IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF TETRAHYDROLIPSTATIN TARGETS IN M. BOVIS BCG AT DIFFERENT METABOLIC STATES MADHU SUDHAN RAVINDRAN M. Sc. Advanced Biochemistry, University of Madras. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 Madhu Sudhan Ravindran Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Madhu Sudhan Ravindran 05 Dec 2012 Madhu Sudhan Ravindran Acknowledgement A wholehearted thanks to my supervisor, Dr. Markus R. Wenk who supported me throughout my graduate study, offered me full freedom to perform my experiments and made himself available throughout. I am grateful to NUS Graduate School for Integrative Sciences and Engineering (NGS) for their generous funding and the staff for all the help. I would like to thank my collaborators Dr. Shao Q. Yao and Dr. Srinivasa Rao, for guidance and experimental support, especially Dr. Yao who pushed me firmly during the early days of graduate study. It was a great experience to work with Chionh Yok Hian, whose innovative (crazy) ideas and immense knowledge provided useful tips for my project. I value the friendship and scientific collaboration with Lukas. Ankit for bearing me as a mentor from the past months and Sudar for finishing the incomplete experiments I leave behind. I am grateful to Amaury, Charmaine, Federico, KL, Lynette, Peng-Yu and Shareef for their technical support. Thanks to Pradeep for making himself available for the most cherished lengthy coffee breaks. I am thankful to Anne, Huimin, Phyliss and Sarah for making the non-scientific management process so much more easier. I would also like to thank other past and present lab members who directly or indirectly helped me especially Aaron, Husna, Jacklyn, Jingyan and Robin. A big thank you to all of you for being such amazing friends. I would like to thank my family members for their affection and understanding and my best friend — my wife for her constant support and encouragement. Madhu Sudhan Ravindran SUMMARY 11 LIST OF TABLES 13 LIST OF FIGURES 14 LIST OF ABBREVIATIONS 17 PUBLICATIONS 20 CHAPTER INTRODUCTION 21 1.1 Biology of Mycobacteria 21 1.1.1 Tuberculosis: the disease 1.1.1.1 Mycobacterium tuberculosis 21 22 1.1.2 History of mycobacteria 23 1.1.3 Diagnosis of TB 24 1.1.4 Prevention and treatment of TB 25 1.1.5 Life cycle of Mtb 27 1.1.5.1 Infection stage 27 1.1.5.2 Immune activation stage 27 1.1.5.3 Latency stage 28 Madhu Sudhan Ravindran 1.1.5.4 Reactivation/resuscitation 29 1.1.6 Granulomas 30 1.1.7 Survival strategies in the host 32 1.1.8 Lipids in mycobacterial survival 33 1.2 Mycobacterial metabolic stages 36 1.2.1 Definitions of dormancy, persistence and latency 36 1.2.2 Generation of dormant bacilli 37 1.2.3 Laboratory model for generating dormant bacilli 37 1.2.3.3 The Wayne model: Hypoxia-induced NRP 40 1.2.4 Lipids: an alternative energy source 42 1.3 Mycobacterial lipid metabolizing genes 44 1.3.1 Lipid droplets: The energy store house of dormant bacilli 44 1.3.2 Lipases and esterases 45 1.3.2.2 Lipolytic esterases 48 1.3.2.3 Hormone-sensitive lipases (HSL) 49 1.3.3 Carboxylester hydrolases in bacterial pathogenesis 51 1.3.4 Lipases and esterases in mycobacteria 52 1.3.4.1 α/β-hydrolase superfamily 52 1.3.5 Carboxylester hydrolase in Mycobacterial life cycle 58 1.3.6 Inhibitors of lipases and esterases 60 1.3.7 Tetrahydrolipstatin (THL) 61 1.3.7.1 Chemistry and mechanism of action 61 1.3.7.2 Anti-obesity and anti-cancer drug 62 Madhu Sudhan Ravindran 1.3.7.3 Anti-mycobacterial drug 63 1.4 Specific Aims of The Study 64 CHAPTER MATERIALS AND METHODS 65 2.1 Bacterial Strains and Growth Media 65 2.2 Mycobacterial cultures 65 2.3 Cloning procedures 67 2.3.1 Mycobacterial genomic DNA isolation 67 2.3.2 Construction of over-expression plasmids 67 2.3.3 Electroporation and selection of transformants 68 2.4 Drug inhibitory concentration 69 2.4.1 Minimal inhibitory concentration (MIC50) 69 2.4.2 Minimal bactericidal concentration (MBC) 70 2.5 Protein extraction 70 2.6 Chemicals, THL analogues and click reagents 71 2.7 Huisgen cycloaddition reaction 73 2.7.1 Fluorescent tagging Madhu Sudhan Ravindran 73 2.7.2 Target enrichment 74 2.8 Western blot 75 2.9 Proteomics 76 2.9.1 Two-dimensional (2D) electrophoresis 76 2.9.2 Peptide MS Analysis 77 2.9.3 Peptide MS data searching and processing 77 2.10 Lipid Extraction 78 2.11 Thin layer chromatography (TLC) 79 2.12 Liquid chromatography and mass spectrometry (LC-MS) of lipids 79 2.13 Enzymatic assay and IC50 80 2.14 RNA isolation and quantitative RT-PCR 81 2.15 Fluorescent labeling of LDs and quantification of esterase activity 82 2.16 Flow cytometry 82 2.17 In silico characterization of lipH 83 2.18 Statistical Analysis 84 Madhu Sudhan Ravindran CHAPTER THL BINDS α/β-HYDROLASE FAMILY PROTEINS 85 3.1 Introduction 85 3.2 Results 88 3.2.1 Efficiency of THL-alkyne analogues in M. bovis BCG 88 3.2.2 THL targets α/β-hydrolase family proteins in M. bovis BCG 94 CHAPTER HYPER-RESISTIVITY OF ESTERASE ENHANCED MYCOBACTERIA TO THL WHEN RESUSCITATED FROM NON-REPLICATIVE STATE 99 4.1 Introduction 99 4.2 Results 101 4.2.1 Total cellular esterase activity is diminished during NRP and revived during regrowth 4.2.2 THL is effective against mycobacteria resuscitating from NRP 101 105 4.2.3 THL targets are regulated in biphasic pattern in different metabolic states 107 4.2.4 Regulation of α/β-hydrolases occurs at protein level Madhu Sudhan Ravindran 112 CHAPTER THE