On a stake-out: Mycobacterial small RNA identification and regulation.

Persistence of mycobacteria in the hostile environment of human macrophage is pivotal for its successful pathogenesis. Rapid adaptation to diverse stresses is the key aspect for their survival in the host cells. A range of heterogeneous mechanisms operate in bacteria to retaliate stress conditions. Small RNAs (sRNA) have been implicated in many of those mechanisms in either a single or multiple regulatory networks to post-transcriptionally modulate bacterial gene expression. Although small RNA profiling in mycobacteria by advanced technologies like deep sequencing, tilling microarray etc. have identified hundreds of sRNA, however, a handful of those small RNAs have been unearthed with precise regulatory mechanism. Extensive investigations on sRNA-mediated gene regulations in eubacteria like Escherichia coli revealed the existence of a plethora of distinctive sRNA mechanisms e.g. base pairing, protein sequestration, RNA decoy etc. Increasing studies on mycobacterial sRNA also discovered several eccentric mechanisms where sRNAs act at the posttranscriptional stage to either activate or repress target gene expression that lead to promote mycobacterial survival in stresses. Several intrinsic features like high GC content, absence of any homologue of abundant RNA chaperones, Hfq and ProQ, isolate sRNA mechanisms of mycobacteria from that of other bacteria. An insightful approach has been taken in this review to describe sRNA identification and its regulations in mycobacterial species especially in Mycobacterium tuberculosis.

Introduction

The genus Mycobacterium generally consists of nonpathogenic environmental bacteria, which are closely related to the soil bacteria Streptomyces and Actinomyces [1]. However, multiple clinically relevant highly successful pathogens like Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium ulcerans, the causative agents of tuberculosis, leprosy, and Buruli ulcers, also constitute the mycobacterium genus. Genus Mycobacteria are classified into three classes: M. tuberculosis complex, M. leprae, and non-tuberculosis mycobacteria [2]. Profound insight into this genus can reveal plethora of information about various kind of organisms, and can offer the connecting links between them. More than 140 species are known in the mycobacteria genus [3]. Infectious species like M. tuberculosis, M. leprae, M. bovis BCG etc. in this genus have attracted enormous interest of the scientific communities and the healthcare sectors.Successful infection of these microorganisms partially lies in their ability to escape the macrophage engulfment and successful proliferation inside macrophages [4,5].Identification of non-tuberculosis mycobacteria is intricate since they are diverse in growth temperatures, growth rates, and drug susceptibility, as well as in clinical relevance [1].

M. tuberculosis alone is contemplated as one of the most successful pathogens for causing tuberculosis in human and animals. It is the predominant causative agent for death than any other microorganism, and furthermore, >30% of the current population of the world is believed to carry this bacterium in its non-proliferative state [6]. Interestingly, the development of effective drugs against this pathogen have always been a challenge because of its distinctive life cycle.Between two of its metabolic states, one is the latent persistent and other being the active replicative state [7]. For the development of drugs against M. tuberculosis, it is crucial to apprehend how does it switch between its two states, and on what factor does this transition depends? Initiation of the infection happens through an asymptomatic latent form, and a small number of individuals develop progressive lung disease. Occurrence of tuberculosis depends on the capability of pathogens to balance the altered environments within the host, and under proper circumstances, the transmission of signals that subvert the immune response in order to cause localized immunopathology [8,9]. Continuous macrophage activation is necessary to preclude reactivation of the infection [10,11]. M. bovis and five other mycobacterial species are also categorized in M. tuberculosis complex based on their high genome sequence similarities. Although these species have high resemblance in their virulence properties, but the host and phenotypic expression differ across species [12,13].M. leprae infection on the contrary develops leprosy, a disease with high inconsistency in clinical symptoms. Tuberculoid type leprosy is associated with granuloma formation and severe nerve damage as a consequence of killing of Schwann cells. In contrast, leprosy in multibacillary condition is recognized with heavily infected and inflamed perineurium [14].A fair extent of effort has been given to develop effective drugs against these microorganisms, however, little attention have been paid to understand how different regulators fine tune various physiological processes of these species right after the initiation of transcription. Small RNA (sRNA) regulators frequently modulate numerous posttranscriptional processes to alter gene expressions and cellular phenotype [15] and thus, the understanding the role and mechanism of these regulators will help manipulate mycobacterium virulence.

Aside from sRNAs, other classes of regulatory RNAs like CRISPR (clustered regularly interspaced short palindromic repeats) RNAs [16,17] and riboswitches [18,19] are also found in mycobacterial species. However, the functions of riboswitches or CRISPR will not be discussed in this review. Primary focus of this review is to comprehensively present an insightful analysis of how different strategies were taken to identify mycobacterial sRNAs and how these sRNAs act in vivo to interact with their targets and subsequently, modulate downstream gene expression.

