Exploring chaperone and immunogical functions of mycobacterium indicus pran II protein mip--05962

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EXPLORING CHAPERONE AND IMMUNOLOGICAL FUNCTIONS OF MYCOBACTERIUM INDICUS PRANII

PROTEIN MIP_05962

ASHISH SHARMA

KUSUMA SCHOOL OF BIOLOGICAL SCIENCES INDIAN INSTITUTE OF TECHNOLOGY DELHI

OCTOBER 2017

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©Indian Institute of Technology Delhi (IITD), New Delhi, 2017

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EXPLORING CHAPERONE AND IMMUNOLOGICAL FUNCTIONS OF MYCOBACTERIUM INDICUS PRANII

PROTEIN MIP_05962

by

ASHISH SHARMA

Kusuma School of Biological Sciences

Submitted

In fulfillment of the requirements of the degree of Doctor of Philosophy to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

OCTOBER 2017

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Dedicated to my beloved family

for their everlasting love, affection and care

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CERTIFICATE

This is to certify that the thesis entitled “Exploring chaperone and immunological functions of Mycobacterium indicus pranii protein MIP_05962” being submitted by Mr. Ashish Sharma to the Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi for the award of the degree of “Doctor of Philosophy” is a record of the bonafide research work carried out by him, prepared under my supervision, in conformity with the rules and regulations of the ‘Indian Institute of Technology Delhi’. The research report and the results present in the thesis have not been submitted to any other University or Institute for the award of any other degree or diploma.

Prof. Tapan K. Chaudhuri Prof. Seyed E. Hasnain

Kusuma School of Biological Sciences Kusuma School of Biological Sciences Indian Institute of Technology Delhi Indian Institute of Technology Delhi Hauz Khas, New Delhi-110016 Hauz Khas, New Delhi-110016

Date:

Place:

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ACKNOWLEDGEMENTS

I am highly grateful to the Almighty who has bestowed me with all his blessings and kindness and made this thesis possible.

PhD is not just a scientific degree towards accomplishing a given project, but is a learning phase in every sense and also the overall growth of an individual scientifically as well as personally.

During my PhD, I have not only enhanced my scientific knowledge and experimental skills but also learned to overcome failures and rejections and have grown as an individual together. The tenure of my PhD has made me more patience, composed, organized and most importantly independent thinker. This Thesis cannot be achieved without the help of many those who have helped me in one or other way to accomplish my goal. Here, I would like to take the opportunity to acknowledge their contributions and express my sincere gratitude.

First and foremost, I would like to express my sincere and heartiest gratitude to my both PhD supervisors Prof. Tapan K. Chaudhuri and Prof. Seyed E. Hasnain for their immense continuous outstanding support, guidance and encouragement which helped me out in bringing the best from me and completing this thesis. I am thankful to them for enabling me to develop the understanding of the research. I am highly grateful to them for their constructive and critical comments which utterly helped me to grow as a researcher and were the base of my research. I am highly thankful to them also for being kind hearted and patience throughout my stay at their laboratories. For me, it’s a great pleasure to work under their esteemed supervision and guidance. I owe them lots of gratitude for providing me freedom of thoughts, great and healthy

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iii work environment and motivations to complete my work. I am privileged to be associated with them and can’t express my gratitude in words.

I am highly indebted to Dr. Sangeeta Bhaskar, National Institute of Immunology, New Delhi, for allowing me to complete a part of my PhD project in her laboratory, without which my thesis work would have been incomplete. Her invaluable assistance and advice have contributed a lot to the successful completion of my PhD project. I wish to thank PhD student Mohd. Saqib in her lab to help in my research work.

I am really grateful to my SRC members, Prof. James Gomes, Prof. S.K. Khare and Dr.

Manidipa Banerjee for monitoring my research progress timely and giving valuable suggestions which have helped me a lot in improving my PhD project. I would also like to thank all the present faculty members of the Kusuma School of Biological Sciences, IIT Delhi for their constants support and encouragement through their classroom programs or through informal discussions.

