A detoxifying oxygen reductase in the anaerobic protozoan Entamoeba histolytica

Abstract

We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

Fig 1

EhFdp1 localizes to the cytoplasm and protein abundance increases upon oxygen exposure. (A) Immunofluorescence microscopy analysis of EhFdp1. Phase-contrast (differential interference contrast [DIC]) transmission images are shown. Antibodies: anti-EhFdp1 (1:500; Open Biosystems), anti-rabbit–Alexa 488 (1:1,000; Molecular Probes), anti-HgL (1:50), and anti-mouse–Alexa 594 (1:500; Molecular Probes). Images were collected with a Zeiss LSM700 confocal microscope and filtered using Volocity software (Improvision). (B) Western blot analysis of the effect of oxygen exposure on EhFdp1 expression in E. histolytica HM-1:IMSS. Anti-EhFdp1 antibody (1:500) and HRP-conjugated anti-rabbit antibody (1:1,000; Cell Signaling) were used (1 h of incubation each), and the signal was detected with ECL+ (GE Healthcare). Primary (anti-actin, 1:1,000; MP Biomedicals) and secondary (HRP-conjugated anti-mouse antibody at 1:1,000; Cell Signaling) antibodies were used for the actin control. Densitometric analysis with ImageJ reveals an increase in EhFdp1 expression with increasing oxygen exposure time from ∼2-fold after 1 h (n = 5; P = 7.0 × 10⁻³) to ∼5-fold after 4 h (n = 5; P =: 0.029). (C) Representative Western blot assay displaying EhFdp1 levels increasing with oxygen exposure time.

Fig 2

Production of functional recombinant EhFdp1. (A) UV-visible spectrum of pure recombinant EhFdp1 (15 μM in 20 mM Tris-HCl–18% glycerol [pH 7.5]; A₂₈₀/A₄₅₇ ratio, 9.1). Gray line, magnified visible region (multiplied by 7 for visualization). Inset, SDS-PAGE (12.5% acrylamide) analysis of purified EhFdp1. (B) EPR spectrum of partially reduced EhFdp1 (190 μM in 50 mM Tris-HCl–18% glycerol [pH 7.5]) showing a rhombic signal attributed to the diiron active site in a mixed-valence (Fe^III-Fe^II) state. Reduction was obtained by sodium dithionite addition in the presence of the redox mediators listed in Table S2 in the supplemental material. The spectrum was recorded with a Bruker EMX spectrometer equipped with an ESR 900 continuous-flow helium cryostat (Oxford Instruments).

Fig 3

EhFdp1 is an oxygen reductase. Oxygen consumption was assayed by spectrophotometric (NADH consumption) and amperometric (O₂ consumption determined with a Clark-type oxygen electrode) measurements. Assays were done at room temperature in 50 mM Tris-HCl–18% glycerol (pH 7.5). Electron delivery to EhFDP1 was achieved by assembling a hybrid ET chain composed of NADH, EcRdR, and EcRd. (A) Spectrophotometric NADH consumption was assayed with a reaction mixture volume of 1 ml and sequential addition of 160 μM NADH, 0.17 μM EcRdR, 2.65 μM EcRd, and 36 nM EhFdp1. The decrease in A₃₄₀ shows NADH oxidation at the expense of oxygen reduction mediated by the ET chain with EhFdp1 as the terminal oxygen reductase. (B) Oxygen consumption was assayed with a reaction mixture volume of 1.5 ml and the sequential addition of 1 mM NADH, 0.2 μM EcRdR, 2 μM EcRd, and 150 nM EhFdp1. Oxygen depletion upon the addition of substoichiometric EhFdp1 is shown; addition of catalase (Kat) yielded no production of oxygen, suggesting that EhFdp1 reduces O₂ directly to water.