Small RNAs and Hfq in pathogenesis

Numerous physiological circuits in bacteria are regulated by sRNA to remodel their gene expression in response to changing environmental conditions [[20], [21], [22], [23], [24], [25], [26], [27]]. These sRNAs are usually 50–400 nt long and act as a linchpin for the regulation of gene expression under virulence, stress, quorum sensing etc. [23,[27], [28], [29]]. They are either expressed from the cognate genes or generated from the nucleolytic processing of larger transcripts [20].Enormous studies on the identification of sRNAs and their functional role in the cell have largely been investigated in Escherichia coli and Salmonella sp. Majority of the sRNAs in these species have been recognized with specific functions. Based on the analyses, small RNAs have been found to be synthesized either as cis or trans relative to their target mRNAs and generally regulate their expression by base pairing mechanisms resulting in altered mRNA translation and stability [30,31].Cis-encoded sRNA genes lie in the DNA strand, complementary to one from which target mRNA is transcribed, and hence, a broad region of complementarity is present between them. In contrast, trans-encoded sRNAs are transcribed from cognate genes, located far away from their target genes, and thus, share limited complementarity with their targets.

In majority of the instances, trans-encoded sRNAs require the assistance of a chaperone like Hfq or ProQ for a stable sRNA:mRNA base pairing [21,27]. RNA chaperone proteins also play a necessary role of protecting sRNA from cellular degradosome. Interestingly, mycobacterium species have been reported to be devoid of Hfq and ProQ-encoding genes in their chromosome [32], which warns to investigate how sRNAs in mycobacterium species acquire stability inside the cells. Although a wide array of sRNA regulators modulate their target gene expression by base pairing, a small number of sRNAs directly bind and regulate their function. These kind of sRNAs often mimic the structure of the proteins’ cognate targets and sequester such RNA binding proteins. The protein once sequestered, it is no longer available to exhibit its specialized function on their targets [33,34].

Discoveries of increasing number of sRNA-mediated regulation in bacteria have intensified the interest to detect the potential link between the regulatory sRNAs and bacterial pathogenesis. Furthermore, RNA binding proteins like Hfq [32], CsrA [35] etc. have been implicated in impaired bacterial virulence [36]. RNA chaperone Hfq in gram negative bacteria including pathogenic species is essential to promote interaction between trans-encoded sRNAs with their targets. In those cases, pathogenic strains lose their virulence character upon deletion of hfq gene, presumably due to loss of sRNA function [32]. However, Hfq function in gram positive bacteria is ambiguous, as some of the pathogenic bacteria lack hfq gene in their chromosome [37,38]. This apprises the question of how is base pairing of trans-acting sRNAs in mycobacteria with their targets achieved and what would be the subsequent fate of sRNA-mRNA duplex. Probable alternatives are either mycobacterial sRNAs can independently interact with their cognate mRNAs without the assistance of Hfq or they possess different RNA chaperone, which has not been discovered yet. Recently reported additional RNA chaperones like ProQ in Salmonella typhimurium and Rv2367, a homologue of Sinorhizobium meliloti YbeY protein etc. [39,40] are also absent in mycobacteria, thus, it is not unlikely that mycobacterium will be evolved with a novel RNA chaperone.Nearly 70% of mycobacterial genome comprises of GC sequence, and hence, it is conceivable that the secondary structures of mycobacterials RNAs are highly stable owing to their higher degree of intramolecular stability due to high GC content. Moreover, the presence of intrinsic terminator and high GC content restrict the prevalence of AU-rich stretch, which are typical site for Hfq binding [41].

Rapid regulatory circuits particularly regulatory RNA networks in pathogenic bacteria are requisite in the changing environmental conditions for their adaptation, which eventually control their virulence. Small RNAs associated with regulatory proteins and two-component systems integrate environmental signals into essential outcome indispensable for bacterial pathogenesis [42]. Small RNAs have several advantages over protein transcription factors (TFs) in a regulatory circuit of a cell. Lower energy cost of sRNA synthesis, as they are much smaller than mRNA and do not generally require to be translated(27, 31), rapid binding to their targets, and faster clearance while they are no longer needed, have established sRNAs as better contender for controlling bacterial gene regulation.

