Báo cáo khoa học: The sigma factors of Mycobacterium tuberculosis: regulation of the regulators potx

REVIEW ARTICLE The sigma factors of Mycobacterium tuberculosis: regulation of the regulators Preeti Sachdeva 1 , Richa Misra 1 , Anil K. Tyagi 2 and Yogendra Singh 1 1 Institute of Genomics and Integrative Biology (CSIR), Mall Road, Near Jubilee Hall, Delhi, India 2 Department of Biochemistry, University of Delhi South Campus, New Delhi, India Introduction The causative agent of tuberculosis (TB), the intracel- lular bacterial pathogen Mycobacterium tuberculosis, latently infects about one-third of the world’s popula- tion and claims one life every 15 s. Although the World Health Organization-recommended treatment strategy for the detection and cure of TB, the ‘Directly Observed Treatment, Short-course’ (DOTS), has reduced the burden of TB to a great extent, the inci- dence of TB has shown an increase in recent years as a result of the emergence of drug-resistant TB and co-infection with the human immunodeficiency virus (http://www.who.int/tb/en/). Despite global efforts, no new drugs for the treatment of TB have been devel- oped successfully for more than four decades. The success of M. tuberculosis as a highly adapted pathogen rests upon its ability to establish a persistent infection in the hostile environment of the host cell through mechanisms involving transcriptional repro- gramming, ensuring metabolic slowdown and the upregulation of virulence and stress-response pathways [1]. In the course of a successful infection, the bacte- rium copes with numerous stresses (reviewed previ- ously [2]) and modulates host responses through coordinated regulation of its gene expression in response to signals encountered in the host body. Gene expression in bacteria is regulated primarily at the level of transcription initiation, which is mediated by the RNA polymerase (RNAP) holoenzyme. The Keywords Mycobacterium; post-translational regulation; sigma factor; tuberculosis Correspondence Y. Singh, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Fax: +11 2766 7471 Tel: +11 2766 6156 E-mail: ysingh@igib.res.in (Received 8 September 2009, revised 27 October 2009, accepted 4 November 2009) doi:10.1111/j.1742-4658.2009.07479.x One of the important determinants of virulence of Mycobacterium tuberculosis is adaptation to adverse conditions encountered in the host cells. The ability of Mycobacterium to successfully adapt to stress condi- tions is brought about by the expression of specific regulons effected by a repertoire of r factors. The induction and availability of r factors in response to specific stimuli is governed by a complex regulatory network comprising a number of proteins, including r factors themselves. A serine– threonine protein kinase-mediated signaling pathway adds another dimen- sion to the mycobacterial r factor regulatory network. This review high- lights the recent advances in understanding mycobacterial r factors, their regulation and contribution to bacterial pathogenesis. Abbreviations ECF, extracytoplasmic function; imp, immunopathology; M. bovis BCG, Mycobacterium bovis Bacille Calmette–Gue ´ rin; PPE, proline- proline-glutamate; RNAP, RNA polymerase; STPK, serine–threonine protein kinase; Tat, twin-arginine translocation; TB, tuberculosis; TCS, two-component system; TSP, transcription start point; ZAS, zinc-associated anti-r factor. FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 605 holoenzyme comprises a five-subunit core RNAP (sub- unit composition, a 2 bb¢x) and a dissociable subunit, sigma (r). The r factor contains many, if not all, of the promoter recognition determinants and confers promoter specificity to the RNAP holoenzyme. The association and dissociation of distinct r factors with core RNAP mediates specific cellular responses through redirection of transcription initiation at vari- ous regulons [3,4]. The temporal expression of specific regulons controlled by the induction or availability of one or more r factors allows M. tuberculosis to sustain multiple stages of host–pathogen interactions, includ- ing adhesion, invasion, intracellular replication and dissemination to other sites [5]. In this review, we sum- marize the current knowledge about mycobacterial r factors and their regulation, and discuss their role and importance in the pathogenesis of mycobacteria. General overview of r factors The r factors have been divided into two main fami- lies, namely the sigma 70 (r 70 ) family and the sigma 54 (r 54 ) family, named after the 70 kDa primary r factor and the 54 kDa nitrogen-regulation r factor both from Escherichia coli, respectively. r 54 -type r fac- tors are structurally different from the r 70 family members and utilize a distinct mechanism of open complex formation. There are no known representa- tives of the r 54 family in any GC-rich, Gram-positive bacteria and cyanobacteria; however, r 70 -related r fac- tors are encoded in all bacterial genomes [4,6]. The r 70 family of proteins contain up to four conserved regions (regions 1, 2, 3 and 4) and have been divided into four groups (groups 1–4) based on their phylogenetic rela- tionships and modular structure [4,7] (Fig. 1). Group 1 comprises essential housekeeping r factors and these r factors contain all the four conserved regions. Group 2 r factors are most closely related to group 1 r factors, but are not essential and lack the subregion 1.1 (Fig. 1). Most group 3 r factors contain conserved regions 2–4. Group 4 includes r factors containing only regions 2 and 4 and accommodates the highly diverged extracytoplasmic function (ECF) subfamily, members of which respond to signals from the extra- cytoplasmic environment [4,8,9]. A r 70 -dependent promoter sequence may comprise the following potential elements that mediate its recog- nition by the RNAP holoenzyme: (a) the -10 element, a hexameric sequence, centered about 10 bp upstream of the transcription start point (TSP) and recognized by the 2.4 subregion; (b) usually an extended -10 motif (TGN motif), situated immediately upstream of the -10 sequence and recognized by an a-helix in the region 3.0; (c) the -35 element, a hexameric sequence centered about 35 bp upstream of the TSP and recognized by the 4.2 subregion [3,10,11]; and (d) an AT-rich UP element, generally located between 61 and 41 