Helicobacter pylori interferes with leukocyte migration via the outer membrane protein HopQ and via CagA translocation.

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Helicobacter pylori interferes with leukocyte migration via the outer membrane protein HopQ and via CagA translocation Benjamin Busch a , Ramona Weimer a , Christine Woischke a , Wolfgang Fischer a,∗ , Rainer Haas a,b a b

Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany German Center for Infection Research, Partner Site München, München, Germany

a r t i c l e

i n f o

Article history: Received 21 August 2014 Received in revised form 23 December 2014 Accepted 9 February 2015 Keywords: Helicobacter pylori Granulocytes Leukocyte migration CagA HopQ fMLP

a b s t r a c t The human gastric pathogen Helicobacter pylori is a paradigm for chronic bacterial infections. Persistent colonization of the stomach mucosa is facilitated by several mechanisms of immune evasion and immune modulation, such as avoidance of Toll-like receptor recognition or skewing of effector T cell responses. Interactions of H. pylori with different immune cells have been described with respect to immune cell activation, cytokine release, or oxidative burst induction. We show here that H. pylori infection of human granulocytes, or of HL-60 cells differentiated to a granulocyte-like phenotype (dHL-60 cells) results in inhibition of cell migration under different conditions. Migration of dHL-60 cells in a three-dimensional collagen gel was found to be inhibited independently of the cag pathogenicity island, whereas migration inhibition in an under agarose assay was dependent on the cag pathogenicity island, on its effector protein CagA, and on the outer membrane protein HopQ. CagA translocation into leukocytes is accompanied by its tyrosine phosphorylation and by proteolytic processing into an N-terminal 100 kDa and a C-terminal 35 kDa fragment at a distinct cleavage site. By using complemented H. pylori strains producing either phosphorylation-resistant or cleavage-resistant CagA variants, we show that CagA tyrosine phosphorylation is required for migration inhibition, but CagA processing is not. Our results suggest that direct contact of H. pylori with immune cells subverts not only their activation characteristics, but also their migratory behaviour. © 2015 Elsevier GmbH. All rights reserved.

Introduction Colonization of the human stomach with H. pylori invariably results in a strong immune response, characterized by neutrophil infiltration of the gastric submucosa during the acute phase of infection, and a predominantly lymphocytic infiltration during chronic infection. Despite this marked inflammatory response and the production of H. pylori-specific antibodies, spontaneous elimination of the bacteria in the absence of antibiotic treatment is very rare. Several persistence strategies are discussed, including an evasion of recognition by the innate immune system, and a suppression of T cell-mediated immunity by several mechanisms (Salama et al., 2013). Remarkably, protective immunity against, or control of, H. pylori infection in animal models crucially depends on the T cell-mediated, rather than the humoral, immune response (Kusters et al., 2006). Although H. pylori is considered as an extracellular pathogen residing at the luminal surface of gastric epithelia,

∗ Corresponding author. Tel.: +49 89 218072877; fax: +49 89 218072923. E-mail address: fi[email protected] (W. Fischer).

the bacteria can also be observed in intracellular compartments (Dubois and Borén, 2007) or in the submucosa, for example as a consequence of impairment or destruction of gastric epithelial integrity after prolonged colonization (Necchi et al., 2007). In the latter case, bacterial cells might be in direct contact with immune cells. On the other hand, there is also histological evidence for the presence of transepithelial dendritic cells in infected human gastric tissue, and for contacts of these cells with H. pylori (Necchi et al., 2009). In H. pylori-infected mice, gastric dendritic cells with extensions across the epithelial layer could be directly detected by two-photon microscopy (Kao et al., 2010). The fate of gastric dendritic cells after contact with H. pylori is not well-understood. Generally, gastric mucosal dendritic cells are more prevalent after H. pylori infection and display more activation markers (CD83, CD86, CCR7) (Hafsi et al., 2004; Hansson et al., 2006; Kranzer et al., 2004). In H. pylori-infected mice, dendritic cells have been shown to migrate to the paragastric lymph nodes (Algood et al., 2007), also indicating their activation in the gastric mucosa. However, activation by H. pylori might be incomplete or inefficient. For example, it has been shown that upregulation of the CCR7 receptor on monocyte-derived dendritic cells upon contact with H.

http://dx.doi.org/10.1016/j.ijmm.2015.02.003 1438-4221/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Busch, B., et al., Helicobacter pylori interferes with leukocyte migration via the outer membrane protein HopQ and via CagA translocation. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.02.003


