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Early secretory antigenic target-6 of Mycobacterium tuberculosis: enigmatic factor in pathogen–host interactions
Microbes and Infection 14 (2012) 1220e1226 www.elsevier.com/locate/micinf
Review
Early secretory antigenic target-6 of Mycobacterium tuberculosis: enigmatic factor in pathogenehost interactions Ramesh Chandra Rai a,1, Ved Prakash Dwivedi a,1, Samit Chatterjee a,1, Durbaka Vijaya Raghava Prasad b, Gobardhan Das a,* a
Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India b Department of Microbiology, Yogi Vemana University, Kadapa 516003, Andhra Pradesh, India Received 28 May 2012; accepted 31 July 2012 Available online 9 August 2012
Abstract T helper (Th) 1 and 17 cells play important roles in host protective responses against tuberculosis. Early Secretory Antigenic Target 6; a Region of Difference 1 (RD1) encoded protein, mounts Th17-responses in the lung. Therefore, lack of RD-1 region makes Bacillus CalmetteeGue´rin (BCG) less vaccine efficacious than parent strains. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Mycobacterium tuberculosis; BCG; RD-1; ESAT6; CFP10; Innate/adaptive immunity
1. Introduction Discovered by Robert Koch in 1882, Mycobacterium tuberculosis (M. tb) remains a leading cause of mortality in humans despite tremendous scientific progress since its discovery. Tuberculosis ranks second only after HIV among all infectious killers worldwide [1]. Globally, around 95% of all deaths due to tuberculosis (TB) are from developing countries [2]. In 2010, there were an estimated 8.5e9.2 million cases of TB and 1.2e1.5 million deaths, including deaths in individuals coinfected with HIV. Although the number of TB cases are falling worldwide, the rapid rise of multiple drug resistant (MDR) and extensively drug resistant (XDR) organisms are alarming [2]. M. tb and HIV co-infection, patient noncompliance with the long treatment regimen, and improper treatment prescriptions by clinicians are some of the reasons for the emergence of drug resistance in M. tb [3]. While the majority of infected individuals remain asymptomatic, active disease
* Corresponding author. E-mail address: [email protected] (G. Das). 1 These authors contributed equally to this work.
ensues during the lifetime of 5e10% of infected individuals due to a perturbation of host immune responses [4]. M. tb is an airborne pathogen and spreads through coughing and sneezing by the infected individuals. While lungs are the primary site of infection, this pathogen can also spread to other parts of the body [5]. Upon inhalation by an individual, the pathogen is engulfed by alveolar macrophages, which provide a suitable niche for this facultative intracellular pathogen [6,7]. As this pathogen has co-evolved with humans, it has attained several immune-evasion tactics to subvert the host immune response that targets its destruction [8]. The pathogen protects itself from destruction by macrophages through various mechanisms such as prevention of phagosome maturation, inhibition of fusion of the phagosome with lysosomes, and interference with the antigen processing and presentation machinery [9]. The cell wall of M. tb, which is abundant in mycolic acids, assists the organism in remaining dormant for many years [7]. M. tb also secretes many proteins that play critical roles in modulating host immune responses that favour its survival inside host macrophages [10,11]. Presentation of M. tb antigens to CD4þ T cells by major histocompatibility complex (MHC) class II molecules, which normally assists in clearing the intracellular pathogens, is also impaired [12].
1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2012.07.019
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Thus, M. tb has evolved multiple mechanisms to promote its survival inside the host. During the early stages of infection, the surface-exposed and secretory proteins of M. tb are the primary targets of the host immune response. Studies on ESAT6, a secretory mycobacterial protein, have suggested that its incorporation in the vaccine strain Bacillus CalmetteeGue´rin (BCG) or as part of a subunit vaccine can improve vaccination strategies against TB. The immunological role of ESAT6 was demonstrated by its capacity to induce gamma interferon (IFN-g) production in T cells isolated from immune mice challenged with M. tb [13]. It is now well established that the BCG vaccine does not fully protect against pulmonary TB in adults. Recent findings from several laboratories, including our own, have suggested that, in addition to T helper (Th) type 1 immune responses, Th17 cell responses are required to induce long-lasting protection against M. tb infection in mice. In light of these experimental findings, the failure of BCG to stimulate Th17 cell responses might well be the major reason for its poor vaccine efficacy in adult pulmonary TB [10]. Thus, incorporating immunodominant surface antigens such as ESAT6 in BCG through genetic engineering to promote the generation of Th17mediated immunity might provide a means to induce sustained protection against TB [14]. 2. Bacillus CalmetteeGue´rin (BCG) e success and failure BCG, the only vaccine against TB that is currently available, was developed by attenuation of Mycobacterium bovis (M. bovis) by French scientists Albert Calmette and Camille Gue´rin at the Pasteur Institute (Paris, France) at the beginning of the 20th century. When administered to infants in France in 1921, BCG reduced the mortality of TB by up to 90% but in adults its efficacy was poor [15]. It was later shown that the vaccine efficacy of BCG also varies with the person’s ethnicity and geographical location [16]. BCG induces an immune response which is able to contain the M. tb infection only partially [17], leaving behind the imminent possibility for pathogen resurgence and active disease. In the face of these disappointing findings, guidelines were developed by the World Health Organization (WHO) to improve the efficacy and compliance of TB treatment. The internationally recognized Directly Observed Treatment Short course (DOTS) programme was adopted and proved to be very effective. Nevertheless, this therapeutic regimen, which involves long-
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term treatment with multiple antibiotics, retained the risk of developing drug resistance in the M. tb organisms [18]. Therefore, alternative therapies and treatment strategies are needed to fight M. tb infections. 3. Genetic organization of the region of difference (RD) 1 Genomic comparison of the vaccine strain BCG with virulent mycobacterial strains revealed deletion of multiple genes encoding open reading frames (ORF) from its genome [19]. It is now broadly accepted that these gene products play critical roles in the virulence of this pathogen and that they are critical for the establishment of protective immunity in the host [10,20,21]. Reports based on genomic comparisons of virulent M. tb H37Rv, virulent M. bovis, and the vaccine strain M. bovis BCG suggested that 129 ORFs have been deleted in BCG [22]. These deletions occurred during repeated, longterm in vitro passage of the BCG strain [19,23]. The regions of the genome that are missing from M. bovis BCG are commonly known as regions of difference (RD) [22]. RD1, which includes 9 ORFs (Rv3871 to Rv3879) within a 9454 bp long segment, is the most critical RD for antigenic determination and is absent from all BCG strains but present in the M. tb complex (Fig. 1) [24,25]. Important proteins encoded within the RD1 region include ESAT6 (Early Secretory Antigenic Target 6, 6-kDa), CFP10 (Culture Filtrate Protein 10, 10kDa), and CFP21 (Culture Filtrate Protein 21) [26]. These secretory proteins are important T cell antigens and their absence from BCG is thought to be the main reason for the dearth of BCG to prime the immune system [19,23]. Deletion of RD1 from virulent M. tb strains leads to impaired growth of the organisms and attenuation of their virulence in vivo, resulting in disease pathogenesis similar to BCG [27]. The RD1 secretion system plays diverse roles in the pathogenesis of TB, from virulence to immune-modulation [21] and includes the formation of granulomas [28], inhibition of phagosomal maturation [29] and trans-migration of bacteria from the phagosome to the cytosol [30]. It also downregulates the functions of dendritic cells, macrophages and T cells by inhibiting secretion of cytokines that are important for immune cell activation. It is now clear that the mycobacterial factors encoded within the RD1 region can modulate early events during infection and ensure survival of the pathogen inside the hostile environment of the cell. While RD1 has emerged as one of the main determinants of the pathogenesis of virulent M. tb, Gene size (bp)
RD1 region
Fig. 1. Diagram of the RD1 region of Mycobacterium tuberculosis along with flanking genes. Arrows show the transcriptional direction of genes. Rv3874 and Rv3875 gene loci encode CFP10 and ESAT6, respectively.
