Functional Characterization of Peroxiredoxins from the Human Protozoan Parasite <i>Giardia intestinalis</i>

<div><p>The microaerophilic protozoan parasite <i>Giardia intestinalis</i>, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O₂ and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O₂, NO and superoxide anion (O₂^−•) have been recently reported for <i>Giardia</i>, but it is yet unknown how the parasite copes with H₂O₂ and peroxynitrite (ONOO⁻). <i>Giardia</i> encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), <i>Gi</i>Prx1a and <i>Gi</i>Prx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. <i>Gi</i>Prx1a and a truncated form of <i>Gi</i>Prx1b (delta<i>Gi</i>Prx1b) were expressed in <i>Escherichia coli</i>, purified and functionally characterized. Both Prxs effectively metabolize H₂O₂ and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and <i>E. coli</i> thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO⁻ rapidly (<i>k</i> = 4×10⁵ M⁻¹ s⁻¹ and 2×10⁵ M⁻¹ s⁻¹ at 4°C, for <i>Gi</i>Prx1a and delta<i>Gi</i>Prx1b, respectively). Consistent with a protective role against oxidative stress, expression of <i>Gi</i>Prx1a (but not delta<i>Gi</i>Prx1b) is induced in parasitic cells exposed to air O₂ for 24 h. Based on these results, <i>Gi</i>Prx1a and delta<i>Gi</i>Prx1b are suggested to play an important role in the antioxidant defense of <i>Giardia</i>, possibly contributing to pathogenesis.</p>