The human epithelial melanoma cell line WM-115 (ATCC, Wesel, Germany; CRL-1675™) is an adherent growing cell line with continuous integrin expression (mainly integrin α₅β₁, α_Vβ_3, and α_Vβ₅) and was cultivated in DMEM medium (PAA, Pasching, Austria) supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin (PAA, Pasching, Austria). The human gastric adenocarcinoma cell line AGS (ATCC CRL-1739™) was cultivated in RPMI 1640 medium, which was supplemented with 10% fetal calf serum (Gibco, Paisley, UK). Cells were cultivated at 37°C and 5.3% (v/v) CO₂ and subcultivated in a ratio of 1:3–1:5 every 2–3 days at a confluence of 70% to 90%. The H. pylori strains P12 WT and P12ΔcagL were generated and grown as described (Kwok et al., 2007). To complement the P12ΔcagL mutant strain, genes encoding CagL^WT or CagL mutant proteins were introduced into the chromosomal ureA locus, using a pAD1-derived plasmid. CagL proteins expressed from the ureA promoter contain a hemagglutinin (HA) tag introduced following the signal sequence at amino acid position 22 (Shaffer et al., 2011). For infection, H. pylori were grown for 2 days in thin layers and added at a multiplicity of infection (MOI) of 50 or 100 (Selbach et al., 2002; Moese et al., 2004). The number of elongated AGS cells in each experiment was quantified in 10 different 0.25 mm² fields (Tegtmeyer et al., 2011b). All experiments were done in triplicate.

CagL^WT and mutants were expressed as C-terminal His-tag fusions in vector pET28a (Novagen^®, Merck, Darmstadt, Germany) and purified as described earlier (Kwok et al., 2007). Briefly, E. coli ER2566 (NEB, Ipswich, USA) transformed with the plasmids were grown in 10 ml LB medium at 37°C. After overnight incubation, 500 ml of fresh LB medium were added and shaken for another 2.5–3 h until reaching OD₆₀₀ = 1. Then 300 μl of a 1-mM IPTG stock solution was added and the bacteria were grown for 1.5 h to induce CagL expression. Bacterial pellets were collected by centrifugation and then resuspended in ice-cold buffer CW (50 mM KH₂PO₄–K₂HPO₄, pH 7.5, 200 mM NaCl) supplemented with 20 μM PMSF. After lysis using a French Press, the overexpressed CagL present in the inclusion bodies was solubilized in buffer LW (50 mM KH₂PO₄–K₂HPO₄, pH 7.5, 200 mM NaCl, 6 M guanidine hydrochloride) and refolded by a rapid dilution approach in ice-cold refolding buffer (50 mM Tris–HCl, pH 8.2, 20 mM NaCl, 0.1 mM KCl, 1 mM EDTA, 2 mM reduced glutathione, 0.2 mM oxidized glutathione) using a dual-channel syringe pump (KD Scientific Inc., Holliston, USA) with a flow of 0.1 ml/min. After refolding, CagL was further purified by metal-chelate affinity chromatography through Talon^® resin (BD Biosciences). Protein concentrations of the resultant samples were determined by NanoDrop 1000 (Thermo Scientific, Waltman, USA) and typically yielded a total amount of about 1.5 mg CagL in 10 ml buffer. Purification of CagL was judged to be of >95% homogeneity by SDS-PAGE/Coomassie Blue staining. The folded conformation of the purified CagL proteins was subsequently confirmed by circular dichroism (CD) as described below.

Site-directed mutagenesis of CagL was performed using the corresponding pET28a or pAD1 vectors as DNA template (Table 1). For amplification, Phusion^® High-Fidelity DNA Polymerase (NEB, Ipswich, USA) was used, followed by PCR purification (MinElute PCR Purification Kit, Qiagen, Hilden, Germany), digestion with DpnI (Promega, Madison, USA), and ligation using T4 DNA Ligase (Promega). Re-sequencing and Western blotting of E. coli or H. pylori strains, respectively, verified the appropriate expression of CagL in the resulting plasmids.

