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Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages

Open AccessPublished:April 28, 2017DOI:https://doi.org/10.1074/jbc.M117.775205
      Mycobacterium tuberculosis is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. M. tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. The M. tuberculosis genome encodes 23 such ESAT-6 family proteins. We herein report that M. tuberculosis and M. bovis bacillus Calmette–Guérin infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 lysine 9 (H3K9me2/3). Further, we showed that M. tuberculosis ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that M. tuberculosis esxL induced the expression of nitric-oxide synthase, NO production, and p38 MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of nitric-oxide synthase, p38 MAPK, and G9a abrogated H3K9me2/3, resulting in increased CIITA expression. A chromatin immunoprecipitation assay confirmed that hypermethylation at the promoter IV region of CIITA is mainly responsible for CIITA down-regulation and subsequent antigen presentation. We found that co-culture of macrophages infected with esxL-expressing M. smegmatis and mouse splenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we demonstrate that M. tuberculosis EsxL inhibits antigen presentation by enhancing H3K9me2/3 at the CIITA promoter, thereby repressing its expression through NO and p38 MAPK activation.

      Introduction

      Pathogenic bacteria employ various host immune evasion strategies to facilitate their survival in the host cells. One such mechanism involves the induction of epigenetic modifications in the host DNA, histone proteins, and RNA by bacterial proteins (
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      Modifiable risk factors in periodontal disease: epigenetic regulation of gene expression in the inflammatory response.
      ,
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      Can the battle against tuberculosis gain from epigenetic research?.
      ). Various bacterial virulent proteins have been shown to promote host chromatin and/or histone modifications via different signaling cascades. For example, Shigella flexneri, Listeria monocytogenes, and Helicobacter pylori regulate the p38 mitogen-activated protein kinase (MAPK) pathway by promoting histone H3 phosphorylation and acetylation processes, which subsequently modulate the secretion of various cytokines and chemokines in infected cells (
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      ). S. flexneri infection inhibited MAPK-dependent histone H3 serine 10 phosphorylation that impaired the recruitment of nuclear factor-κB (NF-κB) at the interleukin-8 (IL-8) promoter (
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      ). On the other hand, L. monocytogenes promoted histone H3 Lys-8 acetylation, resulting in transcriptional activation of IL-8 via the MAPK pathway (
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      ).
      Tuberculosis (TB),
      The abbreviations used are: TB
      tuberculosis
      ROS
      reactive oxygen species
      CIITA
      class-II transactivator
      BCG
      bacillus Calmette–Guérin
      iNOS
      inducible nitric-oxide synthase
      H3K4 and H3K9
      histone H3 Lys-4 and Lys-9, respectively
      me1
      me2, and me3, mono-, di-, and trimethylation, respectively
      PMA
      phorbol 12-myristate 13-acetate
      qRT-PCR
      quantitative RT-PCR
      EHMT2
      euchromatic histone-lysine N-methyltransferase 2
      HDAC
      histone deacetylase
      pERK and pp38
      phosphorylated ERK and p38, respectively
      RIPA
      radioimmune precipitation assay.
      caused by an intracellular pathogen, Mycobacterium tuberculosis, is a life-threatening disease that infects 9 million people and kills more than 1.5 million people every year worldwide (
      • World Health Organization
      ). According to the World Health Organization report, one-third of the global population is latently infected with M. tuberculosis, but only 5–10% of people with latent TB develop into an active TB disease (
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      ). The underlying mechanisms responsible for this adaptation are poorly understood. Although several reports are available on the correlation of other bacterial infections and epigenetics in the disease outcome, very little is known about the dynamics of epigenetic changes during mycobacterial infection. Macrophages, the primary host cells of M. tuberculosis, play a crucial role in recognition, phagocytosis, and killing of mycobacteria. Non-pathogenic mycobacteria, such as M. smegmatis, are readily killed by macrophages, whereas pathogenic mycobacteria (M. tuberculosis) are able to survive for an extended period of time by manipulating the macrophage immune functions (
      • Pieters J.
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      ). These include prevention of phagolysosome fusion, inhibition of phagosome acidification due to depletion of vesicular proton-ATPase, evasion from toxic effects of nitric oxide (NO) and reactive oxygen species (ROS), suppression of protective cytokine synthesis and Th-1 (T-helper-1) responses, and inhibition of apoptosis (