bp up- stream of the TSP [12,13]. The upstream (UP) element binds the C-terminal domain of the a subunit of the RNAP and enhances the promoter activity by two-fold to as much as 90-fold [12,14]. Besides, the presence of GC-rich sequences in the spacer region has been shown to drastically influence the promoter strength [15]. All bacteria have at least one essential r factor that transcribes the genes required for cell viability, and most bacteria harbor alternative r factors that tran- scribe regulons in response to specific stimuli. The number of alternative r factors correlates generally with the variability of the environments encountered by a given bacterial species. The number may range from two alternative r factors in Streptococcus pyoge- nes, whose primary niche is limited to the human oro- pharynx, to more than 60 encoded by the soil actinomycete, Streptomyces coelicolor, whose natural habitat is highly variable in terms of nutrients, stresses and competing microbial flora [16]. Also, while the number of r factors is generally found to increase with the genome size, microorganisms that have developed differentiation programmes (e.g. sporulation) tend to have higher alternative r factor ⁄ genome size ratios than most obligate pathogens or commensal bacteria. Interestingly, M. tuberculosis has the highest alterna- tive r factor ⁄ genome size ratio amongst the obligate pathogens, suggesting a highly complex regulatory mechanism for its transcription [17]. Notably, a recent report suggested endospore formation by mycobacteria and speculated sporulation as a possible mechanism for dormancy [18]. M. tuberculosis encodes a repertoire of 13 r factors [19,20], of which r A , r B and r F are representatives of groups 1, 2 and 3 of the r 70 family, respectively, while the remaining 10 r factors belong to group 4. Amongst the ECF r factors, r G , r I and r J possess an additional carboxy-terminal extension in their struc- ture, which is presumed to provide a surface for inter- action with other regulatory molecules [17]. The gene loci for the principal r factor in M. tuberculosis, sigA, and for the principal factor-like r factor, sigB, are well conserved across all sequenced mycobacterial genomes. The alternative r factors of M. tuberculosis have var- ied representation, in terms of number and functional- ity, across the Mycobacterium genus. sigC orthologs have been found to be present in all pathogenic Myco- bacterium spp. sequenced to date, including Myco- bacterium leprae, but absent in nonpathogenic Mycobacterium spp. sequenced so far. By contrast, The r-factors of M. tuberculosis P. Sachdeva et al. 606 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS sigE is the only ECF r factor conserved across the Mycobacterium genus. Five r factors (sigD, sigF, sigG, sigH and sigJ) are present in all genomes, except for that of M. leprae, where these r factors are present as pseudogenes. The remaining ECF r factors have mixed representation across pathogenic and nonpatho- genic species. The details of the r factor content and the paralogous members of a r subfamily in various sequenced Mycobacterium spp. have already been reviewed [17]. Interestingly, among all bacterial genus types, Mycobacterium exhibits the maximum variation in the number of r factors among its species [21] with the saprophytic species, Mycobacterium smegmatis, possessing 28 r factors and the obligate pathogen, M. leprae, having only four functional r factors [17]. Physiological roles of mycobacterial r factors SigA (r A ) r A , the primary or principal r factor, is indispensable for growth both in M. tuberculosis [2] and in M. smegmatis [22]. The M. tuberculosis H 37 Rv sigA transcript is maintained at a constant level under vari- ous stress conditions; however, there have been a few reports of its upregulation during infection in human macrophages [23] and downregulation during low aera- tion and stationary-phase growth [24]. During condi- tions of low aeration and in the stationary phase, the energy available to the bacteria may be quite low and Fig. 1. Promoter recognition by the RNA polymerase-r 70 holoenzyme. [Correction added on 8 December 2009 after original online publication: in Fig. 1 ‘transcription atrat site’ was changed to ‘transcription start site’, and ‘is acidic an nature’ was changed to ‘is acidic in nature’.] P. Sachdeva et al. The r-factors of M. tuberculosis FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 607 the decreased levels of the sigA transcript might reflect a decrease in the mRNA pool relative to the total RNA of the cell [24]. The sigA transcript continues to be widely used as an internal standard for normaliza- tion in quantitative RT-PCR experiments using M. tuberculosis RNA isolated under all conditions [24,25]. The unusual stability of sigA mRNA (half life > 40 min) [26] makes it a preferred standard for this purpose. A point mutation in the 4.2 domain of r A (R515H) resulted in attenuation of virulence of the M. bovis strain ATCC 35721 in a guinea pig model of infection [27]. The mutated r A was reported to be incapable of interaction with an accessory transcription factor, WhiB3. Inactivation of the whiB3 gene in another M. bovis strain, ATCC 35723, resulted in the attenua- tion of pathological changes in lungs, as reported for the M. bovis sigA mutant strain. However, in a differ- ent genetic background, such as M. tuberculosis H 37 Rv, whiB3 deletion resulted in only partial attenua- tion of virulence in both mice and guinea pig models [28]. A number of such results have been reported in the last few years, establishing the role of r A in host– pathogen interactions, in addition to its housekeeping function. In a clinical strain (the M. tuberculosis 210 isolate, TB294, known for its higher intracellular growth rate compared with other strains), sigA is natu- rally upregulated. Overexpression of sigA in M. tuber- culosis H 37 Rv enhanced its growth in human macrophages and in the lungs of mice after aerosol infection, further suggesting its role in virulence [29]. The same group recently reported that this effect of r A is mediated, in part, by upregulation of one of its target genes, eis (enhanced intracellular survival), which contributes to the enhanced capacity of M. tuberculosis strain 210 to grow in monocytes [30]. The