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a detailed picture of the mechanisms by which factors encoded within the RD1 region influence bacterial pathogenesis and host immunity is not yet available. It is also unclear how the specialized ESAT6 secretion system (ESX)-1 affects interaction of the bacteria with their host. Recent reports have suggested a role for genes flanking the RD1 region in the secretion of ESAT6 and CFP10 [31]. ESAT6, which is recognized by the immune system, has also been proposed as a potential biomarker for M. tb infection [32]. 4. Role of mycobacterial secretory proteins in hostepathogen interactions Due to their production at the early phase of M. tb infection, the arsenal of secretory proteins produced by the bacteria facilitates microbial pathogenesis and intracellular survival in the host. However, these factors also trigger host immune responses. Secretory proteins of M. tb interact with various cells of the immune system, including macrophages, dendritic cells and T cells [13,25]. The paucity of BCG to mount an adequate immune response against M. tb has been postulated to be due to the absence of these immunodominant antigenic molecules [19,23]. Pym et al. (2003) demonstrated that an engineered BCG vaccine strain producing ESAT6 provides superior protection towards M. tb infection as compared with the parental BCG strain. While in complex with ESAT6, the C-terminal region of CFP-10 forms a long flexible arm that binds to the surface of antigen presenting cells, suggesting cooperative functions between ESAT6 and CFP10 in cellular signalling and as modulators of host immune responses to the pathogen [33]. These proteins are able to subvert the antibacterial function of macrophages [34] by impinging on the ERK1/2 MAP kinase signalling pathway [35]. They also down-regulate the production of reactive oxygen species inside macrophages, thus inhibiting NF-kB-dependent gene expression [34]. These findings have provided strong support for a critical role of M. tb secretory proteins in hostepathogen interactions during M. tb infection. 5. Role of early secretory antigenic target-6 (ESAT6) in mycobacterial pathogenesis ESAT6 was first purified by Anne L. Sorensen et al. (1995) from the cytosol of mycobacteria as a protein with a molecular weight of 6 kDa. As its name suggests, ESAT6 is released early during infection by M. tb or M. bovis [36]. Secretory proteins are also thought to be involved in invasion of the mammalian host by the bacterial pathogens. It has been reported that multiple domains of the ESAT6 protein are required for developing antigen-specific immune responses, as both N- and C-terminal truncation of ESAT6 impaired the induction of immune responses [37]. The Rv3874 and Rv3875 genes, which encode the CFP10 and ESAT6 proteins, respectively, are part of the same operon [26]. These genes are transcribed and expressed in a coordinated fashion to form a tight 1:1 protein complex [33]. While mycobacterial strains carrying deletions that overlap with RD1 but do
not include ESAT6 and CFP10 retain expression of these factors, such strains fail to secrete the ESAT6/CFP10 complex. Thus, these findings suggest that secretion of ESAT6/CFP10 is dependent on an intact RD1 region [31,33]. The absence of canonical protein motifs for secretion in both ESAT6 and CFP10 suggests that these proteins are exported from the bacteria with the assistance of a secretion system that involves protein products encoded by other genes of the RD1 region [24,27]. Individual ablation of the RD1 region genes Rv3870, Rv3871 or Rv3877 resulted in blockade in the secretion of ESAT6 and CFP10. These findings therefore suggested that products encoded within the RD1 region form a secretion system, which is now termed ESX-1 [14,20]. The heterodimer formed by ESAT6 and CFP10 might require chaperone activity mediated by the ATP-dependent protein Rv3868, as expression of this protein is a prerequisite for their tight linkage and secretion [14,20]. The secretion of ESAT6 and CFP10 also requires proteins Snm-1, Snm-2 and Snm-4, which are all members of the AAA family of ATPases [38]. It has also been shown that these proteins are secreted through a Sec-independent secretion pathway. Snm mutants of M. tb exhibit growth defects and impaired virulence in the host. These mutants also fail to replicate in macrophages infected in vitro and are unable to block inflammatory responses induced in these macrophages, suggesting that the ESX-1 secretion system plays an important role in subverting immune responses mediated by macrophages [38]. Apart from their role in suppressing immune responses mediated by macrophages and dendritic cells, ESAT6 and CFP10 have been proposed as potential candidates for inclusion in vaccines. It has also been suggested that these secretory proteins could be included in tuberculin tests for diagnosis of TB [21]. While re-introduction of the RD1 region into BCG (designated BCG::RD1) increased its virulence, it also promoted enhanced host immune responses. Thus, BCG::RD1 became more protective and similar to M. tb than the parental BCG strain in terms of immunogenicity [14]. When used as a vaccine, BCG::RD1 induced recruitment of antigen presenting cells and T cells much more efficiently than the parental BCG strain [39]. 6. ESAT6 and host immune responses M. tb infects host phagocytes, which attempt to degrade the bacteria. This is facilitated by fusion of phagosomes with lysosomes whose acidic environment and different hydrolases assist in destroying the pathogens. However, M. tb has evolved many ways to counteract host immune responses [40]. Antigenic molecules of M. tb are presented by MHC molecules to naı¨ve T cells, which lead to the generation of antigen-specific immune responses. Multiple cytokines and chemokines are secreted at the site of infection in response to interaction of the pathogen-associated molecular patterns (PAMPs) of the bacteria with pattern recognition receptors (PRRs) expressed by innate immune cells [41]. In the case of M. tb infection, the main PRRs that contribute to pathogen recognition and subsequent cellular signalling are toll like receptor (TLR) 2 and TLR9 [42]. M. tb also interferes with the expression of MHC class II molecules through ESAT6 [43]. It has been
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shown that ESAT6 interacts with TLR2 and modulates host immune responses [44]. It has further been established that all TLRs, except for TLR3, signal through the adaptor protein myeloid differentiation primary response protein 88 (MyD88), and that MyD88-deficient mice exhibit increased susceptibility to infection by M. tb [45]. Recognition of ESAT6 by T cells isolated from M. tbinfected animals suggests the importance of ESAT6 as a T cell antigen during the establishment of cell-mediated immunity [36]. T cell receptor (TCR) transgenic T cells specific for ESAT6 confer protection against TB [46]. While it is wellestablished that CD4þ T cells play a major role in host resistance against TB, emerging evidence has suggested a critical role for CD8þ T cells as well [47]. CD4þ T cells recognise protein antigens presented by MHC class II molecules on antigen presenting cells. Development of Th1 cells producing IFN-g, lymphotoxin (LT) and tumour necrosis factor (TNF)-a in infected individuals confers resistance to infection, whereas Th2 cells producing interleukin (IL)-4, IL5, and IL-13 increase disease susceptibility [47,48]. Consequently, mice lacking IFN-g exhibit increased susceptibility and mice lacking IL-4 exhibit increased resistance to M. tb infection [49]. These findings therefore suggest that IFN-gmediated immunity is required for disease resistance. However, while mycobacterial antigen-specific Th1 cells are required for clearance of M. tb organisms [50,51], they are not sufficient [52]. Thus, an effective way to prevent TB would be to concomitantly promote Th1 cell responses and suppress Th2 cell responses.