Direct involvement of sRNAs in regulating bacterial pathogenesis stems from the fact that the deletion of sRNA genes resulted in the phenotype with impaired virulence [43,44]. A reasonable number of sRNAs have been implicated in bacterial virulence and it is growing with new discoveries. Functionally redundant quorum regulatory sRNAs, Qrr1-4, in Vibrio harveyi use distinct regulatory mechanisms to control four different target mRNAs; luxM, luxO, luxR and aphA. All of these sRNAs are the members of the quorum-sensing regulatory circuit [45,46] and thus, directly regulate the virulence character of Vibrio sp. One of the small pathogenicity island RNAs SprD in Staphylococcus aureus down regulates the expression of Sbi immune-evasion molecule, impairing both the adaptive and innate host immune responses [47]. Tn-seq transposon screening and targeted genetic approaches demonstrated that majority of the validated 56 sRNAs in Sreptococcus pneumonia have been implicated in important global and niche-specific roles in virulence [48]. Regulatory sRNAs, RivX and FasX,in Sreptococcus pyogeneshave been demonstrated in virulence gene regulation and interactions with host cells, respectively [49].

A novelcis-encoded antisense small RNA 5ꞌ-UreB-sRNA in Helicobacter pylori, a human stomach colonizing pathogen, down regulates the expression of gastric acid acclimatization operon ureABby truncation at neutral pH, but in acidic environments it releases its control to promote survival [9,50]. Small RNAs like RprA, DsrA, ArcZ, in E.coli and Salmonella sp. act as secondary regulator of bacterial pathogenesis by modulating the expression of stationary phage specific σS-factor RpoS [51,52], which transcribes many of sRNAs related to bacterial virulence. RNA thermosensor in Listeria monocytogenes, a bacteria with low GC content, has been widely established as one of the key regulators of bacterial virulence [53]. Recent reviews [48,[53], [54], [55], [56]] on sRNAs will enumerate different types and mechanisms of sRNA to regulate bacterial virulence. Discoveries of new sRNAs in different pathogenic microorganisms are extending the list of sRNAs implicated in pathogenesis.

Identification of sRNAs

Gene-encoded sRNA was first identified in 1984 through the discovery of a 174 nt long MicF RNA in E. coli [57]. However, first dedicated attempt for the identification of sRNA in bacteria through genome-wide analysis using systematic computational approach was carried out by different groups in 2001 to hunt for RNA secondary structures, orphan promoter and terminator sequence in the intergenic region in E. coli [[58], [59], [60], [61]]. Small RNA list is extending with advanced technologies like deep sequencing, high density tilling microarray etc. [62,63]. In recent years, sRNA identification in large scale has been accomplished by a method, RNA-interaction by ligation and sequencing (RIL-seq), which discovered an extensive network of RNA-RNA interaction [64]. A single bacterium can possess hundreds of sRNA upon encountering to external cues. A handful of sRNAs in different microorganisms, largely from enterobacteria E. coli and Salmonella, have been characterized. Hence, the necessity to perceive sRNA function is heightened in bacteria, especially in pathogenic bacteria.

Arnvig et al. first identified a set of nine putative sRNAs in M. tuberculosis apart from their house keeping sRNAs like M1 RNA, tmRNA, 4.5S RNA etc. by screening cDNA libraries prepared from low molecular weight RNA fraction. Clones of sRNAs were validated by northern blotting and were further mapped for their mature 5ꞌ and 3ꞌ end [65]. In addition to the sRNAs transcripts from IGR (trans-encoded), ORF was also found to be the precursors sRNA transcripts (cis-encoded).All of the identified sRNAs in M. tuberculosis are predicted to be folded in a stable structure with a C:G ratio >1. M. tuberculosis sRNAs possess C-rich stretches [66] in their structures, which presumably have enormous significance for interacting with the target RNAs in absence of Hfq [67,68]. A combined computational and experimental approach was utilized to identify sRNAs from M. tuberculosis and/or M. smegmatis and revealed that the expression of many sRNAs are conserved across the mycobacterial species [69]. A study on the total transcriptome of M. tuberculosis uncovered an abundance of noncoding RNAs including antisense transcripts, intergenic sRNAs and cis-regulatory elements [70]. Another automated approach using RNA-seq technology had been followed to globally identify the sRNA-encoding genes in M. tuberculosis [70]. Computational predictions from the sRNA identification protocol using the algorithm of high-throughput technologies (SIPHT) identified 144 sRNAs from M. bovis BCG and 34 of them validated by northern blotting. A combination of SIPHT and large scale northern blot validation additional 17 sRNAs from M. bovis BCG and 23 sRNAs from M. smegmatis were identified [71]. In the similar direction, transcriptome generated from an exponential phase culture was utilized for identification of 192 novel candidate sRNA-encoding regions in IGR. Additional 664 RNA transcripts, which are synthesized from the region complementary to the ORF by transcription, were also recognized and 28 sRNAs among them were validated by northern blotting [72]. M. leprae genome was also investigated for sRNA expression by whole genome tilling microarray [73].