role of r A in the expression of virulence genes is, however, a host-specific feature, because complementa- tion with a functional r A is sufficient for the restora- tion of virulence of M. bovis ATCC 35721 in a subcutaneous guinea pig model but not in an intratrac- heal Australian brushtail possum model of experimen- tal TB [31]. Furthermore, the consensus sequence for a r A -dependent promoter has been predicted upstream of an operon, Rv3134c-devR-devS [32], which encodes proteins involved in establishing and maintaining TB latency under hypoxic conditions [33]. Recently, PE_PGRS33, a cell-surface molecule that plays an important role in TB pathogenesis [34], has also been shown to be transcribed from a r A -dependent pro- moter [35]. For a very long time, genes upregulated in phagocytosed bacteria were considered as potential candidates for virulence over constitutively expressed genes. This perception, however, has been challenged by a study which proposed that the majority of M. tuberculosis genes required for intracellular survival are constitutively expressed rather than regulated by macrophages [36]. Interestingly, the role of r A in viru- lence gene expression goes very well with this report. SigB (r B ) r B , the principal factor-like r factor of M. tuberculosis, is very similar to the C-terminal portion of r A [17,37]. But, unlike r A , it has been found to be dispensable for growth in both M. smegmatis [22] and M. tuberculosis [38]. Moreover, the r A and r B regulons do not overlap much except for a few genes such as those belonging to the PE_PGRS family [35,39]. A comparison of amino acid residues from the 2.4 and 4.2 subregions of r A and r B shows that these r factors differ at four of 23 posi- tions in the 2.4 subregion. More significantly, the 4.2 subregion of r A and r B differs at 15 of 39 positions and nine of these changes are nonconserved. While these differences would definitely reflect on the pro- moter sequences recognized by these two r factors, other aspects that contribute to the nonoverlapping repertoire of genes transcribed by the two r factors remain to be elucidated [40]. Besides, unlike sigA, expression of the M. tuberculosis sigB gene increases upon exposure to various environmental stresses such as low aeration [24], treatment with hydrogen peroxide and heat shock, with a more pronounced effect seen in stationary phase than in logarithmic phase [26]. A recent report demonstrated that inactivation of the sigB gene did not affect the survival of M. tubercu- losis during infection in human macrophages or in mouse and guinea pig models [38]. However, deletion of sigB in M. tuberculosis results in its higher sensitiv- ity to SDS-induced surface stress, heat shock, oxidative stress, exposure to vancomycin and hypoxic conditions [17,38]. Further evidence for the role of r B in adapta- tion to stationary phase and nutritionally poor condi- tions came from a report by Mukherjee et al. [41] This group reported that upon overexpression of M. tuber- culosis sigB in M. smegmatis, the cell-surface glycopep- tidolipids found in the outer layers of M. smegmatis become hyperglycosylated, similarly to what is observed during carbon starvation. Certain metabolic enzymes, namely succinyl-coenzyme A synthetase, glycosyltransferases (encoded by genes in the glycopep- tidolipid locus), b-ketoacyl coenzyme A synthetase, rhamnosyltransferase and acetylcoenzyme A acetyl- transferase, were also found to be induced upon over- expression of r B in M. smegmatis [42]. Overexpression of sigB in M. tuberculosis resulted in a significant The r-factors of M. tuberculosis P. Sachdeva et al. 608 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS upregulation of genes encoding proteins involved in cell wall-related processes, several early culture filtrate antigens (ESAT-6-like proteins), 50S ribosomal pro- teins, PE-PGRS proteins, keto-acyl synthase, KasA and the regulatory proteins, WhiB2, IdeR and r B itself [39]. Also, sigB is transcribed from a predicted r B - dependent promoter in an in vitro assay, further sug- gesting its autoregulation [39]. Moreover, expression profiling of the M. tuberculosis sigB mutant strain revealed regulation of ideR, furA, katG, ppe19 and hsp20 by r B [38]. Previous studies have suggested that there are two more promoters upstream of r B : one is recognized by RNAP containing any of the three r factors, r E [43], r H [44] and r L ; while the other is recognized by RNAP containing r F [45]. The transcription of sigB under stan- dard physiological growth conditions and its induction upon exposure to surface stress is dependent on r E [43], while its induction during heat shock or oxidative stress is r H -dependent [44]. In contrast to the r F -dependent transcription of sigB observed in an in vitro transcrip- tion assay [45], the upregulation of sigB upon overex- pression of sigF was not observed [46]. The possibility of certain r F -dependent genes being missed in the study by Williams et al. cannot be ruled out in view of induc- tion of the anti-r F protein, UsfX, which is expected to significantly reduce the effective concentration of active r F in the cell. The conditions for r F - and r L -dependent transcription of sigB are yet to be identified. r B seems to operate as a downstream response regu- lator in the hierarchy of the r factor regulation net- work, the levels of which are adjusted in response to different environmental conditions brought about by an ensemble of five different r factors, including itself (Fig. 2). The self-regulation of r B is expected to result in its autoamplification and therefore a pronounced effect on its level, even in the presence of minimal changes in the levels of its upstream regulators. Inter- estingly, other than r factors, a two-component system (TCS) response regulator, MprA, also regulates the in vivo expression of sigB in M. tuberculosis under SDS-induced surface stress and exponential growth via its binding to conserved motifs in the upstream region of the gene [47]. SigC (r C ) sigC is conserved across all pathogenic mycobacterial species, including M. leprae [48], and is absent in all nonpathogenic species sequenced to date, such as M. smegmatis [21], Mycobacterium gilvum, Mycobacte- rium vanbaalenii, Mycobacterium sp. MCS, Mycobacte- rium sp. KMS and