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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.

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.

Purpose: MHC class I presentation of peptides allows T cells to survey the cytoplasmic protein milieu of host cells. During infection, presentation of self peptides is, in part, replaced by presentation of microbial peptides. However, little is known about the self peptides presented during infection, despite the fact that microbial infections alter host cell gene expression patterns and protein metabolism. Experimental design: The self peptide repertoire presented by HLA‐A*01;01, HLA‐A*02;01, HLA‐B*07;02, HLA‐B*35;01, and HLA‐B*45;01 (where HLA is human leukocyte antigen) was determined by tandem MS before and after vaccinia virus infection. Results: We observed a profound alteration in the self peptide repertoire with hundreds of self peptides uniquely presented after infection for which we have coined the term “self peptidome shift.” The fraction of novel self peptides presented following infection varied for different HLA class I molecules. A large part (approximately 40%) of the self peptidome shift arose from peptides derived from type I interferon‐inducible genes, consistent with cellular responses to viral infection. Interestingly, approximately 12% of self peptides presented after infection showed allelic variation when searched against approximately 300 human genomes. Conclusion and clinical relevance: Self peptidome shift in a clinical transplant setting could result in alloreactivity by presenting new self peptides in the context of infection‐induced inflammation.