A novel strategy was implemented by Li et al. [74] for the identification of novel sRNAs by expressing a heterologous RNA chaperone Hfq from E. coli in M. smegmatis with the aim that sRNAs especially trans-acting sRNAs in M. smegmatis will be enriched by Hfq interaction. Immunoprecipitation of Hfq divulged the presence of 12 trans-encoded and 12 cis-encoded sRNAs in the immune-precipitated fraction and many of those sRNAs were found to be differentially expressed at exponential compared to stationary phase, which indicates the involvement of sRNAs in the regulation of mycobacterial growth. Intriguingly, phylogenetic conservation analysis showed that five among these cis-encoded sRNAs are related to mycobacterial virulence. Although this approach explored the advantage of intrinsic sRNA-binding capability of Hfq, however, nonexistence of Hfq or any of its homologue in mycobacterium species limits the practicability of this approach [74].

Mode of regulation by sRNAs

Although a huge number of sRNAs have been identified and validated in mycobacteria, only a handful of them have been designated with their in vivo function [75]. Small RNA-mediated regulation of gene expression is largely achieved through base pairing mechanisms. Structural features of these sRNAs comprise of at least a single-stranded stretch with certain degree of complementarity with their target mRNAs for effective base pairing and a Rho-independent terminator, possessing a stable stem-loop with a poly(U) rich extension [76]. In a number of examples, base pairing of sRNA with its target mRNA is initiated by a fast high affinity contacts through a few exposed nucleotides in the seed region of the single stranded stretch of the stem-loop structure. Subsequent base pairing is assisted by initial “kissing” interaction through the rearrangement of sRNA secondary structures [28]. This type of structure-driven base pairing mechanisms are widespread among cis-encoded sRNAs. In contrast, interaction between trans-encoded sRNAs with their target RNAs in bacteria is primarily promoted by RNA chaperone [77,78]. However, mycobacteria lacks any homologue of Hfq or ProQ, and thus, how trans-acting sRNAs in mycobacteria interact with their cognate targets is still elusive and an intriguing area of active research.

Base pairing mechanism

The outcome of sRNA interaction with their targets is either the repression or the activation of target gene expression. Any sRNA pairing at the ribosome binding site (RBS) on mRNA will interfere 30S ribosome loading at the translation initiation region (TIR) on mRNA, which will ultimately inhibit the initiation of translation, the rate-limiting step in bacterial protein synthesis mechanism [79]. Majority of the sRNAs in bacteria have been reported to be induced in altered environmental conditions and occludes TIR. Intercession of ribosome binding mediated through stress induced sRNAs also delineated to promote Rho-dependent transcription termination [80]. sRNA:mRNA pairing surrounding 5ꞌUTR or RBS or downstream coding sequence (CDS) recruits ribonucleases for rapid turnover of both target and sRNA [81] (Fig. 1A). Hfq furthermore protects sRNA, especially trans-acting sRNAs, from ribonuclease-mediated decay [82,83]. Contrarily, a class of sRNAs can anneal to the complementary sequence stretch of RBS or 5ꞌUTR, which under normal condition is sequestered in a secondary structure, and liberate RBS for active translation by ribosome. Unfolding of this intrinsic inhibitory structure on mRNA extricate SD region for 30S ribosome binding for protein synthesis [84,85] (Fig. 1B). A number of sRNA base pairing to either 5ꞌUTR or CDS protects and stabilizes mRNA from ribonucleolytic degradation, thus promoting active translation [86,87]. A set of sRNAs follow a general mechanism of the sequestration of RNA binding proteins’ which culminates in the elimination of their regulatory functions [[88], [89], [90]] (Fig. 1C).

Fig. 1

Modes of sRNA action. General mechanism of repression by antisense pairing at RBS (A), active translation by anti-antisense pairing (B) on target mRNA. (C) Protein sequestration to regulate mRNA translation.

Adaptation of microorganisms to altered environmental conditions is an inherent feature and plays a decisive role in pathogenesis. M. tuberculosis encounters a series of unfavorable conditions like abrupt change in temperature and pH during transmission between the host and external environment, exposure to reactive oxygen and nitrogen species as an outcome of the oxidative burst after being taken up by mammalian phagocytic cells, necessary nutrient and cofactor deprivation, iron homeostasis etc. Adaptation of M. tuberculosis to aforementioned stress conditions implicates the integration of regulation of protein transcription factors with RNA based regulatory network. Initial investigations recognized an analogous set of sRNAs in the transcriptome of M. tuberculosis, which indicates the potential role of these sRNAs in mycobacterial pathogenesis [91,92].

