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Compensatory Functions of Two Alkyl Hydroperoxide Reductases in the Oxidative Defense System of Legionella pneumophila

Departments of Microbiology and Immunology,¹ Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7,² Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada,³ Department of Internal Medicine, Division of Infectious Diseases and International Health,⁴ Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-1340⁵

Received 4 May 2006/ Accepted 16 June 2006

Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H₂O₂). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H₂O₂ resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H₂O₂, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were 7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.

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