Supplementary Information

Genomice and Transcriptomic Analysis for Identification of Genes and Interlinked

Pathways Mediating Artemisinin Resistance in Leishmania donovani

Sushmita Ghosh1,2, Aditya Verma1, Vinay Kumar1, Dibyabhaba Pradhan3, Angamuthu

Selvapandiyan 2, Poonam Salotra1, Ruchi Singh1*

1. ICMR- National Institute of Pathology, Safdarjung Hospital Campus, New Delhi-110029,

India

2. Jamia Hamdard University-Institute of Molecular Medicine, New Delhi-110062, India

3. ICMR-AIIMS Computational Genomics Centre, Indian Council of Medical Research, New

Delhi- 110029

*Correspondence: [email protected]

Supplementary Figures and Tables:

Figure S1.

Figure S1: Comparative transcriptional responses following ART adaptation in L. donovani. Overlap of log2 transformed K133 AS-R and K133 WT expression ratio plotted as a function of chromosomal location of probes representing the full genome microarray. The plot represents the average values of three independent hybridizations for each isolate.

Table S1: List of genes validated for their modulated expression by Quantitative real time- PCR

S.N Primer Gene Name/ Function/relevance Primer Sequence o. Name Gene ID 1 AQP1 Aquaglyceropor Metal ion F- in (LinJ.31.0030) transmembrane 5’CAGGGACAGCTCGAGGGTAA transporter activity, AA3’ integral to membrane; transmembrane R- transport; transporter 5’GTTACCGGCGTGAAAGACAG activity; water TG3’ transport. 2 A2 A2 protein Cellular response to F- (LinJ.22.0670) stress 5’GTTGGCCCGCTTTCTGTTGG3’

R- 5’ACCAACGTCAACAGAGAGA GGG3’ 3 ABCG1 ATP-binding ATP binding, ATPase F- cassette protein activity, coupled to 5’GATCTTCTTGAATGTGCGGGC subfamily G, transmembrane C3’ member 1 movement of (LmjF.06.0080) substances, R- phosphopantetheine 5’CCACCAGCACTGCAAGAATC binding TG3’ 4 GATase Glutamine Putative cysteine F- aminotransferas conjugate beta- 5’GCAGGCTCTCAACGCCATATT e (LinJ.33.1410) ,aminotransferase C3’ - like proteinbiosynthetic R- process 5’AGCTTCCAGTTCTCAGCGGAT T3’ 5 PECH peroxisomaleno Putativeenoyl-CoA F- yl-coahydratase hydratase activity, 5’AGATTATCCAGCGCTCCTCAC (LinJ.18.0580) Fatty acid metabolism C3’

