAI in survival within macrophages, which is in contrast to the conclusions drawn by Allen et al. (2000) suggesting that type I strains show a selective advantage for survival in macrophages.
A characteristic feature of H. pylori infection is its early onset, usually in childhood, and the often lifelong persistence if not treated. The bacteria are living in the mucous layer of the stomach, but a considerable proportion are also found on the epithelial surface. Type I H. pylori strains induce the release of IL-8 from epithelial cells, a chemokine which is involved in the massive infiltration of PMNs into the gastric submucosa of infected patients. When destruction of the gastric epithelium is progressing, the bacteria might be confronted with numerous phagocytes, especially PMNs and macrophages. These phagocytes, especially PMNs, are able to reach the surface of epithelial cells, as demonstrated by electron micrographic studies of the human gastric mucosa ( Steer, 1975). The question arises then as to how H. pylori can persist under these conditions and why the innate immune system is not able to eliminate the infection rapidly.
In contrast to type II strains, the more virulent H. pylori type I strains possess a pathogenicity island, which encodes an active type IV protein secretion system. CagA, a protein encoded by the cag-PAI, is translocated into epithelial cells and becomes tyrosine phosphorylated ( Asahi et al., 2000 ; Backert et al., 2000; Odenbreit et al., 2000 ; Stein et al., 2000 ). A consequence of the CagA translocation is the dephosphorylation of host cell proteins in the size range of 80 kDa and 120–130 kDa. The functional role of CagA in eukaryotic cells is not understood at all. In this study, we analysed whether CagA translocation and the observed dephosphorylation of host cell proteins could be involved in a bacterial signalling, leading to an anti-phagocytosis mechanism. A paradigm for such a process is the species of pathogenic Yersinia. They are able to translocate YopH, a tyrosine-specific phosphatase that is involved in dephosphorylation of host cell proteins and thereby induces anti-phagocytosis mechanisms in professional phagocytes ( Persson et al., 1997 ). YopH dephosphorylates the focal adhesion kinase (FAK), a 125-kDa tyrosine-phosphorylated protein that probably results in a block of phagocytosis of the bacteria by professional phagocytes such as PMNs or macrophages ( Persson et al., 1997 ). It was therefore tempting to speculate that H. pylori might use the type IV secretion system to induce a similar state of anti-phagocytosis in professional phagocytes, which could explain the lifelong persistence of the bacterium despite the presence of phagocytes during active gastritis. An alternative strategy of H. pylori might be to tolerate phagocytosis and to modulate the phagosome by the type IV secretion system, which could allow active survival and probably even replication of the bacteria in the phagosome. Salmonella species and Legionella pneumophila use this last strategy to modulate the phagosome by their cognate type III and type IV secretion systems ( Swanson and Sturgill-Koszycki, 2000).
We therefore analysed first whether professional phagocytes would be targets for CagA protein translocation in vitro. Our experiments show that under the conditions used different types of phagocytes, such as PMNs and macrophages, are targets for translocation and tyrosine phosphorylation of CagA. CagA is transferred rapidly into phagocytes and tyrosine phosphorylation occurs immediately. In contrast to epithelial cells, CagA is processed into a p100 and a p35–p45 fragment. The protease activity involved has not been identified; it might be a protease present in the phagocytic cell, which is not present or is inactive in epithelial cells. The proteolytic cleavage of CagA presumably occurs directly after transfer and tyrosine phosphorylation. Tyrosine phosphorylation must be in the C-terminal portion of CagA as this part contains the size-variable region, which differs between independent H. pylori strains in amino acid sequence and length. This portion of CagA contains one or two putative tyrosine phosphorylation sites. The functional role of tyrosine phosphorylation and cleavage of CagA in epithelial cells and phagocytes is not understood so far. But the question arises of whether or not the cleavage of CagA is part of a functional maturation of CagAP–Tyr in the phagocyte, or whether it is an inactivation of the active CagAP–Tyr molecule. This question can only be answered when more is known about the function of CagAP–Tyr and its interacting partners in both epithelial and phagocytic cells. It will be important to test whether CagA will be delivered into phagocytes in vivo as well when the bacteria colonize the host. It is also unclear why the CagA protein of strain J99 is not processed in phagocytes. Is it because it is not tyrosine phosphorylated or is this just a coincidence? This question will be addressed in future by site-specific mutagenesis of the putative tyrosine phosphorylation sites of CagA.