The cell adhesion assays to immobilized CagL proteins were performed as described previously (Conradi et al., 2011). A 96 well microtiter plate (Nunc Maxisorp™, Thermo Fisher Scientific Inc., Waltham, USA) was coated with 100 μl of protein solution (CagL^WT or mutant; 10–20 μg/ml) per well, and immobilized for 18 h at 37°C. The solution was aspirated and free binding sites were blocked with 100 μl of a solution, consisting of 2% (w/v) fatty acid-free BSA (PAA, Pasching, Austria) in PBS for 1 h at 37°C. The adhesion assays were performed with WM-115 human epithelial cancer cells. This cell line was chosen for the cell adhesion assays due to a constitutive expression of integrin α₅β₁, α_Vβ₃, and α_Vβ₅, and was shown to display reproducible adherence to CagL and fibronectin (Conradi et al., 2011). The WM-115 cells were cultivated to a confluence of 70%, and detached with Trypsin-EDTA (0.05/0.02% in D-PBS; PAA, Pasching, Austria). After washing with DMEM medium, the cells were resuspended in DMEM medium with 1 mg/ml fluorescein diacetate (Sigma-Aldrich, St. Louis, USA), adjusted to a cell density of 1 × 10⁵ cells/ml and incubated at 37°C for 30 min under steady shaking. Two DMEM washing steps were performed to remove excess fluorescein diacetate. The cells were resuspended in DMEM medium containing divalent cations Ca²⁺ and Mg²⁺ (2 nM) to a cell density of 1 × 10⁵ cells/ml and incubated in the dark on ice for 30 min. Subsequently, the cell solution was distributed into solutions of peptides ranging in concentrations of millimolar to nanomolar, and the resulting mixtures were incubated at 37°C for 30 min. The solutions were then dispensed on the coated microtiter plate (5 × 10³ cells/well) and incubated for 1 h at 37°C. After several washing steps the fluorescence of cells, adherent to the immobilized CagL, was measured (λ_ex 485 nm; λ_em 514 nm) using an Infinite 200 Microplate Reader (Tecan, Männedorf, Switzerland). The inhibition concentration IC₅₀, confidence interval CI_95%, and the square of the correlation coefficient R² values were evaluated. To test the accuracy of the fit model for the non-linear regression a “Runs test” was performed and high P-values were obtained for all measurements (data not shown), which support the chosen regression model. All evaluation was performed using the GraphPad Prism 4.03 Software (GraphPad, San Diego, USA).

Infection of AGS at a density of 3.2 × 10⁵ cells/well was performed using a MOI of 50 or 100 per H. pylori strain (Kwok et al., 2002). After infection for 4 h, the AGS-bacterium co-cultures were washed three times with 1 ml of pre-warmed RPMI medium per well to remove non-adherent bacteria. To determine the total number of colony forming units (CFU) corresponding to host-bound H. pylori, the infected monolayers were incubated with 1 ml of 0.1% saponin in PBS at 37°C for 15 min. The resulting suspensions were diluted and incubated on GC agar plates as described (Kwok et al., 2002). The total CFU of cell-bound H. pylori are given as CFU per well of AGS cells.

