The ESX-4 substrates, EsxU and EsxT, modulate Mycobacterium abscessus fitness

Abstract

ESX type VII secretion systems are complex secretion machineries spanning across the mycobacterial membrane and play an important role in pathogenicity, nutrient uptake and conjugation. We previously reported the role of ESX-4 in modulating Mycobacterium abscessus intracellular survival. The loss of EccB4 was associated with limited secretion of two effector proteins belonging to the WXG-100 family, EsxU and EsxT, and encoded by the esx-4 locus. This prompted us to investigate the function of M. abscessus EsxU and EsxT in vitro and in vivo. Herein, we show that EsxU and EsxT are substrates of ESX-4 and form a stable 1:1 heterodimer that permeabilizes artificial membranes. While expression of esxU and esxT was up-regulated in M. abscessus-infected macrophages, their absence in an esxUT deletion mutant prevented phagosomal membrane disruption while maintaining M. abscessus in an unacidified phagosome. Unexpectedly, the esxUT deletion was associated with a hyper-virulent phenotype, characterised by increased bacterial loads and mortality in mouse and zebrafish infection models. Collectively, these results demonstrate that the presence of EsxU and EsxT dampens survival and persistence of M. abscessus during infection.

Fig 1. M. abscessus EsxU and EsxT are co-expressed and secreted by ESX-4.

(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.

Fig 2. ΔesxUT is maintained in an intact phagosome without acidification in macrophages.

(A) Expression of esxU and esxT genes in J774.2 macrophages at 4 hrs and 24 hrs post-infection (black) relative to planktonic growth expression in broth medium (gray). (B) Relative intracellular survival of WT (black), ΔesxUT (red), and ΔesxUT::esxUT (blue) strains as determined by CFU counts during infection in J774.2 macrophages at an MOI of 10:1. (C) Phagosomal rupture detected by CCF-4 FRET-based flow cytometry 24 hrs post-infection. Results are depicted as signal overlays per group with 1,000,000 events per condition acquired in WT (black line), heat-killed (dotted line), ΔesxUT (red line), ΔesxUT::esxUT (blue line) strains and in uninfected cells (NI, gray line). (D) Colocalization of M. abscessus WT, heat-killed, ΔesxUT and ΔesxUT::esxUT strain expressing mCherry with the acidotropic dye LysoTracker Green in infected J774.2 cells 3 hrs post-infection. Arrows indicate intracellular mycobacteria co-localizing with LysoTracker green. (Scale bar, 10 μm). (E) mCherry-labeled strains colocalized with LysoTracker were measured by Pearson correlation of at least 100 infected cells in 10 different fields. Data are representative of three (B and C) or two (D and E) independent experiments and represent means ± SEM. P values were determined by ANOVA with Tukey’s test using GraphPad prism program (A and B) and unpaired t test (E); ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3. Interaction of the M. abscessus EsxU/EsxT complex with DOPC bilayers.

(A) Schematic of the NanoSpot method. DOPC bilayers were built over cavities filled with a fluorescent dye (green). A second fluorescent dye of known size (red) was added to the top of the membrane prior to addition of the protein complex. Fluorescent signals were continuously measured. (B) Dye influx and efflux using the NanoSpot technique. DOPC bilayers were formed at 37°C and pH 7.2 on top of cavities filled with Atto488 carboxy fluorescence dye. Fluorescence (black curve) decreased as more protein was added (final EsxU/EsxT concentration is indicated in blue). Upon addition of Triton X-100, fluorescence decreased abruptly. The red curve represents the trend line of fluorescence loss by photo-bleaching if no protein had been added. Over 300 cavities covered with DOPC membrane are plotted in this graph. (C) 40, 000 MW TexasRed-labelled dextran was added to the media outside the cavities before the EsxU/EsxT complex was supplemented to the well (final concentration: 10 μM). Green dots represent efflux of Atto488 carboxy efflux dye and red dots represent influx of TexasRed dye. Each graph represents one cavity.

Fig 4. ΔesxUT is hypervirulent in zebrafish and C3HeB/FeJ mice.

(A) Zebrafish were injected with 250–300 CFU of M. abscessus strains by tail vein injection and monitored daily for a 12-day infection period to determine relative embryo survival (B) At 6 dpi, zebrafish embryos were imaged under a fluorescent microscope and fluorescent pixel counts (FPC) calculated for each group using ImageJ. Each dot represents an individual embryo. (C) At 2, 4 and 6 dpi, infected zebrafish embryos were imaged using fluorescence microscopy to calculate the number of embryos with granulomas. Granulomas were identified on the basis of colocalisation of several fluorescent macrophages at an infection foci. (D) On days 2, 4, and 6 dpi, infected zebrafish embryos were imaged using fluorescence microscopy to calculate the proportion of embryos with abscesses. Abscesses were identified based on the relative size of infection foci far exceeding the surrounding recruited immune cells, representing uncontrolled bacterial growth. (E) Representative images of zebrafish infected with M. abscessus strains at day 6 post-infection. Red represents bacterial infection foci. Scale bar = 1mm. (F) Survival curves of C3HeB/FeJ mice after tail vein infection with 10⁸ CFU/mouse of WT (black line) and ΔesxUT (red line). (G) CFU counts of WT (black dots) and ΔesxUT (red dots) in lungs, liver, kidneys, and spleen at 1, 7, 14, and 20 dpi. Five mice were used per group and experiment was performed twice. P values were determined by ANOVA with Tukey’s test using GraphPad prism program (B, C and D) and unpaired t test (G); ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The log-rank (Mantel-Cox) test for Kaplan-Meier survival curves was used to assess survival statistics significance.