I express my thanks to all my lab members of TKC and SEH group at KSBS, IIT Delhi for their immense support and encouragement through all these years. I would also like to thank to my dear friends who were always there for me in need.

I am thankful to Prof. Nasreen Z. Ehtesham and her lab members at National Institute of Pathology (NIOP) Safdarjung, New Delhi for constant scientific discussions and motivation.

I am highly thankful to Council of Scientific and Industrial Research (CSIR), Govt. of India for the financial support during the tenure of my PhD.

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iv This acknowledgment would be absolutely deficient without showing gratitude towards my family members. My most sincere gratitude goes to my grandfather late Sh. Radheshyam Sharma and grandmother Smt. Narabda devi and I am indebted to my dear parents Mr.

Chiranji Lal Sharma and Mrs. Shakuntala, who made numerous sacrifices throughout the journey for my different academic endeavors. Since my childhood, they always been the strong pillars of my life and bore all the responsibilities and duties of family, so that I can enable to devote my full time with at most concentration and energy on my PhD project. All I can say that whatever I could do in life is only because of them. My father has been the greatest source of my inspiration to do good karmas and work hard with a strong will and vision to accomplish a goal.

My mother has offered me with great emotional support, affection, love and warmth.

I am highly thankful to my chachaji and chachiji Mr. Hari Prasad Sharma and Mrs. Bidam Devi for their kind affection, support and care.

I totally indebted to my dear younger sisters Anita Sharma and Archana Sharma, cousin brother Anuj Sharma, for their unconditional constant support, love, affection and care.

Heartiest thanks to all my beloved family members for always being there for me. Truly, my family is the God’s biggest blessing to me and there is no substitute for them.

Ashish Sharma

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v ABSTRACT

Tuberculosis (TB) still remains a major global health issue. TB causes the death of 1.5 million people every year. About one-third of the world population is infected with the bacterium that causes TB, Mycobacterium tuberculosis (M.tb). The situation of TB is further aggravated due to lack of proper diagnostic tools and facilities; lack of efficacious vaccine; co-infection with HIV and co-morbidity with diabetes; and the emergence of drug resistant strain of M.tb. The current vaccine against TB is a live-attenuated Mycobacterium bovis Bacillus-Calmette-Guerin (BCG).

BCG confers effective protection to childhood TB as compared to adulthood TB; not efficient enough to prevent reactivation of latent TB infection; efficacy also varies among different populations group in the world. Therefore, extensive research is being carried out all over the world for more effective interventions against TB. Since past two decades, various strategies are being followed to replace BCG with a better organism or boosting BCG with strong immunodominant antigens. Mycobacterium indicus pranii (MIP) is a non-pathogenic, saprophytic organism and displays strong immunomodulatory properties. MIP is currently being used as an intervention against leprosy and also shown protection against M.tb in animal models.

Recently, the whole genome sequence of MIP was completely sequenced and annotated and compared with M.leprae and BCG. Through comparative sequence analysis, it was revealed that there were 36 genes that were present only in MIP and M.leprae but absent in BCG. Most of these genes had significant antigenicity index and many of them had similarity with M.leprae and M.tb antigens. One among them, a putatively immunogenic protein, encoded by MIP_05962 ORF has an alpha-crystallin domain and has a very high amino acid similarity with HSP18 of M.leprae and antigen of M.tb. HSP18 of M.leprae is a prominent immunogenic protein and shared several common T cell epitopes with M.tb and elicits a significant CD4⁺T-cell response.

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vi CD4⁺T cells producing Th1 cytokine response play a pivotal role in the protection of host against intracellular pathogens including M.tb. As Th1-type cell-mediated immune response is very crucial for restricting the multiplication of M.tb infection; therefore, novel subunit vaccines against TB are focusing on activating this arm of the immune system in the host.

