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Antisense RNA Modulation of Alkyl Hydroperoxide Reductase Levels in Helicobacter pylori Correlates with Organic Peroxide Toxicity but Not Infectivity

Departments of Microbiology and Immunology,¹ Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7,² Digestive Health Center of Excellence, Division of Gastroenterology and Hepatology,³ Department of Medicine, Division of Infectious Diseases and International Health,⁴ Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-1340⁵

Received 3 January 2007/ Accepted 23 February 2007

Much of the gene content of the human gastric pathogen Helicobacter pylori (1.7-Mb genome) is considered essential. This view is based on the completeness of metabolic pathways, infrequency of nutritional auxotrophies, and paucity of pathway redundancies typically found in bacteria with larger genomes. Thus, genetic analysis of gene function is often hampered by lethality. In the absence of controllable promoters, often used to titrate gene function, we investigated the feasibility of an antisense RNA interference strategy. To test the antisense approach, we targeted alkyl hydroperoxide reductase (AhpC), one of the most abundant proteins expressed by H. pylori and one whose function is essential for both in vitro growth and gastric colonization. Here, we show that antisense ahpC (as-ahpC) RNA expression from shuttle vector pDH37::as-ahpC achieved an 72% knockdown of AhpC protein levels, which correlated with increased susceptibilities to hydrogen peroxide, cumene, and tert-butyl hydroperoxides but not with growth efficiency. Compensatory increases in catalase levels were not observed in the knockdowns. Expression of single-copy antisense constructs (expressed under the urease promoter and containing an fd phage terminator) from the rdxA locus of mouse-colonizing strain X47 achieved a 32% knockdown of AhpC protein levels (relative to wild-type X47 levels), which correlated with increased susceptibility to organic peroxides but not with mouse colonization efficiency. Our studies indicate that high levels of AhpC are not required for in vitro growth or for primary gastric colonization. Perhaps AhpC, like catalase, assumes a greater role in combating exogenous peroxides arising from lifelong chronic inflammation. These studies also demonstrate the utility of antisense RNA interference in the evaluation of gene function in H. pylori.

Published ahead of print on 2 March 2007.

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