Ultrastructural, Cytochemical, and Comparative Genomic Evidence of Peroxisomes in Three Genera of Pathogenic Free-Living Amoebae, Including the First Morphological Data for the Presence of This Organelle in Heteroloboseans

Abstract

Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

Keywords: Acanthamoeba; Balamuthia; Naegleria; diaminobenzidine; peroxin content; transmission electron microscopy.

Fig. 1

Identification of peroxisomes in three genera of pathogenic free-living amoebae by transmission electron microscopy. Spherical structures with a dark granular content and limited by a single membrane were observed in Acanthamoeba griffini (A), Acanthamoeba polyphaga (B), Acanthamoeba royreba (C), Acanthamoeba castellanii (D), Balamuthia mandrillaris (E), and Naegleria fowleri (F). These structures ranged in size from 0.2 to 0.9 µm and they were morphologically similar to peroxisomes from mouse liver samples, which are shown for comparison (G, H). M, mitochondrion; Nu, nucleus. Bar = 0.5 µm.

Fig. 2

Peroxisome identification in three genera of pathogenic free-living amoebae by cytochemical staining. Thin sections of trophozoites were treated with diaminobenzidine and hydrogen peroxide to detect catalase activity. Positive reaction products were clearly seen in Acanthamoeba griffini (A), Acanthamoeba polyphaga (B), Acanthamoeba royreba (C), Acanthamoeba castellanii (D), Balamuthia mandrillaris (E) and Naegleria fowleri (F). In all of these amoebae, the labeling was deposited in round structures with a uniform electrondense content. Mouse liver samples were used as a positive control for the cytochemical staining (G). As a negative control for the cytochemical reaction, a mouse liver sample was incubated without H₂O₂ (H). ER, endoplasmic reticulum; M, mitochondrion; Nu, nucleus. Bar = 0.5 µm.

Fig. 3

Phylogenetic analysis of the peroxisomal biogenesis protein Pex1. The consensus tree was obtained with RAxML-HPC2 8.2.12. Bootstrap values >80% are indicated. The tree is rooted midpoint and the scale bar indicates the mean number of amino acid substitutions per site. Sequences are colored to denote the different taxonomic lineages (see box).

Fig. 4

Phylogenetic analysis of the peroxisomal biogenesis protein Pex5. RAxML-HPC2 8.2.12 was used to obtain the consensus tree. Bootstrap values >80% are shown. The tree is midpoint rooted and the scale bar shows the mean number of amino acid substitutions per site. Sequences are colored to indicate their taxonomic lineage (see box).