Mycobacterium sp. JLS. Despite the sigC transcript being the most abundant transcript of all r factors, most of the core RNAP during exponen- tial phase has been found to be associated with either r A or r B . Therefore, it was speculated that M. tubercu- losis sigC is either translated at a very low efficiency or has a low affinity for RNAP [24]. M. tuberculosis sigC is downregulated during stationary phase and in response to heat shock and SDS-induced surface stress [24]. Interestingly, sigC was also found to be downregu- lated in the M. tuberculosis CDC1551 sigF mutant strain [49], adding another r factor in the hierarchical regulatory network of mycobacterial r factors (Fig. 2). r C is not required for the survival of Mycobacterium in murine bone marrow-derived macrophages or in activated J774A.1 macrophages [50]. In a mouse model of infection, the sigC mutant in both the CDC1551 and H 37 Rv backgrounds grows and persists in lungs but shows attenuated disease progression and fails to elicit the same degree of lethal immunopathology as the wild-type strain [50,51]. This phenotype of bacterial persistence at high colony counts with reduced host mortality is designated as the immunopathology (imp) defect and is associated with a significant reduction in the number of infiltrating neutrophils and with the production of pro-inflammatory cytokines such as tumor necrosis factor- a-a, interleukin-1b, interleukin-6 and interferon-c in the lungs of the infected animal [51]. The attenuation of the sigC mutant may result from dysregulation of expression of several key viru- lence-associated genes, such as hspX (encoding an a-crystallin homolog), senX3 (a sensor kinase), mtrA (a response regulator), polyketide synthases and fbpC (antigen 85C) [50]. Most importantly, in a guinea pig model of infection that best mimics the granuloma for- mation and disease progression in humans, the M. tuberculosis sigC mutant displayed delayed growth with fewer caseating lesions compared with the wild- type strain [51]. The two promoter-recognition domains of r C (r C 2 and r C 4 ) have been crystallized [52] and interact in vitro involving occlusion of the Pribnow box recognition region. This interdomain interaction is suggestive of an alternate mechanism of regulation of r C activity via autoinhibition in the absence of a cog- nate anti-r factor [53]. SigD (r D ) r D is expressed at a moderately high and constitutive level during exponential and stationary growth phases and declines significantly, following hypoxia, in a pat- tern very similar to that of r A in an in vitro culture [54,55]. The stringent response is modulated by the relA gene product via the synthesis of a key signaling P. Sachdeva et al. The r-factors of M. tuberculosis FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 609 molecule, guanidine tetraphosphate (ppGpp) [56]. relA deletion in Mycobacterium results in a loss of virulence accompanied with a significant decrease in sigD expres- sion during the logarithmic phase [57]. sigD is upregu- lated during nutrient starvation [58] (a condition that is at least partly Rel-dependent), further suggesting the role of r D in physiological adaptations such as stringent response and starvation. The intracellular r D levels decrease, following infection, in both quiescent and acti- vated macrophages [59] and the loss of sigD does not affect the ability of M. tuberculosis CDC1551 to survive in J774A.1 macrophages. However, the mutant strain induced a lower level of tumor necrosis factor- a produc- tion by macrophages relative to the wild-type strain. The M. tuberculosis H 37 Rv sigD mutant showed a mod- erate loss of virulence with less extensive inflammation and histopathological changes in BALB ⁄ c mice [54], while deletion of sigD in the CDC1551 background resulted in significant attenuation of lethality in a C3H:HeJ mouse model of infection [60]. Some of the important r D -regulated genes include those encoding proteins involved in lipid metabolism, cell wall-related processes, stress response and DNA binding and repair. Several genes, such as rpfC (impli- cated in the revival of dormant mycobacteria), mce1 (associated with the entry of Mycobacterium into non- phagocytic cells), pks10 (a polyketide-like chalcone synthase), recR and those encoding several chaperones, ribosomal proteins, elongation factors and ATP syn- thase subunits were also reported to be downregulated in the M. tuberculosis sigD mutant in the late station- ary phase [54,60]. Notably, several r D -regulated genes were reported to be highly expressed in the MprAB TCS mutant strain under SDS stress, indicating that many of the r D -regulated genes are under the repres- sive effect of MprA [61]. SigE (r E ) r E is one of the two ECF r factors encoded by the M. leprae genome [48]. sigE is upregulated in myco- bacteria grown within human macrophages compared with those grown in an in vitro culture [23,62]. Its expression also increases following exposure to heat Fig. 2. Regulatory network of Mycobacterium tuberculosis r factors. Color coding and symbols for the various regulators are mentioned in the key. The red horizontal line indicates blockade; (?) indicates that the significance of the phosphorylation is not known. The r-factors of M. tuberculosis P. Sachdeva et al. 610 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS shock, SDS-mediated surface stress [24], isoniazid [63] and vancomycin [64]. Although sigE is not essential for growth of M. smegmatis, its deletion results in increased susceptibility to oxidative stress and acidic pH [65,66]. The M. tuberculosis CDC1551 sigE mutant exhibits the imp phenotype in the C3H ⁄ HeJ mouse model of lung infection [67]. The M. tuberculosis H 37 Rv sigE mutant showed decreased viability in mac- rophages [43] and is strongly attenuated for virulence in both BALB ⁄ c and severe combined immunodeficient (SCID) mice. Moreover, the sigE mutant manifested the formation of granulomas with characteristics dif- ferent from those induced by the wild-type strain [68] and a reduced ability to grow at 4 days postinfection, as well as impaired CXCL10 expression in monocyte- derived dendritic cells. The impaired CXCL10 expres- sion is thought to inhibit the recruitment of activated effector cells involved in the formation of granulomas [69]. Also, the global