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pylori is less efficient than that induced by E. coli (Hansson et al., 2006). Nevertheless, H. pylori-activated DCs acquire the ability to migrate towards CCL19 (Hansson et al., 2006), consistent with their later arrival in gastric lymph nodes. Upon coincubation with naive CD4+ T cells, H. pylori-exposed DCs are able to induce IFN-␥, resulting in a polarization towards a Th1 or Th17 phenotype (Bimczok et al., 2010; Hafsi et al., 2004). However, the T cell priming capabilities of DCs may depend on the tissue environment; for instance, H. pylori-pulsed monocyte-derived DCs induce much less IFN-␥ production in T cells when they are incubated with gastric or intestinal stroma-conditioned media prior to T cell interaction (Bimczok et al., 2011). Recent studies have also shown that H. pylori infections are characterized by a significant infiltration of regulatory T cells in the gastric submucosa, which counteract the proinflammatory activity of Th1 or Th17 cells (Hitzler et al., 2011; Lundgren et al., 2003; Oertli et al., 2012; Rad et al., 2006). T cell priming towards these regulatory effector functions depends on a tolerogenic reprogramming of DCs by the H. pylori virulence factors VacA, ␥-glutamyltransferase, or CagA (Kaebisch et al., 2013; Oertli et al., 2013). The gastric immune response to H. pylori infection and the development of gastritis thus involves chemotactic migration of several leukocyte types. Apart from dendritic cells migrating towards the lymph nodes, neutrophils are recruited to the gastric submucosa via chemotaxis towards CXCL1–3 (GRO␣, ␤ and ␥) and CXCL8 (IL-8) (Yamaoka et al., 1998), and via endothelial cell activation characterized by upregulation of E-selectin, VCAM-1 and ICAM-1 (Innocenti et al., 2002; Svensson et al., 2009). Moreover, the bacterial protein HP-NAP (H. pylori neutrophil-activating protein) has been shown to attract and to activate neutrophils (Evans et al., 1995; Polenghi et al., 2007; Satin et al., 2000). The activity of HP-NAP might also be involved in priming of T cells to a proinflammatory phenotype (Amedei et al., 2006). Infiltrating T lymphocytes have been reported to be recruited to the gastric mucosa via CCL20 binding to their CCR6 chemokine receptors (Cook et al., 2014; Wu et al., 2007), via L-selectin receptors (CD62L) binding to peripheral lymph node addressin (PNAd) (Kobayashi et al., 2004), and via ␣4 ␤7 integrins, which are known to bind to MAdCAM-1 adhesion molecules expressed by endothelial cells in the gastrointestinal tract (Michetti et al., 2000; Quiding-Järbrink et al., 2001). While the influence of H. pylori virulence factors on activation of dendritic cells and on their acquisition of distinct T cell-priming capacities has been examined in several studies, a direct impact of virulence factors on leukocyte migration has not been determined so far. We show here that one of the major H. pylori pathogenicity factors, the cag pathogenicity island-encoded type IV secretion system with its effector protein CagA, has an inhibitory activity on leukocyte migration. The inhibitory activity of CagA depends on its tyrosine phosphorylation, but not on its processing into two fragments, which is a characteristic feature of CagA translocated into leukocytes. We can also show that the outer membrane protein HopQ is involved in migration inhibition, suggesting an unprecedented role of the HopQ–CagA axis in immune modulation. Materials and methods Bacterial and cell culture H. pylori strains were grown on GC agar plates (Oxoid) supplemented with vitamin mix (1%), horse serum (8%), vancomycin (10 mg/l) and trimethoprim (5 mg/l) (serum plates), and incubated for 16 to 60 h in a microaerobic atmosphere (85% N2 , 10% CO2 , 5% O2 ) at 37 ◦ C. E. coli strains Top10 (Invitrogen) and DH5␣ (BRL) were grown on Luria–Bertani (LB) agar plates or in LB liquid medium supplemented with ampicillin (100 mg/l), chloramphenicol (30 mg/l), or kanamycin (40 mg/l), as appropriate. For the generation of isogenic mutants in H. pylori strain P12, plasmids were introduced by

