A novel regulatory interplay between atypical B12 riboswitches and uORF translation in Mycobacterium tuberculosis.

Vitamin B12 is an essential cofactor in all domains of life and B12-sensing riboswitches are some of the most widely distributed riboswitches. Mycobacterium tuberculosis, the causative agent of tuberculosis, harbours two B12-sensing riboswitches. One controls expression of metE, encoding a B12-independent methionine synthase, the other controls expression of ppe2 of uncertain function. Here, we analysed ligand sensing, secondary structure and gene expression control of the metE and ppe2 riboswitches. Our results provide the first evidence of B12 binding by these riboswitches and show that they exhibit different preferences for individual isoforms of B12, use distinct regulatory and structural elements and act as translational OFF switches. Based on our results, we propose that the ppe2 switch represents a new variant of Class IIb B12-sensing riboswitches. Moreover, we have identified short translated open reading frames (uORFs) upstream of metE and ppe2, which modulate the expression of their downstream genes. Translation of the metE uORF suppresses MetE expression, while translation of the ppe2 uORF is essential for PPE2 expression. Our findings reveal an unexpected regulatory interplay between B12-sensing riboswitches and the translational machinery, highlighting a new level of cis-regulatory complexity in M. tuberculosis. Attention to such mechanisms will be critical in designing next-level intervention strategies.

Premature termination of transcription is shaped by Rho and translated uORFS in Mycobacterium tuberculosis.

Little is known about the decisions behind transcription elongation versus termination in the human pathogen Mycobacterium tuberculosis (M.TB). By applying Term-seq to M.TB we found that the majority of transcription termination is premature and associated with translated regions, i.e., within previously annotated or newly identified open reading frames. Computational predictions and Term-seq analysis, upon depletion of termination factor Rho, suggests that Rho-dependent transcription termination dominates all transcription termination sites (TTS), including those associated with regulatory 5' leaders. Moreover, our results suggest that tightly coupled translation, in the form of overlapping stop and start codons, may suppress Rho-dependent termination. This study provides detailed insights into novel M.TB cis-regulatory elements, where Rho-dependent, conditional termination of transcription and translational coupling together play major roles in gene expression control. Our findings contribute to a deeper understanding of the fundamental regulatory mechanisms that enable M.TB adaptation to the host environment offering novel potential points of intervention.

Using a whole genome co-expression network to inform the functional characterisation of predicted genomic elements from Mycobacterium tuberculosis transcriptomic data.

A whole genome co-expression network was created using Mycobacterium tuberculosis transcriptomic data from publicly available RNA-sequencing experiments covering a wide variety of experimental conditions. The network includes expressed regions with no formal annotation, including putative short RNAs and untranslated regions of expressed transcripts, along with the protein-coding genes. These unannotated expressed transcripts were among the best-connected members of the module sub-networks, making up more than half of the 'hub' elements in modules that include protein-coding genes known to be part of regulatory systems involved in stress response and host adaptation. This data set provides a valuable resource for investigating the role of non-coding RNA, and conserved hypothetical proteins, in transcriptomic remodelling. Based on their connections to genes with known functional groupings and correlations with replicated host conditions, predicted expressed transcripts can be screened as suitable candidates for further experimental validation.

Challenges in defining the functional, non-coding, expressed genome of members of the Mycobacterium tuberculosis complex.

A definitive transcriptome atlas for the non-coding expressed elements of the members of the Mycobacterium tuberculosis complex (MTBC) does not exist. Incomplete lists of non-coding transcripts can be obtained for some of the reference genomes (e.g., M. tuberculosis H37Rv) but to what extent these transcripts have homologues in closely related species or even strains is not clear. This has implications for the analysis of transcriptomic data; non-coding parts of the transcriptome are often ignored in the absence of formal, reliable annotation. Here, we review the state of our knowledge of non-coding RNAs in pathogenic mycobacteria, emphasizing the disparities in the information included in commonly used databases. We then proceed to review ways of combining computational solutions for predicting the non-coding transcriptome with experiments that can help refine and confirm these predictions.

The Mycobacterium tuberculosis sRNA F6 Modifies Expression of Essential Chaperonins, GroEL2 and GroES.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host.

Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

OBJECTIVES: Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. METHODS: Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. RESULTS: Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified. CONCLUSIONS: Understanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

Coupling of Peptidoglycan Synthesis to Central Metabolism in Mycobacteria: Post-transcriptional Control of CwlM by Aconitase.

