Abstract
Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90–100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
Highlights
Entamoeba histolytica is a causative agent of amoebiasis, one of the most prevalent parasitic diseases of humans
We identified seven proteins involved in peroxisome biogenesis, the peroxins, and our investigations of their cellular localization indicated that E. histolytica possesses the anaerobic form of peroxisomes that we recently discovered in free-living anaerobic relatives
Proteomic analysis of anaerobic peroxisomes revealed the presence of highly active homodimeric myo-inositol dehydrogenase, which catalyzes NADdependent oxidation of myo-inositol and other cyclitols
Summary
Entamoeba histolytica is a causative agent of amoebiasis, one of the most prevalent parasitic diseases of humans. The organelles do not contain DNA or exhibit classical energy metabolism, yet they are surrounded by a double membrane [6,7,8]. The E. histolytica cytosol contains numerous vesicles, lysosomes, endosomes, and multivesicular bodies, whereas the endoplasmic reticulum (ER) and Golgi apparatus (GA) are difficult to recognize and, in the case of the Golgi, were once thought to be absent [12]. Some specialized forms of the organelle do not contain these key pathways and play various different roles: glycosomes of trypanosomatids are known to contain the first six or seven glycolytic enzymes [19], and plant glyoxysomes engage in the glyoxalate cycle, whereas Woronin bodies of filamentous fungi serve as a physical barrier between two cells upon hyphal wounding [20]
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