All Fmoc-α-amino acids (9-fluorenylmethylcarbonyl-protected α-amino acids) were purchased from IRIS Biotech (Marktredwitz, Germany) and Advanced ChemTech (Louisville, USA). MALDI-ToF MS analyses were performed on a Voyager-DE (Perseptive Biosystems, Foster City, USA) using 2,5-dihydrobenzoic acid as the matrix. The analytical RP-HPLC was performed with UV detection at 220 nm and the following elution gradients: eluent A: 95% water, 5% acetonitrile, 0.1% TFA; Eluent B: 95% acetonitrile, 5% water, 0.1% TFA; 0.7 ml min⁻¹, 0–5 min 100% A → 100% B; 5–6 min 100% B → 100% A; 6–6.5 min 100% A (Thermo Separation Products apparatus equipped with a Hypersil Gold (3 μm, 150 mm × 2.1 mm) column (Thermo Fisher Scientific, Waltham, USA). Preparative RP-HPLC was performed on a Thermo Separation Products apparatus equipped with a Jupiter C18 (350 Å, 10 μm, 250 mm × 21.2 mm) efficiency column (Phenomenex, Torrance, USA) with water/acetonitrile gradients as the eluent and UV detection at 220 nm. Linear peptides were synthesized by solid phase peptide synthesis on a Liberty 12 channel microwave-assisted automated peptide synthesizer (CEM, Matthews, USA) according to a Fmoc-protocol with 2-chlorotrityl resin (IRIS Biotech) as solid support. The resin loading was 0.8 mmol/g. The C-terminal resin bound amino acids were Phe for cyclopeptides-2 to -5, d-Phe for cyclopeptide-1, and Val for the linear peptide 6. Peptide coupling was performed with three equivalents of Fmoc-amino acid (0.5 M in DMF), three equivalents TBTU (3-[bis-(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide; 0.5 M in DMF; dimethylformamide), and six equivalents DIPEA [diisopropylethylamine; 2 M in NMP (1-methyl-2-pyrrolidone)]. After washing with DMF the Fmoc group was cleaved with a solution of 20% piperidine in DMF. After synthesis of the bound linear peptides, resin cleavage was performed with 1% TFA (trifluoroacetic acid) in dichloromethane (10 times for 5 min each). The peptides one to five were cyclized under pseudo-high dilution conditions (Malesevic et al., 2004). For slow reagent addition a dual-channel syringe pump (KD Scientific Inc., Holliston, USA) was used. 200 μmol of linear precursor were dissolved in 20 ml DMF, and 600 μmol HATU (1-[bis-(dimethylamino) methyliumyl]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide) were dissolved in the same volume of DMF. Each solution was transferred into a syringe, and both solutions simultaneously were added to a stirred solution of 1200 μmol DIPEA and 20 μmol HATU in 10 ml DMF at a rate of 1.00 ml/h (Malesevic et al., 2004). Finally, the mixture was stirred for further 15 min, and the solvent was evaporated under reduced pressure at a temperature below 30°C. Peptide purification was carried out using preparative RP-HPLC. Deprotection of the cyclic peptides took place in a mixture of TFA (95%), TIS (triisopropylsilane; 2.5%), and water (2.5%), with shaking at room temperature for 2 h. The solvent was evaporated and cold diethyl ether (30 ml) was added to the residue. After centrifugation for 1 h at 0°C and 4000 × g, diethyl ether was decanted and the residue was dissolved in water, lyophilized, and purified by preparative RP-HPLC. The yield and purity of the synthesized peptides are given in Table 2.

The CagL peptide arrays were generated by the SPOT-synthesis technique as described earlier (Beutling et al., 2008). Briefly, the indicated peptides in Figure 5 were synthesized on an amino-functionalized cellulose membrane using Fmoc/tert-butyl chemistry. The spots consist of ∼5 nmol of each peptide (Dikmans et al., 2006). For the binding assays, the peptide arrays were blocked overnight at room temperature with blocking buffer consisting of 2× blocking buffer concentrate (Sigma-Aldrich, St. Louis, MO/USA) and 5% (w/v) sucrose in TBS-T (0.02 M sodium phosphate buffer with 0.15M sodium chloride (pH 7), 0.05% Tween 20). Approximately 1 μg of purified integrins α₅β₁, α_Vβ₃, and α_Vβ₅ (Chemicon-Millipore, Billerica, MA, USA) in blocking buffer was added to CagL peptide arrays for 4 h at room temperature. Next, the arrays were washed with a 10-fold volume of TBS-T three times and then incubated with α-integrin-β₁, α-integrin-α_v, or α-integrin-β₅ antibodies (Santa Cruz, Santa Cruz, USA). Finally, a chemiluminescence reaction using the ECL Plus kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) was performed as described below for Western blotting.

Circular dichroism spectroscopy was performed on a Jasco J-810 spectrometer (Jasco, Groß-Umstadt, Germany). For the CD measurements of the CagL proteins a buffer containing 5 mM NaCl and 10 mM Na₂HPO₄ (pH 7.4) was used. The CagL^WT and all mutants were measured in a 1-mm quartz cuvette, adjusted to a concentration of 3.9 nM. The secondary structure was evaluated using the deconvolution function of the Spectra Manager II Software (Jasco) based on CDPro structure analysis methods using Yang’s references (Yang et al., 1986; Sreerama and Woody, 2004).