SHORT-CHAIN ESTERASE ACTIVITY OF LIPH IS INHIBITED BY THL 116 5.1 Introduction 116 5.2 117 Results 5.2.1 Over-expression of tesA and lipH leads to increased bacterial resistance to THL 117 5.2.2 LipH over-expression causes reduced utilization of TAG during regrowth 122 5.2.3 Short-chain esterase activity of lipH is inhibited by THL 126 CHAPTER 134 CONCLUSION AND PERSPECTIVE 134 6.1 Summary and key findings 134 6.2 THL binds α/β-hydrolase family proteins 136 6.3 Esterase enhanced resuscitating mycobacteria is sensitive towards THL 139 6.4 A short chain esterase, lipH is an opportunistic carbon supplier for 6.5 resuscitating BCG 144 Future perspective 148 Madhu Sudhan Ravindran 6.5.1 Identification of THL targets in NRP bacilli membrane and culture filtrate fractions 148 6.5.2 PTM-mediated protein regulation 149 6.5.3 Elucidating the substrate of lipH 149 6.5.4 Validation and characterization of BCG2654c and lipM 150 6.5.5 Carboxylester hydrolases in the mycobacterial starvation model 150 6.5.6 THL targets in human macrophages 151 6.5.7 Mechanism of THL bacteriostatic and bactericidal activity on BCG 151 BIBLIOGRAPHY 153 ANNEXES 167 Annex 1: THL targets identified in mycobacterial logarithmic state Annex 2: THL targets identified in mycobacterial NRP state Annex 3: THL targets identified in mycobacterial regrowth state Annex 4: Effect of THL on mycobacterial PDIM and PGL Annex 5: Ethambutol is insensitive towards NRP and resuscitating cells Annex 6: Two dimensional (2D) electrophoresis Madhu Sudhan Ravindran 10 Madhu Sudhan Ravindran 169 Rv3338 Rv0129c Rv1400c Rv2284 Rv2970c Rv2928 Rv1923 Rv2224c Rv0646 Rv2681 Rv1869c Rv2150c Rv2715 Rv1399c Rv1426c Rv1192 Rv1071c Rv0242c Rv1698 Rv1454c Rv2095c Rv3684 Rv3203 Rv3775 Rv3802c Rv0859 Rv3464 BCG_3408 BCG_0163c BCG_1461c BCG_2299 BCG_2991c BCG_2950 BCG_1962 BCG_2241c BCG_0695c BCG_2694 BCG_1905 BCG_2167c BCG_2728 BCG_1460c BCG_1487c BCG_1252 BCG_1129c BCG_0280c BCG_1727 BCG_1515c BCG_2115c BCG_3743 BCG_3229 BCG_3837 BCG_3864c BCG_0911 BCG_3529 rmlB1 Probable dTDP-Gluocse 4,6-dehydratase fadA Possible acyl-CoA thiolase BCG_3864c Hypothetical protein lipE Putative lipase lipV Possible lipase BCG_3743 Probable lyase BCG_2115 Conserved hypothetical protein qor Probable quinone reductase tyrS (TYRRS) Probable Tyrosyl-tRNA synthase fabG4 Probable 3-oxoacyl-[ACP] reductase echA9 Probable enoyl-CoA hydratase BCG_1252 Hypothetical protein lipO Probable esterase lipH Probable lipase BCG_2728 Possible hydrolase ftsZ Cell division protein BCG_1905 Probable oxidoreductase BCG_2694 Conserved hypothetical alanine rich protein lipG Probable lipase/esterase BCG_2241c Probable exported protease lipD Probable lipase tesA Probable thioesterase lipN Probable lipase/esterase lipM Probable esterase lipI Probable lipase A85c, fbpC Secreted antigen 331 403 336 415 224 246 316 328 424 454 345 275 420 319 341 379 411 438 301 520 647 261 376 431 320 340 297 aa 37.6 42.5 35.6 45.5 23.7 37.9 34.0 34.0 46.3 46.9 36.4 30.4 46.2 34.0 37.0 38.7 43.6 43.6 33.0 56.5 47.2 29.3 40.3 46.7 34.1 36.8 32.0 461 465 471 471 472 489 491 493 493 504 569 687 687 796 950 952 1088 1088 1092 1092 1174 1248 1291 1446 1498 1503 2280 20 14 16 18 13 16 16 15 15 12 15 26 39 29 31 28 31 31 47 33 41 106 41 62 47 73 86 Mw (kD) Ion score Queries match WCL 4.2.1.46 M, WCL 2.3.1.- M, WCL, CF - M, WCL 3.1.-.- WCL 3.1.-.- M, WCL, CF 4.-.-.- WCL, M - M, WCL, CF 1.6.5.5 M - CF,M,WCL 1.1.1.100 CF,M,WCL 4.2.1.17 - - M 3.1.-.- WCL 3.1.-.- WCL 3.-.-.- M - CF,M,WCL 1.-.-.- - - - 3.1.-.- M,WCL, CF 3.4.-.- M 3.1.-.- WCL 3.1.2.- M, WCL 3.1.1.- WCL 3.1.-.- WCL 3.1.-.- CF 2.3.1.- - - Location Family Annex 1: THL targets identified in mycobacterial logarthimic state BCG_3408 Conserved hypothetical protein Protein Function Proteins in blue are a/b-hydrolase H37Rv BCG - - + - - - + - - - - - - - - - + - - + - - - + - + - Continued in next page - - +1 - - - - - - - - - - - - - - - - +1 - - - +3 - +1 - Memb dom. Sec signal Madhu Sudhan Ravindran 170 Rv3774 Rv2841c Rv3057c Rv1323 Rv0467 Rv0906 Rv0548c Rv3042c Rv2751 Rv0427c Rv0400c Rv1912c Rv1812c Rv3651 Rv1423 Rv3057c Rv1023 Rv0530 Rv3570c Rv1655 Rv1367c Rv0547c Rv3084 Rv1438 Rv0645c Rv1446c Rv1679 BCG_3836 BCG_2861c BCG_3082c BCG_1385 BCG_0507 BCG_0958 BCG_0592c BCG_3066c BCG_2767 BCG_0466c BCG_0437c BCG_1951c BCG_1846c BCG_3709 BCG_1484 BCG_3844 BCG_1080 BCG_0573 BCG_3635c BCG_1694 BCG_1429c BCG_0591c BCG_3109 BCG_1499 BCG_0694c BCG_1507c BCG_1717 fadE16 Possible acyl-CoA dehydrogenase opcA Putative OXPP cycle protein mmaA1 Methoxy mycolic acid synthase tpi Probable triosephosphate isomerase lipR Probable acetyl-hydrolase/esterase BCG_0591c Possible oxidoreductase BCG_1429c Conserved hypothetical protein argD Probable acetylornithine aminotransferase BCG_3635c Possible oxidoreductase BCG_0573 Conserved hypothetical protein eno Probable enolase BCG_3844 Possible L-rhamnosyltransferase whiA Transcription regulatory protein BCG_3709 Conserved hypothetical protein BCG_1846c Probable dehydrogenase fadB5 Possible oxidoreductase fadE7 Probable acyl-CoA dehydrogenase xthA Probable exodeoxyribonuclease III protein BCG_2767 Conserved hypothetical protein serB2 Probable phosphoserine phosphatase menB Probable napthoate synthase BCG_0958 Conserved hypothetical protein icl Isocitrate lyase fadA4 Probable acetyl-CoA acetyltransferase BCG_3082c Short chain alcohol dehydrogenase/reductase nusA Probable N utilization substance protein A echA21 Putative enoyl CoA hydratase BCG_1386 Possible thioredoxin Proteins in blue are a/b-hydrolase Rv1324 BCG_1386 373 303 286 261 308 294 377 400 394 405 429 287 325 345 400 334 395 291 296 409 314 372 38.0 31.0 33.3 27.5 33.0 32.0 41.4 41.0 43.2 43.2 44.8 34.2 35.0 38.0 42.2 35.8 42.6 32.2 33.7 43.3 34.8 40.7 47.2 40.1 389 428 31.2 37.7 29.2 32.2 287 347 274 304 306 307 307 310 311 320 328 329 332 335 339 346 348 348 352 367 371 374 374 379 389 389 395 399 403 424 433 447 10 12 12 12 11 10 11 11 10 12 13 14 15 12 15 11 11 15 18 12 12 M,WCL - M,WCL 1.3.99.