natural transformation, as described (Haas et al., 1993), and transformants were selected on serum agar plates containing 6 mg/l chloramphenicol, 10 mg/l erythromycin, or 8 mg/l kanamycin, as appropriate. AGS gastric epithelial cells and J774A.1 cells were cultivated under standard conditions as described previously (Odenbreit et al., 2000, 2001). HL-60 cells were cultivated in RPMI medium (Life Technologies) supplemented with 10% FCS and kept at a density of 1 × 105 to 2 × 106 cells/ml. Differentiation of HL-60 cells to a granulocyte-like phenotype (dHL-60 cells) was performed as described (Collins et al., 1978). Briefly, 107 HL-60 cells were harvested by centrifugation at 100 × g for 5 min at room temperature, resuspended in 10 ml RPMI supplemented with 10% FCS and 1.3% dimethylsulfoxide, and cultivated for further 6 days without medium exchange. Differentiation was controlled by flow cytometry using CD11b and CD66acde markers (data not shown). Isolation of human granulocytes Human PMNs were isolated from blood donors by density gradient centrifugation. Briefly, 20 ml of blood were drawn into a syringe coated with heparin (10 U/ml blood) and diluted with PBS to a final volume of 50 ml. Subsequently, 25 ml of diluted blood were layered on top of 15 ml Ficoll (Biochrom) in a 50 ml tube. The Ficoll gradient was centrifuged at 400 × g for 30 min at room temperature without brake. The clear supernatant including an opaque layer was removed and the remaining pellet was resuspended in 50 ml PBS. Cells were collected by centrifugation at 350 × g for 10 min at 4 ◦ C without brake, the supernatant was removed, and the pellet resuspended in erythrocyte lysis buffer (10 mM KHCO3 , pH 7.4; 155 mM NH4 Cl, 0.12 mM EDTA) and incubated for 30 min at 4 ◦ C. PMNs were harvested by centrifugation and resuspended after an additional erythrocyte lysis step in RPMI medium with 10% FCS. Plasmid constructions For construction of the cagA 3 deletion plasmid pCW1, a 600 bp cagA downstream region of strain P12 was amplified by PCR using primers WS390 (5 -CGGGATCCTA AAGGATTAAG GAATACC-3 ) and WS391 (5 -ACCTGCGGCC GCTAAAGTGG AATTTCATGC G-3 ) and cloned into the BamHI and NotI sites of pBluescript II KS. Subsequently, an internal 500 bp cagA fragment (codons 613–781) amplified with primers WS452 (5 -GCGGTACCGT CGACGATCTT GAAAAATCTC TAAAGAAAC-3 ) and WS359 (5 -ACCGCTCGAG GTTATCTTTT GATTGATGAT C-3 ) was cloned together with a SalI/BamHI-restricted rpsL-erm cassette (Fischer et al., 2010) into the KpnI and BamHI sites of the resulting plasmid to obtain plasmid pCW1. The cognate cagA reconstitution plasmid pCW2 was generated by subcloning the cagA 3 region (codons 613–1214) amplified with primers WS452 and WS425 (5 -ACCGCTGCAG GGTACCTTAA GATTTTTGGA AACCAC-3 ) and restricted with SalI and PstI together with a BamHI/SacI-restricted cagA downstream fragment as described above and a PstI/BamHI-restricted terminatorless cat cassette (Fischer et al., 2001) into the SalI and SacI restriction sites of pUC18. Transformation of an H. pylori strain, which carries a cagA 3 deletion introduced by plasmid pCW1, with plasmid pCW2 thus replaces the rpsL-erm cassette with a complete cagA 3 region and introduces a downstream chloramphenicol resistance cassette for selection. For generation of a processing-resistant CagA variant, plasmid pCW2 was subjected to an inverse PCR with primers RW1 (5 -GCTGCGGCCG CTGCTGCTGG ACTCAAAAAC GAACCCATTT ATG-3 ) and WS454 (5 -GAAATTTCCA AGTTTTGCAT TC-3 ), and the resulting PCR product was religated after DpnI digestion of template DNA to obtain pRW2, in which the codons for asparagine residues 880 to 885 are thus replaced with six alanine codons. A phosphorylation-resistant CagA




variant was generated along with a phosphorylation-competent control using plasmids pWS520 and pWS521. These plasmids were constructed by cloning an XhoI/NdeI-restricted 800 bp cagA upstream fragment obtained by PCR using primers hp546r (5 -ACCGCTCGAG TGTAAAAAAT TTCATGCGTT-3 ) and WS242 (5 ACCGCTCGAG CATATGTTCT CCTTACTAAC TAGTTTC-3 ) together with an NdeI/BglII-restricted gsk-cagA (5 ) fragment (codons 1–613) from plasmid pIP9 (Hohlfeld et al., 2006) and a BglII/KpnI-restricted cagA (3 ) fragment from either pHel3-Flag-CagY5 (pWS521) or pHel3-Flag-Cag-F5 (pWS520) (Mimuro et al., 2002) into the SalI and KpnI sites of pCW2. To generate the corresponding H. pylori strains, plasmids pWS520 and pWS521 were transformed into H. pylori P12 containing the 3 cagA deletion introduced by pCW1. To obtain green fluorescent H. pylori, the wild-type strain P12 and its isogenic cagA and cagPAI mutants were transformed with plasmid pDH80 (Heuermann and Haas, 1998) and checked for GFP production by fluorescence microscopy. Antisera and immunoblotting Antisera against CagA, RecA, and H. pylori (AK175) have been described previously (Fischer and Haas, 2004; Kutter et al., 2008). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blotting was performed as described (Fischer et al., 2001). For the development of immunoblots, polyvinylidene difluoride (PVDF) filters were blocked with 5% nonfat milk powder in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl), 0.1% (v/v) Tween 20, and incubated with the respective antisera at a dilution of 1:1000–1:5000. Alkaline phosphatase-conjugated protein A or horseradish peroxidase-conjugated anti-rabbit IgG antiserum was used to visualize bound antibody.

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containing 10% FCS, and filled into the dishes. After solidification and further incubation at 37 ◦ C for 1 h, slots with a diameter of approximately 3.5 mm and a distance of 2.2 mm were formed using a template. Chemoattractant solutions (100 nM fMLP) were filled in the slots 1 h before infected or uninfected cells were filled into the neighbouring slots. After additional incubation at 37 ◦ C for 1 h, cell migration was monitored by time lapse microscopy using a Leica SP5 confocal microscope with a HCX PL APO CS 10× or a 40×/1.25 oil immersion objective. Migrating cells were manually tracked using the Manual Tracking Plugin for ImageJ and further analyzed with the ‘Chemotaxis and Migration Tool’ (Ibidi, Martinsried). Migration assay in a 3D collagen matrix Migration assays in three-dimensional collagen matrices were performed with ␮-slides chemotaxis3D (Ibidi, Martinsried) according to the manufacturer’s instructions. Collagen gels were prepared by mixing 20 ␮l 10× MEM (Life Technologies), 10 ␮l NaHCO3 (7.5%, Life Technologies), 20 ␮l H2 O, 50 ␮l RPMI + 10% FCS (Life Technologies), 150 ␮l collagen type I (3 mg/ml; Purecol, Advanced Biomatrix) and 50 ␮l RPMI + 10% FCS containing 1 × 106 cells, and filling 6 ␮l of this solution into the slides. The collagen gel was allowed to solidify for 1 h at 37 ◦ C, 5% CO2 . Subsequently, the reservoirs were filled with medium with or without chemoattractant, and the slides were further incubated for 1 h at 37 ◦ C, 5% CO2 in order to allow chemoattractant gradients to be formed. Cell migration was examined by time lapse microscopy and analyzed as described above. For better visualization of cells within the collagen gel, cells were stained with TAMRA (Sigma) at a concentration of 1 ␮M for 10 min prior to casting the collagen gel. Flow cytometry