6.1. Interaction of ESAT6 with immune pathway components Metazoans express evolutionary conserved PRRs that recognise invading pathogens. Cells of the innate immune system express multiple TLRs that are specific for a variety of conserved molecules expressed by microbes. TLR signalling links innate immunity to adaptive immunity as it can directly regulate phagocytosis or can trigger the release of different cytokines that regulate dendritic cell differentiation, which in turn instructs adaptive immune responses. It is now widely accepted that early innate recognition of pathogens shapes the ensuing adaptive immune responses. TLR2 and TLR4 have been reported to play important roles in the interaction of M. tb with its hosts. TLR2 can form heterodimers with TLR1 or TLR6 and is able to recognise a variety of microbial products [53]. It has been shown that TLR2 and its adaptor protein, MyD88 play important roles in the expression of MHC class II molecules and, thus the presentation of mycobacterial antigen to CD4þ T cells [12]. Consequently, mice deficient in MyD88 exhibit increased susceptibility to M. tb infection [45]. The mycobacterial antigens that acts as agonists for TLR2 and inhibit MHC class II-restricted antigen presentation include lipoproteins such as LpqH, LprG and LprA, as well as some non-lipoproteins [54].
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6.2. Th17 cells are required for long-lasting immunity against M. tb infection In recent years several new subset of T cells have been discovered. Although the role of these new T cell subsets in the pathogenesis of TB has not been extensively investigated, recent studies have suggested a role for IL-17-producing CD4þ T helper cells (Th17 cells). Th17 cells are a distinct subtype of differentiated CD4þ T cells that play crucial functions in host defence. They were also shown to provide protection against certain bacterial and fungal pathogens [55]. Dysregulated Th17 cell responses mediate a variety of autoimmune inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease (IBD) and multiple sclerosis (MS). While Th17 cells were increased in BCG-infected IFN-g-deficient mice there was no change in bacterial clearance [56]. One study suggested that IL-17 produced in response to M. tb infection during the primary immune response inhibited effective secondary immune responses [57]. Although their protective role in primary immunity against TB is unclear, several studies have provided evidence for a protective role of Th17 cells in secondary immune responses against M. tb [58,59]. For example, in IFN-g-deficient mice, BCG infection induces strong IL-17-dependent memory responses, which provides protection against disease upon subsequent challenge with M. tb [59]. 6.3. Modulation of host miRNA expression by ESAT6 MicroRNAs (miRNAs) are endogenous regulators of gene expression and function. They are highly conserved small (18e25 nucleotides) non-coding RNAs that bind with the 30 un-translated region (UTR) of their target mRNA and thus block its translation. miRNAs regulate the differentiation and activation of monocytes and granulocytes, sense the presence of pathogens, and are involved in inflammatory responses and antiviral immunity [60]. Apart from their broad functions in different biological processes they also play critical regulatory roles in innate and adaptive immunity. Many micro-RNAs such as miR29, miR146a and miR155 have been shown to inhibit crucial molecules of pro-inflammatory signalling pathways. It has been clearly demonstrated that M. bovis upregulates miR29 resulting in the suppression of IFN-g production in mice [61]. A recent report indicated that miR146a is upregulated in dendritic cells in response to infection by M. tb [62]. miR146a regulates production of IL-6 in dendritic cells and functions as a negative feedback regulator in TLR signalling by targeting the IL-1 receptor-associated kinase (IRAK) 1 and the TNF receptor-associated factor (TRAF) 6. Inhibition of IRAK1 and TRAF6 expression by miR146a suppresses NF-kB activity and induction of a variety of cytokines such as IL-6, IL-8, IL-1b and TNF-a [63]. Recently, it has been shown that ESAT6 upregulates the expression of miR155 affecting expression of BTB and CNC homology 1 (Bach1) and SH2-containing inositol 5phosphatase (SHIP1), which is further linked with the intracellular survival of M. tb in RAW264.7 and murine bone marrow derived macrophages [64]. miR155 also regulates IL-6
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production from the macrophage infected with M. tb. It is important to note here that IL-6 deficient animals are hyper susceptible to M. tb infection [65]. Therefore, it is highly likely that M. tb regulates multiple miRNAs viz. miR155 and miR146a through ESAT6 to control IL-6 production as a survival strategy in the phagocytes of susceptible hosts [10,64]. 6.4. Changes in the cytokine production profile of antigen presenting cells induced by ESAT6 Coordination between cells of the innate and adaptive immune systems is mediated by secretion of distinct cytokines. Upon interaction with mycobacteria, cells of the innate immune system secrete a variety of pro- and anti-inflammatory cytokines that direct the differentiation of distinct effector T cell subsets from naı¨ve CD4þ T cell precursors. The simultaneous presence of both IL-6 and TGF-b has been shown to be required for the differentiation of Th17 cells [66,67]. Production of large amounts of both of these cytokines has been observed by macrophages infected with M. tb, suggesting that infection with this pathogen aids in Th17 cell differentiation [68]. Findings from our laboratory have suggested that the ESAT6 protein induces IL-6 and TGF-b in dendritic cells in a TLR2- and MyD88-dependent manner, and that this generates an environment that is conducive to the differentiation of Th17 cells in the lung [10]. We recently also reported that TGF-b is dispensable for the Th17 cell differentiation [69] but instead promotes Th17 cell responses by inhibiting Th1 and Th2 cell differentiation. Consistent with this notion, IL-6deficient mice showed a marginally increased bacterial burden in the initial phase of M. tb infection, suggesting a minor protective role of IL-6 confined to cells of the innate immune response [69]. Taken together, these findings suggested that Th1 and Th17 cells are protective whereas Th2 cells assist in the progression of TB disease. Both the virulent M. tb strain H37Rv and BCG induce production of IL-12p40, a cytokine required for the differentiation of Th1 cells. In sharp contrast, IL-6 and TGF-b were induced by H37Rv but not BCG, suggesting that BCG is unable to induce Th17 cell differentiation in the infected mice [10]. Studies from our laboratory have provided evidence that H37Rv and BCG::RD1 induce both Th1 and Th17 cell responses whereas an H37Rv mutant with a deletion of the RD1 region (H37RvDRD1) selectively induces Th1 cell responses [10]. Consequently, inclusion of the RD1 region in BCG improved its vaccine efficacy. When a neutralizing antibody against IL-17 was used, the vaccine potential of both H37Rv and BCG::RD1 were drastically reduced. Chatterjee, S. et al. (2011) further showed that IL-6 and TGF-b production were also diminished when H37RvDRD1 or BCG were used to infect dendritic cells, whereas infection with BCG::RD1 induced levels of these cytokines similar to H37Rv. This report also showed that production of these cytokines by dendritic cells upon infection with H37Rv or BCG::RD1 was dependent on activation of the TLR2/MyD88 signalling pathway. Chatterjee, S. et al. (2011) observed lower
