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Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host.

ABSTRACT: The Mycobacterium tuberculosis genome harbors an unusually high number of toxin-antitoxin (TA) systems. These TA systems have been implicated in establishing the nonreplicating persistent state of this pathogen during latent tuberculosis infection. More than half of the M. tuberculosis TA systems belong to the VapBC (virulence associated protein) family. In this work, we first identified the RNA targets for the M. tuberculosis VapC-mt11 (VapC11, Rv1561) toxin in vitro to learn more about the general function of this family of toxins. Recombinant VapC-mt11 cleaved 15 of the 45?M. tuberculosis tRNAs at a single site within their anticodon stem loop (ASL) to generate tRNA halves. Cleavage was dependent on the presence of a GG consensus sequence immediately before the cut site and a structurally intact ASL. However, in striking contrast to the broad enzyme activity exhibited in vitro, we used a specialized RNA-seq method to demonstrate that tRNA cleavage was highly specific in vivo. Expression of VapC-mt11 in M. tuberculosis resulted in cleavage of only two tRNA isoacceptors containing the GG consensus sequence, tRNAGln32-CUG and tRNALeu3-CAG. Therefore, our results indicate that although in vitro studies are useful for identification of the class of RNA cleaved and consensus sequences required for accurate substrate recognition by endoribonuclease toxins, definitive RNA target identification requires toxin expression in their native host. The restricted in vivo specificity of VapC-mt11 suggests that it may be enlisted to surgically manipulate pathogen physiology in response to stress.

SUBMITTER: Cintron M

PROVIDER: S-EPMC6459853 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host.

Cintrón Melvilí M Zeng Ju-Mei JM Barth Valdir C VC Cruz Jonathan W JW Husson Robert N RN Woychik Nancy A NA

Scientific reports 20190411 1

The Mycobacterium tuberculosis genome harbors an unusually high number of toxin-antitoxin (TA) systems. These TA systems have been implicated in establishing the nonreplicating persistent state of this pathogen during latent tuberculosis infection. More than half of the M. tuberculosis TA systems belong to the VapBC (virulence associated protein) family. In this work, we first identified the RNA targets for the M. tuberculosis VapC-mt11 (VapC11, Rv1561) toxin in vitro to learn more about the gen ...[more]

PMID: 30976025

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Project description:Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-? and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR), also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti-tuberculosis agents that work by modulating host pathways. Given the existing experience with some of our identified compounds for other therapeutic indications, further clinically-directed study of these compounds is merited.

Dataset Information

Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host.

Publications

Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host.

Similar Datasets

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