Tyrosine phosphorylation assay Standard infections of AGS cells with H. pylori strains and subsequent preparations for phosphotyrosine immunoblotting were performed as described previously (Odenbreit et al., 2000). Briefly, cells were infected with bacteria at a multiplicity of infection of 60 for 4 h at 37 ◦ C, washed three times and suspended in PBS containing 1 mM EDTA, 1 mM Na3 VO4 , 1 mM PMSF, 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin. Cells with adherent bacteria were collected by centrifugation and resuspended in sample solution. Tyrosinephosphorylated proteins were analyzed by immunoblotting with the phosphotyrosine antibody PY99 (Santa Cruz Biotechnologies).

Flow cytometry analysis was conducted with a BD FACSCanto II flow cytometer. Data were evaluated using the FlowJo software (Tree Star). Statistical analysis Statistical analysis was performed using the GraphPad Prism 5 software. Data sets shown are average values resulting from at least three independent experiments with standard errors of the mean, and significances were determined by one-way ANOVA analysis with Tukey’s multiple comparison test.

Boyden chamber experiments

Results

Chemotaxis of dHL-60 cells and human PMNs towards fMLP was investigated in Transwell chambers using 8 ␮m BD Falcon cell culture inserts in 24-well plates, as described by the manufacturer. RPMI medium containing 100 nM fMLP as a chemoattractant, or RPMI alone as a control, was filled into the bottom chambers, and infected or uninfected dHL-60 cells or PMNs were filled into the upper chambers onto the cell culture inserts. The Boyden chambers were incubated at 37 ◦ C and 5% CO2 for 2 h, and transmigrated cells were subsequently counted with a Neubauer hemocytometer or by flow cytometry.

Migration of leukocytes is impaired after exposition to H. pylori

Under agarose migration assay Under agarose assays using dHL-60 cells were performed as described previously (Heit and Kubes, 2003). Briefly, microscopy dishes (35 mm ␮-Dishes; Ibidi, Martinsried) were blocked with 0.1% BSA in HBSS, pH 7.2 (Sigma), for 1 h at 37 ◦ C and subsequently washed twice with PBS. Ultra-pure agarose (Invitrogen) was dissolved at a concentration of 1.6% in a 1:1 mixture of HBSS and RPMI

It has been shown before that H. pylori infection induces dendritic cells to express higher levels of activation markers such as CD80, CD86, and CCR7, and to migrate towards the chemokine CCL19 (Hansson et al., 2006). However, the migration capability of infected DCs was dependent on the H. pylori strain used, suggesting that individual virulence factors might contribute to migration behaviour. To address the question whether H. pylori is able to influence leukocyte migration in general, we used a Boyden chamber assay in which HL-60 cells that had been differentiated to a granulocyte-like phenotype (dHL-60 cells), or freshly isolated human granulocytes (see the Materials and Methods section for details), were infected with different H. pylori strains, or left uninfected, and subsequently exposed to gradients of formyl-methionyl-leucyl-phenylalanine (fMLP) as a chemoattractant. Control experiments indicated that fMLP concentrations of 100 nM were useful for inducing migration to the lower compartment for both cell types (data not shown). While freshly isolated


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human granulocytes showed an enhanced migration towards the lower compartment in the presence of fMLP, there was only a trend towards the same behaviour in dHL-60 cells (Fig. 1A and B). However, both cell types exhibited reduced levels of migration after infection with wild-type H. pylori strain P12 for 1 h (Fig. 1A and B). This phenotype was observed at a multiplicity of infection (MOI) of 1–5 in granulocytes, and at an MOI of 20 in dHL-60 cells, respectively. To examine if inhibition of migration depends on major H. pylori virulence factors, we infected dHL-60 cells or human granulocytes with isogenic P12vacA and P12cagPAI deletion mutants. Interestingly, while vacA mutant-infected cells exhibited the same migration inhibition as wild type-infected cells (data not shown), cells infected with the cagPAI deletion strain seemed to migrate towards fMLP more efficiently than wild type-infected cells in this assay, although this trend did not reach statistical significance and migration was still clearly reduced in comparison to uninfected cells (Fig. 1A and B). To rule out a general migration inhibition after contact with bacteria, we infected the cells with Campylobacter jejuni, which had a certain inhibitory effect as well, but to a lesser extent than H. pylori wild-type or cagPAI mutant strains (Fig. 1B). Thus, we conclude that coincubation with H. pylori generally seems to reduce migration of granulocytes, and that part of this reduction might be due to a cagPAI-dependent activity.

