Substrate recognition and cryo-EM structure of the ribosome-bound TAC toxin of Mycobacterium tuberculosis.

Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo. mRNA cleavage by the toxin occurs after the second nucleotide of the ribosomal A-site codon during translation, with a strong preference for CCA codons in vivo. Finally, we report the cryo-EM structure of the ribosome-bound TAC toxin in the presence of native M. tuberculosis cspA mRNA, revealing the specific mechanism by which the TAC toxin interacts with the ribosome and the tRNA in the P-site to cleave its mRNA target.

TASmania: A bacterial Toxin-Antitoxin Systems database.

Bacterial Toxin-Antitoxin systems (TAS) are involved in key biological functions including plasmid maintenance, defense against phages, persistence and virulence. They are found in nearly all phyla and classified into 6 different types based on the mode of inactivation of the toxin, with the type II TAS being the best characterized so far. We have herein developed a new in silico discovery pipeline named TASmania, which mines the >41K assemblies of the EnsemblBacteria database for known and uncharacterized protein components of type I to IV TAS loci. Our pipeline annotates the proteins based on a list of curated HMMs, which leads to >2.106 loci candidates, including orphan toxins and antitoxins, and organises the candidates in pseudo-operon structures in order to identify new TAS candidates based on a guilt-by-association strategy. In addition, we classify the two-component TAS with an unsupervised method on top of the pseudo-operon (pop) gene structures, leading to 1567 "popTA" models offering a more robust classification of the TAs families. These results give valuable clues in understanding the toxin/antitoxin modular structures and the TAS phylum specificities. Preliminary in vivo work confirmed six putative new hits in Mycobacterium tuberculosis as promising candidates. The TASmania database is available on the following server https://shiny.bioinformatics.unibe.ch/apps/tasmania/.

Inhibition of the inflammatory response to stress by targeting interaction between PKR and its cellular activator PACT.

PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.