Organelles in Blastocystis that blur the distinction between mitochondria and hydrogenosomes

Abstract

Blastocystis is a unicellular stramenopile of controversial pathogenicity in humans. Although it is a strict anaerobe, Blastocystis has mitochondrion-like organelles with cristae, a transmembrane potential and DNA. An apparent lack of several typical mitochondrial pathways has led some to suggest that these organelles might be hydrogenosomes, anaerobic organelles related to mitochondria. We generated 12,767 expressed sequence tags (ESTs) from Blastocystis and identified 115 clusters that encode putative mitochondrial and hydrogenosomal proteins. Among these is the canonical hydrogenosomal protein iron-only [FeFe] hydrogenase that we show localizes to the organelles. The organelles also have mitochondrial characteristics, including pathways for amino acid metabolism, iron-sulfur cluster biogenesis, and an incomplete tricarboxylic acid cycle as well as a mitochondrial genome. Although complexes I and II of the electron transport chain (ETC) are present, we found no evidence for complexes III and IV or F1Fo ATPases. The Blastocystis organelles have metabolic properties of aerobic and anaerobic mitochondria and of hydrogenosomes. They are convergently similar to organelles recently described in the unrelated ciliate Nyctotherus ovalis. These findings blur the boundaries between mitochondria, hydrogenosomes, and mitosomes, as currently defined, underscoring the disparate selective forces that shape these organelles in eukaryotes.

Figure 1

Confocal Immunofluorescence Microscopy Images Showing the Subcellular Localization of [FeFe] Hydrogenase in Blastocystis The images show a Blastocystis cell with one nucleus and two mitochondrion-like organelles. (A) DAPI staining of the nucleus (bright) and mitochondrial DNA (faint; see arrows); (B) immunolocalization of [FeFe] hydrogenase using anti-BlastocystisHyd antibody; (C) MitoTracker orange staining of the mitochondrion-like organelles; (D) colocalization of MitoTracker orange, anti-BlastocystisHyd antibody, and DAPI in the mitochondrion-like organelles; and (E) DIC image of the Blastocystis cell.

Figure 2

Analysis of the Organellar Genome Fragment (A) Schematic representation of an ∼6 kb fragment from the Blastocystis organellar genome. Black boxes represent tRNA genes, which are identified by the amino acid they incorporate. nad3 and nad6 are NADH dehydrogenase subunits. M^e is the elongator methionine tRNA, and M^f is the initiator (formyl-) methionine tRNA. rns and rnl are the small and large subunit ribosomal RNA genes, respectively. Genes are not drawn according to scale. (B) Maximum-likelihood tree derived from analysis of six concatenated mitochondrial genome-encoded proteins (Nad2, Nad3, Nad4, Nad6, Nad9, and Rps10). The clade containing eukaryotes is shaded. The analysis was based on 1137 unambiguously aligned positions. The tree was rooted arbitrarily with Dehalococcoides and Cyanobacteria. Numbers at nodes represent ML bootstrap values calculated with IQPNNI. Black dots on branches indicate bootstrap values ≥90%. Values below 50% are not shown.

Figure 3

Proposed Metabolic Map of the Blastocystis Mitochondrion-like Organelles Proteins with predicted leader peptides have solid black outlines and protein complexes for which leader peptides for only some of the subunits have been predicted have dashed black outlines. The blue pathway represents the conversion of pyruvate to acetyl-CoA via the pyruvate dehydrogenase complex (PDH) or pyruvate:ferredoxin oxidoreductase (PFO). PFO reduces flavodoxin (Fld) and the [FeFe] hydrogenase (HYD) oxidizes flavodoxin with the concomitant production of molecular hydrogen; the question mark indicates that hydrogen production has not been demonstrated for Blastocystis. Acetyl-CoA is converted by the acetate:succinate CoA transferase (ASCT) to acetate (acetate production has not been tested for Blastocystis, but we identified an acetyl-CoA hydrolase like that known to function as ASCT in Trichomonas hydrogenosomes [35]). Transporters and translocators are shown in yellow. Components of the ETC and the partial TCA cycle are orange. Proteins involved in DNA transcription and translation are light pink. The mitochondrial part of the urea cycle is green. Pathways for the metabolism of the amino acids leucine, valine, and isoleucine are purple. Protein import and folding are light green. Pathways of the iron-sulfur cluster assembly are depicted in brown. Light blue represents the glycine-cleavage-system pathway. The question mark next to propionate shows that propionate production has not been assayed for Blastocystis; propionate is a metabolic end product of succinate degradation. For a complete list of abbreviations and numbers used in this figure see the Supplemental Data.