migrating cells were tracked. Similar to our observations in the Boyden chamber experiments, we found that H. pylori infection inhibits dHL-60 migration (Fig. 2A). Infected cells displayed both a decreased average migration velocity, and an impaired directionality, as shown in a forward migration index towards the chemoattractant (Fig. 2B). However, we did not find a significant difference in either parameter when cells were infected with P12 wild-type or P12cagPAI, suggesting that the activity of the Cag type IV secretion system has no impact on integrin-independent migration in a three-dimensional collagen matrix. Two-dimensional migration of leukocytes is inhibited by H. pylori in a cagPAI-dependent manner Leukocyte migration on two-dimensional surfaces was simulated by under agarose assays, in which dHL-60 cells were infected with H. pylori for 1 h, or left uninfected, and subsequently allowed to migrate underneath an agarose gel (see the Materials and Methods section for details). In this assay, cells can move either dependent on (fast migration), or independent of (slow migration), contractile forces (Jacobelli et al., 2009; Lämmermann and Sixt, 2009). Again, we found that cells infected with wild-type H. pylori strain P12 were significantly less able to migrate underneath the agarose layer than uninfected cells (Fig. 3A and B). When cells were infected with an isogenic cagA mutant or an isogenic cagPAI mutant, the velocity of migration was still reduced in comparison to uninfected cells, but much less than in wild type-infected cells (Fig. 3A and B). To exclude that different bacterial loads are responsible for the observed differences in migration, we performed control infections under the same conditions with gfp-expressing H. pylori strains and visualized fluorescent bacteria associated with or taken up by dHL-60 cells directly under the agarose layer. Comparison of randomly chosen visual fields showed that >95% of all migrating cells were associated with bacteria for all strains used (data not shown). Flow cytometric analysis of dHL-60 cells infected with the wild-type strain P12 or its isogenic cagA and cagPAI mutants also showed no significant difference in their bacterial loads, arguing against varying capacities of the mutants to interact with dHL-60 cells as a reason for the observed differences in their migration behaviour (Fig. 3C and D). These results suggest that in this model of (preferentially) two-dimensional leukocyte migration, dHL-60 cells are specifically inhibited in their migratory capacity in a cagPAI- and cagA-dependent manner.

Leukocyte migration in a three-dimensional collagen matrix is inhibited in a cagPAI-independent manner Leukocytes can migrate by two mechanistically different modes, depending on their local environment. Migration on twodimensional surfaces, such as vascular endothelia, depends on surface anchoring, usually mediated by integrin interactions with endothelial surface molecules (e.g., ICAM-1). In contrast, migration in three-dimensional environments, for instance through interstitial tissues, is independent of integrins, but rather relies on actomyosin contractile forces and possibly membrane blebbing (Lämmermann and Sixt, 2009). To differentiate between these modes of migration, we employed two further assays. Migration in a three-dimensional environment was examined by embedding dHL-60 cells with or without prior H. pylori infection in a collagen matrix, and migration on two-dimensional surfaces was simulated by under agarose assays (see below). Migration in a collagen matrix was stimulated by addition of fMLP as a chemoattractant and monitored by time-lapse microscopy. For each experiment, at least 20

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Fig. 1. Migration of dHL-60 cells and granulocytes in a Boyden chamber experiment. Differentiated HL-60 cells (A) or freshly isolated human granulocytes (B) were infected with different H. pylori strains, or left uninfected, and incubated for 1 h in transwell chambers in the absence or presence of 100 nM fMLP, as indicated. Subsequently, cells migrated to the lower compartment were counted using flow cytometry. Numbers of migrated cells are shown as average values with standard errors of the mean from three independent experiments. p Values are indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.0001); n.s., not significant.




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Fig. 2. Migration of dHL-60 cells in a three-dimensional collagen gel. (A) Cells were infected with H. pylori P12 for 1 h, or left uninfected, embedded in a three-dimensional collagen gel, and exposed to a gradient of fMLP. Individual tracks of representative migration experiments are shown. Directionality of migration (forward migration index; (B)), as well as migration velocities in (C) were calculated from the tracks and are shown for a representative experiment. p Values are indicated by asterisks (*** p < 0.0001); n.s., not significant.

Inhibition of two-dimensional migration depends on CagA phosphorylation We and others have shown previously that CagA can be translocated via the Cag type IV secretion system into phagocytic cells, such as macrophages or granulocytes, where it is tyrosinephosphorylated and additionally processed into an N-terminal fragment of approximately 100 kDa, and a C-terminal fragment of approximately 35 kDa which contains the phosphorylated tyrosine residues (Moese et al., 2001; Odenbreit et al., 2001). Translocation monitoring with an N-terminal phosphorylatable tag (GSK tag) suggested that CagA translocated into phagocytic cells is completely converted into these proteolytic fragments (TorruellasGarcia et al., 2006). However, it is currently unclear at which stage during the translocation process proteolysis occurs and what functional significance, if any, it has. In order to be able to examine CagA tyrosine phosphorylation and CagA processing and their impact on migration independently, we decided to construct a phosphorylation-resistant CagA variant and a CagA variant with a defective processing site. To examine if CagA phosphorylation is involved in migration inhibition, we generated a pair of P12 strains producing a hybrid CagA protein containing five EPIYA motifs (ABCCC; CagAY5), or a variant of the same CagA protein in which all five tyrosine residues were replaced by phenylalanine residues (CagAF5). Tyrosine phosphorylation assays in both AGS (Fig. 4A) and dHL-60 cells (Fig. 4B) confirmed that CagAY5 was strongly phosphorylated and processed, whereas CagAF5 was processed in dHL-60