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      ).
      Recently, a few reports have demonstrated that M. tuberculosis infection induces epigenetic modifications in host cells to aid its replication, propagation, and protection from host immune responses (
      • Esterhuyse M.M.
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      ,
      • Kumar P.
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      • Siddiqui I.
      • Vora H.
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      ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner.
      ,
      • Siddle K.J.
      • Deschamps M.
      • Tailleux L.
      • Nédélec Y.
      • Pothlichet J.
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      ). Mycobacterial cell wall protein, LpqH, was shown to block interferon-γ (IFN-γ)-induced transcription of class-II transactivator (CIITA) by SWI/SNF binding and histone deacetylation at the CIITA promoter (
      • Pennini M.E.
      • Pai R.K.
      • Schultz D.C.
      • Boom W.H.
      • Harding C.V.
      Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling.
      ). IFN-γ induces the expression of major histocompatibility complex class II (MHC class II) by activating the transcription of CIITA (
      • Boehm U.
      • Klamp T.
      • Groot M.
      • Howard J.C.
      Cellular responses to interferon-γ.
      ). Another study has shown that M. tuberculosis down-regulates HLA-DR transcription and MHC-II by inhibiting IFN-γ-dependent histone acetylation and by recruiting mSin3A repressor at the HLA-DR promoter (
      • Wang Y.
      • Curry H.M.
      • Zwilling B.S.
      • Lafuse W.P.
      Mycobacteria inhibition of IFN-γ induced HLA-DR gene expression by up-regulating histone deacetylation at the promoter region in human THP-1 monocytic cells.
      ).
      Inducible nitric-oxide synthase (iNOS) catalyzes the formation of nitric oxide (NO), which helps in bacterial clearance, including M. tuberculosis (
      • Rich E.A.
      • Torres M.
      • Sada E.
      • Finegan C.K.
      • Hamilton B.D.
      • Toossi Z.
      Mycobacterium tuberculosis (MTB)-stimulated production of nitric oxide by human alveolar macrophages and relationship of nitric oxide production to growth inhibition of MTB.
      ,
      • Shiloh M.U.
      • Nathan C.F.
      Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria.
      ). It has been shown that NO knock-out mice were more susceptible to M. tuberculosis infection (
      • Yang C.S.
      • Yuk J.M.
      • Jo E.K.
      The role of nitric oxide in mycobacterial infections.
      ). In addition to its antibacterial properties, NO also mediates nitration, nitrosation, and nitrosylation of key signaling molecules that determine the fate of macrophages and dendritic cells during bacterial infection (
      • Ehrt S.
      • Schnappinger D.
      • Bekiranov S.
      • Drenkow J.
      • Shi S.
      • Gingeras T.R.
      • Gaasterland T.
      • Schoolnik G.
      • Nathan C.
      Reprogramming of the macrophage transcriptome in response to interferon-γ and Mycobacterium tuberculosis: signaling roles of nitric-oxide synthase-2 and phagocyte oxidase.
      ,
      • Jagannath C.
      • Actor J.K.
      • Hunter Jr., R.L.
      Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis.
      ,
      • MacMicking J.
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      • Nathan C.
      Nitric oxide and macrophage function.
      ,
      • Nicholson S.
      • Bonecini-Almeida Mda G.
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      • Nathan C.
      • Xie Q.W.
      • Mumford R.
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      • Calaycay J.
      • Geng J.
      • Boechat N.
      • Linhares C.
      • Rom W.
      • Ho J.L.
      Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis.
      ,
      • Serbina N.V.
      • Salazar-Mather T.P.
      • Biron C.A.
      • Kuziel W.A.
      • Pamer E.G.
      TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection.
      ). NO was shown to induce CIITA and MHC-II inhibition by signaling cross-talks along NOTCH-PKCδ-MAPK-NFκB-KLF4 pathway during M. bovis BCG infection (
      • Ghorpade D.S.
      • Holla S.
      • Sinha A.Y.
      • Alagesan S.K.
      • Balaji K.N.
      Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress major histocompatibility complex II expression during Mycobacterium bovis bacillus Calmette-Guerin infection.
      ).
      M. tuberculosis early secretory antigenic target protein-6 (ESAT-6; esxA) is a known virulent protein as well as T-cell antigenic determinant (
      • Brodin P.
      • Rosenkrands I.
      • Andersen P.
      • Cole S.T.
      • Brosch R.
      ESAT-6 proteins: protective antigens and virulence factors?.
      ). M. tuberculosis ESAT-6 protein is involved in the cytosolic escape of bacteria by inducing pore formation in the phagosomal membrane (

