Post-translational knockdown and post-secretional modification of EsxA determine contribution of EsxA membrane permeabilizing activity for mycobacterial intracellular survival

Current genetic studies (e.g. gene knockout) have suggested that EsxA and EsxB function as secreted virulence factors that are essential for Mycobaterium tuberculosis (Mtb) intracellular survival, specifically in mediating phagosome rupture and translocation of Mtb to the cytosol of host cells, which further facilitates Mtb intracellular replicating and cell-to-cell spreading. The EsxA-mediated intracellular survival is presumably achieved by its pH-dependent membrane-permeabilizing activity (MPA). However, the data from other studies have generated a discrepancy regarding the role of EsxA MPA in mycobacterial intracellular survival, which has raised a concern that genetic manipulations, such as deletion of esxB-esxA operon or RD-1 locus, may affect other codependently secreted factors that could be also directly involved cytosolic translocation, or stimulate extended disturbance on other genes' expression. To avoid the drawbacks of gene knockout, we first engineered a Mycobacterium marinum (Mm) strain, in which a DAS4+ tag was fused to the C-terminus of EsxB to allow inducible knockdown of EsxB (also EsxA) at the post-translational level. We also engineered an Mm strain by fusing a SpyTag (ST) to the C-terminus of EsxA, which allowed inhibition of EsxA-ST MPA at the post-secretional level through a covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and functional inhibition of EsxA resulted in attenuation of Mm intracellular survival in lung epithelial cells or macrophages, which unambiguously confirms the direct role of EsxA MPA in mycobacterial intracellular survival.