In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site.

Since its discovery in 1982 by Marshall and Warren [1], Helicobacter pylori has been shown to be the causative agent of chronic gastritis, gastric and duodenal ulcers, and mucosa-associated lymphoid tissue (MALT) lymphoma [2,3]. Many infected individuals fail to develop clinical symptoms of disease, although all exhibit histologic evidence of pathology [4,5]. However, the exact mechanisms employed by H. pylori to cause such varied host pathology is unknown. Research on H. pylori pathogenesis has been partially hampered by insufficient and suboptimal complementation systems. Complementation is the process of introducing a wild-type copy of a gene into a mutant strain disrupted for the same gene, leading to restoration of phenotypes that were lost in the mutant. Current methods used for gene complementation in H. pylori include shuttle plasmids and chromosomally based systems. Shuttle plasmids for H. pylori are hybrids comprised of H. pylori cryptic plasmids and plasmids isolated from Escherichia coli, and they contain antibiotic resistance markers for selection [6–10]. Unfortunately, these shuttle plasmids transform some H. pylori strains at low frequencies and completely fail to transform other H. pylori strains [6,8]. Chromosomally based complementation systems in H. pylori have been used to complement genes such as luxS, recG, rdxA, comH, and ureB, but these systems interrupt important chromosomal segments or can be susceptible to polar effects that may influence expression of other H. pylori genes [11–15]. Therefore, improved genetic complementation tools are needed for confirming the function of H. pylori genes. This study was undertaken to develop a different complementation system and was validated by complementing the gene encoding H. pylori arginase, rocF. Arginase is an enzyme that hydrolyzes L-arginine to L-ornithine and urea. The urea is then hydrolyzed by urease to produce CO2 and ammonia, the latter serving to neutralize harmful stomach acid for H. pylori [16,17]. Arginase-mediated hydrolysis of arginine consumes the same substrate required for nitric oxide (NO) synthesis [18], an important innate host defense mechanism. The consumption of host L-arginine by H. pylori arginase can lead to dysregulation of the host immune response through inhibition of T-cell proliferation and CD3ζ chain expression on the T-cell surface [19]. Herein, we describe a gene complementation system that utilizes a suicide plasmid vector containing a specific intergenic region (between genes hp0203 and hp0204) from the H. pylori chromosome. These regions were separated into two fragments, IR203 and IR204, flanking a multicloning site. Once introduced into the bacterium by electroporation, these intergenic regions can undergo double cross-over homologous recombination into the bacterial chromosome, thereby inserting any genes cloned within the multicloning site and eliminating extraneous plasmid sequences. IR203 and IR204 regions were initially selected because the region represents one of the largest intergenic regions in the H. pylori genome. Moreover, this region is reasonably well conserved between the sequenced H. pylori strains (26695 and J99) [20,21]. Additionally, the hp0203 and hp0204 hypothetical genes are adjacent in 26695 and J99 strains. The hp0203 and hp0204 genes are transcribed towards each other, greatly minimizing possible polar effects, and avoiding insertion of DNA fragments into putative promoter regions. Finally, no annotated H. pylori genes will be disrupted by this chromosomally based strategy. By using this plasmid vector system, the arginase mutant was complemented for the first time in H. pylori, and phenotypic effects lost in the rocF mutant H. pylori could be restored via complementation as determined by arginase activity, Western blot, and L-arginine consumption. It was also shown that the complementation system can be used for in vivo experiments without supplying antibiotics to animals. This technology provides a general method for complementing H. pylori mutants.