Enzymatic and Structural Characterization of Proteins Linked to Mycobacterium tuberculosis Pathogenicity

Tuberculosis (TB) is the leading cause of death by any treatable infectious disease.¹ An estimated 2 billion people are infected by the etiological agent Mycobacterium tuberculosis worldwide.² In 2006, 9.2 million new cases were reported and 1.7 million deaths from TB occurred globally.¹ The strong resurgence of the disease is due to HIV coinfection and the appearance of drug-resistant strains of the mycobacterium. In addition to the typical bacterial drug-resistance mechanisms,³ M. tb possesses a physical barrier that promotes resistance due to its thick hydrophobic cell envelope. The importance of the cell envelope was identified early on and its structure has been well characterized. Molecules involved in the cell wall maintenance are of great interest in tuberculosis research. The antigen 85 complex (Ag85), responsible for the attachment of fatty acid moieties to carbohydrate components of the cell wall, is a validated drug target.⁴ The existing radiometric assay developed for testing the activity of Ag85 is time consuming, thus not readily adapted to the high throughput format required in the drug discovery process. The first part of this work describes the development of a colorimetric enzymatic assay adapted to drug screening. The coupled assay uses a glycoconjugate, p-nitrophenyl 6-O-octanoyl-β-D-glucopyranoside, as a substrate. Ag85 transfers the octyl moiety to an acceptor and β-glucosidase releases the chromophore, p-nitrophenol. After optimization of the assay parameters, the assay was used to screen libraries of synthetic and natural compounds. In addition to its utility for screening applications, the assay was also used for the kinetic characterization of the Ag85 family of enzymes.

Another interesting feature of M. tb is its ability to interfere with the host immune response. For example, the maturation of the phagosome is arrested in the early stages after phagocytosis of the mycobacterium and this process allows the intracellular survival of the pathogen. One protein promoting intracellular survival was identified and called the enhanced intracellular survival (Eis) protein.⁵ The pathological effects of Eis have been characterized, however little is known about the exact function of the protein. This work presents crystallization studies of Eis. The putative enzyme was successfully crystallized; however the diffraction achieved did not result in exploitable data.