- WCL - CF,M,WCL 2.1.1.- M,WCL 5.3.1.1 - 3.1.1.- WCL 1.-.-.- WCL - CF,M,WCL 2.6.1.11 M,WCL 1.-.-.- - - CF,M,WCL 4.2.1.11 WCL 1.1.-.- WCL - - - WCL 1.-.-.- - 1.6.5.5 M,WCL 1.3.99.- WCL 3.1.11.2 WCL - WCL 3.1.3.3 WCL, M 4.1.3.36 M,WCL - CF, WCL 4.1.3.1 CF,M,WCL - WCL 1.1.-.- M,WCL - M, WCL, CF - - - - - + - - - - - - - - - + - - - - - - + - - - - - - Continued in next page - - - - - - - - - - - - - - - - - - - - - - - - - - - - Madhu Sudhan Ravindran 171 Rv2495c Rv2344c Rv0482 Rv3804c Rv3262 Rv3373 Rv1202 Rv2498c Rv3028c Rv2139 Rv2580c Rv0830 Rv1479 Rv2953 Rv3010c Rv3683 Rv0620 Rv3692 Rv3372 Rv0931c Rv3294 Rv0217c Rv2605c Rv1649 Rv2952 Rv1383 Rv3256c BCG_2515c BCG_2367c BCG_0523 BCG_3866c BCG_3291 BCG_3445 BCG_1262 BCG_2518c BCG_3051c BCG_2156 BCG_2603c BCG_0883 BCG_1541 BCG_2974 BCG_3032c BCG_3742 BCG_0666 BCG_3751 BCG_3444 BCG_0984c BCG_3294c BCG_0254c BCG_2630c BCG_1688 BCG_2973 BCG_1444 BCG_3285c BCG_3285c Conserved hypothetical protein carA Carbamoyl-phosphate synthase 346 376 270 341 pheS Phenylalanyl-tRNA synthetase, α chain BCG_2973 Possible methyltransferase 281 302 211 664 391 358 363 141 343 418 377 301 423 tesB2 Probable acyl-CoA thioesterase II lipW Possible esterase wbbL1 Possible L-rhamnosyltransferase pknDa Transmembrane serine/threonine-protein kinase D otsB2 Possible trehalose 6-phosphate phosphatase moxR2 Putative methanol dehydrogenase galK Probable galactokinase BCG_3742 Conserved hypothetical protein pfkA Probable 6-phosphofructokinase Rv2953 Conserved hypothetical protein moxR1 Probable transcriptional regulatory protein BCG_0883 Conserved hypothetical protein hisS Histidyl-tRNA synthetase 357 318 fixB Probable electron transfer flavoprotein (α-subunit) BCG_2156 Dihydroorotate dehydrogenase 273 354 213 448 338 369 431 393 373 citE Probable citrate (pro-3s)-lyase (beta subunit) dapE Probable succinyl-diaminopimelate desuccinylase echA18 Enoyl-CoA hydratase fbiB Probable F420 biosynthesis protein A85a, fbpA Secreted antigen murB UDP-N-acetylenolpyruvoylglucosamine reductase dgt Deoxyguanosine triphosphate triphosphohydrolase pdhC Probable dihydrolipoamide S-acetyltransferase BCG_2474c Probable oxidoreductase (β subunit) Proteins in blue are a/b-hydrolase Rv2454c BCG_2474c 35.3 40.0 30.8 37.4 31.7 32.4 33.0 31.5 41.8 38.0 37.7 34.6 37.1 45.3 40.7 33.4 45.2 37.9 31.7 29.0 37.5 31.6 47.6 35.8 38.7 47.0 41.3 40.4 255 255 256 260 261 263 263 264 264 265 268 274 276 276 277 278 280 281 282 286 289 290 291 299 299 302 303 305 10 17 12 10 16 10 12 13 11 15 13 12 12 10 11 11 13 WCL - WCL, M 6.3.5.5 WCL, M 2.1.1.- M, WCL 6.1.1.20 M, WCL 3.1.2.- - 3.1.1.- - 2.-.-.- M, WCL 2.7.1.- CF,WCL 3.1.3.12 M,WCL - - 2.7.1.6 WCL - CF,M, WCL 2.7.1.11 - - M - M,WCL 2.1.1.- WCL 6.1.1.21 M 1.3.98.1 M, WCL, CF - M,WCL 4.1.3.6 WCL 3.5.1.18 - 4.2.1.17 M 6.3.2.- M, WCL, CF 2.3.1.- - 1.1.1.158 WCL - WCL 2.3.1.12 M,WCL 1.-.-.- - - - - - - - - - - - - + - - - - - - - - - - - + - - - - Continued in next page - - - - - - +1 - - - +1 - - - - - - - - - - - +1 - - - - Madhu Sudhan Ravindran 172 Rv2981c Rv1010 Rv2161c Rv0480c Rv3505 Rv3439c Rv3276c Rv3838c Rv2744c Rv3224 Rv1299 Rv3708c Rv1078 Rv2296 Rv3569c Rv0439c Rv1099c Rv0215c Rv0889c Rv3680 Rv0770 Rv3881c Rv0510 Rv1901 Rv0707 Rv3502c Rv1480 BCG_3002c BCG_1067 BCG_0994c BCG_0521c BCG_3569 BCG_3505c BCG_3305c BCG_3901c BCG_2760c BCG_3252 BCG_1359 BCG_3768c BCG_1136 BCG_2312 BCG_3634c BCG_0478c BCG_1159c BCG_0252c BCG_0941c BCG_3739 BCG_0822 BCG_3936c BCG_0553 BCG_1940 BCG_0757 BCG_3566c BCG_1542 BCG_1542 Conserved hypothetical protein fabG Probable short-chain type dehydrogenase/reductase rpsC Probable 30S ribosomal protein S3 cinA cinA-like protein hemC Probable porphobilinogen deaminase BCG_3936c Hypothetical alanine and glycine rich protein BCG_0822 Probable dehydrogenase/reductase BCG_3739 Probable anion transporter atpase citA Citrate synthase II fadE3 Probable acyl-CoA dehydrogenase glpX Fructose-1,6-bisphosphatase class BCG_0478c Putative dehydrogenase/reductase bphD 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase BCG_2312 Probable haloalkane dehalogenase pra Probable Proline-rich antigen homolog asd Aspartate dehydrogenase prfA Probable peptide chain release factor BCG_3252 Possible short-chain dehydrogenase/reductase BCG_2760 35 kDa alanine rich protein pheA Possible prephenate dehydratase purK Phosphoribosylaminoimidazole carboxylase ATPase BCG_3505c Hypothetical alanine and proline rich protein fadE27 Probable acyl-CoA dehydrogenase BCG_0521c Conserved hypothetical protein Possible oxidoreductase ksgA Probable dimethyladenosine transferase ddlA Probable D-alanine--D-alanine ligase arcA Arginine deiminase Proteins in blue are a/b-hydrolase Rv1058 BCG_1058 317 317 274 430 309 460 295 386 373 388 362 311 291 300 240 345 357 282 270 321 429 467 373 340 288 317 373 543 34.5 32.8 30.0 45.7 32.1 47.8 30.4 41.5 40.2 42.2 38.4 33.6 32.0 33.3 25.2 36.0 39.0 30.0 29.2 33.8 45.6 45.8 45.8 32.5 31.8 34.8 39.8 43.2 210 215 219 219 221 224 226 226 227 227 229 232 233 233 236 237 237 247 248 251 251 251 251 252 253 253 253 254 10 10 10 10 12 11 11 M - WCL 1.