Mycobacterium tuberculosis causes human tuberculosis, and a better understanding of its biology is required to identify vulnerabilities that might be exploited in developing new therapeutics. The iron-sulfur cluster of the essential M. tuberculosis central metabolic enzyme, aconitase (AcnA), disassembles when exposed to oxidative/nitrosative stress or iron chelators. The catalytically inactive apo-AcnA interacts with a sequence resembling an iron-responsive element (IRE) located within the transcript of another essential protein, CwlM, a regulator of peptidoglycan synthesis. A Mycobacterium smegmatis cwlM conditional mutant complemented with M. tuberculosis cwlM with a disrupted IRE is unable to recover from combinations of oxidative, nitrosative, and iron starvation stresses. An equivalent M. tuberculosis cwlM conditional mutant complemented with the cwlM gene lacking a functional IRE exhibits a growth defect in THP-1 macrophages. It appears that AcnA acts to couple peptidoglycan synthesis and central metabolism, and disruption of this coupling potentially leaves mycobacteria vulnerable to attack by macrophages.

Riboswitches: choosing the best platform.

Riboswitch discovery and characterisation have come a long way since the term was first coined almost two decades ago. Riboswitches themselves are likely derived from ancient ligand-binding transcripts, which have evolved into sophisticated genetic control elements that are widespread in prokaryotes. Riboswitches are associated with a multitude of cellular processes including biosynthetic pathways, transport mechanisms and stress responses leading to an ever-increasing appreciation for an in-depth understanding of their triggers and functions in order to address physiological and regulatory questions. The majority of riboswitches exert their control via transcriptional or translational expression platforms depending on their genetic context. It remains, however, to be determined precisely why one platform is favoured over another. Is this a question of the layout of the gene expression machinery, ligand availability, the degree of control required, serendipity or various combinations of these? With this review, rather than providing answers, I am hoping to plant a seed for further scientific discussions about this puzzle.

Dual RNA-Seq of Human Leprosy Lesions Identifies Bacterial Determinants Linked to Host Immune Response.

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type I IFNs and antibody production. Dual RNA-seq on patient lesions identifies two independent molecular measures of M. leprae, each of which correlates with distinct aspects of the host immune response. The fraction of bacterial transcripts, reflecting bacterial burden, correlates with a host type I IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial mRNA:rRNA ratio, reflecting bacterial viability, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for the interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

Regulatory RNA in Mycobacterium tuberculosis, back to basics.

Since the turn of the millenium, RNA-based control of gene expression has added an extra dimension to the central dogma of molecular biology. Still, the roles of Mycobacterium tuberculosis regulatory RNAs and the proteins that facilitate their functions remain elusive, although there can be no doubt that RNA biology plays a central role in the baterium's adaptation to its many host environments. In this review, we have presented examples from model organisms and from M. tuberculosis to showcase the abundance and versatility of regulatory RNA, in order to emphasise the importance of these 'fine-tuners' of gene expression.

Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition between these states. Rpf expression is tightly regulated as these enzymes are able to degrade the cell wall, and hence potentially lethal to the bacterium itself. We have identified a regulatory element in the 5' untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which appears to be restricted to pathogenic mycobacteria, suggesting a role in virulence. We have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an rpfB antisense RNA. Finally, we show that rpfB is co-transcribed with ksgA and ispE downstream. ksgA encodes a universally conserved methyltransferase involved in ribosome maturation and ispE encodes an essential kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch.

NLRP3 inflammasome activation by mycobacterial ESAT-6 and dsRNA in intraocular tuberculosis.

The molecular basis of intraocular tuberculosis (TB) is not well understood. In this study, we investigated the role of two constituents of viable Mycobacterium tuberculosis - Early Secreted Antigenic Target-6 (ESAT-6), and mycobacterial RNA- in inflammasome activation in the retinal pigment epithelium (RPE), a key site of inflammation in intraocular TB. We found that ESAT-6 induced caspase-1 activation and inflammasome priming in mouse RPE cells, substantially more in wild-type than in Tlr2/3/4/7/9-/-, Myd88-/- or Nlrp3-/- RPE cells. Sub-retinal ESAT-6 injection resulted in greater RPE degeneration in wild-type than in Nlrp3-/- mice. In human ocular TB tissue sections, NLRP3 staining was noted in retina as well as RPE. Mycobacterial RNA, specifically its double stranded component, also induced caspase-1 activation, and the double stranded RNA was immunolocalized to human ocular TB sections. Our observations suggest that inflammasome activation in RPE by viable M. tuberculosis could potentially contribute to human intraocular TB.