The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones needs to be explored. Therefore, in the present study, the chaperone and immunological functions of MIP_05962 protein were thoroughly investigated in-vitro as well as in-vivo. To explore its putative functions, MIP_05962 was cloned, expressed and recombinant protein was purified. Biophysical and biochemical characterization was done on purified recombinant MIP_05962 to prove it to be a molecular chaperone. The observations of formation of large oligomers, thermal aggregation prevention and refolding of substrate proteins maltodextrin glucosidase (MalZ) and citrate synthase (CS) in the presence of MIP_05962 pointed to its chaperone function. Additionally, the interaction with non-native proteins, prevention of thermal inactivation of Nde1, surface hydrophobicity, thermal stability and structural regaining property, in vivo thermal rescue of E.coli expressing MIP_05962, enhancing solubility of insoluble protein MalZ with co-expression of MIP_5962 under in-vivo conditions categorically demonstrated that MIP_05962 was a chaperone-like protein.

Immunological functions of MIP_05962 protein were investigated in mice model. The recombinant MIP_05692 protein showed significant proliferation of splenocytes and B cell response which showed that MIP_05962 protein is highly antigenic in nature. MIP_05962 in mice model induced significant secretion of Th1 cytokines (IFN-γ, TNF-α, IL-12) in-vitro from splenocytes culture supernatant by ELISA as well as intracellularly in both CD4⁺ and CD8⁺ T

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vii cells and also evoked significant effector memory (TEM) in CD4⁺T cells. Based on these observations it can be concluded that MIP_05962 is a molecular chaperone and has a potential to generate a specific Th1 type response and can evoke significant CD4⁺ T helper cells. MIP_05962 seems to have potential to be either as a booster to BCG or as a subunit vaccine.

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सार

क्षयरोग (टीबी) अभीभीएकप्रमुखवैश्ववकस्वास््यमुद्दाबनीहुईहै।टीबीहरसाल 15 लाखलोगोंकीमौतकाकारणबनताहै

दुननयाकीआबादीकाएकनतहाईहहस्साजीवाणुसेसंक्रममतहोताहैजोटीबी, मायकोबैक्टीररयमट्यूबरकुलोमसस (एमआरबी) काकारणबनताहै।उचितनैदाननकउपकरणऔरसुववधाओंकीकमीकेकारणटीबीकीश्स्िनतआगेबढ़रहीहै; प्रभावकारी

टीका कीकमी; एिआईवी केसाि सह-संक्रमण औरमधुमेहके साि सह-रोग; औरएम.टी.बी. कीदवा प्रनतरोधीतनाव का

उद्भव टीबीके खखलाफवततमानटीका एकजीववत-एहटन्यूएटेडमायकोबैक्टीररयम बोववसबैमसलस-कैल्मेटे-ग्यूररन (बीसीजी) है। बीसीजी वयस्कता टीबी की तुलना में बिपन टीबी को प्रभावी संरक्षण प्रदान करता है; अनुपश्स्ित टीबी संक्रमण के

पुनसतक्रक्रयण को रोकने के मलए पयातप्त नहीं; दुननया में ववमभन्न आबादी समूह के बीि प्रभावकाररता भी मभन्न होती है।

इसमलए, टीबी के खखलाफअचधकप्रभावी हस्तक्षेप केमलएपूरी दुननया में व्यापक शोध क्रकया जारहा है। बीतेदोदशकों से

बीसीजी को बदलने के मलए एक बेहतर जीव के साि या बीसीजी को मजबूत इम्यूनोडोमंटेंट एंटीजन के साि ववमभन्न रणनीनतयोंकाअनुसरणक्रकयाजारहाहै।मायकोबैक्टीररयमइंडडकसवप्रनीआई (एमआईपी) एकगैर-रोगजन्य, सापोपायहटक जीवहैऔरमजबूतइम्युनोमोडायलेटरगुणप्रदमशततकरताहै।एमआईपीवततमानमेंकुष्ठरोगकेखखलाफएकहस्तक्षेपकेरूप में इस्तेमालक्रकया जारहाहैऔरपशुमॉडलमें एम.ए.बी. हालहीमें, एमआईपीकेपूरेजीनोमअनुक्रमपूरीतरहअनुक्रममत औरएनोटेटक्रकयागयािाऔरएम।लेप्रेऔरबीसीजीकेसाितुलनाकीगईिी।तुलनात्मकअनुक्रमवववलेषणकेमाध्यम से, यहपतािलािाक्रक 36 जीनिेजोकेवलएमआईपीऔरएम.लेप्रेमेंमौजूदिेलेक्रकनबीसीजीमेंअनुपश्स्ितिे।इनमेंसे

