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The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS–based proteomics, and CCF-4 FRET analysis, we obtained evidence that the N(α)-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded N(α)-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the N(α)-acetylated Thr-2 makes direct and frequent “bind-and-release” contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the N(α)-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Mycobacterium tuberculosis virulence factors, EsxA and EsxB, are secreted as a heterodimer (EsxA:B) and play an essential role in mycobacterial phagosomal escape and cytosolic translocation. Current studies support a model that EsxA must dissociate from its chaperon EsxB at low pH in order for EsxA to interact with host membranes. However, the mechanism of the heterodimer separation is not clear. In the present study, we have obtained evidence that the Nα-acetylation of Thr2 on EsxA, a post-translational modification that is present in mycobacteria, but absent in E. coli, is required for the heterodimer separation. The point mutations at Thr2 without Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the membranes, which resulted in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamic simulation showed that at low pH the Nα-acetylated Thr2 made direct and frequent “bind-and-release” contacts with EsxB, which generates a dragging force to pull EsxB away from EsxA.