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

Abstract

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.

Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions

“…Our findings were contradicted by prior work suggesting that ESAT-6 alone directly lyses membranes by functioning as a secreted pore-forming protein (15,18,28,34). We were puzzled by this discrepancy particularly because we too had previously found that the widely used, commercially prepared recombinant ESAT-6 [from Biodefense and Emerging Infections (BEI) Research Resources Repository] mediated hemolysis and so did the rESAT-6 we prepared using the published protocol (44).…”

“…In contrast, those that did not use the detergent step reported ESAT-6 to be devoid of cytolytic activity (Table 1). Of note, both we and others (34) have found that recombinant CFP-10 preparations (including from BEI Resources) fail to lyse cells despite being subjected to the identical detergent wash step to that for ESAT-6. This result suggests that any residual detergent was removed during the subsequent wash and dialysis steps in the case of CFP-10 but not ESAT-6 because of tighter binding of detergent to the latter protein.…”

“…Prior work had suggested that ESAT-6 mediated ESX-1 membrane lytic activity through pore-forming activity (15,18,28,34). If so, mycobacterial culture supernatants should produce hemolysis.…”

“…4A). While our experiments were in progress, a paper was published that also found that rESAT-6 lysed RBCs only if the preparations were treated with ASB-14 (34). These authors interpreted the data to mean that detergent modifies the structure of ESAT-6, activating its lytic activity.…”

Mechanism of ESAT-6 membrane interaction and its roles in pathogenesis of Mycobacterium tuberculosis

“…The ASB-treated and non-treated ESAT-6 showed two distinct conformational states and presented opposing effects on mycobacterial infectivity, macrophage survival, interferon -γ secretion and pore formation (Refai et al, 2015). This observation suggests that the heterodimer of ESAT-6/CFP-10 may be dissociated by a mechanism other than acidification and N-α-acetylation.…”