Conference Agenda

Mitochondria are central cellular organelles that are often metabolically modified and reduced into various anaerobic types of mitochondria (ATM) in organisms adapted to endobiosis under anaerobic conditions. It is not known whether this trade is reversible as cases of reverse adaptation to a free-living lifestyle are rare. Hence, we decided to analyse ATMs of Hexamita inflata, a diplomonad with potential parasitic ancestry living in anaerobic freshwater sediments on the edge of endobiotic and free-living lifestyles. The genome of H. inflata is about ten times larger compared to genomes of its parasitic relatives. Each cell contains about 20-30 double-membrane ATMs without cristae with a diameter of about 400-600 nm. Genome homology searches identified more than 150 proteins with possible ATM localization. However, similar to ATMs in other diplomonads, the vast majority of nuclear-encoded preproteins lack the mitochondrial N-terminal targeting presequence, which hinders reliable bioinformatic predictions. Therefore, we isolated ATM-enriched fractions and subjected them to a proteomic analysis. The proteome of the organelle contains more than 400 proteins including those involved in the iron-sulphur cluster assembly, pyruvate metabolism, ATP synthesis, hydrogen production, oxygen and ROS detoxification, and amino acid metabolism, indicating the ATM of H. inflata is an ATP-producing hydrogenosome.

Mitochondria and peroxisomes are functionally interrelated oxygen-metabolizing organelles that underwent major functional and structural changes in eukaryotes adapted for life under oxygen-limited or anaerobic conditions. Mitochondria transformed into a range of anaerobic types (ATMs) that reduced the electron transport chain to varying degrees, lost oxidative phosphorylation, and gained components for anaerobic energy metabolism, typically hydrogenase (hydrogenosomes), or lost their energy metabolism completely (mitosomes). In some cases, the organelles themselves were lost. Peroxisomes were proposed to evolve from ER for compartmentalization of a hydrogen peroxide-producing enzyme involved in fatty acid metabolism, although there is a large diversity of other peroxisomal metabolic functions. Based on the absence of peroxisomes in Metamonada, an anaerobic group of protists with ATMs, it was anticipated that peroxisomes would also be lost in other anaerobic eukaryotes. However, the discovery of anaerobic peroxisomes in Archamoebae showed that the presence of peroxisomes is not conditioned by obligater aerobiosis. Through phylogenomic searches, we found that peroxisomes are conserved in some members of anaerobic ciliates, heteroloboseans, and strictly anaerobic fungi of the Neocallimastigomycota group. We attempt to reconstruct the functional losses, gains, and links between anaerobic peroxisomes and ATMs across eukaryotic lineages, providing insights into their co-evolution in anaerobic eukaryotes.

Adaptation of eukaryotes to an anaerobic lifestyle results in reduced forms of mitochondria, such as hydrogenosomes and mitosomes, while peroxisomes were believed to be lost due to the key role of oxygen in peroxisomal metabolism. However, the discovery of anaerobic peroxisomes in Archamoebae challenges this assumption, prompting further research into the evolution of anaerobic eukaryotes. This study focused on Neocallimastix lanati, a strictly anaerobic fungus from the phylum Neocallimastigomycota. Bioinformatic, enzymatic, and proteomic analyses revealed typical hydrogenosomal enzymes related to the production of hydrogen and ATP under anaerobic conditions, components of membrane biogenesis TOM/TIM complexes, iron-sulphur cluster assembly, and amino acid biosynthesis. Notably, we identified components of respiratory complex I and II, dihydroorotate dehydrogenase, and the ATP synthase complex F1. In peroxisomes, 13 common peroxins were identified, covering all functional categories of peroxisomal biogenesis. The search for peroxisomal targeting signals predicted 140 peroxisomal proteins. Functional annotation indicated the presence of multifunctional protein, a typical fatty acid beta-oxidation component, but not acyl-CoA oxidase and catalase. Additionally, enzymes related to amino acid metabolism, cofactor biosynthesis, redox balance, and ROS metabolism were detected without a clear biochemical context. In summary, anaerobic fungi represent a eukaryotic group that possesses both hydrogenosomes and anaerobic peroxisomes.

Trichomonads that possess an undulant membrane, a recurrent flagellum and the costa. It originates at the anterior region of the protozoan and projects towards the posterior region displaying a specific striated pattern produced by the alternation of electron lucent and electron dense bands. Previous cytochemical studies and its isolation with subsequent proteomic analysis showed that the costa contains several proteins. We selected some of the most abundant proteins to produce specific antibodies making it possible to confirm, using immunocytochemistry their presence in the costa. The first protein was designated as costain 1. Here we describe two new proteins who are also localized in the costa, as shown using conventional and expansion immunofluorescence microscopy and transmission electron microscopy. They were designated as costain 2 and 3. In addition, using each protein sequence information, we built models to predict the three-dimensional structure of these proteins using ProtCHOIR software, in the attempt to further elucidate their specific functions in the costa. Our findings showed that the three costa proteins have apparently very different 3-D structures, even though they share some aminoacidic sequences. The characterization of these proteins can help elucidate further the function of this intriguing cytoskeleton structure found in trichomonads.

The internal structure of Giardia flagella, resembling that of other organisms, is characterized by an extensive cytosolic segment before emerging as functional flagella. The region of flagella emergence, known as the flagellar pore, has been a subject of limited study. Here, we analyzed the ultrastructural organization of the Giardia flagella, employing high-resolution microscopy techniques: HR-scanning EM, tomography, atomic force microscopy, and freeze-fracture. Intact cells revealed a prominent surface forming a ring at the flagella exit. The internal organization of this region showed electron-dense plaques on the cytoplasmic side, at the point where the membrane curves at the flagella base. When the plasma membrane was removed, an extra-axonemal structure associated with the outer doublets of the axoneme was visualized in this area. This structure, a novel discovery, is present in all trophozoite flagella pairs and persists even when interconnections between axonemal microtubules are disrupted by detergent treatment. In conclusion, our findings not only reveal a novel structure at the externalizing point of the G. intestinalis flagella but also enhance our understanding of the arrangement of the Giardia flagellar pore. These observations offer valuable insights into the biology, survival, and pathogenesis of this parasite. Supported by CAPES, FAPERJ, CNPq, FINEP and CENABIO.

When Giardia intestinalis differentiates into a cyst, the single cell undergoes extensive rearrangement involving the transport of cyst wall proteins (CWPs) within encystation-specific vesicles (ESVs) to the cell surface. There, the proteins combine with unique β-1,3-N-acetylgalactosamine homopolymer (giardan). By analyzing the phenotypes of Giardia mutant cell lines generated via CRISPR/Cas9—either lacking CWP1 or having defective giardan synthesis—we characterized the structural roles of the protein and sugar components in the cyst wall. Cells lacking CWP1 retained cyst wall material on the surface, but the giardan filaments condensed into thicker structures, suggesting that CWP1 plays a role in fine branching of the polysaccharide. This phenotype was reversed by reintroducing the endogenous gene or by using the CWP homologue from Carpediemonas membranifera, a free-living metamonad discovered in marine sediments of the Great Barrier Reef. Conversely, disrupting giardan synthesis by deleting UDP-N-acetylglucosamine 4-epimerase (ΔUAE) resulted in "naked" cysts surrounded only by the cytoplasmic membrane. Finally, we observed dynamic behavior of a proteasomal component on the ESV surface during encystation, indicating regulated, site-specific protein degradation. In this work, we propose the origin of the cyst wall formation and its evolution in Metamonada.