cells, but not phosphorylated in either cell type. Migration experiments in the under agarose assay showed that infection with the phosphorylation-competent strain P12 (CagAY5) clearly inhibited dHL-60 migration, whereas infection with the phosphorylationresistant strain P12(CagAF5) did not (Fig. 4C). This indicates that the inhibitory activity of CagA depends on CagA tyrosine phosphorylation. CagA processing in leukocytes occurs at a conserved site, but is not required for migration inhibition Processing of CagA has not only been described after translocation into leukocytes, but also for recombinant CagA produced in E. coli, where a stable C-terminal CagA fragment is preceded by a stretch of five or six consecutive asparagine residues which have been suggested as a possible cleavage site (Angelini et al., 2009). For constructing a processing-resistant CagA variant, we employed a two-step procedure, in which the 3 region of the cagA gene (codons 782–1214) was first replaced in strain P12 with an rpsL-erm resistance cassette, using plasmid pCW1. The resulting mutant was subsequently transformed with plasmid pCW2, reintroducing the cagA 3 region (codons 613–1214) and replacing the rpsL-erm cassette with a cat resistance cassette downstream of cagA. This strain producing a reconstituted CagA was checked in a phosphotyrosine assay and found to have CagA translocation fully recovered (data not shown). Next, we transformed the P12[pCW1] mutant with plasmid pRW2, in which the six asparagine codons (amino acids 880–885) of CagA had been replaced with six


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Fig. 3. Migration of dHL-60 cells in an under agarose assay. (A) Cells were infected with the indicated H. pylori strains for 1 h, or left uninfected, and incubated in a slot within an agarose slab. Migration velocities were calculated from individual tracks of cells moving underneath the agarose layer and are shown for a representative experiment. (B) Migration velocities of five independent experiments calculated from cell tracks as in (A) are shown in percent values in relation to velocities of control (not infected) cells. (C) Differentiated HL-60 cells were infected with GFP-producing variants of the indicated strains for 1 h. Subsequently, cells were washed to remove unbound bacteria and assayed by flow cytometry for GFP fluorescence. (D) Mean fluorescence of uninfected or H. pylori-infected cells measured as in (C) was calculated for three independent experiments and is given as average values with standard errors of the mean. p Values are indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.0001); n.s., not significant.

alanine codons (CagAN6). The resulting strain was checked for production of full-length CagA and for translocation of CagA into AGS cells. Immunoblotting with CagA and phosphotyrosine antibodies showed that CagAN6 is translocated and phosphorylated at an efficiency comparable to wild-type CagA (Fig. 5A). AGS cells infected with the CagAN6 strain also showed the hummingbird phenotype (data not shown), indicating that deletion of the asparagine residues had no general effect on CagA function. CagAN6 was also translocated and phosphorylated in the macrophage cell line J774A.1 (Fig. 5B) or in dHL-60 cells (data not shown), but processing did not occur any more, confirming that asparagine residues 880 to 885 constitute at least part of the CagA processing site. To exclude that CagA processing depends on its prior phosphorylation, we treated J774A.1 cells with the tyrosine kinase inhibitor genistein, or left them untreated, and subsequently infected them with the P12 wild-type strain or with P12(CagAN6) and examined CagA processing and tyrosine phosphorylation via immunoblotting. The results show that phosphorylation of the 35 kDa CagA fragment could not be observed any more, but processing still occurred (Fig. 5C), indicating that CagA processing is independent of phosphorylation. To examine an influence of CagA processing on its migration inhibition effect, we performed under agarose assays

with dHL-60 cells infected with P12 wild-type or P12(CagAN6). The processing-defective CagA variant did not induce a significantly different response of dHL-60 cell migration in comparison to the wild type (Fig. 5D), suggesting that CagA processing is not required for its migration inhibition activity. Influence of the H. pylori outer membrane protein HopQ on leukocyte migration CagA phosphorylation in epithelial cells has previously been shown to be significantly reduced when bacteria lack the gene encoding the outer membrane protein HopQ (Belogolova et al., 2013). To examine whether HopQ also influences CagA translocation into leukocytes, and if so, leukocyte migration, we infected dHL-60 cells with an isogenic hopQ deletion mutant of strain P12, or with a complemented hopQ mutant, and determined CagA tyrosine phosphorylation and migration in the under agarose assay. Consistent with the data obtained in AGS cells, the P12hopQ mutant showed a weaker CagA tyrosine phosphorylation of the 35 kDa Cterminal CagA fragment after infection of dHL-60 cells, whereas the complemented hopQ mutant had its CagA phosphorylation ability restored (Fig. 6A). Interestingly, processing of CagA did not seem to be reduced in P12hopQ mutant-infected cells in comparison




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Fig. 4. Influence of CagA phosphorylation on migration inhibition in the under agarose assay. (A) AGS cells were left uninfected, or were infected for 4 h with H. pylori P12 mutants producing hybrid CagA molecules with (EPIYA-ABCCC; Y5) or without (EPIFA-ABCCC; F5) phosphorylatable tyrosine residues. Immunoblot with an antiphosphotyrosine antibody confirms CagA phosphorylation in AGS cells. (B) dHL-60 cells were infected for 4 h with the indicated strains and subjected to a phosphotyrosine immunoblot. (C) Average velocities of dHL-60 cells infected with the indicated strains and measured in the under agarose assay. Values shown are derived from three independent experiments and are given in relation to velocities of control (not infected) cells (in %). p Values are indicated by asterisks (*** p < 0.0001); n.s., not significant.