अचधकांश जीनों में महत्वपूणत एंटीजेनेमसटी इंडेक्स िा और उनमें से बहुत से एम। लेप्रे और एम.टी.बी. प्रनतजनों के साि

समानता िी। उनमें से एक, एक भरोसेमंद प्रोटीन, एमआईपी_05962 द्वारा एनकोड ओआरएफ में एक अल्फा-क्रक्रस्टमलन डोमेनहैऔरएम.एल.बी. केएि.पी. 18 औरएम.ए.बी. केप्रनतजनकेसािबहुतअचधकएममनोएमसडसमानताहै। M.leprae कीएिएसपी 18 एकप्रमुखइम्युनोजेननकप्रोटीनहैऔरएम.ए.बी. केसािकईआमटीसेलएवपटेप्ससाझाकरताहैऔरएक

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महत्वपूणतसीडी 4 + टी-सेलप्रनतक्रक्रयादेता है।सीडी 4 + टीकोमशकाओंकाउत्पादनि 1 साइटोक्रकनप्रनतक्रक्रयाएमआरबीबी

सहहतइंट्रासेल्युलररोगजनकोंकेखखलाफमेजबानकेसंरक्षणमें एकननणातयक भूममकाननभाताहै। एम 1 बीकेसंक्रमणके

गुणनकोसीममत करनेकेमलएि 1-प्रकारसेल-मध्यस्िताप्रनतरक्षाप्रनतक्रक्रयाबहुतमहत्वपूणतहै; इसमलए, टीबीकेखखलाफ उपन्याससबयूनेटटीकेहोस्टपरप्रनतरक्षाप्रणालीकेइसहािकोसक्रक्रयकरनेपरध्यानकेंहितकररहेहैं।

मैकेबैक्टीररयलप्रजानतयोंमेंकईसंरक्षककेस्पष्टववरोधाभासऔरइनसावधानीकेक्लाइंटप्रोटीनोंकेसेलुलरफंक्शंसकेबारे

में पता लगाने की आववयकता है। इसमलए, वततमान अध्ययन में, एमआईपी_05962 प्रोटीन केसंरक्षक औरप्रनतरक्षात्मक कायोंकोइन-ववट्रोऔरसाि हीवववोमें पूरीतरहसेजांिकीगईिी।अपनेपुत्रीयकायोंकापतालगानेकेमलए, एमआईपी

एमआईपी_05962 कोक्लोनक्रकया गया, व्यक्त क्रकयागया औरपुनःसंयोजक प्रोटीनशुद्धक्रकया गया।बायोक्रफश्जकल और बायोकेममकललक्षण वणतनशुद्ध रीकॉ ंबबनेंटएमआईपी_05962 परक्रकया गया िा ताक्रकयह एकआणववक शैक्षकबनसके।

एमआईपी_05962 की उपश्स्िनतमें बडे ऑमलगोमसत, िमतल एग्रीग्रेशनप्रोटेन्सन माल्टोडेक्सहट्रन ग्लुकोमसडेस (माल़्) और साइटट्रेटमसंिेस (सीएस) कीिमतलएग्रीग्रेरणकीरोकिामऔरररफोश्ल्डंगकीहटप्पखणयोंने अपनेसंरक्षकसमारोहकीओर इशारा क्रकया। इसके अनतररक्त, गैर-देशी प्रोटीन के साि बातिीत, एनडी 1 की िमतल ननश्ष्क्रयता की रोकिाम, सतह की

