BACKGROUND: Hydrogenosomes are a specific type of mitochondria that have adapted for life under anaerobiosis. Limited availability of oxygen has resulted in the loss of the membrane-associated respiratory chain, and consequently in the generation of minimal inner membrane potential (Δψ), and inefficient ATP synthesis via substrate-level phosphorylation. The changes in energy metabolism are directly linked with the organelle biogenesis. In mitochondria, proteins are imported across the outer membrane via the Translocase of the Outer Membrane (TOM complex), while two Translocases of the Inner Membrane, TIM22, and TIM23, facilitate import to the inner membrane and matrix. TIM23-mediated steps are entirely dependent on Δψ and ATP hydrolysis, while TIM22 requires only Δψ. The character of the hydrogenosomal inner membrane translocase and the mechanism of translocation is currently unknown. RESULTS: We report unprecedented modification of TIM in hydrogenosomes of the human parasite Trichomonas vaginalis (TvTIM). We show that the import of the presequence-containing protein into the hydrogenosomal matrix is mediated by the hybrid TIM22-TIM23 complex that includes three highly divergent core components, TvTim22, TvTim23, and TvTim17-like proteins. The hybrid character of the TvTIM is underlined by the presence of both TvTim22 and TvTim17/23, association with small Tim chaperones (Tim9-10), which in mitochondria are known to facilitate the transfer of substrates to the TIM22 complex, and the coupling with TIM23-specific ATP-dependent presequence translocase-associated motor (PAM). Interactome reconstruction based on co-immunoprecipitation (coIP) and mass spectrometry revealed that hybrid TvTIM is formed with the compositional variations of paralogs. Single-particle electron microscopy for the 132-kDa purified TvTIM revealed the presence of a single ring of small Tims complex, while mitochondrial TIM22 complex bears twin small Tims hexamer. TvTIM is currently the only TIM visualized outside of Opisthokonta, which raised the question of which form is prevailing across eukaryotes. The tight association of the hybrid TvTIM with ADP/ATP carriers (AAC) suggests that AAC may directly supply ATP for the protein import since ATP synthesis is limited in hydrogenosomes. CONCLUSIONS: The hybrid TvTIM in hydrogenosomes represents an original structural solution that evolved for protein import when Δψ is negligible and remarkable example of evolutionary adaptation to an anaerobic lifestyle.

• Keywords
• Trichomonas vaginalis, Hydrogenosomes, Mitochondria, Parasite, Presequence translocase-associated motor, Protein import machinery, TIM22 complex, TIM23 complex,
• MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondria metabolism   MeSH
• Organelles metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Protein Transport * MeSH
• Trichomonas vaginalis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Protozoan Proteins MeSH

The endobiotic flagellate Monocercomonoides exilis is the only known eukaryote to have lost mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the capability of proteins from this eukaryote to enter emerging mitochondria using a specifically designed in vitro assay. Hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases the import was confirmed by the overexpression of their tagged version in T. vaginalis. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited clear signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishment of the organelle and shaping of its proteome in the initial stages of endosymbiosis.

• Keywords
• evolution of protein targeting, hydrogenosome, mitochondrion-free eukaryote, protein import,
• MeSH
• Eukaryota * metabolism   MeSH
• Mitochondria metabolism   MeSH
• Organelles chemistry   metabolism   MeSH
• Protozoan Proteins * metabolism   MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protozoan Proteins * MeSH

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale's eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

• Keywords
• Caviomonadidae, Fornicata, caviomonads, codon reassignment, hydrogenosome, mitochondrial evolution, mitosome,
• Publication type
• Journal Article MeSH

Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40-70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis.

• Keywords
• TVV, Trichomonasvirus, exosome, extracellular vesicle, proteomics, tsRNA,
• Publication type
• Journal Article MeSH

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.

• MeSH
• Phylogeny MeSH
• Membrane Proteins metabolism   MeSH
• Membrane Transport Proteins metabolism   MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondria metabolism   MeSH
• Organelles MeSH
• Protein Transport physiology   MeSH
• Mitochondrial Membrane Transport Proteins metabolism   MeSH
• Carrier Proteins genetics   metabolism   physiology   MeSH
• Trichomonas vaginalis metabolism   pathogenicity   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• Membrane Transport Proteins MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Membrane Transport Proteins MeSH
• Carrier Proteins MeSH

The majority of established model organisms belong to the supergroup Opisthokonta, which includes yeasts and animals. While enlightening, this focus has neglected protists, organisms that represent the bulk of eukaryotic diversity and are often regarded as primitive eukaryotes. One of these is the "supergroup" Excavata, which comprises unicellular flagellates of diverse lifestyles and contains species of medical importance, such as Trichomonas, Giardia, Naegleria, Trypanosoma and Leishmania. Excavata exhibits a continuum in mitochondrial forms, ranging from classical aerobic, cristae-bearing mitochondria to mitochondria-related organelles, such as hydrogenosomes and mitosomes, to the extreme case of a complete absence of the organelle. All forms of mitochondria house a machinery for the assembly of Fe-S clusters, ancient cofactors required in various biochemical activities needed to sustain every extant cell. In this review, we survey what is known about the Fe-S cluster assembly in the supergroup Excavata. We aim to bring attention to the diversity found in this group, reflected in gene losses and gains that have shaped the Fe-S cluster biogenesis pathways.

• Keywords
• Evolution, Excavata, Fe–S cluster, Mitochondria,
• MeSH
• Eukaryota cytology   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Iron-Sulfur Proteins metabolism   MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Iron-Sulfur Proteins MeSH
• Iron MeSH

The secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host-parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and β-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used β-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two β-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two β-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite.

• MeSH
• beta-Amylase metabolism   MeSH
• Phylogeny MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Trichomonas vaginalis genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Amylase MeSH
• Protozoan Proteins MeSH