Zerumbone Inhibits Helicobacter pylori Urease Activity

Abstract

Helicobacter pylori (H. pylori) produces urease in order to improve its settlement and growth in the human gastric epithelium. Urease inhibitors likely represent potentially powerful therapeutics for treating H. pylori; however, their instability and toxicity have proven problematic in human clinical trials. In this study, we investigate the ability of a natural compound extracted from Zingiber zerumbet Smith, zerumbone, to inhibit the urease activity of H. pylori by formation of urease dimers, trimers, or tetramers. As an oxygen atom possesses stronger electronegativity than the first carbon atom bonded to it, in the zerumbone structure, the neighboring second carbon atom shows a relatively negative charge (δ^-) and the next carbon atom shows a positive charge (δ⁺), sequentially. Due to this electrical gradient, it is possible that H. pylori urease with its negative charges (such as thiol radicals) might bind to the β-position carbon of zerumbone. Our results show that zerumbone dimerized, trimerized, or tetramerized with both H. pylori urease A and urease B molecules, and that this formation of complex inhibited H. pylori urease activity. Although zerumbone did not affect either gene transcription or the protein expression of urease A and urease B, our study demonstrated that zerumbone could effectively dimerize with both urease molecules and caused significant functional inhibition of urease activity. In short, our findings suggest that zerumbone may be an effective H. pylori urease inhibitor that may be suitable for therapeutic use in humans.

Keywords: H. pylori; antimicrobial; dimerization; urease; zerumbone.

Figure 1

Confirmation of minimal inhibitory concentration of zerumbone. (A) H. pylori 60190 was inoculated into Mueller–Hinton agar containing 10% bovine serum and zerumbone at various concentrations (6.25, 12.5, 25, 50 and 100 μM). (B) H. pylori 60190 (1 × 10⁸ CFU/mL) was cultured in Mueller–Hinton broth containing 10% bovine serum and varying concentrations of zerumbone for 3 days (* p < 0.05).

Figure 2

The expressions of urease A and urease B were not affected by zerumbone treatment. (A) H. pylori was cultured in broth with varying concentrations of zerumbone (0, 5, 10, and 20 μM). RT-PCR was performed and the transcription levels of ureA and ureB genes were unchanged regardless of treatment. UDP-galactose 4 epimerase gene (galE) was utilized as a control. (B) Western blot analysis was performed using anti-H. pylori urease antibodies; the expression levels of urease A and urease B were found to be unchanged. To establish a control for comparing the relative change of urease A and urease B with other proteins, we utilized anti-H. pylori whole internal protein antibodies.

Figure 3

Zerumbone inhibited H. pylori urease activity. Urease activity was determined by measuring ammonia production using the indophenol method. H. pylori was grown in culture and treated with 0, 5, 10, or 20 μM zerumbone. Urease activity was found to decrease in a dose-dependent manner to 56%, 66%, and 73% of the untreated control, respectively (* p < 0.05).

Figure 4

Both urease A and urease B formed dimers, trimers, or tetramers with zerumbone. A native-PAGE assay was performed using culture supernatant proteins. (A) The expression of urease A protein (26 kDa) showed almost no change or slightly decreased dose-dependently. On the other hand, anti-urease A antibody-positive proteins were detected around 52 kDa and 78 kDa, which is about two or three times the size of urease A. (B) Urease B also showed dimerization, trimerization, or tetramerization with zerumbone. Anti-urease B antibody-positive proteins were detected around 61 kDa, 121 kDa and 244 kDa, which is once again about twice to three times the size of urease B. (C) To establish a control for comparing the relative change of urease A and urease B with other proteins, anti-H. pylori whole protein antibodies were utilized.

Figure 5

The electrical structure activity relationship of zerumbone. Due to the difference in electronegativity between O and C, the β-position carbon has a positive charge (δ⁺).

Figure 6

Hypothetical principle for the formation of urease–zerumbone dimer, trimer, and tetramer. Due to the electrical gradient of zerumbone’s structure, it is possible that H. pylori urease with its negative charges (such as thiol radicals) might bind to the β-position carbon of zerumbone, sequentially.