Conservation of sRNAs in distantly related species is rare. However, several ubiquitous sRNA regulators like 6S RNA, DsrA, RyhB etc., participate in analogous regulatory network under similar environmental cues [44]. Studies on M. tuberculosis sRNAs confirmed the existence of major housekeeping sRNAs. For instance, C8 RNA in M. tuberculosis is the essential housekeeping 4.5S RNA, a component of signal recognition particle [93]. Arnvig et al. first discovered the existence of sRNAs in M. tuberculosis [65]. ASdes and ASpks have been identified as sRNAs, which are cis-encoded to their target genes, desA1 and pks, both of which have been described to regulate the lipid metabolism. The gene of DesA1, a fatty acid desaturase enzyme, is essential for M. tuberculosis growth [94] and is highly upregulated after being taken up by host macrophages during infection [95]. ASdes also exhibits substantial complementarity to another desaturase (DesA2), and thus, has enormous potential to behave as cis-acting regulator to desA1 and trans-acting to desA2mRNAs [65]. Another cis-encoded sRNA ASpks is transcribed as an antisense RNA within the pks12 gene encoding polyketaid synthase (Pks) 12. Pks is involved in the synthesis of mannosyl-β-1-phosphomycoketide, an immunogenically potent phospholipid component on mycobacterial cell wall, which is recognized as antigen by mammalian CD1 restricted T cell [96]. ASpks also shares significant complementarities to the mRNAs of other ketosynthase domains, and thus, possesses the ability to act as dual function sRNA [65].

A starvation induced sRNA, ncRv12659, in M. tuberculosis has been reported [97] to be synthesized from the complementary strand of Rv2660c mRNA. Transcription of ncRv12659 sRNA starts within region of PhiRv2 prophage and interestingly, is abandoned only in the strains that have retained PhiRv2. Accumulation of ncRv12659 culminates during M. tuberculosis infection. Hence, higher expression of this sRNA bestows a potential biomarker for tuberculosis and also for identification of the cells undergoing nutrient and oxygen starvation. Overexpression of ncRv12659 develops impaired cell growth as well as perturbation in the transcription profile of M. tuberculosis H37Rv. Additionally, the expression of more than fifty genes were affected and majority of them were upregulated. It has also been found that 5ꞌ-end of ncRv12659 act as a useful marker for phenotypic analysis of M. tuberculosis during infection with PhiRv2 positive strain [97]. A different sRNA Mcr11 in both M. tuberculosis and M. bovis with the transcription origin between Rv1264 and Rv1265 genes exhibited differential expression under conditions associated with the status of host macrophage and granulomas during infection [69,98]. Rv1265 is a cyclic AMP induced gene and is upregulated both in M. tuberculosis and M. bovis during macrophage infection [99,100]. Hence, Mcr11 has got particular attention due to its link with cAMP metabolism [101]. The correlation between the growth dependent expressions of Mcr11 and upregulation during host macrophage infection clearly suggest the involvement of this sRNA in the progression of M. tuberculosis infection. However, the mechanism how Mcr11 operates in the cell is yet to be investigated.

Lack of any report regarding the presence any RNA chaperone in mycobacterium species has made the context very difficult to design a strategy to identify potential targets of trans-encoded sRNAs and to speculate their regulatory mechanisms. Phenotypic change as a consequence of the deletion of sRNA gene may reveal an alternate root for target identification. Overexpression of three trans-encoded sRNAs, F6, B11 and G2, showed intense effect on mycobacterial growth. Expression of both B11 and G2 under strong rrnB promoter [102] turned out to be detrimental for M. tuberculosis cell growth, while the outcome of F6 expression was exceptional slow growth of the cell. Strikingly, M. smegmatis upon transformation with B11-harbouring plasmid was grimly affected in their cell division and morphology [65]. The mechanisms of how these trans-acting sRNAs functions to regulate physiological process are yet to be investigated. Expression of another trans-acting sRNA, MTS2823, although abundant in all growth phases of M. tuberculosis cell, is markedly elevated either at the stationary phage of the cells or during infection condition [70]. Higher than the normal expression of this sRNA in the exponential phase caused downregulation of global expression of the genes related to exponential growth. Among the extraordinarily affected genes, methyl citrate network specific genes like prpC in particular and prpD are remarkably downregulated, which suggests the preferential targeting to minimize either the utilization of propionyl-CoA and/or oxaloacetate or the build-up of toxic intermediate like methyl citrate [70]. MTS2823 is not a bona-fide 6S RNA homologue but it exhibits functional similarities to 6S RNA. It accumulates up to 16% of the 16S rRNA level in lungs of mice during acute tuberculosis infection. MTS1338, also a member in trans-encoded sRNA list of mycobacterium, is highly elevated in the stationary phase of cell growth under the control of hypoxia responsive DosRS two component transcriptional regulator.

