Hydrogenosomes in the diplomonad Spironucleus salmonicida

Abstract

Acquisition of the mitochondrion is a key event in the evolution of the eukaryotic cell, but diversification of the organelle has occurred during eukaryotic evolution. One example of such mitochondria-related organelles (MROs) are hydrogenosomes, which produce ATP by substrate-level phosphorylation with hydrogen as a byproduct. The diplomonad parasite Giardia intestinalis harbours mitosomes, another type of MRO. Here we identify MROs in the salmon parasite Spironucleus salmonicida with similar protein import and Fe-S cluster assembly machineries as in Giardia mitosomes. We find that hydrogen production is prevalent in the diplomonad genus Spironucleus, and that S. salmonicida MROs contain enzymes characteristic of hydrogenosomes. Evolutionary analyses of known hydrogenosomal components indicate their presence in the diplomonad ancestor, and subsequent loss in Giardia. Our results suggest that hydrogenosomes are metabolic adaptations predating the split between parabasalids and diplomonads, which is deeper than the split between animals and fungi in the eukaryotic tree.

Figure 1. Spironucleus salmonicida harbours MROs.

(a) A merged immunofluoresence and phase-contrast image of S. salmonicida cells expressing Cpn60-3xHA. The epitope-tagged protein is detected by monoclonal anti-HA Alexa Fluor 488 (green). Nuclei are labelled with DAPI (4',6-diamidino-2-phenylindole, blue). Scale bar, 10 μm. (b) Two double-membraned organelles (H) in a transmission electron micrograph of an S. salmonicida cell expressing Cpn60-3xHA. Scale bar, 500 nm. (c) Enlarged view of the double-membraned organelle marked by a black box in b. Scale bar, 250 nm. (d) Immunoelectron microscopy performed towards the HA epitope in S. salmonicida cells expressing Cpn60-3xHA decorates electron-dense organelles with gold label, consistent with the organelle harbouring Cpn60 and being an MRO (scale bar, 500 nm).

Figure 2. Hydrogen production in Spironucleus and evidence of hydrogenosomes in Spironucleus salmonicida.

(a) Hydrogen production was assayed in three strains of Spironucleus (S. salmonicida, S. barkhanus and S. vortens). The percentage of hydrogen gas in the head-space of cultures (%H₂) after 48 h of growth from 10⁵ cells per ml at the prefered growth temperature (S. salmonicida 16 °C, S. barkhanus 4 °C and S. vortens 22 °C) was recorded. The log₂(%H₂) is plotted for each strain. All three strains produce hydrogen, with S. vortens producing eight- to tenfold higher amounts than S. salmonicida and S. barkhanus. Error bars indicate s.d. (n=3). An S. salmonicida cell expressing (b) Cpn60-3xHA (green) and (c) PFOR5-2xOLLAS (red). (d) Merged imaged of b and c, with DAPI (4',6-diamidino-2-phenylindole)-labelled nuclei (blue) and phase-contrast image show colocalization of the green and red label. An S. salmonicida cell expressing (e) HydF-3xHA (green) and (f) [FeFe]-hydrogenase 5-2xOLLAS (red). (g) Merged imaged of e and f show colocalization of the green and red label. An S. salmonicida cell expressing (h) HydF-3xHA (green) and (i) [FeFe]-hydrogenase 6-2xOLLAS (red). (j) Merged imaged of h and i display colocalization of the green and red label. An S. salmonicida cell expressing (k) Cpn60-3xHA (green) and (l) HydE-2xOLLAS (red). (m) Merged imaged of k and l demonstrate colocalization of the green and red label. An S. salmonicida cell expressing (n) Cpn60-3xHA (green) and (o) HydF-2xOLLAS (red). (p) Merged imaged of n and o show colocalization of the green and red label. An S. salmonicida cell expressing (q) Cpn60-3xHA (green) and (r) HydG-2xOLLAS (red). (s) Merged imaged of q and r show colocalization of the green and red label. (all scale bars 5 μm).

Figure 3. Phylogenies of hydrogenase maturases HydEFG.

Phylogenies of (a) HydE, (b) HydF and (c) HydG inferred from ML analyses. S. salmonicida homologues that localize to the hydrogenosome are indicated by bold font. ML bootstrap values and Bayesian inference posterior probabilities are shown for bipartitions with support greater than 50% and 0.5, respectively. Excavata and Plantae sequences are coloured brown and green, respectively. Black branches indicate prokaryotic sequences. See Supplementary Figs S9-S11, S13 and S15 for detailed phylogenies.

Figure 4. Proteomic analyses of a hydrogenosome-enriched fraction.

(a) Nycodenz discontinuous gradient for the preparation of a hydrogenosome-enriched fraction starting from fraction P50. The Nycodenz interfaces are indicated along with the corresponding fraction or pellet designation. (b) Western Blot of recovered fractions from a in addition to the P50 fraction, which is the starting material for the Nycodenz discontinuous gradient. Hydrogenosomes are detected in P50 and in fractions N2 and N3, with N3 containing the largest amount of epitope-tagged Cpn60. (c) Venn-diagram showing common and differential distribution of proteins identified by LC–MS/MS in fractions P50, N1 and N3. The total number of proteins identified in each fraction is displayed below the fraction designation in bold. The distribution of verified hydrogenosomal proteins in the fractions is indicated below the total number of identified proteins if applicable. (d) A S. salmonicida cell expressing 3xHA-tagged HydF (green). Nuclei are labelled with DAPI (4',6-diamidino-2-phenylindole, blue). (e) Same cell as in d, stained for 2xOLLAS-tagged acetyl-CoA synthetase. (f) Merged image of d and e. (g) DIC image merged with d–f. HydF and acetyl-CoA synthetase colocalize to the hydrogenosome. The cell was imaged by laser-scanning confocal microscopy. Scale bar, 5 μm.

Figure 5. The minimal hydrogenosomal proteome in S. salmonicida indicate a hypothetical organellar pyruvate metabolism.

Proteins identified in both the mitosome of G. intestinalis and the hydrogenosome of S. salmonicida are shown in red. Proteins identified in the S. salmonicida hydrogenosome but not in the G. intestinalis mitosome are shown in yellow. Potential end products of hydrogenosome metabolism are in boxes. Fd_OX, Fd_RED, oxidized and reduced ferredoxin; AcCoA, acetyl-CoA, PFOR, pyruvate ferredoxin oxidoreductase; SHMT, serine hydroxylmethyltransferase; GDC, glycine decarboxylase complex; PYR, pyruvate.