transcription profile of macro- phages infected with the sigE mutant showed upregula- tion of a number of components of the host defense system, such as CCL4 chemokine, prostaglandin E, toll-like receptor-2 and defensins, indicating the role of r E in suppressing the immune system and the antibac- terial response of the host [70]. In M. tuberculosis, sigE is transcribed from three dif- ferent promoters: promoter P1, utilized during growth under normal physiological conditions [71]; promoter P2, regulated by MprAB TCS, induced under surface stress and alkaline pH [47]; and a third, r H -dependent promoter, P3, induced under the conditions of oxida- tive stress and heat shock. The r H -dependent promoter is also considered to be responsible for increased tran- scription of sigE in macrophages [72]. The lack of a functional sigH gene in M. leprae has in fact been implicated in the defective response of the organism to heat stress, despite the presence of a functional r E pro- tein [73]. Various different sigE start codons have also been characterized, which may give rise to different r E isoforms, depending on which promoter is used for transcription [71]. Moreover, in both M. smegmatis and M. bovis Bacille Calmette–Gue ´ rin (BCG), sigE is transcribed from two TSPs, each preferred under dif- ferent temperature conditions [65]. However, despite the presence of identical upstream sequences, these TSPs could not be detected in M. tuberculosis [71]. r E -dependent genes encode proteins belonging to dif- ferent classes, such as transcriptional regulators (includ- ing r B , Rv3050c, MprA and MprB), enzymes involved in fatty acid metabolism (most importantly isocitrate lyase) and the classical heat shock proteins [43,70]. As stated above, the MprAB TCS encoded by the r E - dependent genes mprA and mprB regulates the in vivo expression of sigE as well as another stress-responsive r factor, sigB,inM. tuberculosis. This regulation is medi- ated by binding of the response regulator, MprA, to conserved motifs in the upstream regions of r E and r B [47]. A number of stress-responsive genes have been reported to be downregulated in the mprA mutant strain, some of which may actually be targets of these r factors [61]. In addition, the induction of mprA follow- ing exposure to stress also suggests a direct role of this regulatory system in the stress-response pathways in M. tuberculosis [47]. Surprisingly, some of the r E -depen- dent genes, such as Rv1129c, Rv1130 and Rv1131 (cit- rate synthase; gltA), are also under an indirect repressive effect of MprA, suggesting the MprAB–r E regulation network to be highly complex [43,61]. Under conditions of stress (such as ATP depletion), inorganic polyphosphate polyP (synthesized by the enzyme PPK1) serves as a preferred donor for the MprB-mediated phosphorylation of MprA. This results in MprA-regulated transcription of the mprAB operon, which thereby facilitates r E -mediated transcription of a stringent response gene, rel [74]. As sigD is suggested to be part of the RelA regulon [57], its expression is also likely to be under the indirect control of r E . The positive regulation of r E by MprAB, which results in bimodal rel gene expression and thereby a phenotypic heterogeneity in the bacterial population, may play a role in the devel- opment of persistence in Mycobacterium [75]. The bind- ing affinity of MprA for its promoter increases upon phosphorylation and is required for the upregulation of the mprA gene in vivo [76]. By contrast, the binding of MprA to sigB and sigE upstream regions can occur, even in the absence of phosphorylation [47]. In this view, the downregulation of sigE and sigB and their corre- sponding regulons in the ppk1 deletion mutant [74] is likely to be an indirect consequence of diminished poly- phosphate levels. The direct effect of phosphorylation of MprA on its binding to sigB and sigE upstream regions remains to be studied. The co-dependent transcriptional regulation of r E and MprA [43,61], as well as the autore- gulation of MprA [76], may result in the maintenance of the levels of r E and MprA in a cyclic manner during stress. r E , in addition to a complex transcriptional regu- lation, is also subjected to translational regulation as well as to post-translational regulation by a zinc-associ- ated anti-r factor (ZAS) family protein [71]. SigF (r F ) M. tuberculosis r F , the only group 3 r factor represented in the Mycobacterium genus, bears significant homology to the stress response r factors in Bacillus subtilis, Staph- ylococcus aureus and Listeria monocytogenes and to the P. Sachdeva et al. The r-factors of M. tuberculosis FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 611 sporulation r factors in B. subtilis and S. coelicolor [77]. A study elucidating alignment of the sigF orthologs across sequences obtained from various mycobacterial species revealed a clustering pattern that differentiates slow-growing and fast-growing species [78]. When introduced into M. bovis, the expression of M. tuberculosis sigF is induced under a variety of stress conditions, most notably antibiotic stress (rifampin, ethambutol, streptomycin and cycloserine), nutrient depletion, oxidative stress, cold shock and anaerobic metabolism, particularly in the presence of metronida- zole and during stationary-phase growth [79]. How- ever, no such marked change in the transcript level of sigF was seen in M. tuberculosis H 37 Rv following exposure to cold shock, hypoxia, oxidative stress and entry into stationary phase [24], suggesting a differential regulation pattern of sigF expression in M. tuberculosis and M. bovis BCG. Similarly, the M. tuberculosis CDC1551 sigF mutant strain showed no significant difference in the in vitro survival rate in response to temperature shift, oxidative stress and long-term stationary phase growth. However, the mutant displayed increased susceptibility to rifampin and rifapentine, as well as reduced uptake of a hydro- phobic solute, chenodeoxycholate, suggesting that the sigF deletion produces structural alterations in the mycobacterial cell envelope [80]. In contrast to the findings of Chen et al. [80] and their speculation for the role of r F in bacterial growth during stationary phase and under stress conditions, as well as suscepti- bility to antibiotics, the