Date of Award

2017-01-01

Degree Name

Master of Science

Department

Biological Sciences

Advisor(s)

Jianjun Sun

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent for the disease Tuberculosis (TB) in humans, is present as a latent infection in approximately one third of the world's population. Mtb has become more resilient over the years. The vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), loses effectiveness 10 years after initial vaccination. Recent research has found 6-kD early secretory antigenic target (ESAT-6) and 10-kD culture filtrate protein (CFP-10) are secreted as a heterodimer by Mtb and play important roles in virulence. Additionally, ESAT-6 has been determined to contain membrane lytic activity while CFP-10 has been suggested to be a molecular chaperone. Studies suggest ESAT-6 dissociates from CFP-10 at low pH to interact with the phagosomal membrane, which facilitates the translocation of Mtb into the cytosol of a macrophage. Furthermore, membrane lytic ability of ESAT-6 only occurs after it dissociates from CFP-10. However, the mechanism of heterodimer dissociation remains elusive. Previous studies have identified and isolated an N-α-terminally acetylated ESAT-6 protein. Binding affinity between CFP-10 and ESAT-6 is greatly reduced after acetylation, suggesting that N-α-terminal acetylation could be the cause for the dissociation of the ESAT-6/CFP-10 heterodimer. In this study, we aim to determine whether N-α-terminal acetylation of Threonine-2 (T2) on ESAT-6 is required for dissociation of the heterodimer. To test this, we replaced T2 with three different amino acids alanine (T2/A), glutamine (T2/Q), and arginine (T2/R). We hypothesized T2/A and T2/Q would function as acetylation-mimicking residues with similar activity to wild-type ESAT-6, while T2/R serves as acetylation negative control with less activity. Preliminary data, a liposome leakage assay with the dye/quencher pair, 8-aminonaphthalene-1,3,6 trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX), revealed that all of these mutations did not affect pore formation of ESAT-6 alone, but diminished pore formation of the ESAT-6 complexed with CFP-10. This data suggested T2/A, T2/Q and T2/R mutations inhibited heterodimer dissociation, prompting generation of a new T2/S mutant. Cytotoxicity analysis of macrophages infected by Mycobacterium marinum revealed decreased activity in the mutants while complemented strains with WT ESAT-6 or T2/S ESAT-6 recovered activity. As expected, the mutations inhibited the cytosolic translocation as measured by CCF4 fluorescence resonance energy transfer (FRET). The change in virulence was presumed to be due to the lack of acetylation in the T2/Q, T2/R, and T2/A mutants. Isolation of acetylated ESAT-6 was achieved via ASB-14 or 6M guanidine and was confirmed via 4-chloro-7-nitrobenzofurazan (NBD-Cl) and mass spectrometry. Native gel shift assay revealed un-acetylated ESAT-6 to form a complex with CFP-10 more efficiently than acetylated ESAT-6. Furthermore, preliminary studies using surface plasmon resonance revealed differential binding affinity between either specie of ESAT6. This study has revealed the physiological importance of N-α-acetylation of ESAT-6 in Mtb infection.

Language

en

Provenance

Received from ProQuest

File Size

62 pages

File Format

application/pdf

Rights Holder

Javier A. Aguilera

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Biology Commons

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