Fig 1

(A) Schematic representation of the M. abscessus esx-4 gene cluster. (B) Model of M. abscessus EsxU/EsxT made using ProMod version 3 3.2.10 and based on the NMR structure of M. tuberculosis EsxAB (PDB code: 1wa8.1). EsxU and EsxT are shown in orange and green, respectively, and the essential residues of the WXG motifs and the secretion sequence are represented in boxes. (C) Representative confocal microscopy images of ΔesxUT strains expressing either GFP, EsxT-GFP, EsxU-GFP or EsxUT-GFP to illustrate protein localization. A series of Z-stacks were collected with a Zeiss LSM880 Airyscan confocal microscope and the images were assembled and reconstructed with Zen blue software. (D) Immunoblotting showing the localization of EsxU and EsxT in different fractions: culture filtrate (CF), total lysate (TL), cell wall (CW), plasma membrane (PM) and cytosol (Cyt). Western blotting of subcellular fractions from the ΔesxUT strain show the lack of EsxU and EsxT subunits. Asterisks indicate non-specific bands. The cytosolic marker GroEL1 and the secreted protein Antigen 85B were included as controls. MW, molecular weight marker. (E, left) Co-purified M. abscessus EsxU/EsxT showing bands at approximately 10 kDa in 15% Coomassie-stained SDS-PAGE. Both genes were cloned and expressed in M. smegmatis. (Right) EsxU/EsxT heterodimerization were observed by SEC-MALLS. (F) Ni-NTA column purification of His-tagged EsxT alone in denaturing condition (with urea) and co-purification of EsxU and EsxT in native condition (without urea) in E. coli.