Gamma-glutamyltranspeptidase expression by Helicobacter saguini, an enterohepatic Helicobacter species isolated from cotton top tamarins with chronic colitis.

Anthony Mannion,Yan Feng,Kodihalli Ravindra,Zhongming Ge,Zeli Shen,Stephen C Artim,James G Fox

doi:10.1111/cmi.12968

Anthony Mannion, Yan Feng + Show 5 more

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https://doi.org/10.1111/cmi.12968

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Abstract

Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H.saguini infection in gnotobiotic IL-10-/- mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H.saguini expresses an enzymatically active GGT homologue with virulence properties. Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H.saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments. Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H.saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H.saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H.saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells. This study shows that H.saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.

Highlights

Enterohepatic Helicobacter species (EHS) are gram‐negative, spiral‐ shaped bacteria that colonise the lower intestine, liver, and gall bladder of mammals, birds, and reptiles and are associated with the occurrence of gastrointestinal inflammatory diseases and cancers (Hansen, Thomson, Fox, el‐Omar, & Hold, 2011; Mitchell et al, 2014; Thomson et al, 2011; Whary & Fox, 2004)
The protected status of cotton top tamarins (CTTs) has prevented establishing a direct etiological relationship between Helicobacter infection and inflammatory bowel disease (IBD), we recently demonstrated that monoassociated infection of gnotobiotic C57BL/6 IL‐10−/− mice with H. saguini induces IBD and procarcinogenic changes in the large intestine (Shen et al, 2016)
To exclude that glutamate produced by HSGGT1 activity (Figure S4B) affected cell proliferation, we found that incubating HT‐29 cells with up to 10 mM of glutamate for 72 hr had no effect on proliferation

Summary

| INTRODUCTION

Enterohepatic Helicobacter species (EHS) are gram‐negative, spiral‐ shaped bacteria that colonise the lower intestine, liver, and gall bladder of mammals, birds, and reptiles and are associated with the occurrence of gastrointestinal inflammatory diseases and cancers (Hansen, Thomson, Fox, el‐Omar, & Hold, 2011; Mitchell et al, 2014; Thomson et al, 2011; Whary & Fox, 2004). The protected status of CTTs has prevented establishing a direct etiological relationship between Helicobacter infection and IBD, we recently demonstrated that monoassociated infection of gnotobiotic C57BL/6 IL‐10−/− mice with H. saguini induces IBD and procarcinogenic changes in the large intestine (Shen et al, 2016). These data suggest that H. saguini infection may influence the onset and progression of chronic colitis and colon cancer in CTTs. Though a substantial body of evidence supports a causative association between EHS infection and IBD, the pathogenic mechanisms of these organisms remain incompletely defined. As part of our efforts to further understand the pathogenic potential of H. saguini, the aim of this study was to test the hypothesis that H. saguini expresses an enzymatically functional GGT with virulence properties

| RESULTS

| DISCUSSION

Findings

| EXPERIMENTAL PROCEDURES