हाइड्रोफोबबमसटी, िमतल श्स्िरताऔरसंरिनात्मकपुनगतठन कीसंपश्त्त, ईकोलीकेवववोिमतल बिावमें एमआईपी_05962 कोव्यक्तकरना, एमआईपी_05962 केसह-अमभव्यश्क्त केसािअघुलनशीलप्रोटीनमाल़् कीववलेबबमलटी कोबढ़ानेइन- वववोशतोंकेतहतस्पष्टरूपसेदशातयागयाहैक्रकएमआईपी_05962 एकप्रोपेनप्रोटेन्टिे

िूहों केमॉडलमें एमआईपी 165962 प्रोटीन केइम्यूनोलॉश्जकलफंक्शनकीजांि कीगई।पुनःसंयोजकएमआईपी_05962 प्रोटीननेस्प्लेनोसाइट्सऔरबीसेलप्रनतक्रक्रयाकामहत्वपूणतप्रसारहदखाया, श्जसमेंपतािलाक्रकएमआईपी_05962 प्रोटीन प्रकृनत में अत्यचधक प्रनतजनहै। िूहों मॉडल में एमआईपी_05962, एमलसाद्वारा स्प्लेनोसाइट्स सोमलटेन्टाटंट केसाि ही

सीडी 4 + औरसीडी 8 + टीकोमशकाओं दोनोंमें इंट्रासेल्युलर सेथ्रीसाइटोक्रकन्स (आईएफएन-γ, टीएनएफ-α, आईएल -12)

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इन-ववट्रोकेमहत्वपूणतस्रावकोप्रेररतकरताहैऔरइसकेसािहीमहत्वपूणतप्रभावकारीस्मृनतभीपैदाहोतीहै ( TEM) सीडी 4 + टी कोमशकाओंमें इन हटप्पखणयों के आधार पर यह ननष्कषत ननकाला जा सकता है क्रक एमआईपी_05962 एकआणववक

संरक्षक है औरइसमें एक ववमशष्ट Th1 प्रकारकी प्रनतक्रक्रया उत्पन्न करने कीक्षमता है और यह महत्वपूणत सीडी 4 + टी

सहायककोमशकाओंकोपैदाकरसकताहै।एमआईपी_05962 मेंबीसीजीकोबूस्टरकेरूपमेंयाएकसबयूनेटवैक्सीनकेरूप मेंहोनेकीसंभावनाहै।

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2.3.3 Elucidation of functional form of native MIP_05962 protein 44 2.3.4 MIP_05962 prevents thermal aggregation of CS and MalZ in vitro 46 2.3.5 MIP_05962 prevents inactivation of thermally denatured NdeI 47 2.3.6 MIP_05962 assists in refolding of chemically denatured MalZ and CS 48 2.3.7 MIP_05962 interacts with only non-native substrate proteins 50

2.3.8 Surface hydrophobicity of MIP_05962 protein 52

2.3.9 Thermal stability of MIP_05962 protein 53

2.3.10 Thermal Structural regain of MIP_05962 protein 55

2.3.11 Heat shock survivability of E. coli expressing MIP_05962 protein 56 2.3.12 MIP_05962 assists in folding of MalZ under in vivo condition 57

2.4 Discussion 60

Chapter 3: Characterization of antigenic potential of MIP_05962 68

3.1 Introduction 69

3.2 Materials and Methods 71

3.2.1 Construction, expression and purification of recombinant MIP_05962 protein 71 3.2.2 Immunization of mice and preparation of splenocytes single cell suspension 71

3.2.3 Lymphocyte proliferation assay 72

3.2.4 Estimation of cytokines by ELISA 72

3.2.5 Intracellular cytokine (ICC) staining and extracellular staining of surface markers 73

3.2.6 Memory T cell analysis. 74

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3.3 Results 74

3.3.1 Cloning, expression and purification of MIP_05962 74

3.3.2 Prediction of B- and T-cell epitopes of MIP_05962 74 3.3.3 Mice immunized with MIP_05962 protein elicit significant IgG response 75 3.3.4 Proliferative response of splenocytes from mice immunized with MIP_05962 77 3.3.5 MIP_05962 protein induces pro-inflammatory cytokine (Th1) response 78 3.3.6 Intracellular staining of cytokines to study the pro-inflammatory cytokines secretion response of MIP_05962 on CD4⁺/CD8⁺ T cells 79 3.3.7 T cell memory response against MIP_05692 81