expression levels of miR146a, a negative regulator of IL-6 production, in dendritic cells infected with H37Rv or BCG::RD1 as compared with H37RvDRD1 or BCG. The differential production of IL-6 and TGF-b were thought to be due to the PAMPs encoded within the RD1 region. Since ESAT6 induces protective immune responses and because the H37RvDRD1 mutant fails to induce Th17 cell responses, it is likely that ESAT6 is responsible for inducing Th17 cellpolarizing cytokines in antigen presenting cells. In agreement with these findings, dendritic cells treated with purified ESAT6 protein produced IL-6 and TGF-b in a TLR2/MyD88 dependent manner, and this promoted Th17 cell differentiation (Fig. 2). Infection of ESAT6-treated dendritic cells with BCG or stimulation of these cells with lipopolysaccharide (LPS) downregulated miR146a expression. However, these experiments were unable to exclude a contribution of other gene products encoded within the RD1 region in mounting protective immune responses. Overall these findings indicated that ESAT6 interacts with TLR2 [10,44] and creates a cytokine environment that aids in the differentiation of Th17 cells that provide protection against TB. 7. Conclusions and future perspectives It is now well established that vaccines based on single antigens are unable to provide adequate protection against TB. Therefore, multi-subunit vaccines containing multiple epitopes are needed to provide improved protection against the disease. Such an approach might be able to provide protection in diverse and genetically heterogeneous human populations. Immune responses could be boosted by supplementing the live attenuated BCG vaccine with antigenic, recombinant proteins encoded within the RD1 region of M. tb. Incorporation of ESAT6 in BCG improves the efficacy of this vaccine. Yang X et al. 2011, showed that recombinant BCG strains expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and ESAT6 induce increased antibody titres. They also showed that recombinant BCG is able to enhance protective immune responses by increasing proliferation of splenocytes and thus enhancing the percentage of antigen-specific CD4þ and CD8þ T cells [70]. Being a target of both CD4þ and CD8þ T cells, ESAT6 has been proposed as a potential vaccine candidate. ESAT6 also induces secretion of IFN-g by the peripheral blood mononuclear cells isolated from TB patients [32]. As the RD1 region does not BCG
RD1
H37Rv miR146a RD1
ESAT-6
IL-6 β TGF-β
Th17
Fig. 2. Schematic diagram for the differentiation of Th17 cells after Mycobacterium tuberculosis infection. The ESAT6 protein, which is present in H37Rv but not in BCG, directs Th17 cell differentiation by inducing IL-6 and TGF-b production in dendritic cells by downregulating miR146a.
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have a homologous counterpart in humans, immunization with ESAT6 as part of a vaccine against TB should not have the risk of inducing autoimmunity. Due to its specificity for active TB, i.e. its capacity to differentiate TB patients from BCG-vaccinated persons, and its ability to induce IFN-g secretion by T cells, measuring immune responses against ESAT6 could be employed for diagnosis of M. tb infection [32]. Acknowledgements This work was supported by grants from The WellcomeDBT India Alliance and the Department of Biotechnology, Government of India; and by the International Center for Genetic Engineering and Biotechnology, New Delhi, India. References [1] G.L. Mandell, Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases, Elsevier, Churchill, Livingstone, 2010. [2] WHO, Global Tuberculosis Control (2011). [3] R.J. O’Brien, Drug-resistant tuberculosis: etiology, management and prevention, Semin. Respir. Infect. 2 (1994) 104e112. [4] B.R. Bloom, P.M. Small, The evolving relation between humans and Mycobacterium tuberculosis, N. Engl. J. Med. 338 (1998) 677e678. [5] V. Kumar, A.K. Abbas, N. Fausto, R.N. Mitchell, Robbins Basic Pathology, Saunders/Elsevier, Philadelphia, 2007. [6] E.N. Houben, L. Nguyen, J. Pieters, Interaction of pathogenic mycobacteria with the host immune system, Curr. Opin. Microbiol. 9 (2006) 76e85. [7] P. Peyron, J. Vaubourgeix, Y. Poquet, F. Levillain, C. Botanch, F. Bardou, M. Daffe, J.F. Emile, B. Marchou, P.J. Cardona, C. de Chastellier, F. Altare, Foamy macrophages from tuberculous patients’ granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence, PLoS Pathog. 4 (2008) e1000204. [8] D. Brites, S. Gagneux, Old and new selective pressures on Mycobacterium tuberculosis, Infect. Genet. Evol. 12 (2012) 678e685. [9] N.W. Schluger, W.N. Rom, The host immune response to tuberculosis, Am. J. Respir. Crit. Care Med. 157 (1998) 679e691. [10] S. Chatterjee, V.P. Dwivedi, Y. Singh, I. Siddiqui, P. Sharma, L. Van Kaer, D. Chattopadhyay, G. Das, Early secreted antigen ESAT6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner, PLoS Pathog. 7 (2011) e1002378. [11] X. Wang, P.F. Barnes, K.M. Dobos-Elder, J.C. Townsend, Y.T. Chung, H. Shams, S.E. Weis, B. Samten, ESAT6 inhibits production of IFNgamma by Mycobacterium tuberculosis-responsive human T cells, J. Immunol. 182 (2009) 3668e3677. [12] R.K. Pai, M. Convery, T.A. Hamilton, W.H. Boom, C.V. Harding, Inhibition of IFN-gamma-induced class II transactivator expression by a 19kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion, J. Immunol. 171 (2003) 175e184. [13] A.L. Sorensen, S. Nagai, G. Houen, P. Andersen, A.B. Andersen, Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis, Infect. Immun. 63 (1995) 1710e1717. [14] A.S. Pym, P. Brodin, L. Majlessi, R. Brosch, C. Demangel, A. Williams, K.E. Griffiths, G. Marchal, C. Leclerc, S.T. Cole, Recombinant BCG exporting ESAT6 confers enhanced protection against tuberculosis, Nat. Med. 9 (2003) 533e539. [15] C. Bonah, The ’experimental stable’ of the BCG vaccine: safety, efficacy, proof, and standards, 1921e1933, Stud. Hist. Philos. Biol. Biomed. Sci. 36 (2005) 696e721. [16] P.E. Fine, J.M. Ponnighaus, N. Maine, J.A. Clarkson, L. Bliss, Protective efficacy of BCG against leprosy in Northern Malawi, Lancet 2 (1986) 499e502.
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Review
Early secretory antigenic target-6 of Mycobacterium tuberculosis: enigmatic factor in pathogenehost interactions Ramesh Chandra Rai a,1, Ved Prakash Dwivedi a,1, Samit Chatterjee a,1, Durbaka Vijaya Raghava Prasad b, Gobardhan Das a,* a
Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India b Department of Microbiology, Yogi Vemana University, Kadapa 516003, Andhra Pradesh, India Received 28 May 2012; accepted 31 July 2012 Available online 9 August 2012
Abstract T helper (Th) 1 and 17 cells play important roles in host protective responses against tuberculosis. Early Secretory Antigenic Target 6; a Region of Difference 1 (RD1) encoded protein, mounts Th17-responses in the lung. Therefore, lack of RD-1 region makes Bacillus CalmetteeGue´rin (BCG) less vaccine efficacious than parent strains. Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Mycobacterium tuberculosis; BCG; RD-1; ESAT6; CFP10; Innate/adaptive immunity
1. Introduction Discovered by Robert Koch in 1882, Mycobacterium tuberculosis (M. tb) remains a leading cause of mortality in humans despite tremendous scientific progress since its discovery. Tuberculosis ranks second only after HIV among all infectious killers worldwide [1]. Globally, around 95% of all deaths due to tuberculosis (TB) are from developing countries [2]. In 2010, there were an estimated 8.5e9.2 million cases of TB and 1.2e1.5 million deaths, including deaths in individuals coinfected with HIV. Although the number of TB cases are falling worldwide, the rapid rise of multiple drug resistant (MDR) and extensively drug resistant (XDR) organisms are alarming [2]. M. tb and HIV co-infection, patient noncompliance with the long treatment regimen, and improper treatment prescriptions by clinicians are some of the reasons for the emergence of drug resistance in M. tb [3]. While the majority of infected individuals remain asymptomatic, active disease
* Corresponding author. E-mail address: [email protected] (G. Das). 1 These authors contributed equally to this work.
ensues during the lifetime of 5e10% of infected individuals due to a perturbation of host immune responses [4]. M. tb is an airborne pathogen and spreads through coughing and sneezing by the infected individuals. While lungs are the primary site of infection, this pathogen can also spread to other parts of the body [5]. Upon inhalation by an individual, the pathogen is engulfed by alveolar macrophages, which provide a suitable niche for this facultative intracellular pathogen [6,7]. As this pathogen has co-evolved with humans, it has attained several immune-evasion tactics to subvert the host immune response that targets its destruction [8]. The pathogen protects itself from destruction by macrophages through various mechanisms such as prevention of phagosome maturation, inhibition of fusion of the phagosome with lysosomes, and interference with the antigen processing and presentation machinery [9]. The cell wall of M. tb, which is abundant in mycolic acids, assists the organism in remaining dormant for many years [7]. M. tb also secretes many proteins that play critical roles in modulating host immune responses that favour its survival inside host macrophages [10,11]. Presentation of M. tb antigens to CD4þ T cells by major histocompatibility complex (MHC) class II molecules, which normally assists in clearing the intracellular pathogens, is also impaired [12].
1286-4579/$ - see front matter Ó 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2012.07.019
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Thus, M. tb has evolved multiple mechanisms to promote its survival inside the host. During the early stages of infection, the surface-exposed and secretory proteins of M. tb are the primary targets of the host immune response. Studies on ESAT6, a secretory mycobacterial protein, have suggested that its incorporation in the vaccine strain Bacillus CalmetteeGue´rin (BCG) or as part of a subunit vaccine can improve vaccination strategies against TB. The immunological role of ESAT6 was demonstrated by its capacity to induce gamma interferon (IFN-g) production in T cells isolated from immune mice challenged with M. tb [13]. It is now well established that the BCG vaccine does not fully protect against pulmonary TB in adults. Recent findings from several laboratories, including our own, have suggested that, in addition to T helper (Th) type 1 immune responses, Th17 cell responses are required to induce long-lasting protection against M. tb infection in mice. In light of these experimental findings, the failure of BCG to stimulate Th17 cell responses might well be the major reason for its poor vaccine efficacy in adult pulmonary TB [10]. Thus, incorporating immunodominant surface antigens such as ESAT6 in BCG through genetic engineering to promote the generation of Th17mediated immunity might provide a means to induce sustained protection against TB [14]. 2. Bacillus CalmetteeGue´rin (BCG) e success and failure BCG, the only vaccine against TB that is currently available, was developed by attenuation of Mycobacterium bovis (M. bovis) by French scientists Albert Calmette and Camille Gue´rin at the Pasteur Institute (Paris, France) at the beginning of the 20th century. When administered to infants in France in 1921, BCG reduced the mortality of TB by up to 90% but in adults its efficacy was poor [15]. It was later shown that the vaccine efficacy of BCG also varies with the person’s ethnicity and geographical location [16]. BCG induces an immune response which is able to contain the M. tb infection only partially [17], leaving behind the imminent possibility for pathogen resurgence and active disease. In the face of these disappointing findings, guidelines were developed by the World Health Organization (WHO) to improve the efficacy and compliance of TB treatment. The internationally recognized Directly Observed Treatment Short course (DOTS) programme was adopted and proved to be very effective. Nevertheless, this therapeutic regimen, which involves long-
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term treatment with multiple antibiotics, retained the risk of developing drug resistance in the M. tb organisms [18]. Therefore, alternative therapies and treatment strategies are needed to fight M. tb infections. 3. Genetic organization of the region of difference (RD) 1 Genomic comparison of the vaccine strain BCG with virulent mycobacterial strains revealed deletion of multiple genes encoding open reading frames (ORF) from its genome [19]. It is now broadly accepted that these gene products play critical roles in the virulence of this pathogen and that they are critical for the establishment of protective immunity in the host [10,20,21]. Reports based on genomic comparisons of virulent M. tb H37Rv, virulent M. bovis, and the vaccine strain M. bovis BCG suggested that 129 ORFs have been deleted in BCG [22]. These deletions occurred during repeated, longterm in vitro passage of the BCG strain [19,23]. The regions of the genome that are missing from M. bovis BCG are commonly known as regions of difference (RD) [22]. RD1, which includes 9 ORFs (Rv3871 to Rv3879) within a 9454 bp long segment, is the most critical RD for antigenic determination and is absent from all BCG strains but present in the M. tb complex (Fig. 1) [24,25]. Important proteins encoded within the RD1 region include ESAT6 (Early Secretory Antigenic Target 6, 6-kDa), CFP10 (Culture Filtrate Protein 10, 10kDa), and CFP21 (Culture Filtrate Protein 21) [26]. These secretory proteins are important T cell antigens and their absence from BCG is thought to be the main reason for the dearth of BCG to prime the immune system [19,23]. Deletion of RD1 from virulent M. tb strains leads to impaired growth of the organisms and attenuation of their virulence in vivo, resulting in disease pathogenesis similar to BCG [27]. The RD1 secretion system plays diverse roles in the pathogenesis of TB, from virulence to immune-modulation [21] and includes the formation of granulomas [28], inhibition of phagosomal maturation [29] and trans-migration of bacteria from the phagosome to the cytosol [30]. It also downregulates the functions of dendritic cells, macrophages and T cells by inhibiting secretion of cytokines that are important for immune cell activation. It is now clear that the mycobacterial factors encoded within the RD1 region can modulate early events during infection and ensure survival of the pathogen inside the hostile environment of the cell. While RD1 has emerged as one of the main determinants of the pathogenesis of virulent M. tb, Gene size (bp)
RD1 region
Fig. 1. Diagram of the RD1 region of Mycobacterium tuberculosis along with flanking genes. Arrows show the transcriptional direction of genes. Rv3874 and Rv3875 gene loci encode CFP10 and ESAT6, respectively.
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a detailed picture of the mechanisms by which factors encoded within the RD1 region influence bacterial pathogenesis and host immunity is not yet available. It is also unclear how the specialized ESAT6 secretion system (ESX)-1 affects interaction of the bacteria with their host. Recent reports have suggested a role for genes flanking the RD1 region in the secretion of ESAT6 and CFP10 [31]. ESAT6, which is recognized by the immune system, has also been proposed as a potential biomarker for M. tb infection [32]. 4. Role of mycobacterial secretory proteins in hostepathogen interactions Due to their production at the early phase of M. tb infection, the arsenal of secretory proteins produced by the bacteria facilitates microbial pathogenesis and intracellular survival in the host. However, these factors also trigger host immune responses. Secretory proteins of M. tb interact with various cells of the immune system, including macrophages, dendritic cells and T cells [13,25]. The paucity of BCG to mount an adequate immune response against M. tb has been postulated to be due to the absence of these immunodominant antigenic molecules [19,23]. Pym et al. (2003) demonstrated that an engineered BCG vaccine strain producing ESAT6 provides superior protection towards M. tb infection as compared with the parental BCG strain. While in complex with ESAT6, the C-terminal region of CFP-10 forms a long flexible arm that binds to the surface of antigen presenting cells, suggesting cooperative functions between ESAT6 and CFP10 in cellular signalling and as modulators of host immune responses to the pathogen [33]. These proteins are able to subvert the antibacterial function of macrophages [34] by impinging on the ERK1/2 MAP kinase signalling pathway [35]. They also down-regulate the production of reactive oxygen species inside macrophages, thus inhibiting NF-kB-dependent gene expression [34]. These findings have provided strong support for a critical role of M. tb secretory proteins in hostepathogen interactions during M. tb infection. 5. Role of early secretory antigenic target-6 (ESAT6) in mycobacterial pathogenesis ESAT6 was first purified by Anne L. Sorensen et al. (1995) from the cytosol of mycobacteria as a protein with a molecular weight of 6 kDa. As its name suggests, ESAT6 is released early during infection by M. tb or M. bovis [36]. Secretory proteins are also thought to be involved in invasion of the mammalian host by the bacterial pathogens. It has been reported that multiple domains of the ESAT6 protein are required for developing antigen-specific immune responses, as both N- and C-terminal truncation of ESAT6 impaired the induction of immune responses [37]. The Rv3874 and Rv3875 genes, which encode the CFP10 and ESAT6 proteins, respectively, are part of the same operon [26]. These genes are transcribed and expressed in a coordinated fashion to form a tight 1:1 protein complex [33]. While mycobacterial strains carrying deletions that overlap with RD1 but do
not include ESAT6 and CFP10 retain expression of these factors, such strains fail to secrete the ESAT6/CFP10 complex. Thus, these findings suggest that secretion of ESAT6/CFP10 is dependent on an intact RD1 region [31,33]. The absence of canonical protein motifs for secretion in both ESAT6 and CFP10 suggests that these proteins are exported from the bacteria with the assistance of a secretion system that involves protein products encoded by other genes of the RD1 region [24,27]. Individual ablation of the RD1 region genes Rv3870, Rv3871 or Rv3877 resulted in blockade in the secretion of ESAT6 and CFP10. These findings therefore suggested that products encoded within the RD1 region form a secretion system, which is now termed ESX-1 [14,20]. The heterodimer formed by ESAT6 and CFP10 might require chaperone activity mediated by the ATP-dependent protein Rv3868, as expression of this protein is a prerequisite for their tight linkage and secretion [14,20]. The secretion of ESAT6 and CFP10 also requires proteins Snm-1, Snm-2 and Snm-4, which are all members of the AAA family of ATPases [38]. It has also been shown that these proteins are secreted through a Sec-independent secretion pathway. Snm mutants of M. tb exhibit growth defects and impaired virulence in the host. These mutants also fail to replicate in macrophages infected in vitro and are unable to block inflammatory responses induced in these macrophages, suggesting that the ESX-1 secretion system plays an important role in subverting immune responses mediated by macrophages [38]. Apart from their role in suppressing immune responses mediated by macrophages and dendritic cells, ESAT6 and CFP10 have been proposed as potential candidates for inclusion in vaccines. It has also been suggested that these secretory proteins could be included in tuberculin tests for diagnosis of TB [21]. While re-introduction of the RD1 region into BCG (designated BCG::RD1) increased its virulence, it also promoted enhanced host immune responses. Thus, BCG::RD1 became more protective and similar to M. tb than the parental BCG strain in terms of immunogenicity [14]. When used as a vaccine, BCG::RD1 induced recruitment of antigen presenting cells and T cells much more efficiently than the parental BCG strain [39]. 6. ESAT6 and host immune responses M. tb infects host phagocytes, which attempt to degrade the bacteria. This is facilitated by fusion of phagosomes with lysosomes whose acidic environment and different hydrolases assist in destroying the pathogens. However, M. tb has evolved many ways to counteract host immune responses [40]. Antigenic molecules of M. tb are presented by MHC molecules to naı¨ve T cells, which lead to the generation of antigen-specific immune responses. Multiple cytokines and chemokines are secreted at the site of infection in response to interaction of the pathogen-associated molecular patterns (PAMPs) of the bacteria with pattern recognition receptors (PRRs) expressed by innate immune cells [41]. In the case of M. tb infection, the main PRRs that contribute to pathogen recognition and subsequent cellular signalling are toll like receptor (TLR) 2 and TLR9 [42]. M. tb also interferes with the expression of MHC class II molecules through ESAT6 [43]. It has been
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shown that ESAT6 interacts with TLR2 and modulates host immune responses [44]. It has further been established that all TLRs, except for TLR3, signal through the adaptor protein myeloid differentiation primary response protein 88 (MyD88), and that MyD88-deficient mice exhibit increased susceptibility to infection by M. tb [45]. Recognition of ESAT6 by T cells isolated from M. tbinfected animals suggests the importance of ESAT6 as a T cell antigen during the establishment of cell-mediated immunity [36]. T cell receptor (TCR) transgenic T cells specific for ESAT6 confer protection against TB [46]. While it is wellestablished that CD4þ T cells play a major role in host resistance against TB, emerging evidence has suggested a critical role for CD8þ T cells as well [47]. CD4þ T cells recognise protein antigens presented by MHC class II molecules on antigen presenting cells. Development of Th1 cells producing IFN-g, lymphotoxin (LT) and tumour necrosis factor (TNF)-a in infected individuals confers resistance to infection, whereas Th2 cells producing interleukin (IL)-4, IL5, and IL-13 increase disease susceptibility [47,48]. Consequently, mice lacking IFN-g exhibit increased susceptibility and mice lacking IL-4 exhibit increased resistance to M. tb infection [49]. These findings therefore suggest that IFN-gmediated immunity is required for disease resistance. However, while mycobacterial antigen-specific Th1 cells are required for clearance of M. tb organisms [50,51], they are not sufficient [52]. Thus, an effective way to prevent TB would be to concomitantly promote Th1 cell responses and suppress Th2 cell responses.
