Post-translational knockdown and post-secretional modification of EsxA unambiguously determine the role of EsxA membrane permeabilizing activity in mycobacterial virulence

Abstract 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) virulence, 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 virulence is presumably achieved by its pH-dependent membrane-permeabilizing activity (MPA). However, the data from recent studies have generated a discrepancy regarding to the role of EsxA MPA in mycobacterial virulence with a major concern that genetic manipulations, such as deletion of esxB-esxA operon, may stimulate genetic compensation to produce artifacts and/or affect other co-dependently secreted factors that could be directly involved cytosolic translocation. 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 a Mm strain by fusing a SpyTag to the C-terminus of EsxA, which allows inhibition of EsxA-ST MPA at the post-secretional level through a covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and post-secretional inhibition of EsxA resulted in attenuation of Mm intracellular survival and virulence in macrophages and lung epithelial cells, which unambiguously confirms the role of EsxA MPA in mycobacterial virulence. Author Summary Genetic studies, such as loss of function by gene deletion and disruption, have suggested that EsxA is a virulence factor essential for mycobacterial virulence. However, its role is questioned because knockout of esxA gene may affect the function or secretion of other related genes. Here, we employed two methods other than gene deletion and disruption to determine EsxA role in mycobacterial virulence. First, we added a degradation signal peptide DAS4+ tag to the C-terminus of EsxB, the chaperon of EsxA so that EsxB-DAS4+ could be degraded by protease ClpXP, whose function can be induced by an inducer, ATC. By this way, we were able to control the amount of EsxB and EsxA at the post-translational level. The results showed that ATC inhibited mycobacterial intracellular survival through down-regulating EsxA and EsxB. Second method is to take advantage of SpyTag(ST) and SpyCatcher(SC) system. Like DAS4+, ST was fused to C-terminus of EsxA without affecting its expression, secretion and MPA. After secretion, EsxA-ST can be specifically recognized by SC-GFP and form a covalent bond between ST and SC, which blocks the MPA, an activity that directly related to mycobacterial virulence. Endogenous expression of SC-GFP in the infected cells inhibited mycobacterial intracellular survival. In summary, our results demonstrate that knockdown of EsxA at the post-translational level or inhibition of EsxA MPA at the post-secretional level, attenuate mycobacterial virulence, and this attenuation is solely attributed to EsxA, not to other factors.