Evolution matters: Chloroplasts as targets for antiparasitic drugs

1. Evolution matters: Chloroplasts as targets for antiparasitic drugs

2. Reviewing grants – what happens after your submit? • We will split into two review panels (called study section at the NIH) • Each grant will receive a formal written review from two reviewers assigned by a Scientific Review Officer (me) • The panel meets and discusses each application (in real life there is often some kind of triage process to reduce the work load during the meeting • Every member of the panel has an equal vote in assigning a score • (We will simplify the process: each panel member provides a ranking 1: best proposal)

3. Reviewing grants – typical discussion • Reviewer state preliminary scores (we won’t do that) • The primary reviewer provides the panel with an overview of the grant (quickly summarizes the main ideas & approaches), then he/she discusses the main strength and weaknesses of the proposal • The secondary reviewer provides his/her review focusing in particular on areas that differ from the primary review • Panel members ask questions and voice opinions • Reviewers address those • Chair summarizes discussion • Panel votes

4. Categories addressed in NIH reviews (limit your review to two pages max) • SIGNIFCANCE: Is this important? • APPROACH: Is the approach sound? • INNOVATION: Does this bring anything new to the table? • INVESTIGATOR: Is the PI and his/her team qualified to pursue this project? • ENVIRONMENT: How good is the environment for this type of work? • OVERALL EVALUATION: A paragraph that brings it all together

5. Reviewing grants • Being asked to review grants is an honor, can be a pain, and almost always is a lot of work • You should take your role as reviewer (in this class and elsewhere) seriously • Try to be candid yet polite • Edit your review for emotionally colored language (on page 9 finally an original idea, the lack of data is annoying …)

6. The convergence of three (initially) independent observations • Apicomplexa appear to have three genomes • Apicomplexa are susceptible to certain antibiotics that usually only affect bacteria • Apicomplexa have a subcellular structure in the vicinity of the nucleus that is surrounded by multiple membranes

7. How many genomes do we have? • DNA can be separated by gradient centrifugation • The image shows yeast DNA the bright lower band are the nuclear chromosomes the higher band is the mitochondrial genome • Apicomplexa have a nuclear, and surprisingly two additional genomes www.cc.ysu.edu/~helorime/cscl.html

8. The third genome looks like a chloroplast genome and localizes outside of the nucleus Small dot: apicoplast genome, large spot nuclear genome; red label: specific probe for apicoplast genome

9. The chloroplast genome is localized in an organelle with four membranes

10. What do we know about chloroplasts? • Home of photosynthesis • Have their own genome • Surrounded by two membranes • Evolved by endosymbiosis from a cyanobacteria

11. The apicoplast is the product of secondary endosymbiosis Apicomplexa are not plants – but they ate a plant

12. The apicoplast is the product of secondary endosymbiosis

13. What does the apicoplast do?

14. What does the apicoplast do?

15. What does the apicoplast do?

16. The apicoplast makes fatty acids, isoprenoids and heme • Fatty acids • Major component of lipids that form cellular membranes and serve as energy reservoir • Isoprene • Precursor for many important molecules including cholesterol and many hormones • Heme • Prosthetic group for important enzymes that catalyze oxidation/reduction reactions

17. Due to the evolutionary history of the apicoplast its pathways are “bacterial”

18. Mammals and bacteria (and plastids)synthesize isoprenes differently • Isoprenoid synthesis is rich in proven drug targets • Animals use the mevalonate pathways • Bacteria and plastids use the deoxy-xylulose-P pathway • These pathways use different enzymes, different intermediates and are susceptible to different drugs

19. Mammals and bacteria (and plastids)synthesize isoprenes differently • Apicomplexa have exclusively bacterial type isoprene synthesis • The enzymes for this pathway are localized in the apicoplast

20. Fosmidomycin targets isoprenoid synthesis and is an effective antimalarial, but … • Malaria parasites in culture are killed by fosmidomycin (IC50 ~1 uM) • Malaria and Babesia infection can be cured in animals and people with fosmidomycin • However, most other apicomplexan parasites including Toxoplama and Theileria appear completely resistant Fos