-.-.- M, WCL - WCL - CF, M, WCL 2.5.1.61 M,WCL, CF - - 1.1.-.- WCL 3.6.1.- WCL 2.3.3.1 - 1.3.99.- M, WCL - M, WCL 1.-.-.- M, WCL 3.7.1.- M, WCL 3.8.1.5 M,WCL - M 1.2.1.11 M, WCL - CF,M,WCL 1.-.-.- WCL - M,WCL 4.2.1.51 CF,M,WCL 4.1.1.21 - - M, WCL 1.3.99.- M, WCL - M, WCL 1.-.-.- - 2.1.1.- WCL 6.3.2.4 WCL 3.5.3.6 - - - - + - - - - - - - - - - - - - - - - - - + - - - - Continued in next page - - - - - - - - - - - - - - +2 - - - - - - - - - - - - - Madhu Sudhan Ravindran 173 Rv2192c Rv3725 Rv3227 Rv3206c Rv3767c Rv0183 Rv3556c Rv3523 Rv2982c Rv3634c Rv3149 Rv3546 Rv2298 Rv1708 Rv1082 Rv0247c Rv2373c Rv0542c Rv0154c Rv3075c Rv2696c Rv2045c Rv3915 Rv3168 Rv3218 Rv0553 Rv1309 BCG_2208c BCG_3785 BCG_3257 BCG_3232c BCG_3826c BCG_0220 BCG_3620c BCG_3587 BCG_3003c BCG_3692c BCG_3172 BCG_3610 BCG_2314 BCG_1747 BCG_1140 BCG_0285c BCG_2387c BCG_0586c BCG_0190c BCG_3100c BCG_2709c BCG_2064c BCG_0021 BCG_3192 BCG_3245 BCG_0598 BCG_1369 326 305 atpG Probable ATP synthase gamma chain 321 378 406 511 259 307 403 362 382 248 288 318 323 391 252 314 334 34.0 34.2 34.6 42.5 44.1 56.5 27.2 33.1 44.6 36.7 41.0 29.3 33.0 34.6 35.0 41.6 27.5 33.6 34.4 41.5 41.0 386 394 30.3 34.8 279 314 42.3 46.5 450 392 34.8 37.8 31.7 309 370 297 menC Probable muconate cycloisomerase BCG_3245 Conserved hypothetical protein BCG_3192 Conserved hypothetical protein BCG_0021 Putative hydrolase lipT Probable carboxylesterase BCG_2709c Hypothetical alanine & glycine and valine rich protein BCG_3100c Conserved hypothetical protein fadE2 Probable acyl-CoA dehydrogenase menE Possible o-succinylbenzoic acid--CoA ligase dnaJ2 Probable chaperone protein BCG_0285c Probable succinate dehydrogenase [iron-sulfur subunit] BCG_1140 Mycothiol conjugate amidase BCG_1747 Putative initiation inhibitor protein BCG_2314 Conserved hypothetical protein fadA5 Probable acetyl-CoA acetyltransferase nuoE Probable NADH dehydrogenase I (chain E) galE1 UDP-glucose 4-epimerase gpdA2 Glycerol-3-phosphate dehydrogenase [NAD(P)+ BCG_3587 Probable lipid carrier protein or keto acyl-coa thiolase fadA6 Probable acetyl-CoA acetyltransferase BCG_0220 Possible lysophospholipase BCG_3826c Conserved hypothetical protein moeB1 Probable molybdenum cofactor biosynthesis protein aroA 3-phosphoshikimate 1-carboxyvinyltransferase BCG_3785 Putative oxidoreductase trpD Probable anthranilate phosphoribosyltransferase BCG_2650 Conserved hypothetical protein Proteins in blue are a/b-hydrolase Rv2623 BCG_2650 177 177 177 177 178 178 179 179 179 181 181 182 186 189 189 189 195 195 196 196 200 202 202 202 204 206 208 210 M - M,WCL 3.6.3.14 M,WCL 4.2.1.113 WCL - M,WCL - M,WCL 3.-.-.- - 3.1.1.- M,WCL - M,WCL - M, WCL 1.3.99.- - 6.2.1.26 WCL - M,WCL 1.3.99.1 WCL - WCL - M,WCL 1.-.-.- CF,M,WCL 2.3.1.9 M,WCL 1.6.5.3 M,WCL 5.1.3.2 M,WCL 1.1.1.94 - 2.3.1.16 CF,M,WCL 2.3.1.9 M, WCL 3.1.-.- M,WCL 2.1.1.- WCL - CF,M,WCL 2.5.1.19 WCL 1.-.-.- M, WCL 2.4.2.18 - - - - - - - - - - - - - - - - - + + - - - - - - - - - Continued in next page - - - - - - - - - - - - - - - - - - - - - - - + - - - - Madhu Sudhan Ravindran 174 Rv1112 Rv1336 Rv1086 Rv3205c Rv1605 Rv1124 Rv0772 Rv2927c Rv3161c Rv3575c Rv0989c Rv2564 Rv2857c Rv2135c Rv2153c Rv3283 Rv1855c Rv1497 Rv3305c Rv0840c Rv1612 Rv2188c Rv2230c Rv2090 Rv2427c Rv3215 Rv0562 BCG_1172 BCG_1398 BCG_1144 BCG_3231c BCG_1643 BCG_1185 BCG_0824 BCG_2949c BCG_3185c BCG_3640c BCG_1044c BCG_2586 BCG_2879c BCG_2152c BCG_2170c BCG_3312 BCG_0844c BCG_1560 BCG_3370c BCG_0892c BCG_1650 BCG_2204c BCG_2248c BCG_2110 BCG_2444c BCG_3242 BCG_0607 cysS2 Cysteinyl-tRNA synthetase grcc1 Probable polyprenyl-diphosphate entC Probable isochorismate synthase proA Probable γ-glutamyl phosphate reductase protein BCG_2110 Probable 5'-3' exonuclease BCG_2248c Conserved hypothetical protein BCG_2204c Glycosyltransferase trpB Probable tryptophan synthase pip Probable proline iminopeptidase amiA1 Possible N-acyl-L-amino acid amidohydrolase lipL Probable esterase BCG_0844c Conserved hypothetical protein sseA Probable thiosulfate sulfurtransferase MurG UPD-N-acetylglucosamine- transferase BCG_2152c Conserved hypothetical protein BCG_2879c Probable short-chain type dehydrogenase/reductase glnQ Glu-transport ATP-binding protein ABC transporter grcC2 Probable polyprenyl-diphosphate synthase BCG_3640c Transcriptional regulatory protein BCG_3185c Possible dioxygenase (Phenylpropionate dioxygenase) DivIVA Cell division initiation protein purD Probable phosphoribosylamine--glycine ligase ephC Probable epoxide hydrolase hisF Cyclase subunit-imidazoleglycerol phosphate synthase BCG_3231c Conserved hypothetical protein BCG_1144 Short (C15) chain Z-Isoprenyl diphosphate synthase cysM Probable cysteine synthase b BCG_1172 Probable GTP binding protein Proteins in blue are a/b-hydrolase Rv2130c BCG_2147c 335 372 415 355 379 385 410 286 389 429 347 297 410 236 258 330 325 359 382 245 422 316 267 292 262 323 357 414 35.6 39.3 43.8 38.0 39.9 41.3 43.1 32.2 41.2 45.9 38.0 33.4 42.0 25.2 27.0 35.5 34.8 38.3 42.6 27.1 43.7 34.7 27.3 31.3 29.6 34.5 38.2 45.9 142 142 143 145 145 146 146 148 148 149 152 153 154 155 156 156 158 158 159 163 163 164 167 168 169 172 174 174 19 11 10 WCL 2.5.1.- WCL 5.4.4.2 M, WCL, CF 1.2.1.41 WCL 3.1.11.- M, WCL - M 2.4.1.57 CF,M,WCL 4.2.1.20 WCL 3.4.11.5 M, WCL 3.5.1.