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis.

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.

A new spanner in the works of bacterial transcription.

A promising molecular target that is unlikely to develop antibiotic resistance has been identified in bacteria.

Noncoding RNA in Mycobacteria.

Efforts to understand the molecular basis of mycobacterial gene regulation are dominated by a protein-centric view. However, there is a growing appreciation that noncoding RNA, i.e., RNA that is not translated, plays a role in a wide variety of molecular mechanisms. Noncoding RNA comprises rRNA, tRNA, 4.5S RNA, RnpB, and transfer-messenger RNA, as well as a vast population of regulatory RNA, often dubbed "the dark matter of gene regulation." The regulatory RNA species comprise 5' and 3' untranslated regions and a rapidly expanding category of transcripts with the ability to base-pair with mRNAs or to interact with proteins. Regulatory RNA plays a central role in the bacterium's response to changes in the environment, and in this article we review emerging information on the presence and abundance of different types of noncoding RNA in mycobacteria.

A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.

Genome-wide mapping of transcriptional start sites defines an extensive leaderless transcriptome in Mycobacterium tuberculosis.

Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades. We have combined RNA sequencing (RNA-seq) of 5' triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5' UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.

Definition and annotation of (myco)bacterial non-coding RNA.

RNA in bacteria may be broadly classified into coding and non-coding types. The prior, also known as messenger RNA, encode proteins as their final product. The non-coding RNA include all RNAs that are not translated into a protein. Examples of extensively studied and therefore prominent non-coding RNAs include rRNA, tRNA, tmRNA, whose designations reflect the functions performed by these RNAs. Discoveries of non-coding RNAs in mycobacteria have been reported in the recent years. At this early stage of this discipline of mycobacterial research, there is an opportunity for the scientific community to establish a consistent, systematic and objective approach to annotation of these RNAs. We are providing recommendations for this systematic annotation that we hope will be adopted by the mycobacterial research community. These may also serve as templates for annotation of non-coding RNAs in other bacteria.

Global analysis of the regulon of the transcriptional repressor LexA, a key component of SOS response in Mycobacterium tuberculosis.

The DNA damage response is crucial for bacterial survival. The transcriptional repressor LexA is a key component of the SOS response, the main mechanism for the regulation of DNA repair genes in many bacteria. In contrast, in mycobacteria gene induction by DNA damage is carried out by two mechanisms; a relatively small number of genes are thought to be regulated by LexA, and a larger number by an alternate, independent mechanism. In this study we have used ChIP-seq analysis to identify 25 in vivo LexA-binding sites, including nine regulating genes not previously known to be part of this regulon. Some of these binding sites were found to be internal to the predicted open reading frame of the gene they are thought to regulate; experimental analysis has confirmed that these LexA-binding sites regulate the expression of the expected genes, and transcriptional start site analysis has found that their apparent relative location is due to misannotation of these genes. We have also identified novel binding sites for LexA in the promoters of genes that show no apparent DNA damage induction, show positive regulation by LexA, and those encoding small RNAs.

Long-range transcriptional control of an operon necessary for virulence-critical ESX-1 secretion in Mycobacterium tuberculosis.

The ESX-1 secretion system of Mycobacterium tuberculosis has to be precisely regulated since the secreted proteins, although required for a successful virulent infection, are highly antigenic and their continued secretion would alert the immune system to the infection. The transcription of a five-gene operon containing espACD-Rv3613c-Rv3612c, which is required for ESX-1 secretion and is essential for virulence, was shown to be positively regulated by the EspR transcription factor. Thus, transcription from the start site, found to be located 67 bp upstream of espA, was dependent upon EspR enhancer-like sequences far upstream (between 884 and 1,004 bp), which we term the espA activating region (EAR). The EAR contains one of the known binding sites for EspR, providing the first in vivo evidence that transcriptional activation at the espA promoter occurs by EspR binding to the EAR and looping out DNA between this site and the promoter. Regulation of transcription of this operon thus takes place over long regions of the chromosome. This regulation may differ in some members of the M. tuberculosis complex, including Mycobacterium bovis, since deletions of the intergenic region have removed the upstream sequence containing the EAR, resulting in lowered espA expression. Consequent differences in expression of ESX-1 in these bacteria may contribute to their various pathologies and host ranges. The virulence-critical nature of this operon means that transcription factors controlling its expression are possible drug targets.