Prof. A.S. Kolaskar Vice Chancellor University of Pune

1. Bioinformatics for Parasitic diseases: Malaria Prof. A.S. Kolaskar Vice Chancellor University of Pune

2. Mosquito Human Life cycle of Plasmodium falciparum

3. Countries endemic to Malaria

4. Drug-Resistance in Malaria endemic-countries Source: National Centre for Infectious Diseases, CDC,Atlanta

5. Information & Resources for Malaria: 1 WHO/TDR: Focus on Malaria

6. Information & Resources for Malaria: 2 Malaria Focus: Bill & Melinda Gates Foundation $50 million grant for malaria research

7. Malaria Focus: Wellcome Trust Foundation Partly funded the Plasmodium sequencing project

8. Information & Resources for Malaria: 3

9. MR4@ATCC Information & Resources for Malaria: 4 Deposit OR Order Culture • Text search • Sequence search

10. Information & Resources for Malaria: 5 National Institute of Allergy & Infectious Diseases

11. Information & Resources for Malaria: 6 CDC Home

12. Division of Parasitic Diseases: Information on Malaria

13. CDC: Division of Vector-borne infectious diseases Complete details regarding the life-history of mosquito, the vector for many infectious diseases

14. Genomes: the current status Highly voluminous data: Needs to be analyzed for Knowledge Generation • Published complete genomes: 169 • Archaeal: 17 • Bacterial: 131 • Eukaryal: 21 • Completed Viral genomes: >1400 • Prokaryotic ongoing genomes: 428 • Eukaryotic ongoing genomes: 360 As of January 13, 2004

15. Genome database: Plasmodium

16. Genome database: Anopheles

17. Genome Organisation of Homo sapiens, Anopheles gambiae and Plasmodium falciparum

18. Approaches to mine genomes of host, vector & parasite • Chromosome-wise comparison • Comparison of pathway-specific genes • Stage-specific comparison • Rate limiting factors: • Extent of annotation of genomic data • Lack of complete connectivity between • genomic and derived databases • Need to define appropriate cutoffs to detect • similarities between phylogenetically diverse • organisms

19. Chr Size (bp) Chromosomes of Homo sapiens

20. Chromosomes of Plasmodium falciparum

21. Chromosomes of Anopheles gambiae

22. Chromosome-wise Comparisons of Proteomes: Program & parameters • Data sources: • P. falciparum: PlasmoDB • H. sapiens : RefSeq@NCBI • A. gambiae : ENSEMBL • Program : BLASTP • Sequence identity: >20% • Alignment length: >50aa • E value: zero or with negative powers

23. Comparison of Proteomes of H. sapiens & A. gambiae

24. Comparison of Proteomes of P.falciparum & A. gambiae

25. Comparison of Proteomes of Homo sapiens & Plasmodium falciparum Pf chr1 vsHs chr2 28 significant matches

26. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Significant matches Chr. in Hs Chr. in Pf

27. List of significant matches • Proteins that are part of eukaryotic transcriptional and translational machinery • Heat shock proteins: molecular chaperones • Histones • Actin and tubulin: cytoskeletal proteins • Ornithine aminotransferase: Involved in the inter-conversion of arginine, proline and glutamate residues and the synthesis of polyamines. Polyamines are implicated to have a role in cell proliferation.

28. List of significant matches contd., • Polyubiquitin: involved in the ATP-dependent selective degradation of cellular proteins, maintenance of chromatin structure, regulation of gene expression, stress response and ribosome biogenesis • Proteasomeare large barrel-like bodies which contain proteolytic enzymes in their inner surface. • DEAD family RNA helicases. • Histone deacetylase: critical mediators of transcription repression.

29. Clustered asparagine rich protein (CARP) Function not clearly known Expressed in different stages of the life-cycle of the parasite Immunogenic (Kuma et.al,1990) Bruno like 4 RNA binding protein Transcriptional regulator Case study: 1 Pf_c12 with Hs_c18 A search against Pfam database revealed that the CARP has matches both at its N and C terminii to RNA binding domains. A protein with a probable house-keeping gene activity is known to be immunogenic….