      Manzanillo, P. S., Shiloh, M. U., Portnoy, D. A., and Cox, J. S., Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages. Cell Host Microbe, 11, 469–480.

      ,
      • Simeone R.
      • Bobard A.
      • Lippmann J.
      • Bitter W.
      • Majlessi L.
      • Brosch R.
      • Enninga J.
      Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death.
      ). Previously, ESAT-6 protein was also reported to decrease histone H4 acetylation and H3K4 methylation at the CIITA promoter (pI) (
      • Kumar P.
      • Agarwal R.
      • Siddiqui I.
      • Vora H.
      • Das G.
      • Sharma P.
      ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner.
      ). There are at least 23 such ESAT-6 family proteins present in the M. tuberculosis genome. However, the functions of many of them are still unknown. Herein, we show that M. tuberculosis EsxL, a previously uncharacterized member of the ESAT-6-like family proteins encoded by Rv1198, suppresses antimycobacterial defense mechanisms of macrophages by inhibiting the expression of CIITA and subsequently MHC-II molecules. Further mechanistic studies revealed that CIITA and MHC-II down-regulation by EsxL was due to induction of H3K9 hypermethylation in the CIITA-IV promoter region (pIV), as determined by Western-blotting and chromatin immunoprecipitation (ChIP) assays. We further show that recombinant M. smegmatis expressing esxL (M. smegmatis esxL) induced the synthesis of p38 MAPK, iNOS, and NO that promoted H3K9me2/3 at the CIITA promoter via up-regulation of G9a (also known as euchromatic histone-lysine N-methyltransferase 2, EHMT2), whereas the MtbΔesxL mutant down-regulated H3K9me2/3. EsxL-mediated H3K9me2/3 also resulted in inhibition of antigen presentation and secretion of interleukin-2 (IL-2), a key cytokine involved in T-cell activation. In summary, we identified another mechanism by which M. tuberculosis aids its persistence by repressing CIITA/MHC-II via G9a-, p38-, and NO-dependent H3K9me2/3 at promoter IV of CIITA.

