Nα-Acetylation of the virulence factor EsxA is required for mycobacterial cytosolic translocation and virulence

Javier Aguilera,Chitra B Karki,Lin Li,Salvador Vazquez Reyes,Igor Estevao,Brian I Grajeda,Qi Zhang,Chenoa D Arico,Hugues Ouellet,Jianjun Sun

doi:10.1074/jbc.ra119.012497

Javier Aguilera, Chitra B Karki + Show 8 more

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https://doi.org/10.1074/jbc.ra119.012497

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Abstract

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 Nα-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 Nα-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 Nα-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 Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Highlights

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
3 The abbreviations used are: Mycobacterium tuberculosis (Mtb), Mycobacterium smegmatis (Ms). tuberculosis; Ms, M. smegmatis; Mm, M. marinum; Ec, E. coli; Ec-heterodimer, heterodimer prepared from E. coli; Ms-heterodimer, heterodimer prepared from M. smegmatis; MD, molecular dynamic; ANTS, 8-aminonapthalene-1,3,6 trisulfonic acid; DPX, pxylene-bis-pyridinium bromide; NBD-Cl, 4-chloro-7-nitrobenzofurazan; MOI multiplicity of infection; VMD, visual molecular dynamics
Using the previously established CCF4-FRET assay, we found that the nonacetylated mutations T2A, T2Q, and T2R abolished mycobacterial cytosolic translocation, whereas T2S maintained a similar activity as the WT (Fig. 6B)

Summary

Introduction

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. Substitutions at Thr-2 that precluded N␣-acetylation inhibited the heterodimer separation and prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Our findings provide evidence that the N␣-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence. S.) and National Institutes of Health Grant R25GM069621 The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M. marinum; Ec, E. coli; Ec-heterodimer, heterodimer prepared from E. coli; Ms-heterodimer, heterodimer prepared from M. smegmatis; MD, molecular dynamic; ANTS, 8-aminonapthalene-1,3,6 trisulfonic acid; DPX, pxylene-bis-pyridinium bromide; NBD-Cl, 4-chloro-7-nitrobenzofurazan; MOI multiplicity of infection; VMD, visual molecular dynamics

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