Evolutionary analysis of cellular reduction and anaerobicity in the hyper-prevalent gut microbe Blastocystis.

Blastocystis is the most prevalent microbial eukaryote in the human and animal gut, yet its role as commensal or parasite is still under debate. Blastocystis has clearly undergone evolutionary adaptation to the gut environment and possesses minimal cellular compartmentalization, reduced anaerobic mitochondria, no flagella, and no reported peroxisomes. To address this poorly understood evolutionary transition, we have taken a multi-disciplinary approach to characterize Proteromonas lacertae, the closest canonical stramenopile relative of Blastocystis. Genomic data reveal an abundance of unique genes in P. lacertae but also reductive evolution of the genomic complement in Blastocystis. Comparative genomic analysis sheds light on flagellar evolution, including 37 new candidate components implicated with mastigonemes, the stramenopile morphological hallmark. The P. lacertae membrane-trafficking system (MTS) complement is only slightly more canonical than that of Blastocystis, but notably, we identified that both organisms encode the complete enigmatic endocytic TSET complex, a first for the entire stramenopile lineage. Investigation also details the modulation of mitochondrial composition and metabolism in both P. lacertae and Blastocystis. Unexpectedly, we identify in P. lacertae the most reduced peroxisome-derived organelle reported to date, which leads us to speculate on a mechanism of constraint guiding the dynamics of peroxisome-mitochondrion reductive evolution on the path to anaerobiosis. Overall, these analyses provide a launching point to investigate organellar evolution and reveal in detail the evolutionary path that Blastocystis has taken from a canonical flagellated protist to the hyper-divergent and hyper-prevalent animal and human gut microbe.

The free-living flagellate Paratrimastix pyriformis uses a distinct mitochondrial carrier to balance adenine nucleotide pools.

Paratrimastix pyriformis is a free-living flagellate thriving in low-oxygen freshwater sediments. It belongs to the group Metamonada along with human parasites, such as Giardia and Trichomonas. Like other metamonads, P. pyriformis has a mitochondrion-related organelle (MRO) which in this protist is primarily involved in one-carbon folate metabolism. The MRO contains four members of the solute carrier family 25 (SLC25) responsible for the exchange of metabolites across the mitochondrial inner membrane. Here, we characterise the function of the adenine nucleotide carrier PpMC1 by thermostability shift and transport assays. We show that it transports ATP, ADP and, to a lesser extent, AMP, but not phosphate. The carrier is distinct in function and origin from both ADP/ATP carriers and ATP-Mg/phosphate carriers, and it most likely represents a distinct class of adenine nucleotide carriers.

Reduced mitochondria provide an essential function for the cytosolic methionine cycle.

The loss of mitochondria in oxymonad protists has been associated with the redirection of the essential Fe-S cluster assembly to the cytosol. Yet as our knowledge of diverse free-living protists broadens, the list of functions of their mitochondrial-related organelles (MROs) expands. We revealed another such function in the closest oxymonad relative, Paratrimastix pyriformis, after we solved the proteome of its MRO with high accuracy, using localization of organelle proteins by isotope tagging (LOPIT). The newly assigned enzymes connect to the glycine cleavage system (GCS) and produce folate derivatives with one-carbon units and formate. These are likely to be used by the cytosolic methionine cycle involved in S-adenosyl methionine recycling. The data provide consistency with the presence of the GCS in MROs of free-living species and its absence in most endobionts, which typically lose the methionine cycle and, in the case of oxymonads, the mitochondria.

The Genome of the Mitochondrion-Related Organelle in Cepedea longa, a Large Endosymbiotic Opalinid Inhabiting the Recta of Frogs.

Mitochondrion-related organelles (MROs) are loosely defined as degenerated mitochondria in anaerobic and microaerophilic lineages. Opalinids are commonly regarded as commensals in the guts of cold-blooded amphibians. It may represent an intermediate adaptation stage between the conventional aerobic mitochondria and derived anaerobic MROs. In the present study, we sequenced and analyzed the MRO genome of Cepedea longa. It has a linear MRO genome with large inverted repeat gene regions at both ends. Compared to Blastocystis and Proteromonas lacertae, the MRO genome of C. longa has a higher G + C content and repeat sequences near the central region. Although three Opalinata species have different morphological characteristics, phylogenetic analyses based on eight concatenated nad genes indicate that they are close relatives. The phylogenetic analysis showed that C. longa clustered with P. lacertae with strong support. The 18S rRNA gene-based phylogeny resolved the Opalinea clade as a sister clade to Karotomorpha, which then further grouped with Proteromonas. The paraphyly of Proteromonadea needs to be verified due to the lack of MRO genomes for key species, such as Karotomorpha, Opalina and Protoopalina. Besides, our dataset and analyses offered slight support for the paraphyly of Bigyra.

