CB8500 Intro

1. CB8500 Intro http://www.striepen.uga.edu/biopara/schedule.html

2. Biology of ParasitismCB8500 • We will meet three times a week: Mondays: 3:35 p.m., Wednesdays: 9 a.m., Fridays: 9 a.m. • Two lectures (usually Mon/Fri) and one seminar/journal club (usually Wed) • You find the class schedule & lecture notes on the class web site at: http://www.striepen.uga.edu/biopara/schedule.html (note that this is not web ct)

3. The schedule ahead • Parasite cell & molecular biology (evolution, unique organelles, host cell invasion, control of gene expression, RNA metabolism, signaling pathways, host cell manipulation) • Immunity to parasites (crash course intro, innate immunity to trypanosomes, immunity to various protozoans & worms, intracellular versus extracellular pathogens) • Anti-parasitic drug treatments (parasite metabolism, drugs and their modes of action, mechanisms of drug resistance, potential targets for new drugs) • Control of parasites as public health problems (epidemiology, public health, control programs, mass drug treatments, vaccines)

4. CB8500 a class for loafers without exams, but … • Grading for this class is A-F • To get credit for this class you are expected to: • Show for the lectures and seminars • Present original research papers in the class seminar (one per student in the class) • Participate in the discussions within the class (which implies that you read all papers not only the one you present) • Develop an original new research project in form of a (short version) NIH grant proposal • Write a primary and a secondary peer review of your fellow students research grants • Participate in the review session at the end of the semester • On the upside - this class does not have exams

5. The schedule ahead • How are we getting the three classes back that we lost in the first week? • Could schedule on Saturday as suggested by provost • Could try to find time in the week (rooms are the biggest challenge) • Think about it and let’s make a decision when we have the final number of students in this class

6. Introduction into parasitology • Straight forward concepts and terminology that you are likely already aware off, lecture will be posted and you can review • Most important concept: Parasitism is a way of life and parasites are evolutionary tremendously diverse • Parasitism sprang up independently in many, many taxa across the tree of life

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

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

9. From letter case of life to the tree of life (Linneus to Haeckel) • Taxonomy classifies organisms into meaningful groups that help to conquer and understand the massive diversity • The tree concept uses evolution as guiding principle of taxonomy • No evolution – no tree. • Choosing the tree metaphor makes several important assumptions • All life is related • Life diversifies • Life has a common origin

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

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

12. Molecular phylogeny • Uses the sequence of macromolecules (RNA, DNA & proteins) to measure similarity, and deduce phylogenetic relation • The molecule has to present in all the organisms you want to compare • Multiple 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.

13. Molecular phylogeny

14. Molecular phylogeny • Molecular 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 • A 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 • If you are interested in how this really works we could ask Jessie Kissinger for a primer when she comes to lecture

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

16. The archezoa hypothesis • Several early branching protozoa appear to lack classical mitochondria • These organisms were grouped as “archezoa” • They were hypothesized to represent the eukaryotic root predating the acquisition of mitochondria and certain other ‘advanced’ eukaryotic organelles • How do you acquire an organelle?

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

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

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

20. Is the absence of mitochondria a primary of secondary trait? • The genomes of most important protozoan parasites are now fully sequenced • This provides the opportunity to hunt for ‘molecular fossils’ • No trace of a mitochondrial genome has been found in Entamoeba, Giardia or Trichomonas • However, 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) • So are there remnants of mitochondrial protein genes in the nuclear genome?

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

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

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

24. 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)

25. Trichomonas hydrogenosomes • 0.5-2 m double membrane organelle • no genetic material • Present in anaerobic/ aerotolerant organisms • (Trichomonas, rumen-dwelling ciliates and several other apparently unrelated species)

26. Hydrogenosomes use protons as terminal electron acceptors Pyruvate from the cytosol is oxidized to 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). Int. J. Parasitol. 1999, 29: 199

27. PFO activates the prodrug metronidazole 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–NO2-) 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

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

29. Is (was) the hydrogenosome a mitochondrion or not? • Overall, the mitochondrial origin hypothesis seems to gain more and more support • It is the most parsimonious, explaining emergence of hs in different unrelated taxa • Also 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)

30. The archezoa hypothesis is dead • Lack of mitochondria in “archezoa” is secondary not primary • Recent 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

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

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

33. Boris simplified summary of it all • Note that this is only a schematic tree • Eubacteria, archea & eukaryotes remain three clearly distinguished groups • Eukaryotes have archeal & eubacterial features • Mitochondria evolved by endosymbiosis, we don’t know of any true amitochondriate eukaryotes – there might never have been one • The root of the eukaryotic tree remains in the dark • There 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 • Protozoa are not little animals, they are very diverse and highly divergent from us and each other