Complete understanding of the regulatory function of a sRNA in mycobacterium was first accomplished by Solans et al. [103]. A PhoP regulated expression of a novel 350 nt long sRNA Mcr7 [69] in M. tuberculosis was observed from the IGR between rv2395 and PE_PGRS41. PhoP is one of the components of PhoPR regulatory system [104], which is an essential factor for M. tuberculosis virulence where it regulates expression of nearly 2% of the genes including the genes involved in ESX-1 secretion apparatus, a crucial determinant for virulence [105]. PhoP protein, a part of PhoB/OmpR subfamily, was investigated by chromatin immunoprecipitation followed by high throughput sequencing for the identification of potential genes, which are regulated by this protein. PhoP was found to be capable of binding as much as 35 different loci on the M. tuberculosis genome. Mutation of phoP gene resulted in impaired production of pathogen specific cell wall components and slow growth [106]. PE_PGRS41 gene, which is associated with the IGR carrying mcr7 gene (Fig. 2), was found to be highly deregulated in phoP knock out strain. Among mycobacterial species the expression of mcr7 was found to be prominent particularly in M. tuberculosis and its expression profile coincides with the proposed evolutionary pathway of the tubercle bacilli [107]. A highly structured fold with a 33 nt predicted free loop of Mcr7 was reported to interact with the 5ꞌ-end of tatC mRNA, encoded protein of which is a member of Twin Arginine Translocation (Tat) protein secretion apparatus [106]. This posttranscriptional interaction covers the region of putative RBS and first six codons of tatC mRNA. Thus, an antisense mechanism is followed by Mcr7 RNA to occlude RBS and subsequently to downregulate of tatC gene expression (Fig. 3). A transmembrane protein, encoded by essential tatC gene in M. tuberculosis [108], is a component of TatABC general secretary apparatus, which is indispensable for twin arginine motif containing protein export in their signal peptide [109]. Deletion of phoP gene, which lacks mcr7 expression, resulted in the enrichment of the Tat-dependent substrates in the secretome. Reestablishment of plasmid vector-dependent mcr7 expression in phoP mutant restores the secretion of Tat-dependent proteins, which emulate the wild type expression level. Complementation of mrc7 alone in phoP-deleted strain did not reconstitute the wildtype virulence [106].

Fig. 2

. The figure represents associated components of PhoPR-regulated operon and Mcr7. Acid-stress induction of AprA happens through a suggested mechanism of tatC and Mcr7 interaction.

Fig. 3

PhoP-mediated regulation of . mcr7 transcription is upregulated by PhoP and subsequent antisense binding of Mcr7 to the 5ꞌ-end of tatC mRNA occludes its RBS for active translation. However, absence of Mcr7 in ΔphoP mutant cells does not repress TatC translation.

A novel DosR regulated 109-nt long sRNA DrrS in M. tuberculosis has been reported to be unusually stable with a half-life of several hours [110] due to its stable stem loop structure at its 5ꞌ-end. However, the addition of two or more unpaired nucleotides at the 5′end of that loop drastically decreases its stability (Fig. 4A). Accumulation of this sRNA is slow but strong from the onset of the stationary phase of growth. DrrS expression in the exponential phase of cell growth is low (<1 copy per 10 cells) [6,70]. Extraordinary stability of DrrS play prime role in its accumulation in the stationary phase. Nitric oxide stress, a known condition for DosR induction, was found to upregulate drrS expression in a dose-dependent manner. DosR, which is induced by infection-associated stresses, is a response regulator of the two-component DosRS system [111]. Other stress conditions like oxidative stress, DNA damage, alteration in pH did not cause any change in the expression of drrS. However, DrrS appeared to be expressed at elevated level during chronic mouse infection [65]. Transcription of DrrS happens from two start sites: a weaker one at T1960601 and a stronger at A1960667. Transcriptional analysis reveals that DrrS transcription primarily happens from strong promoter site. Predicted structure of DrrS contains a short five base pair GC rich stem loop at 5′-end and a long imperfect stem with a short CU or CUC tail at its 3′-end. DrrS is transcribed as a precursor (>300 nt) molecule (DrrS+) with extra residues at the 3′-end. Maturation of DrrS involves a rapid 3′ processing of a longer transcript, which lacks a canonical intrinsic terminator structure (Fig. 4B). DrrS + climaxes at early stationary phase and DrrS108 accumulates continuously for three weeks into stationary phase [110]. Differential expression and considerable size differences of DrrS+ and mature DrrS signify that they might have divergent role at different phases of growth.

Fig. 4

(A) Stability of DrrS with varying length of unpaired residues at its 5ꞌ-end.Number of unpaired nucleotides at 5′ is inversely related to the stability of transcript.(B) DosR activation by hypoxia or NO stress leads to increased expression of DrrS+. Maturation of this transcript involves rapid processing at multiple places by ribonucleases. The resulting mature form DrrS is highly stable in the stationary phase of growth.

