Effects of epigallocatechin gallate on Helicobacter pylori gamma-glutamyl transpeptidase

An emergence of antibiotic resistance of H. pylori, a major cause of dyspepsia and a carcinogenic agent of gastric cancer, leads to searches for new natural antibacterial compounds. Catechins, a major bioactive ingredient of green tea (Camellia sinensis L.), shows the capability to inhibit growth of a number of microorganisms including H. pylori. For H. pylori, a possible target of the catechins was reported to be the bacterial urease. Recently, it was demonstrated that epigallocatechin gallate (EGCG), the most potent catechin, showed an ability to inhibit the human liver gamma-glutamyl transpeptidase (GGT) activity with less effect on cell viability. Since GGT is one of H. pylori virulence factors, it is likely that the EGCG can inhibit the growth of H. pylori through the bacterial GGT as another target. To extend our knowledge, the effects of the EGCG on the expression of the H. pylori ggt gene and the transpeptidation reaction of H. pylori GGT were investigated.Initially, the minimal inhibitory concentration (MIC) of the EGCG against H. pylori was determined and found to be 250 µg/ml. Then, the ggt gene expression level of the H. pylori grown on a media containing EGCG at sub-MIC (125 µg/ml) and no EGCG was studied using the quantitative RT-PCR with SYBR green fluorescein method. The result showed that the ggt gene expression of H. pylori exposed to the EGCG was up-regulated by over 12 fold (p < 0.01). This information was contrary to what was expected in that it was likely to be reduced as a result of antibacterial property. An increased transcriptional level of the bacterial ggt gene observed in this study suggested an increased bacterial GGT production to replace the inhibited enzyme for maintaining normal function, and to get rid of EGCG by detoxification role of the enzyme, besides that in bacterial colonization.The inhibition of H. pylori GGT activity in the bacterial lysate by EGCG was evaluated using the substrate L-gamma-glutamyl-p-nitroanilide (l-GpNA), the accepter glyclyglycine (glygly) and different concentration of EGCG as an inhibitor. The data in this study showed that EGCG can inhibit the transpeptidation reactions of the bacterial enzyme with the IC50 of 1.47 mM. The double reciprocal plot of the initial velocity of the transpeptidation reaction with varying concentrations of l-GpNA and 100 mM glygly or varying concentrations of glygly with 2.9 mM l-GpNA in the presence of the EGCG inhibitor were created to determine the type of the EGCG inhibitor. It was showed that the EGCG was a noncompetitive inhibitor of the H. pylori GGT transpeptidation reaction, one of the reactions catalyzed by GGT.