- - 3.1.-.- M,WCL 1.-.-.- WCL 2.8.1.1 - 2.4.1.227 M,WCL - M,WCL 1.-.-.- M,WCL - - 2.5.1.- M,WCL - M,CF 1.-.-.- WCL - CF,M,WCL 6.3.4.13 M,WCL 3.3.2.3 WCL 4.1.3.- M, WCL - WCL 2.5.1.10 M, WCL 2.5.1.47 WCL - M, WCL 6.1.1.16 - - - - + - - - - - - - - - - - - - - - - - - - - - - - Continued in next page - - - - - - - - - - - - - - - - - - - - - - - - - - - - Madhu Sudhan Ravindran 175 Rv1315 Rv0926c Rv2426c Rv2243 Rv2701c Rv1026 Rv3837c Rv0533c Rv0685 Rv2889c Rv1184c Rv2603c Rv3306c Rv3044 Rv1538c Rv2781c Rv1245c Rv3603c Rv2413c Rv1045 Rv1062 Rv3169 Rv2121c Rv1592c Rv0498 Rv2413c Rv1775 BCG_1376 BCG_0978c BCG_2443c BCG_2260 BCG_2714c BCG_1083 BCG_3900c BCG_0577c BCG_0734 BCG_2910c BCG_1246c BCG_2628c BCG_3371c BCG_3068 BCG_1590c BCG_2799c BCG_1305c BCG_3667c BCG_2429c BCG_1103 BCG_1120 BCG_3193 BCG_2138c BCG_1630c BCG_0540 BCG_2592c BCG_1808 BCG_1808 Conserved hypothetical protein BCG_2592c Conserved hypothetical protein BCG_0540 Conserved hypothetical protein BCG_1630c Conserved hypothetical protein HisG Probable ATP phosphoribosyltransferase BCG_3193 Conserved hypothetical protein BCG_1120 Conserved hypothetical protein BCG_1103 Hypothetical protein BCG_2429c Conserved hypothetical protein BCG_3667c Hypothetical alanine and leucine rich protein BCG_1305c Probable short-chain type dehydrogenase/reductase BCG_2799c Possible alanine rich oxidoreductase ansA Probable L-aparaginase fecB Probable Fe III-dicitrate-binding periplasmic lipoprotein amiB1 Probable amidase BCG_2628c Highly conserved hypothetical protein BCG_1246c Possible exported protein EF-TU Probable elongation factor EF-TS Probable elongation factor fabH 3-oxoacyl-[ACP] synthase III BCG_3900c Probable phosphoglycerate mutase BCG_1083 Conserved hypothetical protein suhB Possible extragenic suppressor protein fabD Malonyl CoA-acyl carrier protein transacylase BCG_2443c Conserved hypothetical protein BCG_0978c Conserved hypothetical protein murA UDP-N-acetylglucosamine 1-carboxyvinyltransferase BCG_0181 Conserved hypothetical protein Proteins in blue are a/b-hydrolase Rv0145 BCG_0181 272 316 280 446 284 374 285 293 316 303 276 344 326 359 394 251 359 374 396 335 232 319 290 302 291 362 418 317 29.8 34.6 30.6 48.0 30.6 41.8 29.4 32.0 33.2 31.3 29.2 35.4 33.1 37.1 41.0 26.8 37.7 28.8 43.5 35.0 25.1 33.8 30.2 31.0 32.0 38.0 44.5 34.4 108 108 110 110 111 111 112 112 112 115 116 119 123 123 123 127 127 130 130 131 132 134 135 137 138 138 140 142 - 2.1.1.- - - - - - - - - M, WCL 2.4.2.17 M, WCL - WCL - - - - WCL - M, WCL 1.-.-.- - 1.-.-.- M, WCL 3.5.1.1 CF,WCL - M, WCL 3.5.1.- M,WCL - M - CF,M,WCL - M,WCL - M,WCL 2.3.1.180 WCL 5.4.2.- WCL - WCL 3.1.3.25 - 2.3.1.39 M - WCL - WCL 2.5.1.7 - - - - - + + - + - - - - + - - + - - - - - - - - - - - Continued in next page - - - - - +7 - - - - - - - - - - + - - - - - - - - - - - Madhu Sudhan Ravindran 176 Rv1502 Rv1391 Rv2457c Rv1106c Rv1422 Rv0281 Rv0780 Rv0303 Rv3457c Rv2750 Rv3772 Rv2859c Rv0561c Rv2727c Rv0281 Rv0655 Rv3312c Rv2334 Rv1021 Rv3866 Rv1596 Rv2026c Rv1108c Rv0975c Rv1681 Rv1061 Rv2305 BCG_1566 BCG_1452 BCG_2477c BCG_1166c BCG_1483 BCG_0321 BCG_0832 BCG_0343 BCG_3522c BCG_2766 BCG_3833 BCG_2881c BCG_0606c BCG_2740c BCG_0321 BCG_0704 BCG_3377c BCG_2356 BCG_1078 BCG_3929 BCG_1634 BCG_2045c BCG_1168c BCG_1029c BCG_1719 BCG_1119 BCG_2321 302 342 370 438 418 299 273 BCG_2321 Hypothetical protein BCG_1119 Conserved hypothetical protein moeX Possible molybdopterin biosynthesis protein fadE13 Probable acyl-coa dehydrogenase xseA Exodeoxyribonuclease VII large subunit BCG_2045c Conserved hypothetical protein nadC Probable nicotinate-nucleotide pyrophosphatase BCG_3929 Conserved hypothetical protein BCG_1078 Conserved hypothetical protein cysK1 Probable cysteine synthase A BCG_3377c Conserved hypothetical protein mkl Possible ribonucleotide-transport ATP-binding protein BCG_0321 Conserved hypothetical protein miaA tRNA delta(2)-isopentenylpyrophosphate transferase BCG_0606c Possible oxidoreductase BCG_2881c Possible amidotransferase hisC2 Probable histidinol-phosphate aminotransferase BCG_2766 Probable dehydrogenase rpoA Probable DNA-directed RNA polymerase (alpha chain) BCG_0343 Probable dehydrogenase/reductase 429 287 330 382 415 294 285 283 325 310 308 359 302 314 408 308 353 272 347 302 purC Phosphoribosylaminoimidazole-succinocarboxamide syn 297 BCG_0321 Conserved hypothetical protein BCG_1483 Conserved hypothetical protein BCG_1166c Probable cholesterol dehydrogenase clpx ATP-dependent clp protease ATP-binding subunit dfp DNA/pantothenate metabolism flavoprotein homolog BCG_1566 Hypothetical protein BCG_2518c Probable citrate (pro-3s)-lyase (β subunit) Proteins in blue are a/b-hydrolase Rv2498c BCG_2518c 46.2 32.0 35.8 41.0 44.8 31.5 30.0 30.1 35.4 32.8 34.0 39.4 33.0 34.4 44.0 32.5 38.2 28.3 37.7 33.0 33.0 33.0 35.9 41.0 47.1 43.8 33.7 29.0 93 94 94 94 94 95 96 96 96 98 98 98 99 99 99 100 100 101 101 102 102 102 102 102 102 103 104 105 18 M, WCL - M,WCL - - - - 1.3.99.- M 3.1.11.6 WCL - M,WCL 2.4.2.19 WCL - WCL 3.6.1.8 - 2.5.1.47 - - M,WCL - CF,M,WCL 2.1.1.- - 2.5.1.75 M 1.-.-.- - 2.-.-.- CF,M,WCL 2.6.1.- WCL 1.-.-.- CF,M,WCL 2.7.7.6 WCL 1.-.-.- WCL 6.3.2.6 M, WCL, CF 2.1.1.- - - M,WCL 1.1.1.