3.4 Discussion 83 Chapter 4: Conclusions 87 References 91 Appendix 112

Appendix I: List of chemicals and equipments 113

Appendix II: Media and antibiotic preparation 118

Appendix III: Solutions and buffers 121

Annexure IV: Standard BSA curve 127

Annexure V: Gene and protein sequences 129

APPENDIX VI: Gating strategies for FACS analysis. 130

Author’s Resume 132

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LIST OF FIGURES

Number Figure Titles Page no.

Figure 1.1 Progression of the Tuberculosis disease in Human 3 Figure 1.2 Charles II of England touching for scrofula 5 Figure 1.3 Cellular components of the immunity 8 Figure 1.4 Innate immune response against TB 12 Figure 1.5 Cells involved in immune response during M.tb infection 16

Figure 1.6 Adaptive immunity to TB infection 19

Figure 2.1 Multiple sequence alignment of MIP_05962 with other sHSPs 42 Figure 2.2 Cloning of MIP_05962 into Pet28a expression vector 43 Figure 2.3 Expression, solubility and Purification of MIP_05962 protein 44 Figure 2.4 MIP_05962 has ability to form large oligomer as other chaperones 45 Figure 2.5 MIP_05962 prevents thermal aggregation of MalZ and CS 47 Figure 2.6 MIP_05962 prevents thermal inactivation of Nde1

restriction enzyme activity 48

Figure 2.7 MIP_05962 assists in the refolding of GdnHCl-denatured MalZ and CS 50 Figure 2.8 MIP_05962 only interacts with non-native proteins 51

Figure 2.9 Surface hydrophobicity of MIP_05962 52

Figure 2.10 MIP_05962 is thermally stable at elevated temperatures 54 Figure 2.11 Ability of MIP_05962 for structural regain after thermal denaturation 55

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xiii Figure 2.12 E. coli expressing MIP_05962 is rescued from thermal shock 57 Figure 2.13 Co-expression of MalZ with MIP_05962 or GroELS in E.coli

BL21 (DE3) 59

Figure 3.1 Prediction of B- and T- cells epitopes of MIP_05962 75 Figure 3.2 MIP_05962 protein elicits strong B cell response in immunized mice 76 Figure 3.3 MIP_05962 induces significant proliferation of splenocytes 77 Figure 3.4 MIP_05962 protein induces robust pro-inflammatory cytokines response 78 Figure 3.5 MIP_05962 protein elicits CD4⁺ and CD8⁺T cells to induce Th1

cytokine response in immunized mice 80

Figure 3.6 MIP_05962 protein induces memory responses in CD4/ CD8T cells in

immunized mice 82

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LIST OF TABLES

Number Table Title Page no.

Table 2.1 Secondary structural composition of MIP_05962

showing marginal changes up to 60°C 54

Table 2.2 Regain of secondary structural composition of MIP_05962 56 Table 2.3 Enhancement of solubility and activity of MalZ with