R- 5’ TTGATGGCAGCTTGGTACTCGA 3’ 6 ATPP autophagocytosi autophagy; cytoplasm; F- s protein transport 5’CCCTGAGAGCGTGCAAGACT protein 3’ (LinJ.33.0320) R- 5’CGCCAGAGTCATCGTCGTCT 3’ 7 Tpx Tryparedoxin- Hypothetical protein, F- like protein involved in redox 5’CTCATCTGCTTCTCCGCCCA 3’ (LinJ.31.2010) metabolism in trypanosomatids R- 5’CAGTCTCGGCCGATCGTCTG3 ’ 8 VPS vacuolar protein vesicle docking F- sorting-like involved in exocytosis 5’GTTCTTAGCAGGGTGTACGA protein vesicle-mediated G3’ (LinJ.25.2280) transport R- 5’ GGAAAGCAAGTGAACGACAA C3’ 9 HDH haloacid activity F- dehalogenase- 5’TGACCAGAATGCCCATCAAG like hydrolase 3’ (LinJ.28.1480) R- 5’GCTGGTGATAATGTAGTCCGG 3’ 10 AUT Autophagy- Autophagy-related F- related protein protein 5’CCTCTCCAGACCACCAATGT (LinJ.19.0860) ATG8/AUT7/APG8/PA CC3’ Z2, putative (ATG8B.3) R- 5’GGCGAGCGTCACCTTCTTGA3 ’ 11 gMDH glycosomal glycolysis, malate F- malate metabolic process 5’GCGTGGATGTGTTTGTGATGG dehydrogenase T3’ (LinJ.19.0710) R- 5’TTCGTCACAATGCAGAACAC CG3’ 12 ItRS Isoleucyl-tRNA isoleucyl-tRNA F- synthetase aminoacylation, 5’CCGCTGGATCGACTTTGACA (LinJ.36.5870) tRNA aminoacylation AC 3’ for protein translation, translation R- 5’AGGAAGTATGTGTTGGGGTC GG3’ 13 AC Adenylate cyclic nucleotide F- cyclase-like biosynthetic process, 5’CGGTATGGAAAGGTGTACGA protein intracellular signaling AG3’ (LinJ.28.0090) pathway, transport R- Purine metabolism 5’TCGATGTATGACTGCAAGAC G3’ 14 CYPs Cytochrome Electron carrier F- p450-like activity, heme binding, 5’TACATCCCCTTCAGCTGCGG3’ protein monooxiginase activity (LinJ.34.3110) R- 5’CGCTCGTAAACCTCGCCAAC3 ’ 15 CBS cystathionine Endogenous control F- beta-synthase gene 5’GAAGTACACGGTGGAGGCTG (LinJ.17.0300) 3’ R- 5’CGCTGATCACGACCTTCTTC3’ 16 GAPDH Glyceraldehyde Endogenous control F- s-3 gene 5’CGCCGATGTCAACTGGATG3’ Phosphatedehy R- drogenase 5’GCTCCTTCTTCAGCGTGTCG3’ (LinJ.30.2990)

Table S2: Pattern of up-regulated and down-regulated gene expression in K133 AS-R parasite: Fold changes AS-R, Up-regulated AS-R, Down regulated >2.0-3.0 70 85 >3.0 – 4.0 20 16 >4.0 12 5 Total genes 102 106 Percent of modulated genes 1.11% 1.15%

Footnote: The percent modulated genes calculated from the total 9,170 genes obtained in QC after filter

Table S3: Genes differentially modulated with their functional categories in K133

AS-R L. donovani parasites.

FOLD CHANG S.N E REGULA GENE ID DESCRIPTION O K133AS- -TION R/ K133WT

NUCLEIC ACID METABOLISM

LmjF23.snRNA. 1. 5.49 UP small nuclear RNA, U1 snRNA 01 Hypothetical protein , conserved LinJ.32.2210 2.81 2. UP catalytic activity, hydrolase activity, ion

binding LinJ.35.2650 Hypothetical protein , unknown function 3. 2.49 UP DNA binding LmjF23.snRNA. 4. 2.36 UP small nuclear RNA, U3 snRNA 02 LinJ.06.0750 Hypothetical protein , conserved 5. 2.30 UP Regulation of transcription , DNA binding LinJ.15.0140 hypothetical protein, conserved 6. 2.27 UP nucleic acid binding, zinc ion binding Methylthioadenosinephosphorylase, putative LinJ.05.0830 7. 2.12 UP activity, transferring pentosyl

groups LinJ.33.0580 Hypothetical protein , conserved 8. 2.09 UP RNA activity LinJ.25.1350 DNA-directed RNA ii 9. 2.03 UP Transcription LmjF31.snRNA. 10. 2.01 UP small nuclear RNA, U2 snRNA 01 LinJ.36.1680 3.79 Universal minicircle sequence binding protein, 11. DOWN putative; nucleic acid binding, zinc ion binding LmjF27.rRNA.25 3.76 12. DOWN 28S ribosomal RNA (LSU-beta)

LinJ.28.1000 3.30 Endonuclease/exonuclease/phosphatase-like 13. DOWN protein; endonuclease &exonuclease activity Nucleoside transporter 1, putative LinJ.36.2040 3.20 14. DOWN nucleoside transmembrane transporter

activity; guanosine salvage; inosine salvage. LinJ.33.3390 2.93 h1 histone-like protein 15. DOWN

