The heparin-binding hemagglutinin adhesin (HBHA) is protein virulence factor that enables the Mycobacterium tuberculosis (Mtb) to attach to human epithelial cells to commence extrapulmonary pathogenesis, which leads to disseminated tuberculosis. The glycosaminoglycan-binding domain at the N-terminal of wild-type HBHA was found to contain methylation at the ζN position of lysine residues and the post-translational modification was crucial for its antigenic properties that can be used as immunoprotection against tuberculosis and as a diagnostic marker for active tuberculosis. Previously, the protein was expressed in Escherichia coli (Eco) and which structure and interaction of its glycosaminoglycan-binding domain with heparan sulfate oligosaccharide was characterized by multidimensional NMR. In this study, M. tuberculosis H37Rv HBHA and its glycosaminoglycan-binding domain, which is within residues 110–199, was expressed in Mycolicibacterium smegmatis (Msm) mc24517 to resemble the native protein, which is post-translationally methylated. Unlike Eco HBHA, the full length and truncated glycosaminoglycan-binding domain of methylated Msm HBHA was recognized by the anti-Mtb HBHA monoclonal antibody. 13C–1H HSQC NMR spectroscopy, HCN 3D NMR, 13C-edited NOESY-HSQC 3D NMR, and 1H–1H 2D NOESY identified the resonances of the novel peaks that belong to mono and di-methylated lysine εCH, δCH, ηCH, and ζN of Msm HBHA. 13C–1H HSQC NMR spectroscopy and liquid chromatography–tandem mass spectrometry revealed the overall relative abundance, relative abundance per residue, residue localization, and distribution of mono, di, and tri-methyl lysines within the protein amino acid sequence. Di and tri-methylation occurred more frequently at repeating lysine sequences at C-terminal. Protein interaction studies using isothermal titration calorimetry revealed that Msm HBHA110–199 was six-fold stronger in binding heparan sulfate oligosaccharides. Gel electrophoresis of tryptic digests revealed Msm HBHA was more resistant to trypsin cleavage than unmodified Eco HBHA. The positive effects of HBHA methylation to these interactions enable tuberculosis bacteria to survive in its host environment; at the same time, the effects can also be advantageous as methylated HBHA may serve as a good candidate for host immunoprotection.

Acknowledgment i
Abstract iii
Table of Contents v
List of Tables and Figures vii
1. Introduction 1
1.1. Tuberculosis and the search for an effective vaccine 2
1.2. The heparin-binding hemagglutinin adhesin of M. tuberculosis 3
1.3. Objectives of the Study 5
1.4. Scope and Limitation 6
2. Materials and Methods 7
2.1. Expression and Purification of recombinant HBHA in E. coli 8
2.2. Chemical methylation of E. coli expressed recombinant HBHA 9
2.3. Preparation of chemically competent E. coli cells for cloning Mycobacterium expression plasmids 10
2.4. Preparation of M. smegmatis electrocompetent cells 10
2.5. Cloning and extraction of plasmid vectors 11
2.6. Construction of plasmid vectors with hbhA inserts 12
2.7. Transformation of M. smegmatis mc24517 14
2.8. Extraction and Purification of HBHA and HBHA110–199 from M. smegmatis 15
2.9. Dot blot immunoassay with anti-Mtb H37Rv HBHA monoclonal antibody 17
2.10. Nuclear magnetic resonance spectroscopy of methylated recombinant
HBHA 19
2.11. Multidimensional NMR studies and structure calculation of methylated HBHA-heparin complexes 24
2.13. Mass analysis of proteolyzed methylated HBHA by liquid chromatography and tandem mass spectrometry 25
2.14. Qualitative trypsin digestion assay 27
2.15. Isothermal titration calorimetry of methylated HBHA-enoxaparin versus non-methylated HBHA-enoxaparin 28
2.16. Circular dichroism spectroscopy of M. smegmatis expressed HBHA 29
3. Results 31
3. 1. Cloning of hbhA to pYUB28b vector 32
3.2. Transformation of M. smegmatis mc24517, expression, and purification of HBHA 33
3.3. Dot blot assay with anti-Mtb H37Rv HBHA monoclonal antibody 42
3.4. NMR Spectroscopy of methylated HBHA 46
3.5. LC-MS/MS of M. smegmatis expressed recombinant HBHA 68
3.6. Qualitative trypsin digestion assay of HBHA 75
3.7. Circular dichroism spectroscopy of M. smegmatis expressed HBHA 71
3.8. Isothermal titration calorimetry of HBHA with heparan sulfate hexadecasaccharide 79

4. Discussion 83
4.1. Plasmid construction, cloning, and expression of HBHA in M. smegmatis 84
4.2. Dot blot assay with anti-Mtb H37Rv HBHA monoclonal antibody 85
4.3. Nuclear magnetic resonance spectroscopy of methylated recombinant HBHA 87
4.4. LC-MS/MS of M. smegmatis expressed recombinant HBHA 88
4.5. Qualitative trypsin digestion assay of HBHA 90
4.6. Interaction of HBHA with heparin-like hexadecasaccharides 91
5. Conclusion and Recommendations 93
4.1. Conclusion 94
4.2. Recommmendations 95
5. References 97
6. Appendices 103

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