An RGD helper sequence in CagL of Helicobacter pylori assists in interactions with integrins and injection of CagA

Abstract

Helicobacter pylori is a specific gastric pathogen that colonizes the stomach in more than 50% of the world's human population. Infection with this bacterium can induce several types of gastric pathology, ranging from chronic gastritis to peptic ulcers and even adenocarcinoma. Virulent H. pylori isolates encode components of a type IV secretion system (T4SS), which form a pilus for the injection of virulence proteins such as CagA into host target cells. This is accomplished by a specialized adhesin on the pilus surface, the protein CagL, a putative VirB5 ortholog, which binds to host cell β(1) integrin, triggering subsequent delivery of CagA across the host cell membrane. Like the human extracellular matrix protein fibronectin, CagL contains an RGD (Arg-Gly-Asp) motif and is able to trigger intracellular signaling pathways by RGD-dependent binding to integrins. While CagL binding to host cells is mediated primarily by the RGD motif, we identified an auxiliary binding motif for CagL-integrin interaction. Here, we report on a surface exposed FEANE (Phe-Glu-Ala-Asn-Glu) interaction motif in spatial proximity to the RGD sequence, which enhances the interactions of CagL with integrins. It will be referred to as RGD helper sequence (RHS). Competitive cell adhesion assays with recombinant wild type CagL and point mutants, competition experiments with synthetic cyclic and linear peptides, and peptide array experiments revealed amino acids essential for the interaction of the RHS motif with integrins. Infection experiments indicate that the RHS motif plays a role in the early interaction of H. pylori T4SS with integrin, to trigger signaling and to inject CagA into host cells. We thus postulate that CagL is a versatile T4SS surface protein equipped with at least two motifs to promote binding to integrins, thereby causing aberrant signaling within host cells and facilitating translocation of CagA into host cells, thus contributing directly to H. pylori pathogenesis.

Keywords: CagL; ERK kinase; binding motifs; cortactin; integrin interaction; α5β1.

Figure 1

Computational 3D-structure model of CagL^WT with highlighted motifs of interest. The ribbon diagram of CagL shown in this figure consists of three major helices and a globular domain with the exposed RGD motif highlighted in the front. The second highlighted FEANE sequence (here called RHS motif) is shown in a flexible loop, also exposed at the surface. The CagL homology model is derived from the VirB5 ortholog TraC protein (PDB: 1R8I) encoded in plasmid pKM101 (Yeo et al., ; Backert et al., 2008).

Figure 2

Dose–response curve of a linear RHS motif-containing peptide, inhibiting binding of WM-115 cells to immobilized CagL^WT and mutants. The WM-115 cells were pre-incubated in different concentrations of the linear 15-mer peptide ANFEANELFFISEDV, which mimics the exposed RHS motif of CagL. Purified CagL^WT and the CagL^RAD and CagL^RGA mutants were immobilized on plastic surface followed by addition of the WM-115 cells. After 1 h of co-incubation, the amounts of attached cells were determined. The data were evaluated using non-linear fit for the evaluation of the potency of peptide ANFEANELFFISEDV to inhibit the binding of WM-115 cells to immobilized CagL.

Figure 3

Structural evaluation of purified CagL proteins with CD spectroscopy. (A) The CagL^WT secondary structure was evaluated as described previously (Yang et al., 1986). (A–C) The temperature-dependent structure stability of the mutants CagL^E87A, CagL^E90A, and the CagL^WT protein was evaluated in CD measurements. (D) Secondary structure of CagL^WT and mutants CagL^F86A, CagL^E87A, CagL^E87A/A88E, and CagL^E90A were evaluated to control correct folding of the proteins after expression and purification. (E) pH stability of CagL^WT in the pH range four to nine.