LinJ.29.0260 2.67 Thymine-7-hydroxylase, putative 16. DOWN Iron ion binding; activity. LinJ.36.1710 2.29 Poly-zinc finger protein 2, putative 17. DOWN Nucleic acid binding ; Zinc ion binding LinJ.33.3340 2.22 Small nuclear ribonucleoprotein SmD2 18. DOWN RNA splicing LinJ.36.0800 2.22 Uncharacterized protein 19. DOWN Metal ion binding LinJ.30.3630 2.12 Zinc finger-domain protein, putative 20 DOWN nucleic acid binding ; zinc ion binding

PROTEIN , METABOLISM LinJ.33.0320 3.57 Autophagy-related protein 3 21. UP autophagy ; protein transport LinJ.18.1130 Hypothetical protein, conserved 22. 2.95 UP zinc ion binding Tubulin-tyrosine ligase-like protein LinJ.32.3080 2.91 23. UP tubulin-tyrosine ligase activity

protein modification process Putative lipoate protein ligase LinJ.36.3230 2.72 ligase activity ; octanoyltransferase activity ; 24. UP cellular protein modification process ; lipoate biosynthetic process Ubiquitin-like protein, putative LinJ.36.0590 2.66 damaged DNA binding 25. UP nucleotide-excision repair, proteasomal ubiquitin-dependent protein catabolic process LinJ.27.0310 2.45 26. UP Methylmalonyl-coenzyme a mutase, putative

LinJ.19.1560 2.34 Peptidylprolylisomerase-like protein 27. UP activity ; protein folding Hypothetical protein, LinJ.31.0290 28. 2.16 UP L-ascorbic acid binding, iron ion binding,

oxidoreductase activity LinJ.33.1410 Cysteine conjugate beta-lyase, 29. 2.07 UP aminotransferase- like protein lyase activity ; pyridoxal phosphate binding ; transaminase activity ; biosynthetic process LinJ.04.0160 7.11 Cell adhesion ; 30. DOWN activity LinJ.19.0860 5.32 Autophagy-related protein 31. DOWN autophagy LinJ.09.0180 4.28 Autophagy-related protein 32. DOWN autophagy calpain-like cysteine peptidase, LinJ.20.1320 3.86 33. DOWN putative,calpain-like cysteine peptidase, Clan

CA, family C2 LinJ.19.0820 3.83 Autophagy-related protein 34. DOWN Autophagy Putative calpain-like cysteine peptidase LinJ.20.1220 2.47 35. DOWN calcium-dependent cysteine-type

activity ;. Putative calpain-like cysteine peptidase LinJ.20.1210 2.37 36. DOWN calcium-dependent cysteine-type

endopeptidase activity ; proteolysis. Putative 2.18 aminopeptidase activity ; manganese ion 37. LinJ.11.0640 DOWN binding ; metalloexopeptidase activity ; proteolysis. Ubiquitin-protein ligase, putative LinJ.36.6600 2.13 acid-amino acid ligase activity ; protein 38. DOWN modification process ; ubiquitin-protein transferase activity LinJ.33.2670 2.03 Putative 39. DOWN metallocarboxypeptidase activity ;proteolysis. Putative N-acyl-L-amino acid amidohydrolase LinJ.31.1140 2.01 40. DOWN metallopeptidase activity ;protein dimerization

activity;proteolysis ;aminoacylase activity TRANSLATION LmjF21.0960 3.18 Hypothetical protein, conserved 41. UP ATP binding ; aminoacyl-tRNA ligase activity LmjF27.rRNA. 8.27 42. 32 DOWN 28S ribosomal RNA (LSU-delta, M2)

Translation initiation factor IF-2, putative LinJ.33.2880 2.54 43. DOWN GTP binding ;translation initiation factor

activity; GTPase activity. Isoleucyl-tRNAsynthetase, putative LinJ.36.5870 2.43 ATP binding ;aminoacyl-tRNA ligase activity ; 44. DOWN isoleucine-tRNA ligase activity ; nucleotide binding; zinc ion binding. LinJ.36.1490 2.14 Translation elongation factor 1-beta, putative 45. DOWN translation elongation factor activity LIPID METABOLISM Myo-inositol-1-phosphate synthase,inositol-3- LinJ.14.1450 2.59 phosphate synthase, putative (INO1) 46. UP Inositol biosynthetic process, metabolic process, phospholipid biosynthetic process Hypothetical protein, triglyceride lipase LinJ.13.0200 2.25 47. UP activity, zinc ion binding hydrolase activity ;

lipid metabolic process . Glycerol uptake protein, putative LmjF19.1345 2.64 48. DOWN integral component of membrane

