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

Abstract

9Current genetic studies (e.g. gene knockout) have suggested that EsxA and EsxB function as secreted 10 virulence factors that are essential for Mycobaterium tuberculosis (Mtb) virulence, specifically in mediating 11 phagosome rupture and translocation of Mtb to the cytosol of host cells, which further facilitates Mtb 12 intracellular replicating and cell-to-cell spreading. The EsxA-mediated virulence is presumably achieved by 13 its pH-dependent membrane-permeabilizing activity (MPA). However, the data from recent studies have 14 generated a discrepancy regarding to the role of EsxA MPA in mycobacterial virulence with a major concern 15 that genetic manipulations, such as deletion of esxB-esxA operon, may stimulate genetic compensation to 16 produce artifacts and/or affect other co-dependently secreted factors that could be directly involved cytosolic 17 translocation. To avoid the drawbacks of gene knockout, we first engineered a Mycobacterium marinum 18 (Mm) strain, in which a DAS4+ tag was fused to the C-terminus of EsxB to allow inducible knockdown of 19 EsxB (also EsxA) at the post-translational level. We also engineered a Mm strain by fusing a SpyTag to the 20 C-terminus of EsxA, which allows inhibition of EsxA-ST MPA at the post-secretional level through a 21 covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and post-secretional inhibition of 22 EsxA resulted in attenuation of Mm intracellular survival and virulence in macrophages and lung epithelial 23 cells, which unambiguously confirms the role of EsxA MPA in mycobacterial virulence.24 2Genetic studies, such as loss of function by gene deletion and disruption, have suggested that EsxA is a 28 virulence factor essential for mycobacterial virulence. However, its role is questioned because knockout of 29 esxA gene may affect the function or secretion of other related genes. Here, we employed two methods other 30 than gene deletion and disruption to determine EsxA role in mycobacterial virulence. First, we added a 31 degradation signal peptide DAS4+ tag to the C-terminus of EsxB, the chaperon of EsxA so that EsxB-32 DAS4+ could be degraded by protease ClpXP, whose function can be induced by an inducer, ATC. By this 33 way, we were able to control the amount of EsxB and EsxA at the post-translational level. The results 34 showed that ATC inhibited mycobacterial intracellular survival through down-regulating EsxA and EsxB. 35Second method is to take advantage of SpyTag(ST) and SpyCatcher(SC) system. Like DAS4+, ST was 36 fused to C-terminus of EsxA without affecting its expression, secretion and MPA. After secretion, EsxA-ST 37 can be specifically recognized by SC-GFP and form a covalent bond between ST and SC, which blocks the 38 MPA, an activity that directly related to mycobacterial virulence. Endogenous expression of SC-GFP in the 39 infected cells inhibited mycobacterial intracellular survival. In summary, our results demonstrate that 40 knockdown of EsxA at the post-translational level or inhibition of EsxA MPA at the post-secretion...

Mycobacterial surface-associated ESX-1 virulence factors play a role in mycobacterial adherence and invasion into lung epithelial cells

“…Thus, deletion of EsxAB may also affect the secretion of these factors. In order to determine the exact role of EsxAB in adherence and invasion without apparently affecting other factors, we used the ATC inducible protein knockdown system that has been established in the lab and reported before (44). This system allows inducible knockdown of EsxAB at the post-translational level.…”

“…To further confirm if reduction of EsxAB could affect mycobacterial adherence and invasion, Tween-80 was used to reduce mycobacterial surface-associated EsxA (26,45). In order to efficiently and specifically label and quantify the surface associated EsxA, we used the SpyTag (ST)-SpyCatcher (SC) system that has been described in our previous report (44). Briefly, the 13-amino acid SpyTag is engineered into the C-terminus of EsxA, which produces an EsxA-ST fusion protein that has normal expression, secretion and membrane permeabilization activity as EsxA wild type.…”

“…The A549 cell line was generously offered by Dr. Jianying Zhang from University of Texas at El Paso. The strains of Mm(EsxA-ST) and Mm(EsxB-DAS4+)|pGMCKq1 were generated and preserved in our lab as described before (44).…”

“…The coverslip was immersed in 50% glycerol/water solution and applied to confocal microscopy using a Zeiss LSM 700 (San Diego, CA, USA). Since SC-GFP binds to EsxA-ST with a highly specific covalent bond, the intensity of GFP signal represents the amount of EsxA-ST. To quantify the surface-associated EsxA-ST, the images were analyzed as described before (44). Briefly, the areas with green or red fluorescent signals were first extracted and calculated, respectively.…”

“…This green/red ratio was calculated. Layer extraction and area calculation were achieved with Python 3.7.3, according to equation described elsewhere (44,56).…”