Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

Abstract

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

Immunoproteomic Analysis To Identify Shiga Toxin-Producing Escherichia coli Outer Membrane Proteins Expressed during Human Infection

“…The pooled HUS antiserum also reacted with L-asparaginase II, an enzyme that cata- lyzes asparagine hydrolysis (50), and EF-Tu, one of the most abundant and best-characterized cytoplasmic proteins with a role in protein synthesis (51)(52)(53). Similar to our findings, L-asparaginase II and EF-Tu have been reported to be immunogenic in other pathogenic bacteria (49,(54)(55)(56)(57)(58)(59). In addition, other studies have described the presence of EF-Tu in E. coli OM or OMP extracts (32,(60)(61)(62)(63)(64), as well as the interaction of EF-Tu with fibronectin (65), indicating the participation of EF-Tu in adhesion to human intestinal cells and mucin (66)(67)(68).…”

Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

“…These results demonstrate yet another metabolic pathway influencing acid tolerance: the periplasmic amino acid deamidation reactions carried out by Ggt and AsnB. These two periplasmic enzymes have been previously studied for H. pylori (23)(24)(25)(26); however, their potential role in acid resistance was not addressed. Here we show that both deamidases (Ggt and AsnB) have roles in supporting ureasemediated acid resistance.…”

“…We also studied two amino acid deamidases, glutaminase (Ggt) and asparaginase (AsnB). These enzymes have been studied for their roles in host cell cytotoxicity (23,24) and in suppression of the host immune response (25); however, our interest pertains to their function as periplasmic amino acid deamidases (26). These four enzymes (GS, GDH, Ggt, and AsnB) all appear to be involved in regulating either (i) the hydrolysis of urea inside the cell or (ii) the extent to which this ammonium is extruded versus assimilated.…”

L-Asparaginase II Produced by Salmonella Typhimurium Inhibits T Cell Responses and Mediates Virulence

“…Typhimurium uses L-Asparaginase II to inhibit the response of T cells may be similar to the mechanism by which clinically administered L-asparaginases help eliminate human cancers. The mechanistic details of how inhibition occurs are supported by recent studies demonstrating that purified L-Asparaginase II from S. enteritidis and H. pylori can inhibit protein synthesis, block cell-cycle progression, and suppress replication of cultured cell lines (Iwamaru et al, 2001; Scotti et al, 2010; Shibayama et al, 2011). Additional support comes from our own work indicating that S .…”