[GO:0016021] Fatty acid elongase, putative 49. LinJ.14.0670 2.13 DOWN fatty acid biosynthetic process, long-chain fatty acid biosynthetic process Hypothetical protein, (pseudogene) LinJ.03.0280 2.13 50. DOWN fatty acid biosynthetic process ; iron ion

binding; oxidoreductase activity

CARBOHYDRATE METABOLISM Glucose transporter, lmgt2 fructose transmembrane transporter activity ; LinJ.36.6550 3.48 galactosetransmembrane transporter activity; 51. DOWN glucose transmembrane transporter activity ; mannose transmembrane transporter activity ; glucose import LinJ.22.0002 3.43 Hypothetical protein 52. DOWN

LmjF06.1290 3.37 Hypothetical protein, heme binding, transition 53. DOWN metal ion binding Lipophosphoglycan biosynthetic protein (lpg2) LinJ.34.4290 3.20 GDP-mannose transmembrane transporter 54. DOWN activity nucleotide-sugar transport Glucose transporter, lmgt2 fructose transmembrane transporter activity ; LmjF36.6290 3.15 galactosetransmembrane transporter activity ; 55. DOWN glucose transmembrane transporter activity ; mannose transmembrane transporter activity ; carbohydrate transport Glucose transporter, lmgt1 fructose transmembrane transporter activity ; LinJ.36.6560 3.10 56. DOWN galactosetransmembrane transporter activity;

glucose transmembrane transporter activity; mannose transmembrane transporter activity LinJ.34.4360 2.38 D-isomer specific 2-hydroxyacid 57. DOWN dehydrogenase- like protein LmjF34.1400 2.33 D-isomer specific 2-hydroxyacid 58. DOWN dehydrogenase- protein phosphoacetylglucosaminemutase-like LmjF07.0805 2.01 protein,acetylglucosaminephosphomutase, 59. DOWN putative,N-acetylglucosamine-phosphate mutase, putative CELL MOVEMENT LinJ.25.2050 2.96 Kinesin, putative 60. UP ATP binding, microtubule motor activity LinJ.18.0530 2.15 Hypothetical protein, conserved 61. UP GTPase activator activity ; cell morphogenesis LinJ.06.1070 2.12 Kinesin, putative 62. UP ATP binding, microtubule motor activity Dynein light chain, flagellar outer arm, LinJ.32.0240 2.93 63. DOWN putative

microtubule-based process ARP2/3 complex 16kDa subunit, putative LinJ.05.0290 2.62 64. DOWN Arp2/3 complex-mediated actin nucleation ;

regulation of actin filament polymerization LmjF05.0285 2.58 ARP2/3 complex 16kDa subunit, putative 65. DOWN regulation of actin filament polymerization Tubulin beta chain (Fragment) GTP binding, GTPase activity, structural LinJ.08.1280 2.38 66. DOWN molecule activity ; microtubule-based

movement ; microtubule-based process ; protein polymerization Actin severing and dynamics regulatory LinJ.29.0520 2.17 67. DOWN protein

actin filament depolymerization Putative dynein heavy chain LinJ.23.1570 2.14 68. DOWN ATP binding, ATPase activity, microtubule

motor activity LinJ.05.0380 2.08 69. DOWN Putative microtubule-associated protein

Calpain-like cysteine peptidase, putative small myristoylated protein-2, putative;(SMP- LinJ.20.1340 2.04 70. DOWN 2)

flagellar cell motility ; flagellar pocket membrane. LinJ.35.5310 2.04 71. DOWN Hypothetical protein, conserved

LinJ.33.2770 2.03 72. DOWN Dynein intermediate chain, putative

CELL MEMBRANE PROTEIN LinJ.17.0020 5.56 73. UP integral component of membrane

LinJ.31.0460 5.36 Amastin, putative 74. UP integral component of membrane LmjF31.0450 4.36 75. UP Amastin, putative

