1. Evolution of the eukaryotic cell Protozoa & evolving models on the origin of eukaryotes “Early branching” eukaryotes: primitive or specialized? Primer on Giardia & Trichomonas biology

2. protozoa zPrimary unicellular eukaryotes, often also called protists zMany important human and veterinary pathogens zIt is important to understand that protozoa are mostly a historic grouping and not a cohesive biological group that contains closely related organisms zA very diverse group with a vast variety of morphological and biochemical adaptations to almost any ecological niche

3. From letter case of life to the tree of life (Linneus to Haeckel) zTaxonomy classifies organisms into meaningful groups that help to conquer and understand the massive diversity zThe tree concept uses evolution as guiding principle of taxonomy zNo evolution – no tree. zChoosing the tree metaphor makes several important assumptions zAll life is related zLife diversifies zLife has a common origin

4. the tree of life (Ernst Haeckel, 1874) protozoa reptiles molluscs man crustaceans whales fish worms carnivores ungulates zThe tree of life (who is related and how did they evolve) was initially based on morphological characteristics z“Complex” organisms were viewed as derived and highly evolved “simple” organisms as primitive zThis scheme puts protozoa as a cohesive group to the bottom of the tree

5. the tree of life (Ernst Haeckel, 1866) zMonophyletic tree of organisms again by Haeckel zNote that he divides life into three kingdoms (plants, protists, and animals) zNote also that he hypothesizes a common root (radix) for all organisms zLoss or gain of characters produces branching of the tree zThe advent of electron microscopy brought more morphological characters even for the small protists zHowever, reduction and simplifications (e.g. due to parasitism) pose significant problems for morphology based trees zHomology is not always discernable from analogy, and characters are not always easily quantifiable

6. Molecular phylogeny zUses the sequence of macromolecules (RNA, DNA & proteins) to measure similarity, and deduce phylogenetic relation zThe molecule has to present in all the organisms you want to compare zMultiple sequences are aligned and relatedness is inferred from the simple argument that two molecules from two related organisms are likely to be more similar than from two organisms that branched a long time ago 30S ribosomal subunit, rRNA pink Schluenzen et al. Cell 102 (5): 615–23.

7. Molecular phylogeny

8. zMolecular phylogeny assumes that changes occur over time and that these changes can be modeled and used to infer a process (evolution) out of the current pattern zA large number of statistical approaches has been developed to model and weigh change, build trees that depict the results, and evaluate the significance of the tree topologies obtained zIf you are interested in how this really works we could ask Jessie Kissinger for a primer

9. The three kingdoms of life (Mitch Sogin’s 16s RNA tree)

10. The archezoa hypothesis zSeveral early branching protozoa appear to lack classical mitochondria zThese organisms were grouped as “archezoa” zThey were hypothesized to represent the eukaryotic root predating the acquisition of mitochondria and certain other ‘advanced’ eukaryotic organelles zHow do you acquire an organelle?

11. The Lynn Margulis model of the endosymbiotic origin of mitochondria zA free living alpha proteobacterium was engulfed by a proto- eukaryote and subsequently ‘domesticated’ zThis idea is now very well supported by numerous phylogenetic and biochemical studies that show a clear link between mitochondria and proteobacteria

12. “More good theories for eukaryotic origins than good data” T. Martin Embley and William Martin Nature 440, 623-630 zMost models now assume that eukaryotes are a merger of an archaebacterium and a eubacterium zPhylogenetic analyses of eukaryotes suggest that ‘informational’ proteins (DNA replication, transcription, & translation) are related to archaea while many ‘metabolic’ proteins appear eubacterial zWho ate who and how and when is controversial

13. Archezoa & amoeba the most primitive eukaryotes? zNo mitochondrion and no typical mitochondrial enzymes (Krebs cycle and oxidative phosphorylation is missing) zA fermentative “bacteria-like” anaerobic metabolism zIt was assumed that archezoa and amoeba represent the stage of early eukaryotes before the endosymbiosis event that let to the mitochondrion zAn alternative hypothesis stated that these organisms once had mitochondria and subsequently lost them while adapting to parasitism and life in anaerobic environments

14. Is the absence of mitochondria a primary of secondary trait? zThe genomes of most important protozoan parasites are now fully sequenced zThis provides the opportunity to hunt for ‘molecular fossils’ zNo trace of a mitochondrial genome has been found in Entamoeba, Giardia or Trichomonas zHowever, most proteins that do their job in the mitochondrion are actually encoded in the nucleus and are imported from the cytoplasm (gene transfer from the endosymbiont to the host represents an important element of control and domestication) zSo are there remnants of mitochondrial protein genes in the nuclear genome?

