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.

Figure 1. Isolation of the cell-cycle inhibiting activity from BCF.

(A) Elution profile of BCF (black line) and cell-cycle inhibiting fractions identified by BrdU incorporation in HDF cells (grey histograms). (B) Active fractions separated on a non-reducing SDS gel. The four silver-stained bands of different molecular weights analysed by LC-MS/MS are in rectangles. (C) Stacked line representation of the relative intensities displayed by each of the bands detected in SDS-PAGE in (B). Numbers correspond to molecular masses in kDa. (D) L-asparaginase activity of cell-cycle inhibiting fractions.

Figure 2. Cell-cycle inhibition in HDF cells.

BrdU incorporation in HDF cells incubated with: (A) UBF and BCF treated with the L-asparaginase inhibitor 5-diazo-4-oxo-L-norvaline (DONV, 20 mM) or the GGT inhibitor acivicin (ACI, 5 mM), (B) UBF and BCF derived from different H. pylori strains, (C) different concentrations of recombinant L-asparaginase. Results are expressed as a mean ± SD from 5 independent experiments. ^*P≤0.05, ^**P≤0.01.

Figure 3. Cell-cycle inhibition by L-asparaginase.

BrdU incorporation in cell lines exposed to variable concentration of recombinant H. pylori CCUG 17874 (A) and E. coli L-asparaginase (B) compared to untreated control cells. Boxes: magnification of the corresponding chart for L-asparaginase activities lower than 0.5 U ml⁻¹. The points represent means (n≥3); SD not represented for clarity. (C) IC50 (L-asparaginase concentration inducing 50% cytotoxicity in U ml⁻¹) of H. pylori and E. coli L-asparaginase (MTT assay).

Figure 4. Expression of asparagine synthetase in cultured cell lines.

(A) Western blot analysis of HDF, AGS, MKN28, MKN7 and MKN74 cell lysates for asparagine synthetase (AS). Actin was determined as loading control. (B) Densitometric analysis of protein levels normalized to the internal loading control. Results are expressed as a mean ± SD from 3 independent experiments ^*P≤0.05, ^**P≤0.01.

Figure 5. Subcellular localisation of L-asparaginase and ELISA assays.

(A) Western blot performed on 100 µg of the subcellular fractions of H. pylori CCUG 17874 with an anti-asparaginase antibody. From left to right: 1: pre-lytic fraction; 2: periplasmic fraction; 3 and 4: soluble and insoluble spheroplast fractions, respectively. Molecular mass markers (kDa) are indicated. (B) Total L-asparaginase activity associated with each fraction (U). (C) Catalase, carbonic anhydrase (CA) and malonate dehydrogenase (MDH) were found in the expected H. pylori subcellular fractions: periplasm (2), spheroplast (3) and cytoplasm (4) , respectively. (D) Scatter-plot of ELISA assay results on sera from 42 H. pylori positive and 43 H. pylori negative patients. Serum samples (1∶101 dilution) were tested in parallel using commercial kit 1 and plates pre-coated with recombinant H. pylori L-asparaginase. Medians indicated as horizontal bars. 21% of the total samples were anti-L-asparaginase IgG positive (+). (E) Comparison of the results obtained with commercial kit 1 and L-asparaginase-based ELISA.