LinJ.31.1900 3.51 Hypothetical protein 76. UP integral component of membrane LmjF08.0770 3.44 77. UP Amastin-like protein

LinJ.29.3030 3.37 Amastin, putative 78. UP integral component of membrane LinJ.08.0690 3.35 Amastin-like protein 79. UP integral component of membrane 80. LmjF31.1855 3.35 UP Hypothetical protein integral component of membrane LinJ.08.0680 UP Amastin-like protein 81. 3.30 integral component of membrane LinJ.08.0720 3.29 Amastin-like protein 82. UP integral component of membrane LinJ.24.1300 3.18 Amastin-like surface protein-like protein 83. UP integral component of membrane LinJ.08.0700 2.77 Amastin-like protein 84. UP integral component of membrane LinJ.34.3430 2.62 Hypothetical protein (pseudogene) 85. UP integral component of membrane LmjF34.0500 2.58 86. UP Amastin-like protein

LinJ.26.1440 2.54 Hypothetical protein, integral component of 87. UP membrane LinJ.34.1150 2.39 Amastin-like surface protein, putative 88. UP integral component of membrane LinJ.29.3000 2.38 Amastin, putative 89. UP integral component of membrane Hypothetical protein, conserved, LinJ.26.2590 2.08 ATP binding; D-alanine-D-alanine ligase 90. UP activity; metal ion binding; methyltransferase activity; peptidoglycan biosynthetic process LinJ.30.2810 Hypothetical protein, conserved 91. 2.07 UP integral component of membrane Hypothetical protein, conserved LinJ.25.2390 2.05 protein name -Palmitoyltransferase 92. UP protein-cysteine S-palmitoyltransferase activity ; zinc ion binding Hypothetical protein LmjF31.1165 2.02 93. UP integral component of membrane ; heme

binding LinJ.34.1690 94. 2.01 UP Amastin-like surface protein, putative

LinJ.04.0170 4.68 95. DOWN Surface antigen-like protein

LinJ.21.1090 2.94 96. DOWN integral component of membrane

97. LinJ.05.0900 2.81 DOWN Surface antigen-like protein protein binding Dehydrogenase-like protein LinJ.10.0070 2.52 98. DOWN integral component of membrane ;binding ;

catalytic activity ; oxidoreductase activity. LinJ.04.0180 2.37 99. DOWN Surface antigen-like protein

LinJ.04.0190 2.36 100. DOWN Surface antigen-like protein

LinJ.33.2980 2.34 101. DOWN integral component of membrane

LinJ.14.0500 2.26 102. DOWN integral component of membrane LinJ.34.1050 2.13 103. DOWN Amastin-like protein

LinJ.33.0220 2.12 104. DOWN integral component of membrane

LinJ.08.0600 105. 2.10 DOWN integral component of membrane LinJ.24.1310 2.08 Amastin-like surface protein-like protein 106. DOWN integral component of membrane LinJ.23.1130 107. 2.02 DOWN integral component of membrane CELL SIGNALLING LinJ.35.0780 3.36 108. UP CBS domain protein, conserved

Protein kinase, putative,dual-specificity protein kinase, putative LinJ.33.1930 3.15 109. UP ATP binding, protein kinase activity, protein

serine/threonine kinase activity, protein tyrosine kinase activity LinJ.22.1450 3.11 Ser/thr protein phosphatase, putative 110. UP hydrolase activity LinJ.25.1410 2.99 Hypothetical protein, conserved 111. UP regulation of signal transduction Protein kinase, putative LinJ.17.0440 2.08 112. UP ATP binding, protein kinase activity, protein

serine/threonine kinase activity, protein tyrosine kinase activity

Serine/threonine-protein phosphatase LinJ.34.0840 2.39 113. DOWN metal ion binding; phosphoprotein