The mitosome of the anaerobic parasitic protist Entamoeba histolytica: A peculiar and minimalist mitochondrion-related organelle.

The simplest class of mitochondrion-related organelles (MROs) is the mitosome, an organelle present in a few anaerobic protozoan parasites such as Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium parvum. E. histolytica causes amoebiasis in humans, deemed as one of the important, yet neglected tropical infections in the world. Much of the enigma of the E. histolytica mitosome circles around the obvious lack of a majority of known mitochondrial components and functions exhibited in other organisms. The identification of enzymes responsible for sulfate activation (AS, IPP, and APSK) and a number of lineage-specific proteins such as the outer membrane beta-barrel protein (MBOMP30), and transmembrane domain-containing proteins that bind to various organellar proteins (ETMP1, ETMP30, EHI_170120, and EHI_099350) showcased the remarkable divergence of this organelle compared to the other MROs of anaerobic protozoa. Here, we summarize the findings regarding the biology of the mitosomes in E. histolytica, from their discovery up to the present understanding of its roles and interactions. We also include current advances and future perspectives on the biology, biochemistry, and evolution of the mitosomes of E. histolytica.

Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics.

BACKGROUND: Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. RESULTS: We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5'-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. CONCLUSIONS: This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists.

Adaptations of ciliates to hypoxic environments have arisen independently several times. Studies on mitochondrion-related organelle (MRO) metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes. To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis, mass-culture and single-cell transcriptomes of two anaerobic species, Metopus laminarius (class Armophorea) and Plagiopyla cf. narasimhamurtii (class Plagiopylea), were sequenced and their MRO metabolic maps were compared. In addition, we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes (i.e., Armophorea, Litostomatea, Muranotrichea, Oligohymenophorea, Parablepharismea and Plagiopylea). We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates. The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates, even among closely related species. Notably, our findings indicate the existence of group-specific functional relics of electron transport chains (ETCs). Detailed group-specific ETC functional patterns are as follows: full oxidative phosphorylation in Oligohymenophorea and Muranotrichea; only electron-transfer machinery in Armophorea; either of these functional types in Parablepharismea; and ETC functional absence in Litostomatea and Plagiopylea. These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times. Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00147-w.

The Distribution of Genes Associated With Regulated Cell Death Is Decoupled From the Mitochondrial Phenotypes Within Unicellular Eukaryotic Hosts.

Genetically regulated cell death (RCD) occurs in all domains of life. In eukaryotes, the evolutionary origin of the mitochondrion and of certain forms of RCD, in particular apoptosis, are thought to coincide, suggesting a central general role for mitochondria in cellular suicide. We tested this mitochondrial centrality hypothesis across a dataset of 67 species of protists, presenting 5 classes of mitochondrial phenotypes, including functional mitochondria, metabolically diversified mitochondria, functionally reduced mitochondria (Mitochondrion Related Organelle or MRO) and even complete absence of mitochondria. We investigated the distribution of genes associated with various forms of RCD. No homologs for described mammalian regulators of regulated necrosis could be identified in our set of 67 unicellular taxa. Protists with MRO and the secondarily a mitochondriate Monocercomonoides exilis display heterogeneous reductions of apoptosis gene sets with respect to typical mitochondriate protists. Remarkably, despite the total lack of mitochondria in M. exilis, apoptosis-associated genes could still be identified. These same species of protists with MRO and M. exilis harbored non-reduced autophagic cell death gene sets. Moreover, transiently multicellular protist taxa appeared enriched in apoptotic and autophagy associated genes compared to free-living protists. This analysis suggests that genes associated with apoptosis in animals and the presence of the mitochondria are significant yet non-essential biological components for RCD in protists. More generally, our results support the hypothesis of a selection for RCD, including both apoptosis and autophagy, as a developmental mechanism linked to multicellularity.

Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences.

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.

NommPred: Prediction of Mitochondrial and Mitochondrion-Related Organelle Proteins of Nonmodel Organisms.

To estimate the functions of mitochondria of diverse eukaryotic nonmodel organisms in which the mitochondrial proteomes are not available, it is necessary to predict the protein sequence features of the mitochondrial proteins computationally. Various prediction methods that are trained using the proteins of model organisms belonging particularly to animals, plants, and fungi exist. However, such methods may not be suitable for predicting the proteins derived from nonmodel organisms because the sequence features of the mitochondrial proteins of diversified nonmodel organisms can differ from those of model organisms that are present only in restricted parts of the tree of eukaryotes. Here, we proposed NommPred, which predicts the mitochondrial proteins of nonmodel organisms that are widely distributed over eukaryotes. We used a gradient boosting machine to develop 2 predictors-one for predicting the proteins of mitochondria and the other for predicting the proteins of mitochondrion-related organelles that are highly reduced mitochondria. The performance of both predictors was found to be better than that of the best method available.