title: 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.
authors.affiliation: Institut Pasteur de Tunis, LTCII LR11 IPT02, Tunisia.|Université Tunis El Manar, Tunisia.
authors.forename: Amira
authors.initials: A
authors.lastname: Refai
authors.affiliation: Institut Pasteur de Tunis, LTCII LR11 IPT02, Tunisia.|Université Tunis El Manar, Tunisia.
authors.forename: Meriam
authors.initials: M
authors.lastname: Haoues
authors.affiliation: Université Tunis El Manar, Tunisia.|Institut Pasteur de Tunis, LVBT LR11 IPT08, Tunisia.
authors.forename: Houcemeddine
authors.initials: H
authors.lastname: Othman
authors.affiliation: Institut Pasteur de Tunis, LTCII LR11 IPT02, Tunisia.|Université Tunis El Manar, Tunisia.
authors.forename: Mohamed Ridha
authors.initials: MR
authors.lastname: Barbouche
authors.affiliation: UMR CNRS 6226 ISCR, Plate-forme PRISM, Université de Rennes 1, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Moua
authors.affiliation: UMR CNRS 6226 ISCR, Plate-forme PRISM, Université de Rennes 1, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Bondon
authors.affiliation: UMR CNRS 6226 ISCR, Plate-forme PRISM, Université de Rennes 1, France.
authors.forename: Liza
authors.initials: L
authors.lastname: Mouret
authors.affiliation: Université Tunis El Manar, Tunisia.|Institut Pasteur de Tunis, LVBT LR11 IPT08, Tunisia.
authors.forename: Najet
authors.initials: N
authors.lastname: Srairi-Abid
authors.affiliation: Institut Pasteur de Tunis, LTCII LR11 IPT02, Tunisia.|Université Tunis El Manar, Tunisia.
authors.forename: Makram
authors.initials: M
authors.lastname: Essafi
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D001622:Betaine / Q000031:analogs & derivatives; D016923:Cell Death; D003902:Detergents; D054884:Host-Pathogen Interactions; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000472:pathogenicity* / Q000502:physiology; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014376:Tuberculosis / Q000209:etiology; D014774:Virulence / Q000502:physiology; D037521:Virulence Factors / Q000737:chemistry / Q000502:physiology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; D003902:Detergents; C093276:ESAT-6 protein, Mycobacterium tuberculosis; C505464:ESAT-6-CFP10 fusion protein; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; C506587:amidosulfobetaine-14; D001622:Betaine; D007371:Interferon-gamma
keywords: ESAT-6; biological functions; discrepancy; structure; tuberculosis