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 CalmetteâGuerin (BCG), has been shown to lose effectiveness 10 years after initial vaccination. Recent research found 6âkD early secretory antigenic target (ESATâ6) and 10âkD culture filtrate protein (CFPâ10) are secreted as a heterodimer by Mtb and are important for virulence. Additionally, ESATâ6 has been determined to contain membrane lytic activity, while CFPâ10 has been suggested to be a molecular chaperone. Furthermore, membrane lytic ability from ESATâ6 only occurs after it has dissociated from CFPâ10. Studies suggest ESATâ6 dissociates from CFPâ10 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 Nâterminally acetylated ESATâ6 protein. Binding affinity between CFPâ10 and ESATâ6 is greatly reduced after acetylation, suggesting that Nâterminal acetylation could be the culprit for the dissociation of the ESATâ6 and CFPâ10 heterodimer. In this study, we aim to determine whether Nâterminal acetylation of Threonine 2 on ESATâ6 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 acetylationâmimicking residues, alanine and glutamine would contain similar activity to wildâtype ESATâ6 while acetylation negative control, arginine, would have less activity. Preliminary data, a liposome leakage assay with the dye/quencher pair, 8âaminonaphthaleneâ1,3,6 trisulfonic acid (ANTS) and pâxyleneâbisâpyridinium bromide (DPX), revealed these mutations did not affect pore formation of ESATâ6 alone, but greatly diminished pore formation of the ESATâ6 complexed with CFPâ10, suggesting alanine and glutamine failed to function as acetylationâmimicking residues, highlighting the importance of Thr2. Cytotoxicity analysis of postâmacrophage 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 twoâdimensional gel electrophoresis (2âDE). Additionally, 4âchloroâ7ânitrobenzofurazan (NBDâCl) reacts with unâacetylated proteins, and exhibits measurable fluorescence, while Nâterminally acetylated proteins do not, compromising a reliable method for detection of acetylation in these proteins. This study has revealed the physiological importance of Nâalphaâacetylation of ESATâ6 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 startâup funds.