21. Is the plastid DoxP pathway dispensable? Fos

22. Is Resistance due tolack of drug access?

23. In bacteria fosmidomycin is imported by GlpT

24. Expression of the E. coli GlpT transporter confers Fosm sensitivity to T. gondii

25. What does the apicoplast do? • Apicoplast is home to several biosynthetic pathways that are specific to the parasite • Genetic studies show that some of these pathways are essential to parasite growth • Certain antibiotics that target these pathways in bacteria also inhibit parasites and some show promise in clinical trials • Current work is focused on identifying the best target in the apicoplast and to discovery more potent drugs to inhibit them

26. How does endosymbiosis work? Massive gene transfer from endosymbiont to nucleus, why?

27. The difference between a docile new endosymbiont and hostile take over is control

28. Gene transfer is a hallmark of endosymbiosis • Gene transfer is not only found for the apicoplast but also for the primary chloroplast in plants or our mitochondrion • Gene transfer moves control of gene expression (and therefore essentially everything the symbiont does) to the host • Also note that organellar genomes are only maternally inherited. Transferring to the nucleus makes organellar genes accessible to sexual recombination • What is the problem with transfering genes?

29. Nuclear encoded plastid proteins feature a bipartite targeting signal • Apicoplast proteins that are encoded in the nucleus carry an address tag at the beginning (N-terminus) of the protein • Think of it as a molecular zip code • Conceptually similar tags are found on many organelle protein (e.g. those that are secreted outside or have to go to the mitochondrion or chloroplast) • The apicoplast zip has two elements: the first is a secretory signal peptide resulting into insertion into the endoplasmic reticulum, the second is responsible for the rest of the journey • The tag is removed upon arrival

30. Import across four membranes

31. Import across four membranes

33. Proteins related to protein of the chloroplast Tic are present in the apicoplast membranes ApTic20 Apicoplast merge Van Dooren et al.,(2008) PNAS 105:13574

34. Mutants in apicoplast Tic genes kill parasites and block protein import Wild type Mutant Gene “on” Gene “off” Van Dooren et al.,(2008) PNAS 105:13574

35. That is one how do you cross the rest? TIC20 TIC22

36. Secondary chloroplasts have an ER cleanup system (ERAD) but no ER Chlorarachnion reptans Ludwig & Gibbs J Phycol 1989 Sommer et al. Mol Biol Evol. 2007

37. ERAD: a clean up system for the ER • Many proteins travel through the endoplasmic reticulum – sometimes things get messed up and they don’t fold as they should • Such proteins can cause problems and are pulled back out of the ER into the cytoplasm where they are chucked into the cells trash can for proteins – the proteasome • A protein machine called ERAD (ER associated degradation) forms a pore in the membrane and pulls misfolded proteins back out

38. Apicomplexa have two ERAD systems: one in the ER & cytoplasm HA ER 83.m00015 HA Plastid 46.m01752

39. And a second set in the apicoplast HA 76.m03417

40. Der1Ap KO ablates apicoplast protein import

41. The Apicoplast ERAD system is derived from the red algal endosymbiont

42. A simple & elegant way to engineer protein import

43. A simple & elegant way to engineer protein import

44. A simple & elegant way to engineer protein import

45. A simple & elegant way to engineer protein import

46. Import across four membranes

47. Import across four membranes

48. Conclusions • Apicoplast metabolic pathways are essential and provide excellent highly divergent targets for drug development • Multiple phylogentically independent translocons enable import over four • Plastid division is controlled by the host and depends on “host factors”: the mitotic spindle, and a plastid fission protein

49. Acknowledgements Giel van Dooren, Swati Agraval, Sarah Reiff, Carrie Brooks, Marc-Jan Gubbels, Shipra Vaishnava, Jolly Mazumdar, Lisa Sharling, Mani Muthalagi, Sethu Nair, Sri Ramakrishnan, Nwakaso Umejiego, Carly Jordan, Catherine Li, Marnix Wieffer, Andrea Pruijssers, Adam Stroupe, Chitra Kanchagar, David Morrison, Lilach SheinerColaborators:Cveta Tomova (Nijmejen), Liz Hedstrom (Brandeis), Mike White (MSU), Dubremetz (Montpellier), Chris Hunter (PENN), John Logsdon (Emory). Jessica Kissinger (UGA), Wandy Beatty (Wash U)Funding:National Institutes of HealthAustralian National Health and Medical Research Council American Heart Association Merck Research Laboratories

50. What does the apicoplast do? Modified after Ralph et al., Nat. Rev. Microbiol. 2: 203, Fleige et al., Eukaryot. Cell 6:984