Investigation Into the Role of N‐terminal Acetylation of ESAT‐6 in Pathogenesis of Mycobacterium tuberculosis
Authors: Aguilera Javier Arturo , Sun Jianjun 
Published: November 2023

Mycobacterium tuberculosis (Mtb), the causative agent for the disease Tuberculosis in humans, is present as a latent infection in approximately one third of the world population. Mtb has become more resilient over the years, as the vaccine, Mycobacterium bovis bacillus Calmette‐Guerin (BCG), has been shown to lose effectiveness 10 years after initial vaccination. Recent research 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 are important for virulence. Additionally, ESAT‐6 has been determined to contain membrane lytic activity, while CFP‐10 has been suggested to be a molecular chaperone. Furthermore, membrane lytic ability from ESAT‐6 only occurs after it has dissociated from CFP‐10. Studies suggest ESAT‐6 dissociates from CFP‐10 at low pH to interact with the phagosomal membrane to translocate Mtb into the cytosol of a macrophage, however the mechanism of heterodimer dissociation is unclear. Previous studies have identified and isolated an N‐terminally acetylated ESAT‐6 protein. Binding affinity between CFP‐10 and ESAT‐6 is greatly reduced after acetylation, suggesting that N‐terminal acetylation could be the culprit for the dissociation of the ESAT‐6 and CFP‐10 heterodimer. In this study, we aim to determine whether N‐terminal acetylation of Threonine 2 on ESAT‐6 is required for dissociation of the heterodimer. To test this, three different residues: alanine, glutamine, and arginine, were used to replace Threonine 2. We hypothesized the acetylation‐mimicking residues, alanine and glutamine would contain similar activity to wild‐type ESAT‐6 while acetylation negative control, arginine, would have 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 these mutations did not affect pore formation of ESAT‐6 alone, but greatly diminished pore formation of the ESAT‐6 complexed with CFP‐10, suggesting alanine and glutamine failed to function as acetylation‐mimicking residues, highlighting the importance of Thr2. Cytotoxicity analysis of post‐macrophage infection using Mycobacterium marinum also revealed decreased activity in Thr2 mutants. Accordingly, if these mutations disrupt dissociation, then decreased translocation to the cytosol would be expected, as such fluorescence electron resonance transfer (FRET) was employed to test this theory. Furthermore, a novel in vitro method to dissociate the heterodimer complex was applied to test whether acetylation is present in wild type and not in proteins containing mutations of Thr2. The resultant proteins were analyzed using narrow range pH gradient two‐dimensional gel electrophoresis (2‐DE). Additionally, 4‐chloro‐7‐nitrobenzofurazan (NBD‐Cl) reacts with un‐acetylated proteins, and exhibits measurable fluorescence, while N‐terminally acetylated proteins do not, compromising a reliable method for detection of acetylation in these proteins. This study has revealed the physiological importance of N‐alpha‐acetylation of ESAT‐6 in Mtb infection.Support or Funding InformationThis work was supported by the National Institutes of Health Grant 5G12RR008124 from the National Center for Research Resources and Grant 2G12MD007592 from National Institutes on Minority Health Disparities, a component of National Institutes of Health. This Research is also supported by University of Texas at El Paso new faculty start‐up funds.