Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system.

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.

Comprehensive Definition of the SigH Regulon of Mycobacterium tuberculosis Reveals Transcriptional Control of Diverse Stress Responses.

Expression of SigH, one of 12 Mycobacterium tuberculosis alternative sigma factors, is induced by heat, oxidative and nitric oxide stresses. SigH activation has been shown to increase expression of several genes, including genes involved in maintaining redox equilibrium and in protein degradation. However, few of these are known to be directly regulated by SigH. The goal of this project is to comprehensively define the Mycobacterium tuberculosis genes and operons that are directly controlled by SigH in order to gain insight into the role of SigH in regulating M. tuberculosis physiology. We used ChIP-Seq to identify in vivo SigH binding sites throughout the M. tuberculosis genome, followed by quantification of SigH-dependent expression of genes linked to these sites and identification of SigH-regulated promoters. We identified 69 SigH binding sites, which are located both in intergenic regions and within annotated coding sequences in the annotated M. tuberculosis genome. 41 binding sites were linked to genes that showed greater expression following heat stress in a SigH-dependent manner. We identified several genes not previously known to be regulated by SigH, including genes involved in DNA repair, cysteine biosynthesis, translation, and genes of unknown function. Experimental and computational analysis of SigH-regulated promoter sequences within these binding sites identified strong consensus -35 and -10 promoter sequences, but with tolerance for non-consensus bases at specific positions. This comprehensive identification and validation of SigH-regulated genes demonstrates an extended SigH regulon that controls an unexpectedly broad range of stress response functions.

Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase.

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 (tRNA(Ala2), tRNA(Ser26) and tRNA(Ser24)) 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.

Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site.

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 MazF-mt6-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.

Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin.

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.

Noncognate Mycobacterium tuberculosis toxin-antitoxins can physically and functionally interact.

The Mycobacterium tuberculosis genome harbors a striking number (>40) of toxin-antitoxin systems. Among them are at least seven MazF orthologs, designated MazF-mt1 through MazF-mt7, four of which have been demonstrated to function as mRNA interferases that selectively target mRNA for cleavage at distinct consensus sequences. As is characteristic of all toxin-antitoxin systems, each of the mazF-mt toxin genes is organized in an operon downstream of putative antitoxin genes. However, only one of the seven putative upstream antitoxins (designated MazE-mt1 through MazE-mt7) has significant sequence similarity to Escherichia coli MazE, the cognate antitoxin for E. coli MazF. Interestingly, the M. tuberculosis genome contains two independent operons encoding E. coli MazE orthologs, but they are not paired with mazF-mt-like genes. Instead, the genes encoding these two MazE orthologs are each paired with proteins containing a PIN domain, indicating that they may be members of the very large VapBC toxin-antitoxin family. We tested a spectrum of pair-wise combinations of cognate and noncognate Mtb toxin-antitoxins using in vivo toxicity and rescue experiments along with in vitro interaction experiments. Surprisingly, we uncovered several examples of noncognate toxin-antitoxin association, even among different families (e.g. MazF toxins and VapB antitoxins). These results challenge the "one toxin for one antitoxin" dogma and suggest that M. tuberculosis may enlist a sophisticated toxin-antitoxin network to alter its physiology in response to environmental cues.

Conformational Properties of Helical Protein Polymers with Varying Densities of Chemically Reactive Groups.

Protein engineering strategies have proven valuable for the production of a variety of well-defined macromolecular materials with controlled properties that have enabled their use in a range of materials and biological applications. In this work, such biosynthetic strategies have been employed in the production of monodisperse alanine-rich, helical protein polymers with the sequences [AAAQEAAAAQAAAQAEAAQAAQ](3) and [AAAQAAQAQAAAEAAAQAAQAQ](6). The composition of these protein polymers is similar to that of a previously reported family of alanine-rich protein polymers, but the density and placement of chemically reactive residues has been varied to facilitate the future use of these macromolecules in elucidating polymeric structure-function relationships in biological recognition events. Both protein polymers are readily expressed from E. coli and purified to homogeneity; characterization of their conformational behavior via circular dichroic spectroscopy (CD) indicates that they adopt highly helical conformations under a range of solution conditions. Differential scanning calorimetry, in concert with CD, demonstrates that the conformational transition from helix to coil in these macromolecules can be well-defined, with helicity, conformational transitions, T(m) values, and calorimetric enthalpies that vary with the molecular weight of the protein polymers. A combination of infrared spectroscopy and CD also reveals that the macromolecules can adopt beta-sheet structures at elevated temperatures and concentrations and that the existence and kinetics of this conformational transition appear to be related to the density of charged groups on the protein polymer.