conditional overexpression of sigF during the early exponential growth phase neither resulted in any growth arrest nor reduced the suscepti- bility of the strain to rifampin and isonaizid [46]. Also, M. tuberculosis H 37 Rv sigF was found to be upregulat- ed in a nutrient starvation model of M. tuberculosis [58] and during infection of cultured human macro- phages [62]. However, a study by Williams et al. revealed that M. tuberculosis CDC1551 r F is not required for bacillary survival under nutrient starva- tion conditions and within activated murine macro- phages or for extracellular persistence in an in vivo granuloma model of latent TB infection [46]. The sigF mutant exhibits the imp phenotype in mice [49], as well as reduced lethality in mouse and guinea pig infection models [80,81], relative to the wild-type strain. Unlike M. tuberculosis, M. smegmatis sigF is exp- ressed throughout growth at levels almost comparable to those of sigA. Expression profiling using a recombi- nant M. smegmatis reporter strain revealed significant induction of sigF upon treatment with ethambutol, isoniazid, SDS and cold shock, whereas nutrient deple- tion and salt stress stimulated a lower level of sigF induction compared with the untreated control [78]. Another study with a M. smegmatis mc 2 155 sigF mutant strain demonstrated that r F is required for resistance to heat shock and acid stress, but not for the survival of bacillus in human neutrophils. M. smegma- tis r F also mediates resistance to oxidative stress, prob- ably in a KatG-independent and AhpC-independent manner [82]. As reported for M. tuberculosis [80], M. smegmatis r F is also implicated in the regulation of genes involved in cell wall permeability, as evident by the decreased transformation efficiency in the presence of a functional sigF gene. Also, r F is required for the biosynthesis of carotenoids, complex lipids that act as free-radical scavengers and protect the cells against photodynamic injury in M. smegmatis [83]. In order to identify r F -dependent genes, transcrip- tional profiling of an M. tuberculosis CDC1551 sigF mutant strain at different growth stages and of a sigF- overexpressing strain were carried out independently [46,49]. [Correction added on 8 December 2009 after ori- ginal online publication: in the preceding sentence ‘CDC551’ was changed to ‘CDC1551’.] Disruption of the sigF gene resulted in the downregulation of a signifi- cantly larger number of genes in the late-stationary phase compared with the exponential phase. r F regu- lates the expression of genes involved in the biosynthesis and structure of the mycobacterial cell envelope, includ- ing complex polysaccharides and lipids, particularly vir- ulence-related sulfolipids. In addition to genes involved in energy metabolism, nucleotide synthesis, intermediary metabolism and information pathways, r F regulates genes encoding several transcriptional regulators, for example, MarR, GntR and TetR family regulators, PhoY1 and Rv2884, as well as an ECF r factor, r C [46,49]. Conditional overexpression of sigF during the early exponential phase resulted in the upregulation of several genes encoding cell wall-associated proteins, such as proline-glutamate (PE) and proline-proline-glu- tamate (PPE) family proteins and mmpL family trans- porters (mmpL2, mmpL5 and mmpL11) [46], known to be involved in virulence [84,85]. sigF expression is regulated at the transcriptional level via autoregulation of its promoter. Besides, r F activity is under the control of a complex post-transla- tional regulatory network comprising an array of pro- teins such as anti-r factors, anti-anti-r factors, as well as certain proteins responsible for the modification of these factors [86–88]. SigG (r G ) sigG is one of the most highly induced genes in M. tuberculosis during macrophage infection [23,89] The r-factors of M. tuberculosis P. Sachdeva et al. 612 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS and has been shown, in a macrophage infection model, to be required for survival of the bacterium [90]. How- ever, its expression is downregulated upon exposure to various stress conditions, such as mild cold shock, heat shock, low aeration and SDS-mediated surface stress. sigG is one of the least represented mRNAs among all r factors under normal in vitro growth conditions [24]. The sequence upstream of the M. tuberculosis sigG gene showed similarity with the P1 promoter of a SOS response gene, recA. Also, lexA, the gene encoding a repressor for SOS genes, is regulated by r G . The reduced lexA levels in the sigG mutant strain could account for the upregulation of several SOS genes, including recA. The sigG mutant is resistant to the SOS response inducer, mitomycin C, further support- ing the role of r G in the SOS stress response [90]. Microarray analysis of the mutant strain revealed the downregulation of several genes encoding proteins involved in fatty acid metabolism such as AceA (isoci- trate lyase), FadE5 (acyl-coenzyme A dehydrogenase) and ScoA (succinyl-coenzyme A). Interestingly, several genes reported to be under the control of r H , such as clpB, dnaK and trxB2, were also found to be down-regulated in this study. Moreover, two other r D -regulated genes, Rv1815 and rpfC (one of the five resuscitation-promoting factor-like genes), were found to be upregulated in the sigG mutant. The aforemen- tioned result is expected in view of the downregulation of sigH and the upregulation of sigD upon the deletion of sigG in M. tuberculosis [90]. This finding further corroborates the complex interplay of r factors in M. tuberculosis (Fig. 2). SigH (r H ) The role of r H as a central regulator of oxidative and heat stress responses has been described for M. tuber- culosis as well as for certain other mycobacterial spe- cies, such as M. smegmatis and Mycobacterium avium ssp. paratuberculosis [44,66,72,91]. As mentioned earlier, the lack of a functional sigH plays a role in the unresponsiveness of sigE during heat stress in M. leprae [73]. Although the expression of M. tubercu- losis sigH was found to be induced during macrophage infection [62], the sigH mutant was not attenuated for growth in human macrophages [44]. In a murine model, the M. tuberculosis CDC1551 sigH mutant strain demonstrated a distinctive imp phenotype [92]. To identify r H -regulated genes, microarray experi- ments were carried out at different phases of growth [92] and following diamide stress [44]. The r H regulon includes its own structural gene and genes encoding r B , r E , Rv0142 (putative transcriptional regulator) DnaK, ClpB (heat shock proteins), TrxB and TrxC (thioredoxin reductase ⁄ thioredoxin) [44,92]. r H also induces enzymes involved in cysteine biosynthesis and in the metabolism of ribose and glucose, indicating an increased need for the synthesis of mycothiol precur- sors [44] (mycothiols are known to be involved in cellular protection during oxidative stress in actinomy- cetes [93]). In a recent report on the long-term effects of r H induction following diamide stress, it was observed that in response to oxidative damage, certain virulence ⁄ detoxification genes were induced, while many lipid metabolism genes were repressed, as a part of the stress-defense mechanism in M. tuberculosis.As the effect of stress diminished with time, the expression of lipid metabolism and of cell wall-associated genes resumed, demonstrating a remarkable plasticity in gene expression brought about by a mycobacterial r factor [94]. r H activity is regulated at the transcriptional level via autoregulation of the sigH promoter and post- translationally via interaction with its cognate anti-r factor, RshA [95]. The latter branch of regulation is intersected by PknB, a serine–threonine protein kinase (STPK), which further fine-tunes the stress response regulon controlled by r H [96]. SigI (r I ) and SigJ (r J ) sigI, and to a larger extent, sigJ, genes are expressed at high levels in late stationary phase dormant cultures of M. tuberculosis. The transcription of these two r fac- tors continues following rifampicin treatment of these cultures in an in vitro drug-persistent model of M. tuberculosis [55]. However, no difference in viability was observed between the sigJ mutant and the wild- type strain in a late stationary phase culture following rifampicin treatment, or in an immune stasis murine model. The sigJ mutant is more susceptible to killing by H 2 O 2 than its parental strain. As katG mRNA levels remain unchanged upon deletion of sigJ, r J possibly mediates resistance to H 2 O 2 via a KatG- independent pathway [97]. r J may also contribute to the survival of M. tuberculosis in the host organism, as suggested by an increase in sigJ expression in human macrophages [89]. Using the E. coli two-plasmid system, it was found that the expression of M. tuberculosis sigI is regulated by r J [98], suggesting a possible relationship between the two r factors. It is likely that r I may be involved in regulating stress responses similar to those regulated by r J . Based on the fact that the sigI transcript level increases after mild cold shock (room temperature), the r I regulon has been speculated to be involved in the sur- vival of M. tuberculosis in aerosol particles, where the P. Sachdeva et al. The r-factors of M. tuberculosis FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS 613 ambient temperature is usually lower than 37 °C [24]. The role of sigI in cold shock adaptation has also been suggested in the case of M. avium ssp. paratuberculosis, where it was found to be one of the genes significantly upregulated in cow fecal samples [91]. SigK (r K ) The sigK gene is present in all species of M. tuberculosis complex (MTC, a group of pathogenic organisms in- cluding Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium pinnipedii, Mycobacterium microti and Mycobacterium caprae [100]) and in Mycobacte- rium marinum, while it is absent in certain other myco- bacterial species (e.g. M. avium ssp. paratuberculosis and M. smegmatis) [2]. r K positively regulates the expression of two antigenic proteins, MPB70 and MPB83, in M. bovis BCG. A mutation in the start codon of sigK, and certain mutations in the coding region of its downstream regulator, rskA, account for the variable production of these antigenic proteins in the members of the M. tuberculosis complex [99,100]. The basal expression of mpt70 and mpt83 in M. tuber- culosis is low and induced only during infection of macrophages, while its M. bovis homologs are constitu- tively expressed at high levels. The presence of mutations in RskA, the negative regulator of r K , may have led to a state of constitutive activity of r K and therefore constantly high levels of expression of its reg- ulon observed in the study [100]. Evolutionary analysis indicates that the core regulatory system, r K ⁄ RskA, is conserved across the Mycobacterium genus, whereas the regulon under its control varies considerably across species. Gene alignments indicated insertion, deletion, or re-arrangements within the r K regulon across differ- ent mycobacteria (e.g. mpt83, dipZ and mpt70). It has been suggested that from a minimal module of mpt83 ⁄ rskA ⁄ sigK, a gene-duplication event resulted in two MPT70 ⁄ 83 paralogs. Possibly during evolution, this locus became bifurcated into two regions: the sigK ⁄ rskA locus and the mpt70 ⁄ 83 locus. In slow-grow- ing pathogenic mycobacteria, an additional gene, dipZ, is inserted between the two mpt83 paralogs. The r K regulon is atypically small; however, the number and identity of r K -regulated genes varies across different mycobacterial species [101]. The role and trigger of this regulon in M. tuberculosis pathogenicity merits investi- gation. SigL (r L ) sigL is constitutively expressed at a very low level from a weak r L -independent promoter and is also transcribed from a r L -dependent promoter [102]. In a murine infection model, the sigL mutant exhibited marked attenuation compared with the parental strain, suggest- ing a role of r L in virulence; however, there were no significant differences in the growth rate or in the size and extent of lesions in the infected organs [45,102]. Microarray analysis was carried out by two groups independently in order to characterize the r L regulon [45,102]. In one of the approaches, sigL overexpres- sion from an acetamide-inducible promoter led to the strong upregulation of four small operons: sigL (Rv0735)-rslA (Rv0736); mpt53 (Rv2878c)-Rv2877; pks10 (Rv1660)-pks7 (Rv1661); and Rv1139c-Rv1138c [102]. Mpt53, a DsbE-like protein, possibly acts as an extracellular oxidant required for proper folding of reduced