An in-depth characterization of mycobacterial regulatory sRNA in iron (MrsI) reveals that MrsI or ncRv11846, which is induced under multiple stress conditions like iron starvation, oxidative stress and membrane stress, interacts directly with its target mRNAs encoding nonessential iron-containing proteins to repress their expression [112]. MrsI is ∼100 nt long highly structured sRNA with a predicted rho-independent terminator at the 3ꞌ-end and it is highly conserved across the Mycobacteriaceae and Nocardiaceae. It has been experimentally validated that MrsI directly base pairs through a 6-nt seed region in its 5ꞌ-end with the 5ꞌ-UTR of one of its target mRNA bfrA and upregulates its expression during iron starvation. Knockdown of msrI gene [113] in M. tuberculosis genome culminates in enhanced expression of 118 genes, of which 106 genes have distinctive expression during iron deprivation. Two common gene, bfrA and fprA, were regulated both in M. tuberculosis and M. smegmatis. Iron-depriving circumstances in the macrophage environment generate multiple stress conditions, which seem to trigger MrsI as part of an anticipatory response to iron limitation to help facilitate optimal survival of M. tuberculosis [112].

Protein sequestration

A ∼300 nt long novel sRNA in M. smegmatis termed as Ms1 RNA [114] was identified as the most abundant non-rRNA transcript in stationary phase that sequesters core RNA polymerase(RNAP) lacking σA factors [115,116]. In contrast, 6S RNA, unlike Ms1 RNA, interacts with the RNAP holoenzyme [[117], [118], [119]], although Ms1 shares an analogous predicted secondary structure with 6S RNA-double stranded hairpin with a single stranded bubble in the center. Although Ms1 possesses two additional hairpins at the 5′ and 3′ ends, but the central bubble is essential for the interaction with RNAP core. A homologue (MTS2823) of Ms1 RNA exists in M. tuberculosis and predicted in other mycobacterial and actinobacterial species. Ms1expression level increases ∼115-fold upon entry into the stationary phage [116] and is more stable in the stationary phase compared to exponential phase. Differential accumulation of Ms1 is controlled by an essential ribonuclease PNPase (3′–5′phosphorolytic activity). The build-up of Ms1 RNA in the stationary phase stems from the fact that elevation of its synthesis is integrated with reduced degradation. The activity of Ms1 RNA is regulated by its promoter PMS1 and cis-acting elements. Knockdown of ms1 gene resulted in decreased level of mRNAs encoding β and β′ subunits of RNAP, which was also reflected in the protein level. Thus, during high transcriptional demand ΔMs1 strain provides smaller reservoir of RNAP, which leads to the inability of ΔMs1 strain to respond expeditiously to the changing environment during outgrowth from stationary phase [116] (Fig. 5). Ms1 homologue in M. tuberculosis MTS2823 is highly induced in chronic stages of lung infection, where MTS2823 may promote the pathogen survival in the belligerent environment. Ms1 could also be potentially connected to ethanol stress as under this stress condition Ms1 promoter activity increases [120]. The elevated level of Ms1 improves the cell fitness in the stationary phase where ΔMs1 strain is more vulnerable gamma radiation [116].

Fig. 5

The status of RNAP in wild type (wt) and . Ms1 is unstable in the exponential phase and is degraded rapidly. The degradation is facilitated by PNPase. In ΔMs1, the level of RNAP remains unchanged as compared to wt. In the stationary phase, there is an interaction between RNAP and Ms1 in the wt strain, and a portion of RNAP is also sequestered by Ms1. No Ms1 is present in ΔMs1. Corresponding amount of RNAP is found to be missing when Ms1 is absent. This indicates that the absence of Ms1 is probably being compensated by the proportionate decreased fraction of the RNAP molecules. The group of RNAPs which are specific for transcription are similar in both wt and ΔMs1 strains. During outgrowth, the difference in RNAP levels in both the strains is similar for approximately 30 min, and then the growth of ΔMs1 is slowed down for about 3 h.

Conclusions and perspective

Pathogenic bacteria like M. tuberculosis are exposed to a wide array of stress conditions including oxidative stress, low pH, membrane stress, nutrient limitation etc. while infecting human host. Adaptation to this hostile environment is achieved by fine tuning their stress responsive genes at the transcriptional, posttranscriptional and translational level for survival [[121], [122], [123], [124]]. The role of sRNA in stress response was not anticipated earlier, however, extensive investigations during last decade not only unveil the implication of sRNA in the adaptation to the stress environment, but also established their indispensable role in pathogenesis of different mycobacterial species. The discoveries of the diverse array of mycobacterial sRNAs shaded light on the mechanistic incidents behind how sRNAs exert their function. A systematic approach has been taken in this review to describe different strategies to identify mycobacterial sRNAs and also to comprehensively analyze how these sRNAs exert their regulatory role in different physiological processes.