Treated/infected cells were harvested in ice-cold PBS containing 1 mM Na₃VO₄ (Sigma-Aldrich). Western blotting was done as previously described (Tegtmeyer et al., 2009). Rabbit α-CagL antiserum was raised against the C-terminal peptide (C-RSLEQSKRQYLQER) of the protein and was prepared by Biogenes (Berlin, Germany). The α-HA-tag antibody (Invitrogen, Darmstadt, Germany) was also used to detect tagged CagL. The pan-α-phospho-tyrosine antibody PY-99 (Santa Cruz) and α-CagA (Austral Biologicals, San Ramon, CA, USA) were used to investigate the phosphorylation of CagA. The polyclonal α-phospho-ERK1/2-PT202/PY-204 antibody was purchased from NEB (Frankfurt, Germany). The polyclonal α-phospho-Cortactin-PS-405 antibody was described recently (Tegtmeyer et al., 2011b). The α-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz) served as loading control in each Western blot. As secondary antibodies, horseradish peroxidase conjugated α-mouse, α-rabbit, or α-goat polyvalent sheep immunoglobulin were used and antibody detection was performed with the ECL Plus chemiluminescence kit (Amersham Pharmacia Biotech). Band intensities and corresponding kinase activities were quantitated with the Lumi-Imager F1 (Roche Diagnostics, Mannheim, Germany).

All data were evaluated using Student t-test with SigmaStat statistical software (version 2.0). Statistical significance was defined by P ≤ 0.05 (*) and P ≤ 0.005 (**). All error bars shown in figures and those quoted following the ± signs represent standard deviation.

Previous studies indicated that the RGD sequence in CagL is important but not sufficient to trigger host cell receptor binding and signaling (Kwok et al., 2007; Tegtmeyer et al., 2010; Conradi et al., 2011; Wiedemann et al., 2012). The recently published 3D homology model of CagL (Backert et al., 2008) was screened for other surface exposed amino acid sequences to further explore the hypothesis that other CagL features besides the RGD sequence might be important for integrin interactions. Among other possible motifs that are surface exposed and with an orientation that is directed to the plausible CagL–integrin interaction site, the so-called RHS motif in proximity to the RGD motif (Figure 1) was identified as a candidate for host interaction. A series of five cyclopeptides comprising the RHS sequence was designed and synthesized to mimic the exposed motif in CagL (Figure 1). According to the spatial screening approach each of the five amino acids was mutually replaced by its d-configured counterpart to stabilize the overall conformation and increase the spatial peptide backbone diversity (Tables 2 and 3). None of the cyclopeptides-1 to -5 inhibited WM-115 cell adhesion to immobilized CagL^WT (IC₅₀ > 1 mM; Table 3). However, WM-115 cell adhesion to the mutant CagL^RAD was inhibited by cyclopeptide-2 [c-(-FeANE-)] with an IC₅₀ value in the range of 54–430 μM (Table 3). In addition, cyclopeptides-1 and -2 displayed weak inhibition of WM-115 cell adhesion to the mutant CagL^RGA with IC₅₀ values of 43–340 and 30–530 μM, respectively. The other cyclopeptides-3 to -5 did not display any significant effect on the WM-115–CagL interaction (IC₅₀ > 1 mM; Table 3).

