Evidence for conserved function of γ-glutamyltranspeptidase in Helicobacter genus

Abstract

The confounding consequences of Helicobacter bilis infection in experimental mice populations are well recognized, but the role of this bacterium in human diseases is less known. Limited data are available on virulence determinants of this species. In Helicobacter pylori, γ-glutamyltranspeptidase (γGT) contributes to the colonization of the gastric mucosa and to the pathogenesis of peptic ulcer. The role of γGT in H. bilis infections remains unknown. The annotated genome sequence of H. bilis revealed two putative ggt genes and our aim was to characterize these H. bilis γGT paralogues. We performed a phylogenetic analysis to understand the evolution of Helicobacter γGTs and to predict functional activities of these two genes. In addition, both copies of H. bilis γGTs were expressed as recombinant proteins and their biochemical characteristics were analysed. Functional complementation of Esherichia coli deficient in γGT activity and deletion of γGT in H. bilis were performed. Finally, the inhibitory effect of T-cell and gastric cell proliferation by H. bilis γGT was assessed. Our results indicated that one gene is responsible for γGT activity, while the other showed no γGT activity due to lack of autoprocessing. Although both H. bilis and H. pylori γGTs exhibited a similar affinity to L-Glutamine and γ-Glutamyl-p-nitroanilide, the H. bilis γGT was significantly less active. Nevertheless, H. bilis γGT inhibited T-cell proliferation at a similar level to that observed for H. pylori. Finally, we showed a similar suppressive influence of both H. bilis and H. pylori γGTs on AGS cell proliferation mediated by an apoptosis-independent mechanism. Our data suggest a conserved function of γGT in the Helicobacter genus. Since γGT is present only in a few enterohepatic Helicobacter species, its expression appears not to be essential for colonization of the lower gastrointestinal tract, but it could provide metabolic advantages in colonization capability of different niches.

Figure 1. Multi-alignment of amino acid sequences of different bacterial γGTs, Helicobacter bilis Bgh1 and Bgh2, and class IV Cephalosporin Acylase (CA).

Multi-alignment between residues 379 and 477 (Helicobacter pylori γGT numeration) is shown. Sequences of γGTs of H. pylori (26695; HP1118), H. acinonychis (Sheeba; Hac_0598), H. bizzozeronii (CIII-1;HBZC1_08080), H. salomonis (O6A; EMBL FR821684), H. suis (HS1; HSUHS1_0265), H. felis (ATCC 49179; Hfelis_06880), H. mustelae (12198; HMU08020), Esherichia coli (K12; Swiss-Prot P18956), Campylobacter jejuni (81–176; CJJ81176_0067), Pseudomonas sp. (A14; Swiss-Prot P36267), Bacillus subtilis (168; Swiss-Prot P54422), Arcobacter nitrofigilis (DSM 7299; Arnit_0203), Arcobacter butzleri (RM4018; Abu_0961), H. bilis (Bgh1 = HRAG_01341; Bgh2 = HRAG_01828) as well as class IV CA of Pseudomonas sp. (SE82; Swiss-Prot P15557) are shown. Residues completely conserved among the sequences are indicated with a black background. The catalytic dyad is highlighted with a red box, and the conserved motif GXXGGXXI is enclosed in a black box. The Lid loop consists of the residues G428 to G438 of H. pylori γGT and is indicated in grey. Residues involved in the substrate recognition and the catalytic centre are highlighted in grey. Amino acid substitutions in Bgh1 potentially involved in functional change are indicated by arrows below the sequences.

Figure 2. Unrooted tree based on complete amino acid sequences of different bacterial γGTs, Helicobacter bilis Bgh1 and Bgh2, and Pseudomonas Cephalosporin Acylase (CA).

The evolutionary history was inferred using the Minimum Evolution method and the evolutionary distances were computed using the Dayhoff matrix-based method. Bar indicates amino acid substitutions per position. Numbers at the nodes indicate support for the internal branches within the tree obtained by bootstrap analysis (≥70%; percentages of 500 bootstraps).

Figure 3. Unrooted tree based on the amino acid sequences of: Helicobacter spp. γGTs, Campylobacter jejuni γGTs and Bgh1 and Bgh2 homologues of Helicobacter trogontum and Helicobacter bilis.

The evolutionary history was inferred using the Minimum Evolution method, and the evolutionary distances were computed using the Dayhoff matrix-based method. Bars indicate amino acid substitutions per position. Numbers at the nodes indicate support for the internal branches within the tree obtained by bootstrap analysis (≥70%; percentages of 500 bootstraps). (A) Phylogeny of almost complete pro-enzyme sequences (447 AA). (B) Phylogeny of almost complete N-terminal γGT sequences (Heavy chain; 333 AA). (C) Phylogeny of the almost complete C-terminal γGT sequences (Light chain; 190 AA).

Figure 4. Purity and autoprocessing of recombinant Bgh1 and Bgh2.

(A) SDS-page of the purified proteins after gel filtration: Bgh1 (Lane 1) and fully autoprocessed Bgh2 (Lane 2); (B) time-line for autoprocessing of Bgh1.

Figure 5. Western Blot on whole H. bilis lysate using specific antisera against Bgh1 and Bgh2.

Western blot of whole H. bilis lysate using antisera against (A) Bgh1 and (B) Bgh2.

Figure 6. Comparison of the kinetic constants for Helicobacter bilis γGT (Hb-γGT) at different pH.

Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software, San Diego, CA USA). At indicated pH values, kinetic constants (k_cat in black and KM in grey) for the hydrolysis of L-γ-glutamyl-p-nitroanilide (gGpNA) were determined.

Figure 7. Inhibitory effect on T-cell proliferation (Jurkats) by Helicobacter bilis γGT (Hb-γGT).

Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software). Different letters on the bars indicate significant differences at P<0.05. (A) Inhibitory effect with various amounts of recombinant Hb-γGT (Bgh2) protein; statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test. (B) Inhibitory effect of the culture supernatant of H. bilis wild-type CCUG 23435 (WT) and H. bilis Δggt MR9 (Δggt) with or without pre-treatment with Acivicin (Sigma-Aldrich); statistical analysis was performed using unpaired t-test.

Figure 8. Inhibitory effect on AGS proliferation by Helicobacter bilis γGT (Hb-γGT).

Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software). Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test. Different letters on the bars indicate significant differences at P<0.05.