We used several phagocytic cell lines and fresh human isolates of PMNs and macrophage cell lines to test their phagocytic behaviour towards wt and mutant H. pylori strains. The deletion of the complete cag-PAI (P12ΔPAI) did not have a significant effect on the phagocytosis behaviour of the diverse professional phagocytes tested. Thus, our first hypothesis, saying that the genes on the cag-PAI might be involved in inducing a phagocytotic resistance similar to the pathogenic Yersiniae, could be disproven.
The second hypothesis was based on the recent observations published by Allen et al. (2000) . They described a new mechanism of phagocytosis of type I H. pylori strains by human macrophages, which is characterized by a delayed entry followed by a homotypic phagosome fusion. Early phagosomes containing single bacteria coalesced to generate large phagosomes (megasomes) containing multiple viable organisms. Megasome formation was dependent on active protein synthesis of H. pylori. Type II H. pylori microorganisms were unable to form megasomes, but were rapidly killed in the phagosome. We used the same phagocytic cell line (J774A.1) and similar conditions for phagocytosis as described by Allen et al. (2000) and were able to rescue live H. pylori after 2 h and 20 h from the phagocytic cell line. The absolute number of bacteria surviving a 2 or 20 h period in the phagosome was analysed. Whereas Allen et al. (2000) compared a type I and a type II strain with unrelated genetic background, we used a wt strain and its PAI deletion mutant with otherwise the same genetic background. Our data also show a survival of H. pylori in the phagosome for 20 h, but the deletion mutant behaved in the same way. This clearly shows that the type IV secretion system does not contribute to a prolonged survival of H. pylori in the phagosome. Thus, other differences in addition to the presence or absence of the cag-PAI must be responsible for the different behaviour of the type I and type II strains compared by Allen et al. (2000) .
The mechanism of how H. pylori survives in the phagosome is not yet clear. The type IV secretion system and translocation of CagA into phagocytes seems not to be directly involved in this step. This is in contrast to some facultative intracellular pathogens, such as Legionella pneumophila and Brucella suis, for which cognate type IV secretion systems are essential to survive in macrophage cell lines ( Segal et al., 1998 ; O'Callaghan et al., 1999 ). For H. pylori, it is now important to identify the cellular targets of CagAP–Tyr within epithelial cells and phagocytes. This might be the only way to understand the role of the type IV secretion system of H. pylori and the function of CagA translocation into so many different cell types of its host.
Bacterial strains, cell lines and culture conditions
H. pylori strains were grown on GC agar plates (Gibco) supplemented with horse serum (8%), vancomycin (10 mg l−1), trimethoprim (5 mg l−1) and nystatin (1 mg l−1) (serum plates) and were incubated for 2–3 days in a microaerobic atmosphere (85% N2, 10% CO2, 5% O2) at 37°C. Mutants were selected for growth on chloramphenicol (6 mg l−1) or kanamycin (8 mg l−1). For liquid cultures, the bacteria were grown in Brucella medium containing 10% fetal calf serum (FCS).
Escherichia coli strain DH5α (BRL), E181 and E145 ( Kahrs et al., 1995 ) and Yersinia enterocolitica strains WA-P and WA-C ( Heesemann and Laufs, 1983) were grown on Luria–Bertani (LB) agar plates or in LB liquid medium ( Sambrook et al., 1989 ) supplemented with ampicillin (100 mg l−1), kanamycin (50 mg l−1), chloramphenicol (30 mg l−1) or tetracycline (15 mg l−1), as appropriate.
THP-1 ( Fleit and Kobasiuk, 1991) and J774A.1 (mouse, ATCC TIB-67) macrophage cell lines were grown in RPMI-1640 medium (Gibco) containing l-glutamine and 10% FCS (cell culture medium) in a humid atmosphere at 37°C and 5% CO2.
