Functional Characterization of Peroxiredoxins from the Human Protozoan Parasite Giardia intestinalis

Abstract

The microaerophilic protozoan parasite Giardia intestinalis, 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 Giardia, but it is yet unknown how the parasite copes with H2O2 and peroxynitrite (ONOO−). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H2O2 and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO− rapidly (k = 4×105 M−1 s−1 and 2×105 M−1 s−1 at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O2 for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.

An antioxidant response is involved in resistance of Giardia duodenalis to albendazole

“…G. duodenalis belongs to one of the earliest lines of eukaryotic descent and lacks the catalase, SOD, glutathione and glutathione-dependent reductases and peroxidases. However, Giardia contains alternative antioxidants such as (a) a NADH oxidase (NADHox) and a FDP (A-type flavoprotein), that can detoxify O 2 from trophozoites to form water ( Brown et al, 1996 ; Di Matteo et al, 2008 ); (b) a SOR that converts ⋅O 2 - into H 2 O 2 ( Testa et al, 2011 ); (c) 2-cys peroxiredoxins (Pxr1a and Pxr1b) with ability to detoxify H 2 O 2 to form oxygen and water ( Mastronicola et al, 2014 ); and (d) cysteine as the major molecular antioxidant. Based on a reverse proteomics (effect-to-cause) strategy to search for the expression of the aforementioned enzymes, two of these enzymes, namely NADHox and Pxr1a, could be detected and were found overexpressed in ABZ-resistant clones.…”

Giardia lamblia: missing evidence for a canonical thioredoxin system

“…It has a microaerophilic/anaerobic lifestyle which renders it vulnerable to oxygen and reactive oxygen species (Gillin and Diamond, 1981;Lloyd et al 2000;Paget et al 2004). Like other microaerophilic protist parasites, such as Trichomonas vaginalis (Coombs et al 2004) and Entamoeba histolytica (Arias et al 2007), G. lamblia possesses antioxidant enzyme pathways for the defence against oxidative stress (Mastronicola et al 2014(Mastronicola et al , 2016. It is well established that the thioredoxin-mediated redox system, comprising thioredoxin reductase (TrxR), the electron shuttle protein thioredoxin, and peroxiredoxins (Lu and Holmgren, 2014), plays a central role in the antioxidant defence.…”

Gene expression changes during Giardia–host cell interactions in serum-free medium

“…Genes encoding nitroreductase, peroxiredoxin, FixW-like proteins and thioredoxin reductase were up-regulated after 2.5 h (Table S1 and Fig. S2) and their protein products have been shown to protect against oxidative stress in Giardia and other parasitic protozoa [11][12][13][14]. Glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, key enzymes in the pentose phosphate pathway, show more than 10-fold up-regulation after 2.5 h of interaction (Table S1).…”