Despite many methodological advances that have facilitated investigation of Mycobacterium tuberculosis pathogenesis, analysis of essential gene function in this slow-growing pathogen remains difficult. Here, we describe an optimized CRISPR-based method to inhibit expression of essential genes based on the inducible expression of an enzymatically inactive Cas9 protein together with gene-specific guide RNAs (CRISPR interference). Using this system to target several essential genes of M. tuberculosis, we achieved marked inhibition of gene expression resulting in growth inhibition, changes in susceptibility to small molecule inhibitors and disruption of normal cell morphology. Analysis of expression of genes containing sequences similar to those targeted by individual guide RNAs did not reveal significant off-target effects. Advantages of this approach include the ability to compare inhibited gene expression to native levels of expression, lack of the need to alter the M. tuberculosis chromosome, the potential to titrate the extent of transcription inhibition, and the ability to avoid off-target effects. Based on the consistent inhibition of transcription and the simple cloning strategy described in this work, CRISPR interference provides an efficient approach to investigate essential gene function that may be particularly useful in characterizing genes of unknown function and potential targets for novel small molecule inhibitors.
Toxin–antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Here, we use a specialized RNA-seq approach to identify targets of a Mycobacterium tuberculosis VapC TA toxin, VapC-mt4 (also known as VapC4), which have eluded detection using conventional approaches. Distinct from the one other characterized VapC toxin in M. tuberculosis that cuts 23S rRNA at the sarcin–ricin loop, VapC-mt4 selectively targets three of the 45 M. tuberculosis tRNAs (tRNAAla2, tRNASer26 and tRNASer24) for cleavage at, or adjacent to, their anticodons, resulting in the generation of tRNA halves. While tRNA cleavage is sometimes enlisted as a bacterial host defense mechanism, VapC-mt4 instead alters specific tRNAs to inhibit translation and modulate growth. This stress-linked activity of VapC-mt4 mirrors basic features of eukaryotic tRNases that also generate tRNA halves and inhibit translation in response to stress.
The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three-or five-base RNA sequence and cleaves mRNA. As a result, these toxins selectively target a subset of the transcriptome for degradation and are known as "mRNA interferases." Here we demonstrate that a MazF family member from M. tuberculosis, MazF-mt6, has an additional role-inhibiting translation through targeted cleavage of 23S rRNA in the evolutionarily conserved helix/loop 70. We first determined that MazF-mt6 cleaves mRNA at 5′ UU↓CCU 3′ sequences. We then discovered that MazF-mt6 also cleaves M. tuberculosis 23S rRNA at a single UUCCU in the ribosomal A site that contacts tRNA and ribosome recycling factor. To gain further mechanistic insight, we demonstrated that MazFmt6-mediated cleavage of rRNA can inhibit protein synthesis in the absence of mRNA cleavage. Finally, consistent with the position of 23S rRNA cleavage, MazF-mt6 destabilized 50S-30S ribosomal subunit association. Collectively, these results show that MazF toxins do not universally act as mRNA interferases, because MazF-mt6 inhibits protein synthesis by cleaving 23S rRNA in the ribosome active center.T oxin-antitoxin (TA) systems have the potential to control Mycobacterium tuberculosis growth rate and persistence. One of the best characterized TA modules is mazEF in Escherichia coli (1-7), an autoregulated operon that encodes the intracellular toxin MazF and its antitoxin inhibitor MazE. Under unstressed conditions, the MazE protein forms a stable complex with MazF to neutralize its toxicity (1). During times of stress, however, proteases degrade MazE and allow the relatively stable MazF toxin to disrupt protein synthesis (1, 2), which can induce a state of reversible dormancy (3). Expression of MazF triggers this quasi-dormant state, during which cells stop dividing but are able to transcribe mRNA and synthesize proteins (4). The striking similarities between this state of quasi-dormancy and the slowgrowing or nonreplicating state of M. tuberculosis during latent tuberculosis (TB) have led to the suggestion that TA modules are involved with persistence and dormancy in M. tuberculosis (8).The genome of M. tuberculosis has >80 putative TA pairs (9), a remarkably large number relative to most other prokaryotes. Although TA modules are ubiquitous in bacteria and archaea, few prokaryotes have more than 15 loci (10). Although the physiological role of the large repertoire of TA loci in M. tuberculosis is largely unknown, some clues have emerged. First, there is an inverse correlation between the number of chromosomal TA loci and growth rate (10). Second, more than 60 TA toxins are conserved between five members of the M. tuberculosis complex but not in 13 oth...
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