unfolded secreted proteins [103]. While pks10 and pks7 are polyketide synthase genes, the other r L -regulated gene pair, Rv1139c-Rv1138c, encodes a membrane protein containing an isoprenyl cysteine carboxy methyltransferase motif and a putative oxido- reductase, respectively [102]. In another study, a mutant strain lacking both sigL and rslA was comple- mented by integrating a single wild-type copy of sigL into its chromosome, resulting in its constitutive expression [45]. Some of the genes (pks10, pks7, Rv1138c, mpt53 and Rv2877c) were identified using both approaches. Other genes identified using the lat- ter method included the remaining genes from the pks10 operon and the ppsA gene, involved in the biosynthesis of dimycocerosyl phthiocerol (a cell wall- associated lipid found only in pathogenic mycobacte- ria [104]), mmpL13a and mmpL13b, involved in fatty acid transport [105], another r factor-encoding gene, sigB and two genes thought to be involved in host cell invasion [45]. r L , like r E and r H , is post-translationally regulated by a ZAS family protein with its gene located down- stream of the sigL gene. However, unlike sigE and sigH, sigL does not play a role in the oxidative or nitrosative stress responses [45]. SigM (r M ) sigM expression was found to be induced at high tem- perature and in the stationary phase during in vitro growth of M. smegmatis, M. bovis BCG as well as M. tuberculosis CDC1551 [106,107]. Its induced levels, however, were markedly lower than that of sigA in the late stationary phase in M. tuberculosis [107]. By contrast, Raman et al. did not observe a significant change in sigM expression at any growth stage in M. tuberculosis H 37 Rv [108]. The M. smegmatis sigM mutant is more susceptible to oxidative stress than the The r-factors of M. tuberculosis P. Sachdeva et al. 614 FEBS Journal 277 (2010) 605–626 ª 2009 Council of Scientific and Industrial Research. Journal compilation ª 2009 FEBS [...]... redox conditions Anti-r factors sense external stimuli and release the r factor under stress conditions facilitating the transcription of the stressspecific regulons As most of the r factors exhibit autotranscription, the activation of r factor via this mechanism also results in its transcriptional upregulation Adding to the complexity is the regulation of the anti-r factors themselves, which can be... in the reversal of inhibition [119,120] The regulation of r factors and their regulators in M tuberculosis has not been as extensively studied as that for organisms such as E coli and B subtilis Five anti-r factors have been identified in M tuberculosis to date, of which three, namely regulator of sigmaE A (RseA), regulator of sigmaH A (RshA) and regulator of sigmaL A (RslA) belong to the ZAS family of. .. evidence of the interplay of r factors and their interaction with regulators, how exactly these factors coordinate with one another in the face of an adverse event, and the order of events, still needs to be explored A combination of independent gene mutations that may reduce the virulence of M tuberculosis sufficiently to provide safety in immunocompromised individuals, yet allow the elicitation of an... addition to regulation of r factors by anti-r factors belonging to the redox-sensitive ZAS family and the GHKL family of kinases, fine-tuning of the r–anti-r interaction in certain cases is brought about by STPK-mediated signaling An emerging aspect in the post-translational regulation of mycobacterial r factors is the involvement of STPK-mediated phosphorylation events in the modulation of regulatory... different physiological conditions The latter aspect is essentially governed by proteins such as anti-r factors, which serve to negatively regulate the activity of r factors Generally, the anti-r factors are co-transcribed and induced in the presence of their cognate r factors The overexpression of sigL in a sigL-rslA gene pair knockout strain led to the identification of a number of rL-regulated genes [45],... functional r regulatory GHKL family kinases in the Mycobacterium genus compared with other bacterial genomes (e.g B subtilis and E coli) In view of the abundance of eukaryotic-like STPKs and a significant number of physiological processes, including regulation of rF, being governed by them, the STPKs may compensate for the lacunae created by the dearth of other kinases in Mycobacterium Recently, we identified... robust regulatory mechanism The bacterium responds to environmental cues by recruitment of appropriate r factor(s) to the core RNAP to carry out the coordinate expression of a specific subset of genes The availability of the alternative r factors is controlled at the transcriptional, translational and The r -factors of M tuberculosis post-translational levels Almost all r factors are constitutively expressed... Hence, the criterion of B subtilis rW as a functional counterpart of both rM and rD needs to be further established In view of the significantly higher GC content, higher variability in spacing between the -10 and -35 regions and a low 616 level of general homology of mycobacterial promoters to those of other prokaryotes [112], the use of a consensus template from other unrelated prokaryotes may bias the. .. replication in the lung during the short-term infection of mice [109,110], the negative regulation of their synthesis by rM negates the possibility of its role in virulence during the early course of infection [108] Consistent with this inference, in a guinea pig aerosol model of infection, the sigM mutant strain appeared to be hypervirulent at early time-points, with greater numbers of granulomas and... changed to ‘mpb83’.] However, none of these mutations map to the region required for the interaction of RskA with rK Possibly, these mutations result in major changes in the protein structure and folding [16], thereby making it nonfunctional The stimulus and the mechanism that lead to the release of rK from RskA remain unknown The mode of regulation of M tuberculosis rF and its B subtilis homolog, rB, is . view of the downregulation of sigH and the upregulation of sigD upon the deletion of sigG in M. tuberculosis [90]. This finding further corroborates the. REVIEW ARTICLE The sigma factors of Mycobacterium tuberculosis: regulation of the regulators Preeti Sachdeva 1 , Richa Misra 1 ,