Evolution matters: Chloroplasts as targets for antiparasitic drugs

1. Evolution matters: Chloroplasts as targets for antiparasitic drugs

2. Summary malaria II • Severe forms of malaria include: severe anemia in kids, and cerebral and renal malaria in adults • Severe pathogenesis is related to adherence of infected RBC to entothelia • Adherence is mediated by knobs on the RBC surface made up by parasite proteins (PFEMP1) • PFEMP1 undergoes antigenic variation • Chloroquine accumulates in the food vacuole and prevents heme polymerization (detoxification), resistance is linked to mutations in a transport protein in the food vacuole membrane • Emerging drug resistance is a tremendous challenge to the management of malaria, a constant stream of new drugs with novel modes of action is required to stay ahead of the pathogen

3. Evolution matters: Chloroplasts as targets for antiparasitic drugs • One of the most hotly pursued new targets for drugs is the chloroplast of Apicomplexa, or apicoplast • The surprising discovery of a parasite chloroplast • What does the chloroplast do? • How does endosymbiosis work?

4. The convergence of three (initially) independent observation • 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

5. 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, a mitochondrial and a chloroplast genome www.cc.ysu.edu/~helorime/cscl.html

6. 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

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

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

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

10. What does the apicoplast do?

11. What does the apicoplast do?

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

13. 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

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

15. 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

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

17. The apicoplast isoprenoid pathway is essential for parasite survival DLytB wild type mutant Fos

18. 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

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

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

21. 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?

22. 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

23. Import across four membranes

24. Import across four membranes

26. 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

27. 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

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

29. 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

30. 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

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

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

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

34. A simple & elegant way to engineer protein import

35. A simple & elegant way to engineer protein import

36. A simple & elegant way to engineer protein import

37. A simple & elegant way to engineer protein import

38. Import across four membranes

39. How do host and endosymbiont divide in a coordinated fashion?

40. 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