title: Mycobacterium tuberculosis ESAT-6 exhibits a unique membrane-interacting activity that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis.
abstract: Mycobacterium tuberculosis ESAT-6 (MtbESAT-6) reportedly shows membrane/cell-lysis activity, and recently its biological roles in pathogenesis have been implicated in rupture of the phagosomes for bacterial cytosolic translocation. However, molecular mechanism of MtbESAT-6-mediated membrane interaction, particularly in relation with its biological functions in pathogenesis, is poorly understood. In this study, we investigated the pH-dependent membrane interaction of MtbESAT-6, MtbCFP-10, and the MtbESAT-6/CFP-10 heterodimer, by using liposomal model membranes that mimic phagosomal compartments. MtbESAT-6, but neither MtbCFP-10 nor the heterodimer, interacted with the liposomal membranes at acidic conditions, which was evidenced by release of K(+) ions from the liposomes. Most importantly, the orthologous ESAT-6 from non-pathogenic Mycobacterium smegmatis (MsESAT-6) was essentially inactive in release of K(+). The differential membrane interactions between MtbESAT-6 and MsESAT-6 were further confirmed in an independent membrane leakage assay using the dye/quencher pair, 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX). Finally, using intrinsic and extrinsic fluorescence approaches, we probed the pH-dependent conformational changes of MtbESAT-6 and MsESAT-6. At acidic pH conditions, MtbESAT-6 underwent a significant conformational change, which was featured by an increased solvent-exposed hydrophobicity, while MsESAT-6 showed little conformational change in response to acidification. In conclusion, we have demonstrated that MtbESAT-6 possesses a unique membrane-interacting activity that is not found in MsESAT-6 and established the utility of rigorous biochemical approaches in dissecting the virulence of M. tuberculosis.
authors.affiliation: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas 79968, USA.
authors.forename: Joaquin
authors.initials: J
authors.lastname: De Leon
authors.forename: Guozhong
authors.initials: G
authors.lastname: Jiang
authors.forename: Yue
authors.initials: Y
authors.lastname: Ma
authors.forename: Eric
authors.initials: E
authors.lastname: Rubin
authors.forename: Sarah
authors.initials: S
authors.lastname: Fortune
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000382:microbiology*; D019281:Dimerization; D008969:Molecular Sequence Data; D009164:Mycobacterium Infections / Q000378:metabolism / Q000382:microbiology*; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; C093276:ESAT-6 protein, Mycobacterium tuberculosis