Small RNAs have shown to act on their targets primarily through base pairing mechanism, the outcome of which is either the activation or the repression of their target gene expression [125]. However, it is still beyond our knowledge whether these sRNAs possess any distinctive structural feature to act either as an activator or a repressor of their target gene expression. To date only one protein binding sRNA (Ms1 RNA) has been discovered in mycobacterium [115,116], as the existence of the potential gene for 6S RNA is still controversial [115]. Ms1 is known to be induced in the stationary phase and increases the cell fitness to withstand stress conditions. However, how this sRNA interacts with RNAP core or whether it has any particular sequence motif for binding to RNAP core enzyme have yet to be investigated.

Except several housekeeping sRNAs like M1 RNA, tmRNA, 4.5S RNA etc. in mycobacteria, nearly all sRNAs discovered to date are induced under diverse stress conditions in either of the stationary phase or the macrophage environment. Nearly 20% of total non-rRNA transcripts in exponentially growing M. tuberculosis emanate from IGR and represent sRNAs [6]. A substantial increase of this number to ∼60% in stationary phase cells happens largely due to the accumulation of a highly abundant sRNA MTS2823, a homologue of Ms1 RNA in M. smegmatis [114]. The stockpiling of MTS2823 is even higher than rRNA amount in mice during chronic infection. Artificial overexpression of MTS2823 in the exponential phase of M. tuberculosis severely diminished the transcription of ∼300 mycobacterial genes. All these events suggests that MTS2823 has a possible role during infection [70]. sRNAs in mycobacteria also can be upregulated under multiple stress conditions, which suggests their possible involvement in multiple regulatory network. MsrI in M. tuberculosis is highly induced in three different stresses. MsrI induced under iron starvation condition exhibited the colossal effects on mycobacterium transcriptome profile. Pre-exposure of M. tuberculosis to oxidative or any other stresses results in more rapid inhibition of bfrA expression under Fe-starvation condition. This indicates that M. tuberculosis receives an anticipatory signal upon discerning oxidative or membrane stress and remodels its gene expression for moving into macrophage environment with iron scarcity, priming MsrI to repress its target translation [112]. Studies on the well-characterized sRNAs in E. coli and Salmonella revealed that the function of an individual sRNA within a regulatory circuit differ substantially. A single sRNA can participate in multiple network depending on the interplay between sRNAs and targets [125]. On the contrary, a handful of sRNAs in M. tuberculosis have been validated to interact with their target and mode of their interaction are yet to be enlighten.

Research on identification of protein transcription factors or on small regulatory RNAs incriminated in pathogenesis has increased remarkably after the sequencing of M. tuberculosis genome. Analysis of genome wide mutagenesis discovered that nearly 200 protein encoding genes in M. tuberculosis genome are replaceable without affecting their growth but with attenuated virulence [126,127]. Multiple genetic loci have been denoted to be active in numerous physiological processes during infection e.g. adaptation to altering nutritional and stress conditions, restraining microbial defense against host immune system and production of macromolecules that balance the interaction with host cells [6]. sRNAs regulators have been detected by RNA-seq profiling to be associated with many of these loci [70]. Characterization of different recombinant phenotype of M. tuberculosis strains in terms of their adaptation and growth dynamics in presence of host immune cells was accomplished using a mouse model of infection. M. tuberculosis infection in human induces the generation of granulomatous lesions that provide heterogeneity of bacterial microenvironments [7]. The ability M. tuberculosis to tolerate the resulting truculent host environment and exploit that for bacterial multiplication and transmission will lead to successful pathogenesis. Upregulation of diverse sRNAs in retaliation to different stress conditions is possibly connected to multiple regulatory circuits which control cell survival. Thus, these sRNAs may serve as important biomarkers of the bacterial physiological status, supplying critical information for therapies [6].

Tuberculosis treatment is prolonged and a successful treatment requires medication for at least 6 months or more to remove residual bacterial population, which are resistant to antimicrobial drug. Thus, the salient research objectives in recent times aim to identify and synthesize a useful drug that will eliminate persistent mycobacterial population. Characteristics of resistant M. tuberculosis include the downregulation of a number of genes for their active growth. Profusion of sRNAs in M. tuberculosis cell and its interaction with the genes connected to pathogenesis implies that sRNAs orchestrate gene expression by playing a pivotal role in the posttranscriptional regulation for intracellular survival of this pathogenic bacteria. Phenotypically persistence feature of M. tuberculosis might probably be an outcome of sRNA-mediated modulation of several genes. Thus, the strategies to manipulate sRNA functions, which in consequence will alter the phenotypic properties of M. tuberculosis, will unfold a new dimension in therapeutic regimen for tuberculosis treatment.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Author contribution statement

TD designed the frame of writing. Both TD and ST studied the literature and wrote the paper.