- M, WCL - WCL 4.1.1.36 - - M, WCL 4.1.3.6 - - - - - - - - - - - - + - - - - - - - - - - - - - - - Continued in next page - - - - - - - - - - - - - - - - - - - - - - - - - - - - Madhu Sudhan Ravindran 177 Rv0074 Rv0365c Rv0687 Rv3700c Rv3199c Rv1600 Rv0315 Rv2622 Rv1340 Rv0077c Rv3455c Rv0499 Rv0295c Rv1059 Rv1170 Rv3754 Rv3230c Rv0352 Rv3781 Rv1008 Rv3712 Rv1589 Rv2985 BCG_0105 BCG_0403c BCG_0736 BCG_3759c BCG_3224c BCG_1638 BCG_0355 BCG_2649 BCG_1402 BCG_0108c BCG_3520c BCG_0541 BCG_0335c BCG_1117 BCG_1233 BCG_3813 BCG_3260c BCG_0391 BCG_3843 BCG_1065 BCG_3772 BCG_1627 BCG_3006 mutT1 Possible hydrolase bioB Biotin synthetase BCG_3772 Possible ligase TatD Probable deoxyribonuclease rfbE O-antigen/lipopolysaccharide transport ATP-binding dnaJ1 Probable chaperone protein BCG_3260c Hypothetical oxidoreductase tyrA Prephenate dehydrogenase mshB N-acetyl-myo-Inosityl-2-Glucopyranoside deacetylase BCG_1117 Conserved hypothetical protein BCG_0335c Conserved hypothetical protein BCG_0541 Conserved hypothetical protein truA Probable tRNA pseudouridine synthase A BCG_0108c Probable oxidoreductase rphA Probable ribonuclease BCG_2649 Possible methyltransferase (methylase) BCG_0355 Possible beta-1,3-glucanase precursor hisC1 Probable histidinol-phosphate aminotransferase nudC Probable NADH pyrophosphatase BCG_3759c Conserved hypothetical protein BCG_0736 Putative short-chain type dehydrogenase/reductase BCG_0403c Conserved hypothetical protein BCG_0105 Conserved hypothetical protein BCG_2636c Putative acyltransferase Proteins in blue are a/b-hydrolase Rv2611c BCG_2636c 317 349 413 264 273 395 380 301 303 354 267 291 256 276 259 273 294 380 313 390 275 376 411 316 35.0 38.0 43.6 29.3 30.0 41.7 41.1 31.2 31.7 37.1 29.8 31.0 28.2 29.7 27.6 29.5 32.2 40.6 34.0 40.7 29.0 41.6 43.1 35.4 81 81 82 83 83 83 84 85 85 85 86 86 87 87 88 88 88 88 89 89 90 91 91 93 - 2.3.1.- WCL 3.6.1.- M 2.8.1.- WCL 6.-.-.- M 3.1.21.- M,WCL - M,WCL - - - M,WCL 1.3.1.12 M,WCL M,WCL - WCL - - - M,WCL 5.499.12 M,WCL 1.-.-.- CF,M,WCL 2.7.7.56 WCL 2.1.1.- M,WCL 2.-.-.- M,WCL 2.6.1.9 M 3.6.1.22 - - WCL 1.-.-.- M,WCL - M, WCL - - - - - - - - - - - - - - - - - - - - - - - - - + - + - - - - - - + - - - - - - - - - - - - - - Madhu Sudhan Ravindran 178 H37Rv Rv2284 lipM Probable esterase Rv0896 BCG_0948 ftsZ Cell division protein Rv0818 BCG_0870 Transcriptional regulatory protein RbsK Adenosine kinase Proteins in blue are a/b-hydrolases BCG_0870 BCG_2218c Rv2202c BCG_3124c Conserved hypothetical protein BCG_2167c Rv2150c BCG_3124c Rv3099c fixB Probable electron transfer flavoprotein (α-subunit) BCG_3051c Rv3028c moxR2 Putative methanol dehydrogenase lpqC Lipoprotein, Poly(3-hydroxybutyrate)depolymerase BCG_3327c Rv3298c Rv3692 fadB5 Possible oxidoreductase BCG_3751 echA9 Possible enoyl-CoA hydratase BCG_1951c Rv1912c A85c, fbpC Secreted antigen echA8 Probable enoyl-CoA hydratase EF-TU Probable elongation factor fadE25 Probable acyl-CoA dehydrogenase citA Citrate synthase II BCG_1129c Rv1071c BCG_0163c Rv0129c BCG_1128c Rv1070c BCG_2910c Rv2889c BCG_3303c Rv3274c BCG_0941c Rv0889c fgd1 F420-dependentglucose-6-phosphate dehydrogenase Rv0407 BCG_0446 gltA2 probable citrate synthase i lipN Probable lipase/esterase BCG_3656c Putative hydrolase BCG_2991c Rv2970c BCG_3656c Rv3591c ispG Probable gcpE protein menB Probable napthoate synthase BCG_0592c Rv0548c BCG_2890c Rv2868 lldD2 Possible L-lactate dehydrogenase BCG_3864c Conserved hypothetical protein BCG_1908c Rv1872c BCG_3864c Rv3802c tesA Probable thioesterase BCG_2241c Probable exported protease pfkA Probable 6-phosphofructokinase Rv2928 Protein Function BCG_2654 Conserved hypothetical protein BCG_3032c Rv3010c BCG_2950 BCG_2241c Rv2224c BCG_2299 BCG_2654c Rv2627c BCG 255 324 283 379 318 358 304 334 345 340 257 374 389 373 431 336 376 257 387 314 414 336 343 261 520 431 413 aa 27.6 34.6 30.6 38.7 31.7 38.0 31.7 35.8 36.4 36.8 27.3 43.5 40.2 40.2 48.0 37.0 40.3 28.5 41.0 34.8 45.5 35.6 37.1 29.3 56.5 46.7 46.2 85 85 88 89 91 105 110 111 114 114 119 123 124 128 141 142 143 145 152 157 187 204 207 536 616 701 872 25 20 22 25 Mw (kD) Ion score Queries match M - CF,M, WCL 2.7.-.- M, WCL - M - M, WCL, CF - M,WCL - M, WCL 3.1.-.- - 1.6.5.5 CF,M, WCL 4.2.1.17 CF 2.3.1.- CF,M,WCL 4.2.1.17 CF,M,WCL - CF,M, WCL 1.3.99.- WCL 2.3.3.1 CF,M, WCL 2.3.3.1 M, WCL 1.-.-.- M, WCL 3.1.1.- M, WCL 3.-.-.- M, WCL WCL 4.1.3.36 M, WCL 1.1.2.3 CF - CF,M, WCL 2.7.1.11 WCL 3.1.2.- CF, WCL 3.4.-.- WCL 3.1.-.- M - Location Family Annex 2: THL targets identified in mycobacterial dormancy state - - - - - - - - - +1 - - - - - - - - - - - +1 - - +1 +3 - - - - - - - - - - + - - - - - - - - - - - + - - - + - Memb. dom. Sec. signal Madhu Sudhan Ravindran 179 H37Rv Rv1192 BCG_1252 Rv1923 lipM Probable esterase Rv3338 Rv1484 echA9 Probable enoyl-CoA hydratase BCG_2976c Conserved hypothetical protein BCG_2976c Rv2955c Proteins in Blue are a/b-hydrolases fadE25 Probable acyl-CoA dehydrogenase echA21 Putative enoyl CoA hydratase BCG_1129c Rv1071c Rv3774 EF-TU Probable elongation factor BCG_3656c Putative hydrolase inhA Enoyl-ACP reductase cmaA1 Cyclopropane-fatty-acyl-phospholipid syn BCG_3635c Possible oxidoreductase cmaA2 Cyclopropane fatty acid synthase BCG_3864c Hypothetical protein BCG_3408 Conserved hypothetical protein BCG_3303c Rv3274c BCG_3836 BCG_2910c Rv2889c BCG_3656c Rv3591c BCG_1546 BCG_3461c Rv3392c BCG_3635c Rv3570c BCG_0546c Rv0503c BCG_3864c Rv3802c BCG_3408 BCG_2241c Rv2224c BCG_2241c Probable exported protease BCG_2299 Rv2284 lldD2 Possible L-lactate dehydrogenase BCG_1908c Rv1872c lipI Probable lipase BCG_1461c Rv1400c lipG Probable lipase/esterase lipH Probable lipase BCG_1460c Rv1399c BCG_0695c Rv0646 lipN Probable lipase/esterase fabG4 Probable 3-oxoacyl-[ACP] reductase lipD Probable lipase BCG_2654 Hypothetical protein BCG_1252 Hypothetical protein tesA Probable thioesterase A85c, fbpC Secreted antigen Protein Function BCG_2991c Rv2970c BCG_0280c Rv0242c BCG_1962 BCG_2654c Rv2627c Rv2928 BCG_2950 BCG_0163c Rv0129c BCG 321 345 389 274 374 257 340 287 394 302 336 297 520 431 414 301 320 319 376 454 647 425 275 261 340 aa 36.2 36.4 40.2 29.2 43.5 28.5 28.5 32.6 43.2 34.8 35.6 32.0 56.5 46.7 45.5 33.0 34.1 34.0 40.3 46.9 47.2 46.2 30.4 29.3 36.8 183 189 192 197 212 229 232 236 239 304 310 347 367 373 379 416 438 460 497 504 553 665 687 853 1146 12 11 16 13 11 12 18 12 18 21 26 36 48 Mw (kD) Ion score Queries match WCL, M CF,M,WCL 4.2.1.17 CF,M,WCL 1.3.99.