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

• Published in: Scientific reports Vol. 9; no. 1; p. 5949
• Main Authors: Cintrón, Melvilí, Zeng, Ju-Mei, Barth, Valdir C., Cruz, Jonathan W., Husson, Robert N., Woychik, Nancy A.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11-04-2019; Nature Publishing Group
• Subjects: 45; 45/22; 45/23; 45/77; 45/90; 45/91; 631/45/500; 692/420/254; 82; 82/29; 82/83; Antitoxins; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Conserved sequence; Endoribonucleases - metabolism; Enzymatic activity; Gene Expression Regulation, Bacterial; Genomes; Humanities and Social Sciences; Humans; Isoacceptors; multidisciplinary; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - growth & development; Mycobacterium tuberculosis - metabolism; Pathogens; Ribonucleic acid; RNA; Science; Science (multidisciplinary); Toxin-Antitoxin Systems; Toxins; Trinucleotide repeats; tRNA; Tuberculosis; Tuberculosis - metabolism; Tuberculosis - microbiology; Virulence
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-41548-9

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 (
v
irulence
a
ssociated
p
rotein) 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, tRNA
Gln32-CUG
and tRNA
Leu3-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.