Two distinct conformational states of Mycobacterium tuberculosis virulent factor early secreted antigenic target 6 kDa are behind the discrepancy around its biological functions

Early secreted antigenic target 6 kDa (ESAT‐6) and culture filtrate protein 10 kDa (CFP‐10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT‐6 led to discordant results and the role of ESAT‐6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT‐6. Analysis of a recombinant form of ESAT‐6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT‐6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine‐14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB‐treated versus nontreated ESAT‐6 adopted distinct structural forms but with no well defined tertiary structure. However, protein–protein docking analysis favored a dimeric state of ESAT‐6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon‐γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT‐6/CFP‐10 that ASB was also able to dissociate and which showed functions similar to those of ESAT‐6 dimers/multimers. Our data suggest that, in the absence of CFP‐10, the hydrophobic regions of the ESAT‐6 can form dimers/multimers, mimicking the ESAT‐6/CFP‐10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT‐6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.