- M, WCL, CF - CF,M,WCL - M,WCL 3.-.-.- M,WCL 1.3.1.9 M 2.1.1.79 M,WCL 1.-.-.- WCL, CF 2.1.1.79 M, WCL, CF - - - M,WCL, CF 3.4.-.- WCL 3.1.-.- M,WCL 1.1.2.3 - 3.1.-.- WCL 3.1.-.- WCL 3.1.-.- M, WCL 3.1.1.- CF,M,WCL 1.1.1.100 M 3.1.-.- M - - - WCL 3.1.2.- CF 2.3.1.- Localization Family Annex 3: THL targets identified in mycobacterial regrowth state - - - - - - - - - - + - + + - - - - - - - - - - + continued in next page - - - - - - - - - - +1 - +1 +3 - - - - - - - - - - +1 Memb. Dom. Sec. signal Madhu Sudhan Ravindran 180 Rv1479 Rv0391 Rv0281 Rv0830 BCG_1541 BCG_0428 BCG_0321 BCG_0883 grcc1 Probable polyprenyl-diphosphate BCG_0607 Rv3720 Rv1295 BCG_3780 BCG_1355 pdhC Probable dihydrolipoamide S-acetyltransferase Rv0577 Rv3373 fabG Short-chain type dehydrogenase/reductase echA18 Enoyl-CoA hydratase BCG_3185c Phenylpropionate dioxygenase BCG_0622 Conserved hypothetical protein qor Probable quinone reductase fadE7 Probable acyl-CoA dehydrogenase moxR2 Putative methanol dehydrogenase Proteins in Blue are a/b-hydrolases BCG_3566c Rv3502c BCG_3445 BCG_3185c Rv3161c BCG_0622 BCG_1515c Rv1454c BCG_0437c Rv0400c Rv3692 BCG_2949c Conserved hypothetical protein BCG_3751 BCG_2949c Rv2927c BCG_3709 Conserved hypothetical protein BCG_2276c Possible transcrional regulatory protein Rv3651 thrC Probable threonine synthase BCG_2276c Rv2258c BCG_3709 BCG_2515c Rv2495c lipV Possible lipase Rv3203 BCG_3229 FAS Possible fatty acid synthase ddlA Probable D-alanine--D-alanine ligase BCG_3002c Rv2981c Rv0562 echA8 Probable enoyl-CoA hydratase BCG_1128c Rv1070c BCG_0883 Conserved hypothetical protein BCG_0321 Conserved hypothetical protein metZ O-succinylhomoserine sulfhydrylase moxR1 Probable transcriptional regulatory protein Acg Conserved hypothetical protein Rv2032 BCG_2051 fabG3 Possible 20-beta-hydroxysteroid dehydrogenase GcvT Probable aminomethyltransferase Rv2002 pfkA Probable 6-phosphofructokinase menB Probable napthoate synthase BCG_2227c Rv2211c BCG_2019 BCG_3032c Rv3010c BCG_0592c Rv0548c 317 213 382 261 328 395 358 245 353 345 393 360 420 224 373 335 257 301 302 406 377 331 379 260 343 314 32.8 31.6 42.6 27.3 34.0 42.6 38.0 27.1 37.6 38.0 41.3 37.5 47.0 23.7 39.8 35.6 27.3 33.4 33.0 43.4 40.7 36.7 40.0 27.0 37.1 34.8 111 115 115 118 118 118 119 121 125 127 132 133 134 136 137 142 143 149 153 153 157 158 162 172 175 181 WCL 4.1.3.36 WCL 1.-.-.- - 4.2.1.17 M,CF 1.-.-.- CF,M,WCL - M, WCL, CF 1.6.5.5 M,WCL 1.3.99.- M,WCL - WCL - CF,M,WCL - - - WCL 2.3.1.12 CF,M,WCL 4.2.3.1 M, WCL 2.1.1.- WCL 3.1.-.- WCL 6.3.2.4 WCL 2.5.1.- CF,M,WCL 4.2.1.17 M,WCL 2.1.1.- M, WCL, CF 2.1.1.- CF,M,WCL 4.2.99.- M - WCL 1.-.-.- CF,M,WCL 2.1.2.10 1.1.1.53 CF,M, WCL 2.7.1.11 - - - - - - - - - - - - - - - - - - - - - - - - - - continued in next page - - - - - - - - - - - - - - - - - - - - - - - - - - Madhu Sudhan Ravindran 181 Rv1202 BCG_1262 Rv0687 Rv2952 Rv2296 BCG_0736 BCG_2973 BCG_2312 Rv1093 Rv1309 pyrH UMP kinase (UMPK)-Ec atpG Probable ATP synthase gamma chain BCG_2152c Conserved hypothetical protein glyA1 Probable Serine hydroxymethyltransferase BCG_0279c Conserved hypothetical protein argB Probable Acetylglutamate kinase Proteins in Blue are a/b-hydrolases BCG_2904c Rv2883c BCG_1369 BCG_2152c Rv2135c BCG_1153 BCG_0279c Rv0241c Rv1654 A85a, fbpA Secreted antigen BCG_3866c Rv3804c BCG_1693 BCG_3667c Conserved hypothetical ala & leu rich protein BCG_3667c Rv3603c fadA4 Probable acetyl-CoA acetyltransferase BCG_2628c Highly conserved hypothetical protein BCG_2312 Probable haloalkane dehalogenase BCG_2973 Possible methyltransferase BCG_0736 Short-chain type dehydrogenase/reductase purC Phosphoribosylaminoimidazole-synthase BCG_2972c Possible oxidoreductase dapE Puccinyl-diaminopimelate desuccinylase BCG_3549c Short-chain type dehydrogenase/reductase Rv1323 mapB Methionine aminopeptidase BCG_1542 Conserved hypothetical protein BCG_3549c Rv3485c BCG_1385 BCG_2628c Rv2603c Rv0780 BCG_0832 BCG_2972c Rv2951c Rv1480 BCG_1542 BCG_2883c Rv2861c 261 305 236 426 280 294 338 303 27.4 34.0 25.2 42.0 30.1 31.0 35.8 31.3 33.2 40.1 314 26.8 389 33.3 30.8 29.0 33.0 41.3 37.5 34.5 31.1 251 300 270 275 297 381 354 317 285 83 83 86 86 88 88 88 90 90 91 94 95 99 100 102 102 105 107 108 M,WCL 2.7.4.- M,WCL 3.6.3.14 M,WCL - CF,M,WCL 2.1.2.1 M,WCL - M,WCL 2.7.2.8 M, WCL, CF 2.3.1.- WCL - WCL 1.-.-.- CF,M,WCL - M,WCL - M, WCL 3.8.1.5 WCL, M 2.1.1.- WCL 1.-.-.