Purification of granulocytes (PMNs)
For the isolation of PMNs, 8 ml heparinized blood (25 U ml−1) was applied to a cushion of 4 ml Mono-Poly Resolving Medium (ICN Biomedicals) and centrifuged at 400 g and room temperature for 1 h. A sharp band (containing the PMNs) in the middle of the gradient was transferred to a new centrifuge vial and washed three times (5 min, 400 g) in 6 ml GBSS buffer (Life Technologies). To lyse the remaining erythrocytes, the pellet was shortly suspended in 500 µl water and immediately filled up to 6 ml with GBSS. After a further centrifugation step, the cells were suspended in 1 ml cell culture medium and counted in a Neubauer chamber.
DNA manipulations, construction of plasmids and H. pylori mutant strains
Standard cloning and DNA analysis procedures were performed according to Sambrook et al. (1989) . Genomic DNA was isolated from H. pylori with the QIAamp Tissue Kit (Qiagen). Plasmid DNA was purified from E. coli by the boiling procedure and E. coli cells for electroporation were prepared according to the protocol recommended for the Gene Pulser (Bio-Rad). DNA fragments were separated by preparative agarose gel electrophoresis and isolated from the gel using the GeneClean kit (Bio101). To knockout the hp523 (H. pylori 26695) homologue in the P12 strain, the chromosomal region comprising the open reading frames hp520–523 (546828–550595 bp) ( Tomb et al., 1997 ) was amplified by PCR from H. pylori 26695 chromosomal DNA by using the primers JP1 (ACCGCTCGAG TAAAATTGTG CTTTTTTG) and JP2 (CGGGATCCAA CTTTCTAAGG TATTAG). The resulting 3.8 kb fragment was cloned in the vector pMin1 ( Kahrs et al., 1995 ) and mutated with the transposon TnMax8, as described previously ( Kahrs et al., 1995 ). Individual transposon insertions were transformed into P12, giving rise to the mutant P12(hp523). The cagE knockout mutant P12(cagE) has been described earlier ( Odenbreit et al., 2000 ). To delete the complete cag pathogenicity island (cag-PAI) from the H. pylori chromosome, the regions upstream (hp518–hp519; 545254–547164 bp) and downstream (hp549–hp550; 584570–586563 bp) of the cag-PAI were amplified from the H. pylori 26695 chromosome using the primer pairs JP22 (GGGGTACCTT ACCGGCTTTA TTAATG)/JP23 (GAAGATCTAA GGATCTGACA TGTTTA) and JP24 (GAAGATCTAT CGATTATTTT ATTAGCGTTA C)/JP25 (ACCGCTCGAG CTGCAGCCCA AGAATTCAAA CGAC) respectively. These fragments were separated by a kanamycin resistance cassette (aphA-3), and both were cloned into the pBluescript vector. The resulting plasmid was used for transformation into P12, resulting in the deletion mutant P12ΔPAI ( Fig. 2A). Transformation of H. pylori strains was performed as described previously ( Haas et al., 1993 ).
Phagocytosis. For phagocytosis experiments, the cell lines were usually grown in dishes of 2 cm2 on cover slides for 2 days in cell culture medium. Freshly prepared PMNs were seeded in the same wells to a density of 2 × 105 cells per well and sedimented by centrifugation (5 min, 400 g). Before infection, the bacteria were cultivated in liquid medium for 2–3 h to induce optimal viability. After changing the cell culture medium, cells were infected with bacteria at an appropriate multiplicity of infection (MOI 10–75) and incubated for various times (dependent on the experiment) at 37°C and 5% CO2. After removing free bacteria by washing in PBS (five times), cells were fixed in PBS/3.7% paraformaldehyde for at least 20 min.