15. E. histolytica Cpn60 identifies the ‘mitosome’ zThe E. histolytica genome encodes an ortholog of the mitochondrial chaperon Cpn60 zAntibodies raised against this protein reveal numerous small organelles zThis has now been validated using a number of additional proteins Cpn60 DIC Microbiology 150 (2004), 1245-1250

16. E. histolytica mitosomes do not contain DNA zDNA was detected by in situ nick translation in E. histolytica (a, b) and in mammalian cells (c) zNote absence of labeling in amoeba zDNA is equally absent in Giardia mitosomes and trichomonas hydrogenosomes Microbiology 150 (2004), 1245-1250

17. Mitosomes are also detectable in Giardia (lscU staining) http://www.natur.cuni.cz/~parazit/tachezy_web/mitosome.htm

18. Mitochondrial proteins indentified in ‘amitochondriate’ organisms T. Martin Embley and William Martin Nature 440, 623-630 (blue likely eubacterial, red archaebacterial ancestry, green eukaryotic inventions)

19. Trichomonas hydrogenosomes  0.5-2  m double membrane organelle zno genetic material zPresent in anaerobic/ aerotolerant organisms z(Trichomonas, rumen- dwelling ciliates and several other apparently unrelated species)

20. THE HYDROGENOSOME Tissue and Cell, 1996 28:287

21. Int. J. Parasitol. 1999, 29: 199 Pyruvate from the cytosol is oxidizes do acetyl coA by the Pyruvate Ferredoxin Oxidoreductase (PFO (1)) in the Hydrogenosome. The enzyme Hydrogenase (4) uses the electrons from ferredoxin and transfers them to H + to form hydrogen gas. Acetyl CoA can be further metabolized by the acetate:succinate CoA transferase (2) to form acetate and succiniyl-CoA (2) which could be hydrolyzed into CoA and succinate and the energy released used to make ATP by the succinate thiokinase (3). Hydrogenosomes use protons as terminal electron acceptors

22. In the presence of metronidazole, electrons generated by PFO are transported by ferredoxin [2Fe–2S] to the drug (bold arrow) and not to their natural acceptor hydrogenase (HY). Metronidazole is reduced with one electron forming a nitro anion free radical. The cytotoxic radicals (R–NO 2 - ) are formed as intermediate products of the drug reduction. PFO is not limited to hydrogenosomes but also found in mitosomes and in a variety of anaerobic bacteria Metronidazole (Flagyl) is the standard treatment for Trichomonas, Giardia and invasive amebiasis Int. J. Parasitol. 1999, 29: 199 PFO activates the prodrug metronidazole

23. Is (was) the hydrogenosome a mitochondrion or not? zHydrogenosomes share features with mitochondria zThey have a similar import machinery, they have two membranes and harbor certain mitochondrial proteins (e.g. the mitochondrial iron sulfur cluster assembly machinery zThere are some atypical features like lack of DNA, PFOR, and hydrogenase, which has led some authors to suggest an indpendent origin

24. Is (was) the hydrogenosome a mitochondrion or not? zOverall, the mitochondrial origin hypothesis seems to gain more and more support zIt is the most parsimonious, explaining emergence of hs in different unrelated taxa zAlso recent identification of a hydrogenosome NADH dehydrogenase which shares a common ancestry with mitochondrial enzymes Nature 432, 618-622 (NADH reductase, green; hydrogenosome marker, red)

25. The archezoa hypothesis is dead zLack of mitochondria in “archezoa” is secondary not primary zRecent phylogenies based on multiple concatenated proteins fail to clearly pin the root to one ‘primitive’ eukaryote and rather suggest an explosion of several groups from a common yet unknown ancestor

26. The Baldauf explosion of ‘parallel’ crown groups amoebozoa opisthokonts excavates discicristates chromists alveolates cercozoa plantae rhizaria

27. A similar effort by Simpson showing that after all we might be early branching

28. Boris simplified summary of it all zNote that this is only a schematic tree zEubacteria, archea & eukaryotes remain three clearly distinguished groups zEukaryotes have archeal & eubacterial features zMitochondria evolved by endosymbiosis, we don’t know of any true amitochondriate eukaryotes – there might never have been one zThe root of the eukaryotic tree remains in the dark zThere appears to have been a relatively early split between opisthokonts (animals, fungi & ameba) and plants and the rest of protozoal eukaryotic life on the other branch zProtozoa are not little animals, they are very diverse and highly divergent from us and each other