co-expression of MIP_05962 60

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ABBREVIATIONS AND SYMBOLS

% Percent

λmax Fluorescence emission maxima

~ Approximately

°C Degree Celsius

3D Three dimensional

Å Angstrom

A₂₂₀ Absorbance measured at 220 nm

A280 Absorbance measured at 280 nm

A₄₈₀ Absorbance measured at 480 nm

AIDS Acquired Immunodeficiency Syndrome

Amp Ampicillin

ANS 1-Anilinonaphthalene-8-sulphonate

BCG Bacillus Calmette–Guérin

BLAST Basic Local Alignment Search Tool

BLI Bio-layer Interferometry

BME β Mercaptoethanol

bp Base pair

BSA Bovine serum albumin

CD Circular dichroism

CFP-10 Culture Filtrate Protein of 10 kD

DLS Dynamic light scattering

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DMSO Dimethyl sulfoxide

DNA Deoxy ribonucleic acid

dNTP Deoxyribonucleotide triphosphate

DTT 1, 4-Dithiothreitol

EDTA Ethylene diamine tetra acetic acid

ESAT-6 Early Secreted Antigenic Target

EtBr Ethidium bromide

FPLC Fast protein liquid chromatography

g Gram

GdnCl Guanidine hydrochloride

GFC Gel filtration chromatography

h Hour/hours

His (H) Histidine

HPLC High performance liquid chromatography IPTG Isopropyl- β- D- galactopyranoside

kb Kilobase pair

kDa Kilodalton

LA Luria Bertani Agar

LB Luria Bertani Broth

M Molar

M.tb Mycobacterium tuberculosis

mA Milliampere

MALDI Matrix assisted laser desorption ionization

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MCS Multiple cloning site

MDR Multi Drug Resistant

mg Milligram

min Minute

ml Millilitre

mM Millimolar

MPTR Multiple Polymorphic Tandem Repeats

MRE Molar residue ellipticity

Mw Molecular weight

MW Molecular weight

NCBI National Center for Biotechnology Information

NFW Nuclease Free Water

Ni-NTA Nickel-nitrilotriacetic acid

NK Natural Killer cells

nm Nanometer

OD600 Optical density measured at 600 nm

ORF Open Reading Frame

PAGE Polyacrylamide gel electrophoresis

PAS Para-aminosalisylic acid

PBS Phosphate Buffer Saline

PCR Polymerase Chain Reaction

PDB Protein data bank

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PE Proline-Glutamic acid

PEG Polyethylene glycol

PGRS Poly Glycine Rich Sequence

PMN Polymorphonuclear neutrophils

PPD Purified Protein Derivatives

PPE Proline-Proline-Glutamic acid

ROS Reactive Oxygen Species

rpm Revolution per minute

RT Room temperature

RT-PCR Reverse Transcriptase Polymerase Chain Reaction

SDS Sodium dodecyl sulphate

Sec Second

sHSP Small heat shock protein

SMC Small molecular chaperone

TB Tuberculosis

TBE Tris-borate-EDTA

TCA Trichloroacetic acid

TEMED N,N,N’,N’-Tetramethylethylenediamine

TFA Trifluoroacetic acid

TLR Toll Like Receptor

Tris Tris(hydroxymethyl)aminomethane

Trp (W) Tryptophan

U Unit

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UV Ultra violet

v/v Volume/volume

VDR Vitamin D Receptor

w/v Weight/volume

XDR Extensive Drug Resistant

α Alpha

β Beta

λ Wavelength

λex Excitation wavelength

μg Microgram

μl Microliter

μmol Micromole

Exploring chaperone and immunogical functions of mycobacterium indicus pran II protein mip--05962

EXPLORING CHAPERONE AND IMMUNOLOGICAL FUNCTIONS OF MYCOBACTERIUM INDICUS PRANII

PROTEIN MIP_05962

ASHISH SHARMA

KUSUMA SCHOOL OF BIOLOGICAL SCIENCES INDIAN INSTITUTE OF TECHNOLOGY DELHI

OCTOBER 2017

EXPLORING CHAPERONE AND IMMUNOLOGICAL FUNCTIONS OF MYCOBACTERIUM INDICUS PRANII

PROTEIN MIP_05962

by

ASHISH SHARMA

Kusuma School of Biological Sciences

Submitted

In fulfillment of the requirements of the degree of Doctor of Philosophy to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

OCTOBER 2017

Dedicated to my beloved family

for their everlasting love, affection and care

CERTIFICATE

ACKNOWLEDGEMENTS

Ashish Sharma

CONTENTS

LIST OF FIGURES

LIST OF TABLES

ABBREVIATIONS AND SYMBOLS