phosphatase activity . Protein tyrosine phosphatase-like protein LinJ.16.0240 2.38 protein tyrosine phosphatase activity, protein 114. DOWN tyrosine/serine/threonine phosphatase activity protein dephosphorylation Adenylatecyclase-like protein LinJ.28.0090 2.26 Adenylatecyclase activity ; heme binding ; 115. DOWN oxygen binding ; intracellular signal transduction TRANSPORTER LinJ.31.0030 6.33 Aquaglyceroporin (AQP1) 116. UP metal ion transmembrane transporter activity 2.70 Hypothetical protein, conserved 117. LinJ.18.1270 UP binding; transport. Nucleobase transporter (NT4) LinJ.11.0520 2.60 118. UP Nucleobase transmembrane transporter

activity Transporter, putative,major facilitator LinJ.18.0040 2.54 119. UP superfamily protein (MFS),

putativetransporter activity UDP-galactose transporter (LPG5B) LinJ.18.0400 2.31 120. UP sugar: hydrogensymporter activity;

nucleotide-sugar transport ATP-binding cassette protein subfamily G, member 1, putative (ABCG1), ATP binding, LmjF06.0080 2.27 121. UP ATPase activity, coupled to transmembrane

movement of substances, phosphopantetheine binding LinJ.10.0900 2.17 Nuclear transport factor 2, putative,ntf2-like 122. DOWN transport 2.16 ABC transporter family-like protein 123. LinJ.33.3410A DOWN ATPase activity ; ATP binding LinJ.07.1340 2.15 124. DOWN Amino acid transporter, putative (AAT19)

LinJ.22.0100 2.11 125. DOWN Amino acid permease, putative (AAT22)

STRESS PROTEIN LinJ.22.0670 5.13 A2 protein 126. UP protein binding ;cellular response to stress. Gamma-glutamylcysteinesynthetase (GSH1) glutathione biosynthetic process, response to LinJ.18.1660 4.17 127. UP reactive oxygen species, trypanothione

biosynthetic process glutamate-cysteine ligase activity Hypothetical protein, conserved LinJ.31.0530 2.38 heat shock protein binding ; unfolded protein 128. UP binding; protein folding . dnaj chaperone-like protein LinJ.33.0940 2.25 heat shock protein binding ;unfolded protein 129. UP binding; protein folding. LinJ.28.1480 2.06 Haloaciddehalogenase-like hydrolase, putative 130. UP catalytic activity, hydrolase activity OXIDATION - REDUCTION LinJ.31.1190 2.56 Hypothetical protein, heme binding ; 131. UP transition metal ion binding Hypothetical protein, conserved electron carrier activity, oxidoreductase LinJ.25.2150 2.40 activity 132. UP 2 iron, 2 sulfur cluster binding; nitrite reductase [NAD(P)H] activity ; nitrate assimilation Cytochrome p450-like protein LinJ.34.3110 2.19 133. DOWN electron carrier activity ; heme binding;

monooxygenase activity oxidoreductase-like protein LinJ.19.1490 2.14 electron carrier activity, oxidoreductase 134. DOWN activity

OTHERS LinJ.21.0070 5.64 135. UP Hypothetical protein, conserved

LinJ.18.0610 3.20 Hypothetical protein, conserved 136. UP zinc ion binding LinJ.28.1700 2.52 Hydrolase, alpha/beta fold family, putative 137. UP hydrolase activity LinJ.33.0750 2.46 138. UP Hypothetical protein, conserved

LinJ.29.3020 2.02 139. UP Tuzin like protein, putative

LinJ.06.1350 2.98 Hypothetical protein 140 DOWN heme binding; transition metal ion binding. LinJ.01.0010 2.57 Hypothetical protein 141. DOWN binding Phosphoglyceratemutase family member 5, LinJ.36.4270 2.33 142. DOWN putative

protein serine/threonine phosphatase activity. LinJ.02.0280 2.24 Hypothetical protein, conserved 143. DOWN binding LinJ.33.2460 2.07 Hypothetical protein, 144. DOWN catalytic activity; metabolic process.

Figure S2.

Figure S2: Cytotoxicity of (A) AQP1 inhibitor and (B) Verapamil to host macrophages (mice PECs). % cell viability ± SD with the increasing drug concentration has been plotted here. The data was obtained from three independent experiments.