- WCL 6.3.2.6 M - WCL 3.5.1.18 M - CF 3.4.11.18 - - - - - - +1 - - - - - - - - - - - - - - - - - - + - - - - - - - - - - - - A B Annex 4: Effect of THL on mycobacterial PDIM and Phenolic glycolipid (PGL). A. HPTLC run of mycobacterial TAG and PDIM lipids extracted from THL treated logarithmic phase culture. THL treatment induces accumulation of TAGs, probably due to inhibition on cellular TAG lipases. No significant change in PDIM lipid was observed. B. Two-dimensional HPTLC run of PGLs. Significant change in PGLs were only observed at high concentration of THL. Madhu Sudhan Ravindran 182 Log Dormant Regrowth 1.25 OD 600nm 1.00 0.75 0.50 0.25 0.00 10-2 10-1 100 101 THL conc. (log) 102 103 Annex 5: Ethambutol is ineffective against NRP and resuscitating cells. MIC50 of ethambutol (EMB), a first line anti-tuberculosis drug, did not show significant change in MIC50 value towards M. bovis BCG at different metabolic states. The recorded MIC50 values for various stages are as follows: logarithmic 3.23 µM, NRP 1.80 µM and regrowth 2.21 µM. Madhu Sudhan Ravindran 183 Madhu Sudhan Ravindran 184 [...]... lipoprotein LTQ-FT-MS Linear ion trap-Fourier transform-mass spectrometry m/ z mass by charge ratio MAG Monoacylglycerol MBC Minimal bactericidal concentration MIC50 Minimum inhibitory concentration required to inhibit the growth of 50% of organism Mtb Mycobacterium tuberculosis Mw Molecular weight NaTDC Sodium taurodeoxycholate NRP Non-replicating persistence OD600nm Optical density at 600 nm PBS Phosphate... against contracting pulmonary TB19 The major hurdle in developing anti-TB drugs is due to its complex and rigid outer membrane composition, which hinders the entry of drugs, thereby making many antibiotics ineffective Current TB drugs are mainly effective against growing mycobacteria and ineffective against latent bacilli20 As of 2010, the recommended treatment for active TB is minimum six months of. .. macrophage Infection macrophages Blood vessel Mycobacterium Mononuclear cells Infected macrophage Foamy macrophage Lymphocyte Immune activation Foamy macrophage Lymphocyte Although the contribution of antimicrobi effectors has been established in mice (9, 10 the relative hierarchy of immune-mediated ki ing mechanisms in humans is unclear Howeve from the increased susceptibility of HIV+ h mans, we infer... breakdown of granuloma barrier, leading to release of viable bacteria into the lung and inducing a lethal infection44-46 1.1.6 Granulomas Granuloma formation is a hallmark event in TB infection and its formation is mediated by both innate and acquired immune responses, and bacterial cell wall components such as trehalose dimycocerosate (TDM, also known as cord factor)47 Granuloma formation is initiated... recruitment of immune cells to the site of infection by range of cytokines and chemokines that are released in response to the infection Among the cytokines, tumor necrosis factor (TNF)-α plays a dominant role by elevating the production of the chemokines The granuloma is a complex structure composed of infected macrophages surrounded by lipid droplet (LD) loaded foamy macrophages and other mononuclear... understanding suggests that mycobacteria modulate the host activity and also utilize host lipids for survival 1.1.8 Lipids in mycobacterial survival In Mtb, 30% of the genome codes for genes involved in lipid metabolism, of which 250 genes are involved in fatty acid metabolism and 39 are involved in the polyketide metabolic pathway that generates the unique mycobacterial lipids69 Such enormous dedication... alveolar macrophages The macrophages then invade the subtending epithelial layer, Madhu Sudhan Ravindran 27 which induces a localized inflammatory response that leads to recruitment of mononuclear cells from neighboring blood vessels in order to confine and diminish the pathogen34 From the mycobacterial point of view, however, these immune cells serve as fresh hosts for the expanding bacterial population and. .. example of TB was found in the tubercular decay in the spines of an Egyptian mummy dating back five millennia6,7 The TB discovered was a bovine form, showing that the disease has accompanied mankind since we started domesticating cattle In the earliest known records, the disease had been commonly referred to as ‘phthisis’, or consumption The latter term, in particular, was introduced by Hippocrates,... stress that the balance mycobacteria under the state of dormancy a the recombinant bacterium (22–24) Second, a non-replicating between containment gression to containment occurs earlier, and at do not pose and disease progression is complex and highly lower bacterial load In most animal models, tenuating strains of Mtb through the deletion dynamic and appears to be a local phenomenon bacterial containment... experimental dormancy models87 Mycobacterial persistence is a phenomenon where drug-susceptible mycobacteria survive indefinitely within mammalian host despite continuous exposure to appropriate antibiotic(s) Although, from the host point of view the persistent and dormant bacilli may mean the same, the main distinction in experimental models is the colony forming capability (colony forming unit, CFU) of . Madhu Sudhan Ravindran 1 IDENTIFICATION AND BIOCHEMICAL CHARACTERIZATION OF TETRAHYDROLIPSTATIN TARGETS IN M. BOVIS BCG AT DIFFERENT METABOLIC STATES MADHU SUDHAN RAVINDRAN M. Sc charge ratio MAG Monoacylglycerol MBC Minimal bactericidal concentration MIC 50 Minimum inhibitory concentration required to inhibit the growth of 50% of organism Mtb Mycobacterium tuberculosis. re-emerging from a NRP state. Madhu Sudhan Ravindran 13 List of Tables Table 1.1. List of α/β-hydrolase superfamily in M. bovis BCG 54 Table 1.2. List of ‘Lip family’ proteins in M. bovis