30. Hypothetical protein Blast against nr database shows significant matches towards the N terminus with zinc-finger containing proteins of higher eukaryotes like mammals and fishes. No significant matches to other protozoans Zinc-finger binding domain. Transcriptional factor Binds both to RNA and DNA. Case Study May have acquired from the host…. Pf_c10 vs Hs_c18

31. Expressed in the intraerythrocytic (up to 0.5% of parasite protein) and schizont-stage of the parasite. Dual function of protein folding and signal transduction. Cyclophilin is also present in other parasites like T. gondii, Brugia malayi etc. Receptor for Immuno-supressive drug cyclosporin A Known to be present in higher eukaryotes including plants(involved in handling stress response). CaseStudy Pf_c12 vs Hs_c21

32. Putative helicase Involved in a number of cellular functions including translation, RNA splicing, and ribosome assembly. Located within human major histocompatibility complex class III region. Pf_c2 with Hs_c6

33. Malaria parasite pathways: Hagai Ginsburg URL: http://sites.huji.ac.il/malaria/

34. Metabolome of Plasmodium falciparum • Metabolic pathways of Plasmodium falciparum are known to be stage-specific. • Asexual blood-stage parasites depend on glycolysis and conversion of pyruvate to lactate to derive energy. • MS-MS studies carried out by Florens et.al(2002), revealed that gametocyte and sporozoite stages of the malarial parasite contain peptides of enzymes known to be involved in mitochondrial TCA cycle and oxidative phosphorylation.

35. In Plasmodium falciparum

36. Enzyme Chromosome Hs Ag Pf Citrate synthase 12 3L 10 Aconitase 22 3R 13 Isocitrate dehydrogenase 2 2L 13 Alpha-keto glutarate dehydrogenase (E1) 7 2R 8 Alpha-keto glutarate dehydrogenase (E2) 14 3L 13 Alpha-keto glutarate dehydrogenase (E3) 7 3L 12 Succinyl CoA ligase 13 2L 14 Succinate dehydrogenase (Cyt b560) (SDHA) 1 3L - Succinate dehydrogenase ( Cyt b small) (SDHB) 11 X - Succinate dehydrogenase (flavoprotein) (SDHC) 5 3L 10 Succinate dehydrogenase (iron-sulfur) (SDHD) 1 2L 12 Fumarase 1 * 2R* 9** Malate dehydrogenase 7 3R 6 Chromosomal locations of TCA cycle-enzymes • * Class II non-iron dependent Fumarase • ** Class I iron-dependent Fumarase

37. Comparison of TCA cycle enzymes of Plasmodium falciparum-Anopheles gambiae-Homo sapiens Sequnece identity Enzyme Plasmodium contains only two SDH subunits in contrast to 4 SDH subunits in human & anopheles Fumarase class I is present in Plasmodium whereas Fumarase class II is present in human & anopheles

38. TCA cycle: Comparison of proteome of host,vector and parasite revealed… • TCA cycle-specific enzymes of Homo sapiens and Anopheles gambiae have high degree of sequence identity. • Aconitase and Fumarase enzymes of Plasmodium falciparum show very less similarity with their human and mosquito counterparts. • An iron regulatory protein that has a C terminal domain similar to Aconitase is present in Plasmodium and it likely carries out the function of Aconitase enzyme. • Fumarase (Class I) an iron-dependent enzyme is present in Plasmodium whereas Fumarase (Class II), an non-iron dependent enzyme is present in human and mosquito. • Succinate dehydrogenase in Plasmodium contains only two subunits in contrast to its human & mosquito counterparts, which have four subunits.

39. Homology models of Isocitrate dehydrogenase High sequence identity Structural similarities

41. Chromosomal location of enzymes involved in Purine Biosynthesis

42. Sequence similarity of Enzymes involved in Purine Biosynthesis using Pf as a reference

44. Multi-domain protein in Hs & Ag • Single-domain proteins in Pf • Located on different chromosomes Chromosomal location of enzymes involved in Pyrimidine Biosynthesis

45. Sequence similarity of Enzymes involved in Pyrimidine Biosynthesis using Pf as a reference • Present in Hs, Pf & Ag • No sequence similarity • between Pf vs Hs and Pf vs Ag

47. Chromosomal location of enzymes involved in Hemoglobin degradation pathway

48. Sequence similarity of Enzymes involved in Hemoglobin digestion using Pf as a reference

49. HGPRT: Missing [Hypoxanthine Guanine PhosphoribosylTransferase] • HGPRT: Present • well-studied drug target Purine Biosynthesis • Plasmodium • Salvage • Anopheles • de novo • Salvage Comparing pathways:Lessons learnt • Human: • de novo • Salvage

50. Stage-specific comparison of P.falciparum proteins with Human proteome Transition stage: MosquitoHuman Human: RBC specific Human: Liver specific