to wild type-infected cells (Fig. 6A, upper panel). Since processing is observed only for translocated CagA, but not for CagA inside the bacteria, this suggests that CagA phosphorylation, rather than CagA translocation, is actually impaired in the absence of HopQ. In the under agarose assay, infection with the hopQ mutant still resulted in a slight reduction of dHL-60 migration, but to a significantly lower extent than infection with the wild-type did (Fig. 6B). Infection with the complemented hopQ mutant restored the migration inhibition to wild-type levels. These results indicate that production of HopQ enhances CagA tyrosine phosphorylation after translocation into dHL-60 cells, which in turn results in the CagA phosphorylationdependent migration inhibition described above. However, since CagA phosphorylation is reduced, but still possible in the absence of HopQ, it is likely that HopQ exerts an additional, CagA-independent effect on migration inhibition.

Discussion During chronic infection with H. pylori, direct contacts between the bacteria and different immune cells, for example gastric dendritic cells, are expected to occur frequently. Such direct interactions might not only modulate leukocyte functions such as dendritic cell maturation (Salama et al., 2013; Shiu and Blanchard, 2013), but, as we show here, they might also influence the chemotactic and/or migratory capabilities of leukocytes. A direct inhibition of cell migration has not been shown before for H. pylori, but it has been described for a number of virulence factors in

other bacteria. For example, bacterial toxins such as PMT toxin of Pasteurella multocida (Blöcker et al., 2006) or edema toxin of Bacillus anthracis (Szarowicz et al., 2009) were shown to inhibit migration of dendritic cells or neutrophils, respectively. Several bacteria equipped with type III or type IV secretion systems are also capable of inhibiting leukocyte migration. This has been reported, among others, for Salmonella enterica, where the type III secretion effector SrfH/SseI inhibits macrophage and dendritic cell motility (McLaughlin et al., 2009), for Shigella flexneri, where the type III secretion effector IpgD impairs T cell migration (Konradt et al., 2011), and for Legionella pneumophila, where the Icm/Dot type IV secretion system is responsible for macrophage and neutrophil migration inhibition (Simon et al., 2014). In the same line, we are able to show that the migration inhibition effects exerted by H. pylori in the Boyden chamber and under agarose assays are dependent on the activity of the Cag type IV secretion system and its only known effector protein CagA. In contrast, leukocyte migration inhibition in the three-dimensional collagen matrix was independent of the Cag type IV secretion system (Fig. 2), and also independent of the outer membrane protein HopQ (data not shown). In the under agarose assay, wild type-infected cells were significantly more impaired in their migratory capabilities than cagPAI or cagA mutant-infected cells. These differential effects obtained with isogenic H. pylori mutants also rule out a general unspecific migration inhibition due to the fact that the bacteria produce fMLP or other chemoattractants themselves. An early study has shown that the chemotactic activity exerted by H. pylori on monocytes and neutrophils is mediated,




Fig. 5. Processing of CagA in leukocytes does not influence migration inhibition. (A) AGS cells were infected for 4 h with H. pylori strain P12 and a mutant producing a CagA variant lacking a hexa-asparagine motif upstream of its EPIYA-A motif (P12CagAN6). Immunoblot analysis shows CagA production and tyrosine phosphorylation levels. (B) J774A.1 cells were infected for 4 h with the same strains as in (A) and subjected to a phosphotyrosine assay. Full-length CagA is visible at an apparent molecular weight of 135 kDa, whereas the processed fragments are visible at apparent molecular weights of 100 and 35 kDa. Phosphorylated CagA (fragments) are indicated by arrowheads. (C) J774A.1 cells were infected with H. pylori as in (B), but tyrosine phosphorylation was inhibited by preincubation of cells with genistein 30 min prior to infection. Processed CagA fragments are indicated by arrowheads. (D) Average velocities of dHL-60 cells infected with the indicated strains, as measured in the under agarose assay. Values shown are derived from three independent experiments and are given in relation to velocities of control (not infected) cells (in %). P values are indicated by asterisks (*** p < 0.0001); n.s., not significant.

at least in part, by urease, rather than by fMLP (Mai et al., 1992). Instead, an antibacterial, cecropin-like peptide termed Hp(2-20), which is derived from the ribosomal protein L1 (RplA/RpL1) of H. pylori, has been shown to act as a chemoattractant for neutrophils (Bylund et al., 2001) and basophils (de Paulis et al., 2004) via binding to formyl peptide receptors. Furthermore, the neutrophilactivating protein of H. pylori (HP-NAP) has been described to stimulate chemotaxis in granulocytes (Satin et al., 2000) and to induce their migration through endothelia (Brisslert et al., 2005). One or several of these chemoattractants might be responsible for the cag-independent part of migration inhibition observed in the Boyden chamber assays, and possibly also for the inhibitory effect found in the three-dimensional collagen gel. In contrast, the migration inhibition observed in the under agarose assay was clearly dependent on the cag-PAI and on CagA. It was also dependent on the outer membrane protein HopQ, which influences CagA translocation and/or phosphorylation (Belogolova et al., 2013). Thus, the

