Helicobacter pylori Urease
Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium is the most frequently cited etiologic agent of human gastritis and peptic ulceration. This species, whose niche is highly restricted to the gastric mucosa of humans, has adopted a strategy of survival that includes synthesis of urease as its most abundant cellular protein. This enzyme hydrolyzes urea, releasing ammonia which allows colonization of this acid-sensitive organism at low gastic pH. In addition, urease is a key protein used for detection of the organism by measuring serum antibody to the protein, enzyme activity directly in a gastric biopsy, or a product of hydrolysis (CO 2) using a urea breath test.
The urease of H. pylori is related to that of P. mirabilis but also displays differences. The enzyme is composed of 12 copies of two subunits of 61 kDa and 27 kDa (shown below). Accessory proteins are also required for activation of the apoenzyme by nickel ion insertion.
We have focused on factors that contribute to synthesis of a catalytically active urease. By screening for H. pylori genes that could produce a highly active urease in an E. coli host, clones were isolated that encoded a single component nickel transport system. We designated this protein as NixA (for “nickel crossing”). E. coli expressing this protein demonstrated rapid active transport of nickel ions in to the bacterium (shown below).
A topological model for the insertion of NixA, the high affinity nickel transport protein into the cytoplasmic membrane has been established, and we have identified amino acid residues within the membrane domain that are critical for transport function (shown below). We are also seeking alternate nickel transporters that can complement the activity of NixA. Glutamine synthetase which uses ammonia, the product of urea hydrolysis for production of glutamine from glutamate, has been characterized and demonstrated as essential for H. pylori survival. The role of Hpn, the histidine-rich protein, in bismuth sensitivity has also been elucidated.
We have postulated that NixA (nickel transporter) and other newly identified proteins are necessary for full activation of H. pylori urease. A model for such activation requires recruitment of nickel ions on the cell surface, delivery across the outer membrane and periplasmic space, active transport across the crytoplasmic membrane, establishment of a reservoir of the metal ion in the cytosol, and finally insertion into the catalytic site of the newly synthesized apoenzyme. Since urea hydrolysis is 100%-dependent on nickel incorporation into urease, understanding this process could uncover targets for intervention of colonization of the gastric mucosa.
We are using molecular genetic techniques, protein biochemistry, and bacterial physiology methodology: 1) to determine the mechanisms by which urease activity is modulated; 2) to determine the fine structure of NixA, the mechanism of its gene regulation, and its contribution to virulence; and 3) to determine the gene products that mediate transport of nickel ions across the inner and outer membranes of H. pylori.
H. pylori is dependent upon the production of the highly abundant and active metalloenzyme urease for colonization of the human stomach. Thus, H. pylori has an absolute requirement for the transition metal nickel, a required cofactor for urease. To investigate the contribution of genes that are factors in this process, microarray analysis comparing the transcriptome of wild-type H. pylori 26695 cultured in brucella broth containing fetal calf serum (BBF) alone or supplemented with 100 microM NiCl(2) suggested that HP1512 is repressed in the presence of 100 microM supplemental nickel. When measured by comparative real-time quantitative PCR (qPCR), HP1512 transcription was reduced 43-fold relative to the value for the wild type when cultured in BBF supplemented with 10 microM NiCl 2. When grown in unsupplemented BBF, urease activity of an HP1512::cat mutant was significantly reduced compared to the wild type, 4.9 +/- 0.5 micromol/min/mg of protein (n = 7) and 17.1 +/- 4.9 micromol/min/mg of protein (n = 13), respectively (P < 0.0001). In silico analysis of the HP1511-HP1512 (HP1511-1512) intergenic region identified a putative NikR operator upstream of HP1512. Gel shift analysis with purified recombinant NikR verified nickel-dependent binding of H. pylori NikR to the HP1511-1512 intergenic region.
Furthermore, comparative real-time qPCR of four nickel-related genes suggests that mutation of HP1512 results in reduced intracellular nickel concentration relative to wild-type H. pylori 26695. Taken together, these data suggest that HP1512 encodes a NikR-nickel-regulated outer membrane protein. We propose that nickel can enter H. pylori through HP1512 in the outer membrane and is actively transported across the cytoplasmic in an energy-dependent manner by NixA, where it can then be incorporated into urease (see model below).