In addition, cell adhesion assays were performed to investigate whether the linear peptide 6 (ANFEANELFFISEDV) has effects on the adhesion of WM-115 cells to immobilized CagL^WT and its RGD mutants CagL^RGA and CagL^RAD. Linear peptide 6 mimics the RHS motif with the Phe-Glu-Ala-Asn-Glu sequence and its adjacent amino acids within the CagL protein sequence (Figure 1). According to its CD spectrum, it does not adopt a discrete conformation in solution (data not shown). While linear peptide 6 did not significantly interfere with CagL^WT–WM-115 interaction in the cell adhesion assay (IC₅₀: >1 mM), it exhibited a slight inhibitory activity on the CagL^RAD–WM-115 interaction (IC₅₀: 239 μM; CI_95%: 164–346 μM; R²: 0.88) and the CagL^RGA–WM-115 interaction with an IC₅₀ of 9 μM (CI_95%: 6–15 μM; R²: 0.76; Figure 2; Table 3). In comparison to the properties of the earlier mentioned c-(-RGDlA-) peptide to inhibit the WM-115 cell adhesion to CagL^WT (IC₅₀: 2.31 μM; CI_95%: 1.40–3.82 μM; R²: 0.89) and its RGD mutants CagL^RGA (IC₅₀: 1.63 μM; CI_95%: 1.01–2.63 μM; R²: 0.93) and CagL^RAD (IC₅₀: 0.91 μM; CI_95%: 0.58–1.43 μM; R²: 0.94), the inhibition properties of the linear peptide 6 are obviously reduced (Table 3). Nonetheless the results support involvement of the RHS motif in CagL–WM-115 interaction.

For further investigation of the RHS motif and to reveal the relevance of specific amino acids in this sequence, CagL point mutants were recombinantly produced in E. coli, where individual amino acids were replaced by alanine residues (Table 4). CD spectroscopy was used to verify the correct folding of the mutants CagL^F86A, CagL^E87A, CagL^E87A/A88E, and CagL^E90A in comparison to CagL^WT. The secondary structure composition calculated for CagL^WT is in good accordance with the proposed CagL homology model (Backert et al., 2008) and displays a high content of α-helices (∼35%), accompanied by β-strands (∼29%, Figure 3A). The mutants CagL^F86A, CagL^E87A, CagL^E87A/A88E, and CagL^E90A CD spectra display comparable curve shapes like the CagL^WT (Figure 3D). Unfortunately, the amount of CagL^N89A protein necessary to estimate the secondary structure and to validate the correct protein folding with CD spectroscopy could not be obtained and, therefore, could not be included in the study. However, to obtain information on the structural stability and to further compare the CagL^E87A and CagL^E90A mutants with CagL^WT, temperature-interval dependent CD measurements were performed ranging from 0 to 60°C (Figures 3A–C). In general, all tested proteins behaved in similar manner, exhibiting temperature-dependent denaturation above 40°C. Below 40°C, the secondary structures are comparable, with only minor structural differences. In comparison, the CagL^E90A CD spectrum shows maximum deviations at temperatures below 20°C (Figure 3C). In addition, we performed a pH screening by measuring the CD spectra of CagL^WT over a pH range from four to nine in phosphate buffer and could show that the protein is very stable under the tested conditions (Figure 3E).

Different CagL mutants with amino acid variations in the RHS motif (CagL^F86A, CagL^E87A, and CagL^E90A) were immobilized and investigated in cell adhesion assays. The WM-115 cells were pre-incubated with the previously described cyclopeptide-7 [c-(-Arg-Gly-Asp-d-Leu-Ala-)] in different concentrations from nano- to millimolar ranges to block the RGD-binding site of the integrins. The dose–response curves obtained in these assays are shown in Figure 4. For comparison, the cell adhesion results of the recently published CagL^WT, CagL^RGA, and CagL^RAD mutants are also given in Table 4 (Conradi et al., 2011). Additionally, the CagL^Q40A mutant was included as a negative control to exclude the possibility that genetic mutations in the CagL sequence may generally lead to loss of function. Cyclopeptide-7 inhibited WM-115 cell adhesion to CagL^Q40A with an IC₅₀ value of 1.65 μM (CI_95% = 1.06–2.55; R² = 0.92), while the cell adhesion of WM-115 cells to immobilized CagL^F86A was inhibited with an IC₅₀ value of 2.39 μM (CI_95% = 1.87–3.04 μM; R² = 0.91), a value that is very similar to that of CagL^WT (IC₅₀ = 2.31 μM; CI_95% = 1.87–3.04 μM; R² = 0.91). Interesting results were observed in the inhibition assays for the mutants CagL^E87A and CagL^E90A. WM-115 cell adhesion to immobilized CagL^E87A was inhibited by cyclopeptide-7 with an IC₅₀ of 0.67 μM (CI_95% = 0.38–1.18 μM; R² = 0.72), the lowest value observed for all CagL mutants investigated (Figure 4; Table 4). The IC₅₀ value of 0.88 μM (CI_95% = 0.58–1.35 μM; R² = 0.80) for the inhibition of WM-115 cell adhesion to immobilized CagL^E90A by cyclopeptide-7 is similar, and is also comparable to the values observed for CagL^RGA (0.91 μM; Figure 4; Table 4). In an additional experiment we tested the double mutant CagL^E87A/A88E to reveal more details on the involvement of CagL amino acid Glu⁸⁷ in the interaction with WM-115 cells. The double mutant CagL^E87A/A88E formally is characterized by the shift of an acidic amino acid side chain from position 87 to position 88. The immobilized CagL^E87A/A88E protein displayed no binding to the WM-115 cells, which may indicate a loss of integrin affinity. In conclusion, the results show remarkably reduced adhesion of WM-115 cells to immobilized CagL^E87A and CagL^E90A compared to the CagL^WT, which implies a participation of the glutamates in the CagL–WM-115 interaction.

