Fabrizio Testa

The O2-scavenging flavodiiron protein in the human parasite Giardia intestinalis.

The flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O2-reductase activity (>40 s-1), but very low NO-reductase activity (∼0.2 s-1); O2 reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (Km ≤ 2 μm), producing H2O. The three-dimensional structure of the oxidized protein determined at 1.9Å resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the non-heme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O2, allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.

Redox properties of the oxygen-detoxifying flavodiiron protein from the human parasite Giardia intestinalis.

Flavodiiron proteins (FDPs) are enzymes identified in prokaryotes and a few pathogenic protozoa, which protect microorganisms by reducing O(2) to H(2)O and/or NO to N(2)O. Unlike most prokaryotic FDPs, the protozoan enzymes from the human pathogens Giardia intestinalis and Trichomonas vaginalis are selective towards O(2). UV/vis and EPR spectroscopy showed that, differently from the NO-consuming bacterial FDPs, the Giardia FDP contains an FMN with reduction potentials for the formation of the single and the two-electron reduced forms very close to each other (E(1)=-66+/-15mV and E(2)=-83+/-15mV), a condition favoring destabilization of the semiquinone radical. Giardia FDP contains also a non-heme diiron site with significantly up-shifted reduction potentials (E(1)=+163+/-20mV and E(2)=+2+/-20mV). These properties are common to the Trichomonas hydrogenosomal FDP, and likely reflect yet undetermined subtle structural differences in the protozoan FDPs, accounting for their marked O(2) specificity.

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN=116+/-10s(-1) at 1microM NO, T=37 degrees C). The activity is [O(2)]-dependent and characterized by an apparent K(M,O2)=22+/-7microM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with 5mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.

Giardia intestinalis Escapes Oxidative Stress by Colonizing the Small Intestine: A Molecular Hypothesis

Giardia intestinalis is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. Giardia possesses an O2‐scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O2‐detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O2 to H2O rapidly (∼3 μM O2 × min × 106 cells at 37 °C) and with high affinity (C50 = 3.4 ± 0.7 μM O2). Following a short‐term (minutes) exposure to H2O2 ≥ 100 μM, the O2 consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome‐inhibitor MG132. Instead, H2O2 does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of Giardia to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.

The superoxide reductase from the early diverging eukaryote Giardia intestinalis.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(•-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(•-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

Functional characterization of peroxiredoxins from the human protozoan parasite Giardia intestinalis.

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.

Antioxidant defence systems in the protozoan pathogen Giardia intestinalis

The microaerophilic protist Giardia intestinalis is the causative agent of giardiasis, one of the most common intestinal infectious diseases worldwide. The pathogen lacks not only respiratory terminal oxidases (being amitochondriate), but also several conventional antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase. In spite of this, since living attached to the mucosa of the proximal small intestine, the parasite should rely on an efficient antioxidant system to survive the oxidative and nitrosative stress conditions found in this tract of the human gut. Here, we review current knowledge on the antioxidant defence systems in G. intestinalis, focusing on the progress made over the last decade in the field. The relevance of this research and future perspectives are discussed.

Superoxide reductase from Giardia intestinalis: structural characterization of the first SOR from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage.