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.

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, -A*02;01, -B*07;02, -B*35;01 and -B*45;01 was determined by mass spectrometry 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 (~40%) of the self peptidome shift was composed of peptides derived from type I interferon-inducible genes, consistent with cellular responses to viral infection. Interestingly, ~12% of self peptides presented after infection showed allelic variation when searched against ~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 context of infection-induced inflammation.

Background: Existing anthrax postexposure antibiotic treatments are inadequate because they do not clear the high levels of secreted anthrax toxins. Results: Susceptible cells treated with anthrax toxin receptor-targeted siRNAs became resistant to anthrax toxin-mediated cytotoxicity. Conclusion: RNAi-targeted silencing of anthrax toxin receptors prevents toxins from entering target cells and inducing pathogenesis. Significance: Toxin receptor-targeted RNAi can be developed as a postexposure treatment against anthrax.

There is currently extensive data establishing Mycobacterium tuberculosis (Mtb) uses cholesterol as a carbon and energy source. The ability for Mtb to utilize cholesterol as a carbon source is maintained through transcriptional regulation, yet the mechanisms are not fully understood. The Rv3574 gene encodes for the self‐regulated TetR‐like transcriptional repressor KstR that controls the expression of a large cluster of cholesterol catabolic genes in Mtb. This study aims to provide insight on the regulatory mechanisms of side‐chain cholesterol catabolism and the downstream effect on lipid metabolites, such as phthiocerol pthiocerol dimycocerocate (PDIM), which plays a key role during infection. To study the interaction between KstR, and its physiological ligand(s) in‐vitro, the KstR protein has been expressed in wild‐type and mutant forms and electrophoresis mobility shift assays (EMSA) conducted in the presence or absence of other potential ligands. Surface plasmon resonance (SPR) and X‐ray crystallographic studies of KstR with its corresponding kstR DNA operator sequence as well as with the substrate 3‐oxo‐cholest‐4‐en‐26‐oyl‐CoA ester (25,26‐CoA) have been performed to better understand the conformational changes KstR undergoes in physiological conditions. CDC1551, H37Rv, and Erdman Mtb knockout mutant strains lacking kstR (ΔkstR) and complements have been generated and cultured in the presence of cholesterol. Lipid extractions with these strains and growth conditions have been harvested and analyzed using mass spectrometry. EMSA studies revealed the KstR regulator protein is able to bind to the substrate 25,26‐CoA with apparent higher affinity than that of its 3‐oxo‐4‐cholestenoic acid precursor and SPR data has shown the ability for KstR to interact with both ‐S and ‐R stereoisomers of 25,26‐CoA. Mutant variants of KstR have shown to interact with the DNA operator in different manners. Crystallization of KstR in its ligand bound condition with the 25,26‐CoA, as well as bound to the DNA operator, were accomplished showing different orientations of the repressor. Lipid analysis shows the production of PDIM to be varied dependent on the individual Mtb strains. The characterization of KstR interaction with its corresponding binding DNA motifs in the presence of produced substrates will provide significant and fundamental knowledge of the regulatory mechanisms of cholesterol metabolic processes for Mtb. The information garnered within this and future research will assess KstR as a potential therapeutic drug target for the treatment of tuberculosis disease.Support or Funding InformationThis research was funded by the University of Texas at El Paso Border Biomedical Research Center Grant Support: NCRR (5G12RR008124) and NIMHHD (8G12MD007592) from NIH and by NIH/NIAID/NIGMS SC1 AI116567‐01A1 to HO. CA was supported by the Louis Stokes Alliance for Minority Participation under Grant No. HRD‐1301858This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.