The previously described SPOT technique (Frank, 2002) was applied to identify the CagL amino acid sequence responsible for binding to integrin α₅β₁. Overlapping linear 15-mer peptides derived from the CagL sequence from amino acid position 60–104 were chemically synthesized on a cellulose membrane by the SPOT method (Frank, 1992). As shown in Figure 5A, adjacent peptides share the same sequence of 12 amino acids but differ by three amino acids at the C- or N-terminal ends, respectively. Purified integrin α₅β₁ was incubated with these membranes and the binding was assayed as described in the Section “Materials and Methods.” Figure 5B shows that integrin α₅β₁ binds to the RGD motif-containing arraypeptides-3 and -4. In addition, a very strong signal for arraypeptide-9 covering the RHS motif was recorded (Figure 5B, top and Figure 5C). Interestingly, two other FEANE sequence-containing array peptides (-7 and -8) did not bind integrin α₅β₁, suggesting that the flanking C-terminal sequences are also important for this interaction. As a control, incubation of the membranes with two other integrins, α_Vβ₃ and α_Vβ₅ also revealed signals for the two RGD containing CagL peptides and some weak signals for arraypeptides-10 or -11, respectively (Figure 5B, middle and Figure 5C). The mock control blot revealed no signals as expected (Figure 5B, bottom).

To investigate the importance of the RHS motif in CagL directly during infection with H. pylori, two CagL mutants were generated carrying the A84E/E87A and A88E/L91A point mutations. Based on the above described peptide array the two point mutants were constructed to contain one mutation in the RHS sequence and one either N- or C-terminal, to cover additional amino acids that may be relevant for host cell interactions. CagL^WT and both mutants were chromosomally integrated into a P12ΔcagL deletion mutant and expressed as HA-tag fusions in the ureA locus as described (Shaffer et al., 2011). The correct expression of each of these CagL variants was verified by Western blotting using an α-HA antibody (Figure 6A). The AGS cells were infected with each of these H. pylori strains for 4 h, followed by analysis of viable bacterial binding to cells, as described in Materials and methods. The results show that each of these strains bound to AGS cells with high efficiency. Some minor differences in binding were seen among the different strains but were not statistically significant (Figure 6B). This suggests that mutation of the RHS motif in CagL has no significant inhibitory impact on the overall capacity of the bacteria to bind to AGS host cells.

