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

2. Life cycle of Plasmodium falciparum Human Mosquito

3. Countries endemic to Malaria

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

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

6. Malaria Focus: Bill & Melinda Gates Foundation Information & Resources for Malaria: 2 $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. Text search Sequence search MR4@ATCC Deposit OR Order Culture Information & Resources for Malaria: 4

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

11. CDC Home Information & Resources for Malaria: 6

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  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 Highly voluminous data: Needs to be analyzed for Knowledge Generation

15. Genome database: Plasmodium

16. Genome database: Anopheles

17. Genome Organisation of Homo sapiens, Anopheles gambiae and Plasmodium falciparum OrganismGenome sizeNumber of chromosomes Number of predicted genes Homo sapiens (Hs) 3 GB23~24,000-40,000 Anopheles gambiae (Ag) 0.27 GB3~12,000 Plasmodium falciparum (Pf) 23 MB14~5000

18. Approaches to mine genomes of host, vector & parasite 1.Chromosome-wise comparison 2.Comparison of pathway-specific genes 3.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. Chromosomes of Homo sapiens Chr Size (bp)

20. Chromosomes of Plasmodium falciparum

21. ChrSize (bp) X 24,902,716 2R 78,412,699 2L 52,393,056 3R 64,548,413 3L 56,406,562 Chromosomes of Anopheles gambiae

22. Chromosome-wise Comparisons of Proteomes: Program & parameters Data sources: –P. falciparum: PlasmoDB –H. sapiens : RefSeq@NCBIRefSeq@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 vs Hs 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 Chr. in Pf Chr. in Hs Significant matches

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 Proteasome are large barrel-like bodies which contain proteolytic enzymes in their inner surface. DEAD family RNA helicases. Histone deacetylase: critical mediators of transcription repression.

29. Pf_c12 with Hs_c18 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 gi_pf Seq_ pf Acc_hsSeq_ hs ScoreE value Aln_len%idFunc_hsFunc_pf g23509043459 XP_029431 269713e- 014 37/8444bruno-like 4, RNA binding protein clustered- asparagine- rich protein 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…. Case study: 1

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. Pf_c10 vs Hs_c18 Case Study May have acquired from the host….

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). Case Study Pf_c12 vs Hs_c21

32. Pf_c2 with Hs_c6 Putative helicaseInvolved in a number of cellular functions including translation, RNA splicing, and ribosome assembly. Located within human major histocompatibility complex class III region. gi_pf Seq_ pf Acc_hsSeq_ hs ScoreE value Aln_len%idFunc_hsFunc_pf g16804988457 XP_041840 4285131e- 147 263/45358HLA-B associated transcript-1 eIF-4A-like DEAD family RNA helicase

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. EnzymeChromosome HsAgPf Citrate synthase123L10 Aconitase223R13 Isocitrate dehydrogenase22L13 Alpha-keto glutarate dehydrogenase (E1)72R8 Alpha-keto glutarate dehydrogenase (E2)143L13 Alpha-keto glutarate dehydrogenase (E3)73L12 Succinyl CoA ligase132L14 Succinate dehydrogenase (Cyt b560) (SDHA)13L- Succinate dehydrogenase ( Cyt b small) (SDHB)11X- Succinate dehydrogenase (flavoprotein) (SDHC)53L10 Succinate dehydrogenase (iron-sulfur) (SDHD)12L12 Fumarase1 *2R*9** Malate dehydrogenase73R6 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 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 Sequnece identity Enzyme

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 EnzymeChromosome Hs Ag Pf Adenosine deaminase222L10 Adenylate kinase12L10 Adenylosuccinate lyase12R2 Adenylosuccinate synthetase12R13 DNA polymerase 122L9,10,14, DNA-directed RNA polymerase II113R2 GMP synthetase33R10 Guanylate kinase13L9 Hypoxanthine phosphoribosyltransferaseX-10 Inosine-5'-monophosphate dehydrogenase73L9 Nucleoside diphosphate kinase172L6 Purine nucleoside phosphorylase14add5 Ribonucleotide reductase83R10,14 Thioredoxin reductase22X9

42. EnzymeSequence Identity Hs Ag Adenosine deaminase2630 Adenylate kinase5251 Adenylosuccinate lyase2224 Adenylosuccinate synthetase4645 DNA polymerase 12624 DNA-directed RNA polymerase II5452 GMP synthetase30 Guanylate kinase3740 Hypoxanthine phosphoribosyltransferase49- Inosine-5'-monophosphate dehydrogenase4948 Nucleoside diphosphate kinase6160 Purine nucleoside phosphorylase-- Ribonucleotide reductase6064 Thioredoxin reductase4544 Sequence similarity of Enzymes involved in Purine Biosynthesis using Pf as a reference

44. Chromosomal location of enzymes involved in Pyrimidine Biosynthesis Enzyme Chromosome Hs Ag Pf Aspartate carbamoyltransferase2UNK13 Carbamoyl phosphate synthetase2UNK13 Cytidine triphosphate synthetase13R14 Deoxyuridine 5'-triphosphate nucleotidohydrolase 15UNK11 Thymidylate synthase182L4 Dihydroorotase2X14 Dihydroorotate dehydrogenase162R6 DNA polymerase 132L6 DNA-directed RNA polymerase II163R2 Nucleoside diphosphate kinase172L6 Orotate phosphoribosyltransferase32R5 Orotidine-monophosphate-decarboxylase32R10 Ribonucleotide reductase22L 10,14 Serine hydroxymethyltransferase12X Thioredoxin reductase22X9 Thymidylate kinase22L12 Multi-domain protein in Hs & Ag Single-domain proteins in Pf Located on different chromosomes

45. Enzyme Sequence Identity Hs Ag Aspartate carbamoyltransferase 3738 Carbamoyl phosphate synthetase 4547 Cytidine triphosphate synthetase 4443 Deoxyuridine 5'-triphosphate nucleotidohydrolase 3635 Thymidylate synthase 5542 Dihydroorotase -- Dihydroorotate dehydrogenase 3635 DNA polymerase 1 2624 DNA-directed RNA polymerase II 3052 Nucleoside diphosphate kinase 6160 Orotate phosphoribosyltransferase 2729 Orotidine-monophosphate-decarboxylase 27- Ribonucleotide reductase 6064 Serine hydroxymethyltransferase 4547 Thioredoxin reductase 4544 Thymidylate kinase 3942 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 EnzymeChromosome Hs Ag Pf Aspartyl protease11UNK13,14 Aspartic hemoglobinase--14 Leucine aminopeptidase42R14 Methionine aminopeptidase12,43R,2R10,13,14 O-sialoglycoprotein endopeptidase42L7 Papain family cysteine protease93L9 Pepsinogen11UNK8 SERA antigen/papain-like proteinase with active Cys 93L2 Serine protease4UNK5 Zinc-metallopeptidase17UNK13

48. Enzyme Sequence Identity Hs Ag Aspartyl protease3432 Aspartic hemoglobinase-- Leucine aminopeptidase3930 Methionine aminopeptidase5856 O-sialoglycoprotein endopeptidase3230 Papain family cysteine protease2627 Pepsinogen3130 SERA antigen/papain-like proteinase with active Cys 2324 Serine protease2231 Zinc-metallopeptidase2924 Sequence similarity of Enzymes involved in Hemoglobin digestion using Pf as a reference

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

50. Stage-specific comparison of P.falciparum proteins with Human proteome StageNo. of proteins No. of matches% matches Sporozoite102519819 Merozoite82828434 Trophozoite102433833 Transition stage: Mosquito  Human Human: Liver specific Human: RBC specific