Killing assay. To investigate the survival of H. pylori in macrophages, J774A.1 cells were seeded in 24-well plates to a density of 2 × 105 cells per well. Before infection, the medium was replaced by fresh medium (12°C). Cells were infected with bacteria from a liquid culture at a MOI of 25 (12°C), and infection was synchronized by a short centrifugation step (3 min, 600 g, 12°C). After removing non-adherent bacteria with PBS (12°C), the cells were incubated for 1 h at 37°C. Extracellular bacteria were killed by addition of 100 µg ml−1 gentamicin for another hour. After washing with PBS (five times) and another incubation step in fresh medium for 2 or 20 h, cells were lysed for 5 min with PBS/0.1% saponin. Appropriate dilutions of the supernatant were plated on serum plates and incubated for 2–3 days to count the surviving colony-forming units (cfu). To prevent phagocytosis (negative control), the cell culture medium was supplemented with cytochalasin D (1 µg ml−1) during the phagocytosis assay.
Tyrosine phosphorylation. To investigate the tyrosine phosphorylation of CagA in the infected cells, cells were grown in six-well plates (9.5 cm2) and infected with H. pylori at a MOI of 100. After removing non-adherent bacteria (see above), cells were suspended in 1 ml ice-cold PBS* (PBS, 1 mM EDTA, 1 mM o-vanadate, 1 mM phenylmethane sulphonyl fluoride, 1 µM leupeptin, 1 µM pepstatin) with a cell scraper, collected by centrifugation and resuspended in 30 µl PBS*. The samples were prepared for SDS–PAGE by addition of 40 µl of twofold Laemmli buffer.
Immunoblot analysis of CagA and tyrosine-phosphorylated proteins
Lysates of bacteria or infected cells were subjected to SDS–PAGE using a minigel apparatus (Bio-Rad) and blotted onto PVDF membrane at 1 mA/cm2 using a semidry blot system (Biotec Fischer).
CagA. The filters were blocked with 3% bovine serum albumin (BSA) in TBS (50 mM Tris-HCl, pH 7.5; 150 mM NaCl) and incubated with the antiserum AK257 ( Odenbreit et al., 2000 ). Alkaline phosphatase (AP)-coupled protein A was used to visualize the antibody bound by decomposition of nitroblue tetrazolium.
P-tyrosine. For detection of tyrosine-phosphorylated proteins, the blot was blocked overnight in 0.05 M Tris, pH 7.4, 0.2 M NaCl, 0.1% Tween, 5% milk powder. The P-tyrosine-specific monoclonal antibody PY99 (Santa Cruz Biotechnology) was applied to the blot in washing buffer (10 mM Tris, pH 7.4, 0.9% NaCl, 0.2% Tween-20) supplemented with 1% milk powder (dilution buffer). After extensive washing for 2 h, the blot was incubated for 1 h with horseradish peroxidase-coupled anti-mouse IgG in dilution buffer, washed for an additional 1 h and developed by using the Renaissance detection system (NEN).
To label and distinguish extra- and intracellular bacteria, a sequent double immunofluorescence staining was performed. After removing the fixing solution and blocking with PBS/0.2% BSA for 5 min, the cells were incubated with polyclonal rabbit antisera raised against H. pylori (AK175) ( Odenbreit et al., 2000 ) or Y. enterocolitica (WA-vital) ( Heesemann and Laufs, 1983), as appropriate. After washing three times for 5 min with PBS, the extracellular bacteria were detected with a tetramethyl rhodamine isothyocyanate (TRITC) -conjugated anti-rabbit IgG antibody. After a second washing step (three times), the cells were permeabilized by addition of PBS/0.1% Triton X-100 for another 10 min. By subsequent staining with the primary antibody (AK175 or WA-vital) and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG, extracellular and intracellular bacteria were labelled, resulting in extracellular bacteria in red/yellow (mixed colour between red and green) and intracellular bacteria in green. The fluorescence was evaluated by confocal microscopy (Leica) or by direct fluorescence microscopy using a filter (N2.1, Leica) detecting FITC and TRITC simultaneously. For quantitative assessment, the adherent and intracellular bacteria of 100 eukaryotic cells were counted. The mean values and standard deviations were calculated from three independent experiments and evaluated according to the t-test (P < 0.01).
The authors are grateful for the gift of the THP-1 and J774A1 cell lines by G. Enders and M. Hensel, respectively, as well as Yersinia strains and WA-vital from J. Heesemann. We would like to thank B. P. Burns for constructive criticism on the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to R.H. (HA 2697/2-1).