observed HopQ effect might only be indirect, but it should be noted that CagA tyrosine phosphorylation in dHL-60 cells was still possible in the absence of HopQ, suggesting that HopQ might have a more direct activity as well. CagA is well-known for its multiple effects on proteins regulating host cell polarity and the host cell cytoskeleton (Fischer and Busch, 2013). Since cytoskeletal dynamics are also crucial for cell migration, it is tempting to speculate that one of these specific CagA effects is responsible for the observed migration inhibition. In contrast to epithelial cells as target cells, leukocytes do not only phosphorylate, but also cleave translocated CagA (Moese et al., 2001; Odenbreit et al., 2001). We show here that CagA cleavage occurs at a conserved site containing five or six consecutive asparagine residues, possibly forming an exposed loop between the N-terminal 100 kDa fragment whose structure has been determined (Hayashi et al., 2012; Kaplan-Türköz et al., 2012), and the phosphorylated C-terminal fragment which seems to be




9

Fig. 6. Influence of HopQ on leukocyte migration. (A) Differentiated HL-60 cells were infected with the indicated H. pylori strains for 4 h, and CagA phosphorylation was monitored by immunoblotting with CagA and phosphotyrosine antibodies. The C-terminal 35 kDa CagA fragments are indicated by arrowheads. (B) Average velocities of dHL-60 cells infected with the indicated strains, as measured in the under agarose assay. Values shown are derived from three independent experiments and are given in relation to velocities of control (not infected) cells (in %). P values are indicated by asterisks (** p < 0.01; *** p < 0.0001); n.s., not significant.

intrinsically disordered. However, whereas the inhibitory effect on dHL-60 cell migration in the under agarose assay is clearly dependent on CagA tyrosine phosphorylation, it does not depend on its cleavage to the p100 and p35PTyr fragments. Tyrosine phosphorylation and proteolytic cleavage are furthermore independent of each other, since we found CagA tyrosine phosphorylation in the absence of processing (Fig. 5B), and CagA processing in the absence of tyrosine phosphorylation (Fig. 5C). That migration inhibition by CagA depends on its tyrosine phosphorylation argues against an involvement of the phosphorylation-independent interaction modules binding to MARK kinase or c-Met (Nesic et al., 2010; Suzuki et al., 2009). Instead, CagA interaction partners involved in migration inhibition are expected to be among the numerous SH2 domain-containing proteins that phosphorylated CagA interacts with (Selbach et al., 2009). Further studies are required to identify CagA interaction partners responsible for the observed effects and to elucidate the underlying molecular mechanisms. What consequence might the observed inhibitory effects have in the context of gastric mucosal colonization by H. pylori? While it is unlikely that granulocytes are targets of a migration inhibition activity in vivo, slowing down dendritic cell migration to paragastric lymph nodes might be beneficial for H. pylori. Contact of H. pylori with dendritic cells has been reported to result in their maturation (Bimczok et al., 2010; Hafsi et al., 2004; Kranzer et al., 2004), but this maturation seems to be incomplete (Kaebisch et al., 2013). Although H. pylori-experienced DCs can be found in paragastric lymph nodes (Algood et al., 2007), where they are supposed to activate T cells, it is likely that their manipulation by different H. pylori virulence factors induces immune-modulatory effects (Kaebisch et al., 2013; Oertli et al., 2013). An additional inhibition of the migratory capabilities of dendritic cells, as shown in this study, might result in their prolonged exposure to H. pylori antigens and thus in a differential activation pattern (“DC exhaustion”), which has been suspected previously to influence T cell responses as well (Mitchell et al., 2007). Thus, the effects observed here might be important in conjunction with the skewing of T cell responses, resulting in a synergistic modulation of the host immune response to facilitate persistent colonization. Acknowledgements The authors are grateful to Luisa F. Jiménez Soto for valuable advice during establishing the cell migration protocols, to Evelyn

Weiss for excellent technical assistance, and to members of the Haas laboratory for critical discussion of the data. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB914-TP B5) to RH, and from the FoeFoLe program of the LudwigMaximilians-Universität München (85-2009) to WF.

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Statins Attenuate Helicobacter pylori CagA Translocation and Reduce Incidence of Gastric Cancer: In Vitro and Population-Based Case-Control Studies.

High Prevalence of Helicobacter pylori hopQ II Genotype Isolated from Iranian Patients with Gastroduodenal Disorders.

Phylogenetic analysis of Helicobacter pylori cagA gene of Turkish isolates and the association with gastric pathology.

Novel CagA ELISA exhibits enhanced sensitivity of Helicobacter pylori CagA antibody.

Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR-IκB Kinase-NF-κB Pathways in Dendritic Cells.

CD18- and ICAM-1-dependent mechanism.

Different gastric mucosa and CagA status of patients in India and Japan infected with Helicobacter pylori.

Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.

Prevalence of Helicobacter pylori vacA, cagA, and iceA Genotypes in Cuban Patients with Upper Gastrointestinal Diseases.

Clinical relevance of the cagA, tnpA and tnpB genes in Helicobacter pylori.

Corrigendum: Helicobacter pylori cagA+ Is Associated with Milder Duodenal Histological Changes in Chilean Celiac Patients.

Immunological detection of 34 KDa outer membrane protein as a functional form of OipA in clinical isolates of Helicobacter pylori.