Figure 4

Dose–response curves showing the inhibitory effect of cyclic RGD peptides on WM-115 cell binding to the different immobilized CagL proteins in competitive cell adhesion assays. The purified CagL^Q40A, CagL^F86A, CagL^E87A, and CagL^E90A mutant proteins were immobilized on plastic surface. WM-115 cells were pre-incubated with varying concentrations of cyclic peptide c-(-RGDlA-; d-amino acids in small letter), which is known to inhibit WM-115 cell binding to immobilized CagL in the low μM range (Conradi et al., 2011). Subsequently, the WM-115 cells were incubated with the immobilized CagL mutants. The amount of WM-115 cells bound to immobilized CagL was quantified, and the resulting dose–response curves were evaluated with a non-linear fit to determine the binding inhibition potency of peptide c-(-RGDlA-) for the interaction of WM-115 cells with immobilized CagL.

Figure 5

Mapping of the RHS motif and flanking CagL sequences in binding to integrins using a peptide array. (A) The design of the array of 15-mer peptides and corresponding CagL amino acid sequences from position 60–104 is shown. The RGD and RHS motifs are highlighted with color. (B) Purified integrins α₅β₁, α_Vβ₃, and α_Vβ₅ were incubated with these membranes and bound proteins were identified using antibodies as described in Materials and Methods. As a mock control, buffer-treated membranes followed by incubation with an integrin-β₁ antibody revealed no signals, as expected. (C) Densitometric measurement of individual spot intensities using the Lumi-Imager F1 (Roche) revealed the relative amount of bound binding partner per given peptide in%. The strongest spot intensity seen was set as 100%.

Figure 6

Effect of CagL deletion or point mutations in the CagL RHS motif on binding of H. pylori to AGS cells. (A) A ΔcagL deletion mutant in strain P12 was generated by replacing the cagL gene with an aphA3 cassette. CagL^WT and two CagL mutants carrying either the A84E/E87A or A88E/L91A point mutations were expressed as HA-tag fusion proteins from genes integrated into the ureA locus of the P12ΔcagL strain. The correct expression of each of these CagL variants was verified by Western blotting using an α-HA antibody. The α-CagA Western blot was performed as loading control. (B) AGS cells were infected with each of these strains for 4 h using MOI = 100, followed by determination of the amount of viable bound H. pylori per input strain. The quantification data indicate that each of these strains bound to AGS cells equally well with high efficiency. No significant differences in results were seen when MOI = 50 was used (data not shown).

Figure 7

Effects of CagL point mutations in the RHS motif on CagA injection and phosphorylation during H. pylori infection of AGS cells. (A) The P12ΔcagL deletion mutant was complemented with CagL^WT or various CagL point mutants. AGS cells were infected with the different indicated H. pylori strains (MOI = 100) during a time course of 2 or 4 h, respectively. The resulting protein lysates were subjected to Western blotting using α-phospho-tyrosine (PY-99) and α-CagA antibodies. The α-GAPDH blot served as loading control in each sample. (B) Quantification of CagA phosphorylation. Densitometric measurement of individual band intensities of the α-CagA and α-phospho-tyrosine (PY-99) blots in (A) was performed with the Lumi-Imager F1 (Roche). Quantification revealed the relative amount of phospho-CagA per sample in %. Similar inhibitory activities of all mutants were seen when MOI = 50 was used (not shown).

Figure 8

Mutation of CagL in specific amino acids within the RHS motif results in defects in H. pylori-triggered ERK1/2 activation, cortactin phosphorylation, and AGS cell elongation. (A) AGS cells were infected with the different complemented H. pylori strains (MOI = 100) during a time course of 2 or 4 h, respectively. The same protein lysates as shown in Figure 7 were subjected to Western blotting using the indicated α-phospho-ERK1/2 and α-phospho-cortactin antibodies. (B) Densitometric measurement of individual band intensities of the blots in (A) and comparison to total α-ERK1/2 and α-cortactin blots (not shown) was performed with the Lumi-Imager F1 (Roche). The relative amounts of phospho-ERK1/2 and phospho-cortactin per sample were quantified and given in%. (C) Quantification of the AGS cell elongation phenotype in the same set of experiments. Similar inhibitory activities of all mutants were seen when MOI = 50 was used (not shown).