title: Characterization of Mycobacterium tuberculosis EsxA membrane insertion: roles of N- and C-terminal flexible arms and central helix-turn-helix motif.
abstract: EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed.
authors: From the Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968.
authors: Yue
authors: Y
authors: Ma
authors: From the Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968.
authors: Verena
authors: V
authors: Keil
authors: From the Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas 79968 jsun@utep.edu.
authors: Jianjun
authors: J
authors: Sun
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000382:microbiology; D018360:Crystallography, X-Ray; D019077:Helix-Turn-Helix Motifs; D054884:Host-Pathogen Interactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000472:pathogenicity / Q000502:physiology*; D014376:Tuberculosis / Q000378:metabolism* / Q000382:microbiology*; D037521:Virulence Factors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D008081:Liposomes; D037521:Virulence Factors
keywords: Conformational Change; ESAT-6; EsxA; Membrane; Mycobacterium tuberculosis; Pathogenesis; Virulence Factor
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