AGS cells were infected with the different complemented H. pylori strains during a time course of 2 and 4 h, respectively, to investigate whether strains expressing CagL mutant proteins can trigger the injection and phosphorylation of CagA. The results of the α-phospho-tyrosine and α-CagA specific Western blots show that P12ΔcagL re-expressing CagL^WT can efficiently inject and phosphorylate CagA in a time-dependent fashion (Figures 7A,B). Infection of AGS with P12ΔcagL expressing the CagL^A84E/E87A mutant revealed a ∼46% reduced phospho-CagA signal at 2 h, while very strong phospho-CagA signals were produced at the 4-h time point with almost no difference as compared to the complemented CagL^WT control (Figures 7A,B). This suggests that mutation of A84E/E87A in CagL affects the injection and phosphorylation of CagA at very early times of infection. In contrast, infection with H. pylori expressing the CagL^A88E/L91A mutant revealed a stronger reduction of phospho-CagA signals (∼69%) at 2 h as compared to the complemented CagL^WT control, while this low level of phospho-CagA did only slightly increase after 4 h of infection (Figures 7A,B). This result shows that a strain expressing the CagL^A88E/L91A mutant protein has a significant defect in injection and phosphorylation of CagA at both time points of infection.

Finally the various RHS mutations in CagL were inspected in relation to impacts on cellular downstream signaling involved in the H. pylori-induced AGS cell elongation phenotype. It was recently shown that besides CagA phosphorylation, the activation of the ERK1/2 MAP kinase pathway either by H. pylori infection (Mimuro et al., 2002; Tegtmeyer et al., 2009) or by transfection CagA in the absence of H. pylori (Higashi et al., 2004) is also crucial for phenotypical outcome. Therefore, we tested whether the different CagL-expressing H. pylori strains induced the activation of ERK1/2, using the same conditions as described for Figure 7. The results of the α-phospho-ERK1/2 Western blots show that P12 WT or P12ΔcagL re-expressing CagL^WT (but not P12ΔcagL) can efficiently activate this MAP kinase in a time-dependent fashion (Figures 8A,B). Infection of AGS with H. pylori expressing the complemented CagL^A84E/E87A mutant revealed a significantly (46%) reduced phospho-ERK1/2 signal at 2 h, and a similarly reduced (38%) phospho-ERK1/2 signal at 4 h time point as compared to the complemented CagL^WT control (Figures 8A,B). This suggests that mutation of A84E/E87A in CagL downregulates not only the injection and phosphorylation of CagA, but also reduced the activation of ERK1/2. In addition, infection with H. pylori expressing the CagL^A88E/L91A mutant revealed an even stronger reduction of phospho-ERK1/2 signals by about 63 or 70% at the 2- or 4-h time points, respectively. This result indicates that the CagL^A88E/L91A mutant has a significantly pronounced defect in activating ERK1/2 during infection.

Very recently, we demonstrated that one important downstream target of activated ERK1/2 during infection is the actin-binding protein cortactin, phosphorylated at serine residue 405 (Tegtmeyer et al., 2011b). Hence it was tested whether the various CagL-expressing strains induce the phosphorylation of cortactin at S-405 during a time course of 2 and 4 h infection. The results of the α-phospho-cortactin Western blots show that P12ΔcagL re-expressing CagL^WT but not P12ΔcagL can efficiently phosphorylate cortactin in a time-dependent manner (Figures 8A,B). Infection of AGS cells with H. pylori expressing either CagL^A84E/E87A or CagL^A88E/L917A mutants revealed the induction of phospho-cortactin signals similar to that of CagL^WT with no significant difference at 2 h, but a strongly reduced (about 45–46%) phospho-cortactin signal at the 4-h time point (Figures 8A,B). This suggests that mutation of CagL in the RHS motif (either A84E/E87A or A88E/L91A mutations) downregulates cortactin’s serine phosphorylation to a similar high extent at the 4-h time point, indicating that the CagL^A84E/E87A and CagL^A88E/L91A mutants share a significantly pronounced defect in activating cortactin during infection. Finally, the elongation phenotype was monitored in the same set of experiments. As shown in Figure 8C, mutation of the RHS motif at A84E/E87A or A88E/L91A inhibited the elongation phenotype by more than 50% at the 2-h time point. At the 4-h time point, the CagL^A84E/E87A mutant exhibited a ∼20% reduction and the CagL^A88E/L91A mutant revealed a ∼43% reduction as compared to the complemented CagL^WT control. This set of experiments reveals that mutation of the RHS motif in H. pylori results in significant defects in signaling to cortactin and cell elongation, especially at very early times of infection.