6.1. Interaction of ESAT6 with immune pathway components Metazoans express evolutionary conserved PRRs that recognise invading pathogens. Cells of the innate immune system express multiple TLRs that are specific for a variety of conserved molecules expressed by microbes. TLR signalling links innate immunity to adaptive immunity as it can directly regulate phagocytosis or can trigger the release of different cytokines that regulate dendritic cell differentiation, which in turn instructs adaptive immune responses. It is now widely accepted that early innate recognition of pathogens shapes the ensuing adaptive immune responses. TLR2 and TLR4 have been reported to play important roles in the interaction of M. tb with its hosts. TLR2 can form heterodimers with TLR1 or TLR6 and is able to recognise a variety of microbial products [53]. It has been shown that TLR2 and its adaptor protein, MyD88 play important roles in the expression of MHC class II molecules and, thus the presentation of mycobacterial antigen to CD4þ T cells [12]. Consequently, mice deficient in MyD88 exhibit increased susceptibility to M. tb infection [45]. The mycobacterial antigens that acts as agonists for TLR2 and inhibit MHC class II-restricted antigen presentation include lipoproteins such as LpqH, LprG and LprA, as well as some non-lipoproteins [54].
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6.2. Th17 cells are required for long-lasting immunity against M. tb infection In recent years several new subset of T cells have been discovered. Although the role of these new T cell subsets in the pathogenesis of TB has not been extensively investigated, recent studies have suggested a role for IL-17-producing CD4þ T helper cells (Th17 cells). Th17 cells are a distinct subtype of differentiated CD4þ T cells that play crucial functions in host defence. They were also shown to provide protection against certain bacterial and fungal pathogens [55]. Dysregulated Th17 cell responses mediate a variety of autoimmune inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease (IBD) and multiple sclerosis (MS). While Th17 cells were increased in BCG-infected IFN-g-deficient mice there was no change in bacterial clearance [56]. One study suggested that IL-17 produced in response to M. tb infection during the primary immune response inhibited effective secondary immune responses [57]. Although their protective role in primary immunity against TB is unclear, several studies have provided evidence for a protective role of Th17 cells in secondary immune responses against M. tb [58,59]. For example, in IFN-g-deficient mice, BCG infection induces strong IL-17-dependent memory responses, which provides protection against disease upon subsequent challenge with M. tb [59]. 6.3. Modulation of host miRNA expression by ESAT6 MicroRNAs (miRNAs) are endogenous regulators of gene expression and function. They are highly conserved small (18e25 nucleotides) non-coding RNAs that bind with the 30 un-translated region (UTR) of their target mRNA and thus block its translation. miRNAs regulate the differentiation and activation of monocytes and granulocytes, sense the presence of pathogens, and are involved in inflammatory responses and antiviral immunity [60]. Apart from their broad functions in different biological processes they also play critical regulatory roles in innate and adaptive immunity. Many micro-RNAs such as miR29, miR146a and miR155 have been shown to inhibit crucial molecules of pro-inflammatory signalling pathways. It has been clearly demonstrated that M. bovis upregulates miR29 resulting in the suppression of IFN-g production in mice [61]. A recent report indicated that miR146a is upregulated in dendritic cells in response to infection by M. tb [62]. miR146a regulates production of IL-6 in dendritic cells and functions as a negative feedback regulator in TLR signalling by targeting the IL-1 receptor-associated kinase (IRAK) 1 and the TNF receptor-associated factor (TRAF) 6. Inhibition of IRAK1 and TRAF6 expression by miR146a suppresses NF-kB activity and induction of a variety of cytokines such as IL-6, IL-8, IL-1b and TNF-a [63]. Recently, it has been shown that ESAT6 upregulates the expression of miR155 affecting expression of BTB and CNC homology 1 (Bach1) and SH2-containing inositol 5phosphatase (SHIP1), which is further linked with the intracellular survival of M. tb in RAW264.7 and murine bone marrow derived macrophages [64]. miR155 also regulates IL-6
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production from the macrophage infected with M. tb. It is important to note here that IL-6 deficient animals are hyper susceptible to M. tb infection [65]. Therefore, it is highly likely that M. tb regulates multiple miRNAs viz. miR155 and miR146a through ESAT6 to control IL-6 production as a survival strategy in the phagocytes of susceptible hosts [10,64]. 6.4. Changes in the cytokine production profile of antigen presenting cells induced by ESAT6 Coordination between cells of the innate and adaptive immune systems is mediated by secretion of distinct cytokines. Upon interaction with mycobacteria, cells of the innate immune system secrete a variety of pro- and anti-inflammatory cytokines that direct the differentiation of distinct effector T cell subsets from naı¨ve CD4þ T cell precursors. The simultaneous presence of both IL-6 and TGF-b has been shown to be required for the differentiation of Th17 cells [66,67]. Production of large amounts of both of these cytokines has been observed by macrophages infected with M. tb, suggesting that infection with this pathogen aids in Th17 cell differentiation [68]. Findings from our laboratory have suggested that the ESAT6 protein induces IL-6 and TGF-b in dendritic cells in a TLR2- and MyD88-dependent manner, and that this generates an environment that is conducive to the differentiation of Th17 cells in the lung [10]. We recently also reported that TGF-b is dispensable for the Th17 cell differentiation [69] but instead promotes Th17 cell responses by inhibiting Th1 and Th2 cell differentiation. Consistent with this notion, IL-6deficient mice showed a marginally increased bacterial burden in the initial phase of M. tb infection, suggesting a minor protective role of IL-6 confined to cells of the innate immune response [69]. Taken together, these findings suggested that Th1 and Th17 cells are protective whereas Th2 cells assist in the progression of TB disease. Both the virulent M. tb strain H37Rv and BCG induce production of IL-12p40, a cytokine required for the differentiation of Th1 cells. In sharp contrast, IL-6 and TGF-b were induced by H37Rv but not BCG, suggesting that BCG is unable to induce Th17 cell differentiation in the infected mice [10]. Studies from our laboratory have provided evidence that H37Rv and BCG::RD1 induce both Th1 and Th17 cell responses whereas an H37Rv mutant with a deletion of the RD1 region (H37RvDRD1) selectively induces Th1 cell responses [10]. Consequently, inclusion of the RD1 region in BCG improved its vaccine efficacy. When a neutralizing antibody against IL-17 was used, the vaccine potential of both H37Rv and BCG::RD1 were drastically reduced. Chatterjee, S. et al. (2011) further showed that IL-6 and TGF-b production were also diminished when H37RvDRD1 or BCG were used to infect dendritic cells, whereas infection with BCG::RD1 induced levels of these cytokines similar to H37Rv. This report also showed that production of these cytokines by dendritic cells upon infection with H37Rv or BCG::RD1 was dependent on activation of the TLR2/MyD88 signalling pathway. Chatterjee, S. et al. (2011) observed lower
expression levels of miR146a, a negative regulator of IL-6 production, in dendritic cells infected with H37Rv or BCG::RD1 as compared with H37RvDRD1 or BCG. The differential production of IL-6 and TGF-b were thought to be due to the PAMPs encoded within the RD1 region. Since ESAT6 induces protective immune responses and because the H37RvDRD1 mutant fails to induce Th17 cell responses, it is likely that ESAT6 is responsible for inducing Th17 cellpolarizing cytokines in antigen presenting cells. In agreement with these findings, dendritic cells treated with purified ESAT6 protein produced IL-6 and TGF-b in a TLR2/MyD88 dependent manner, and this promoted Th17 cell differentiation (Fig. 2). Infection of ESAT6-treated dendritic cells with BCG or stimulation of these cells with lipopolysaccharide (LPS) downregulated miR146a expression. However, these experiments were unable to exclude a contribution of other gene products encoded within the RD1 region in mounting protective immune responses. Overall these findings indicated that ESAT6 interacts with TLR2 [10,44] and creates a cytokine environment that aids in the differentiation of Th17 cells that provide protection against TB. 7. Conclusions and future perspectives It is now well established that vaccines based on single antigens are unable to provide adequate protection against TB. Therefore, multi-subunit vaccines containing multiple epitopes are needed to provide improved protection against the disease. Such an approach might be able to provide protection in diverse and genetically heterogeneous human populations. Immune responses could be boosted by supplementing the live attenuated BCG vaccine with antigenic, recombinant proteins encoded within the RD1 region of M. tb. Incorporation of ESAT6 in BCG improves the efficacy of this vaccine. Yang X et al. 2011, showed that recombinant BCG strains expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and ESAT6 induce increased antibody titres. They also showed that recombinant BCG is able to enhance protective immune responses by increasing proliferation of splenocytes and thus enhancing the percentage of antigen-specific CD4þ and CD8þ T cells [70]. Being a target of both CD4þ and CD8þ T cells, ESAT6 has been proposed as a potential vaccine candidate. ESAT6 also induces secretion of IFN-g by the peripheral blood mononuclear cells isolated from TB patients [32]. As the RD1 region does not BCG
RD1
H37Rv miR146a RD1
ESAT-6
IL-6 β TGF-β
Th17
Fig. 2. Schematic diagram for the differentiation of Th17 cells after Mycobacterium tuberculosis infection. The ESAT6 protein, which is present in H37Rv but not in BCG, directs Th17 cell differentiation by inducing IL-6 and TGF-b production in dendritic cells by downregulating miR146a.
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have a homologous counterpart in humans, immunization with ESAT6 as part of a vaccine against TB should not have the risk of inducing autoimmunity. Due to its specificity for active TB, i.e. its capacity to differentiate TB patients from BCG-vaccinated persons, and its ability to induce IFN-g secretion by T cells, measuring immune responses against ESAT6 could be employed for diagnosis of M. tb infection [32]. Acknowledgements This work was supported by grants from The WellcomeDBT India Alliance and the Department of Biotechnology, Government of India; and by the International Center for Genetic Engineering and Biotechnology, New Delhi, India. References [1] G.L. Mandell, Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases, Elsevier, Churchill, Livingstone, 2010. [2] WHO, Global Tuberculosis Control (2011). [3] R.J. O’Brien, Drug-resistant tuberculosis: etiology, management and prevention, Semin. Respir. Infect. 2 (1994) 104e112. [4] B.R. Bloom, P.M. Small, The evolving relation between humans and Mycobacterium tuberculosis, N. Engl. J. Med. 338 (1998) 677e678. [5] V. Kumar, A.K. Abbas, N. Fausto, R.N. Mitchell, Robbins Basic Pathology, Saunders/Elsevier, Philadelphia, 2007. [6] E.N. Houben, L. Nguyen, J. Pieters, Interaction of pathogenic mycobacteria with the host immune system, Curr. Opin. Microbiol. 9 (2006) 76e85. [7] P. Peyron, J. Vaubourgeix, Y. Poquet, F. Levillain, C. Botanch, F. Bardou, M. Daffe, J.F. Emile, B. Marchou, P.J. Cardona, C. de Chastellier, F. Altare, Foamy macrophages from tuberculous patients’ granulomas constitute a nutrient-rich reservoir for M. tuberculosis persistence, PLoS Pathog. 4 (2008) e1000204. [8] D. Brites, S. Gagneux, Old and new selective pressures on Mycobacterium tuberculosis, Infect. Genet. Evol. 12 (2012) 678e685. [9] N.W. Schluger, W.N. Rom, The host immune response to tuberculosis, Am. J. Respir. Crit. Care Med. 157 (1998) 679e691. [10] S. Chatterjee, V.P. Dwivedi, Y. Singh, I. Siddiqui, P. Sharma, L. Van Kaer, D. Chattopadhyay, G. Das, Early secreted antigen ESAT6 of Mycobacterium tuberculosis promotes protective T helper 17 cell responses in a toll-like receptor-2-dependent manner, PLoS Pathog. 7 (2011) e1002378. [11] X. Wang, P.F. Barnes, K.M. Dobos-Elder, J.C. Townsend, Y.T. Chung, H. Shams, S.E. Weis, B. Samten, ESAT6 inhibits production of IFNgamma by Mycobacterium tuberculosis-responsive human T cells, J. Immunol. 182 (2009) 3668e3677. [12] R.K. Pai, M. Convery, T.A. Hamilton, W.H. Boom, C.V. Harding, Inhibition of IFN-gamma-induced class II transactivator expression by a 19kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion, J. Immunol. 171 (2003) 175e184. [13] A.L. Sorensen, S. Nagai, G. Houen, P. Andersen, A.B. Andersen, Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis, Infect. Immun. 63 (1995) 1710e1717. [14] A.S. Pym, P. Brodin, L. Majlessi, R. Brosch, C. Demangel, A. Williams, K.E. Griffiths, G. Marchal, C. Leclerc, S.T. Cole, Recombinant BCG exporting ESAT6 confers enhanced protection against tuberculosis, Nat. Med. 9 (2003) 533e539. [15] C. Bonah, The ’experimental stable’ of the BCG vaccine: safety, efficacy, proof, and standards, 1921e1933, Stud. Hist. Philos. Biol. Biomed. Sci. 36 (2005) 696e721. [16] P.E. Fine, J.M. Ponnighaus, N. Maine, J.A. Clarkson, L. Bliss, Protective efficacy of BCG against leprosy in Northern Malawi, Lancet 2 (1986) 499e502.
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