A Detoxifying Oxygen Reductase in the Anaerobic Protozoan Entamoeba histolytica

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite<jats:named-content content-type="genus-species">Entamoeba histolytica</jats:named-content>. Upon host infection,<jats:named-content content-type="genus-species">E. histolytica</jats:named-content>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<jats:named-content content-type="genus-species">E. histolytica</jats:named-content>. The genome of<jats:named-content content-type="genus-species">E. histolytica</jats:named-content>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<jats:italic>EhFdp1</jats:italic>gene has higher expression in virulent than in nonvirulent<jats:named-content content-type="genus-species">Entamoeba</jats:named-content>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<jats:named-content content-type="genus-species">E. histolytica</jats:named-content>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<jats:named-content content-type="genus-species">E. histolytica</jats:named-content>.</jats:p>

Flavodiiron Oxygen Reductase from Entamoeba histolytica

“…Like the WT protein (11), all the EhFdp1 variants were isolated in the oxidized state with UV-visible absorption bands with maxima at 377 and 456 nm, characteristic of the flavin moiety. The FMN reduction potentials were determined by anaerobic redox titrations (Fig.…”

“…Direct sequencing confirmed that only the desired mutations were inserted. Expression in E. coli BL21(DE3) Gold cells and purification of wild-type (WT) and mutated EhFdp1 were carried out as previously described (11). Because the physiological electron donor to the E. histolytica EhFdp1 is presently unknown, we used an artificial reducing system from E. coli: the flavorubredoxin reductase (FlRd-Red) and the rubredoxin domain (Rd-D) of FlRd, that were expressed and purified as described in Ref.…”

“…FlRd shares a high amino acid sequence identity with the NO-selective FDP from another enterobacterium, Salmonella typhimurium (32), and indeed this type of FDPs (classified as class B (1)) is highly conserved in enterobacteria. On the other end of the substrate specificity spectrum lie the FDPs from G. intestinalis (10) and E. histolytica (11) that have a marked substrate preference toward O 2 , together with the FDPs from T. maritima (25) and the methanogenic Archaeon Methanothermobacter marburgensis (22).…”

“…Although most known FDPs are encoded in the genomes of prokaryotes, several examples are found in multicellular and unicellular eukaryotes (6,10,(17)(18)(19)(20)(21). Among the latter, the enzymes from the anaerobic protozoan pathogens Giardia intestinalis (10), Trichomonas vaginalis (20), and Entamoeba histolytica (Fdp1) (11) have been investigated.…”

“…In pathogens, FDPs were suggested to enable survival in the hostile environment encountered during host infection, contributing to the detoxification of O 2 , particularly in the gut (10,11), and of NO produced by the host immune response (12)(13)(14)(15)(16). Although most known FDPs are encoded in the genomes of prokaryotes, several examples are found in multicellular and unicellular eukaryotes (6,10,(17)(18)(19)(20)(21).…”