      Results

      M. smegmatis EsxL shows prolonged intracellular survival in RAW 264.7 and THP-1 cells

      M. tuberculosis ESAT-6 is known as a potent virulence as well as antigenic determinant (
      • Brodin P.
      • Rosenkrands I.
      • Andersen P.
      • Cole S.T.
      • Brosch R.
      ESAT-6 proteins: protective antigens and virulence factors?.
      ,
      • Geluk A.
      • van Meijgaarden K.E.
      • Franken K.L.
      • Subronto Y.W.
      • Wieles B.
      • Arend S.M.
      • Sampaio E.P.
      • de Boer T.
      • Faber W.R.
      • Naafs B.
      • Ottenhoff T.H.
      Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis.
      ). Recently, we have shown that M. tuberculosis Rv2346c, a member of the ESAT-6 like family proteins, endows bacterial persistence by dampening the antibacterial effector functions through genomic instability and autophagy in macrophages (
      • Mohanty S.
      • Jagannathan L.
      • Ganguli G.
      • Padhi A.
      • Roy D.
      • Alaridah N.
      • Saha P.
      • Nongthomba U.
      • Godaly G.
      • Gopal R.K.
      • Banerjee S.
      • Sonawane A.
      A mycobacterial phosphoribosyltransferase promotes bacillary survival by inhibiting oxidative stress and autophagy pathways in macrophages and zebrafish.
      ). Using M. smegmatis as a surrogate model, we (
      • Mohanty S.
      • Jagannathan L.
      • Ganguli G.
      • Padhi A.
      • Roy D.
      • Alaridah N.
      • Saha P.
      • Nongthomba U.
      • Godaly G.
      • Gopal R.K.
      • Banerjee S.
      • Sonawane A.
      A mycobacterial phosphoribosyltransferase promotes bacillary survival by inhibiting oxidative stress and autophagy pathways in macrophages and zebrafish.
      ,
      • Padhi A.
      • Naik S.K.
      • Sengupta S.
      • Ganguli G.
      • Sonawane A.
      Expression of Mycobacterium tuberculosis NLPC/p60 family protein Rv0024 induce biofilm formation and resistance against cell wall acting anti-tuberculosis drugs in Mycobacterium smegmatis.
      ,
      • Mohanty S.
      • Dal Molin M.
      • Ganguli G.
      • Padhi A.
      • Jena P.
      • Selchow P.
      • Sengupta S.
      • Meuli M.
      • Sander P.
      • Sonawane A.
      Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages.
      ) and several other groups (
      • Yaseen I.
      • Kaur P.
      • Nandicoori V.K.
      • Khosla S.
      Mycobacteria modulate host epigenetic machinery by Rv1988 methylation of a non-tail arginine of histone H3.
      ,
      • Sethi D.
      • Mahajan S.
      • Singh C.
      • Lama A.
      • Hade M.D.
      • Gupta P.
      • Dikshit K.L.
      Lipoprotein, LprI, of Mycobacterium tuberculosis acts as a lysozyme inhibitor.
      ) have proved the functions of several M. tuberculosis proteins in pathogenesis. Similarly, in this study, we ectopically expressed one of the M. tuberculosis ESAT-6 family proteins, EsxL, encoded by Rv1198, in M. smegmatis M. smegmatis esxL) and also constructed M. tuberculosis esxL deletion mutant (MtbΔesxL) and studied its role in pathogenesis. Fig. 1A shows genetic organization of esxL in the M. tuberculosis genome. It is located downstream of another ESAT-6-like protein, EsxK, encoded by Rv1197. EsxL has previously been identified from the membrane fraction (
      • Gu S.
      • Chen J.
      • Dobos K.M.
      • Bradbury E.M.
      • Belisle J.T.
      • Chen X.
      Comprehensive proteomic profiling of the membrane constituents of a Mycobacterium tuberculosis strain.
      ) and culture filtrates (
      • Målen H.
      • Berven F.S.
      • Fladmark K.E.
      • Wiker H.G.
      Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv.
      ) of M. tuberculosis with unknown function. It is reported that immunization of BALB/c mice with Rv1198 induced a pro-inflammatory response with elevated levels of tumor necrosis factor-α (TNF-α) and IL-6, along with low induction of IFN-γ, IL-2, and IL-10 (
      • Pandey H.
      • Tripathi S.
      • Srivastava K.
      • Tripathi D.K.
      • Srivastava M.
      • Kant S.
      • Srivastava K.K.
      • Arora A.
      Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv.
      ). EsxL has been assigned to the ESAT-6 family proteins, with Rv1793, Rv1037c, and Rv2346c as its members (
      • Alderson M.R.
      • Bement T.
      • Day C.H.
      • Zhu L.
      • Molesh D.
      • Skeiky Y.A.
      • Coler R.
      • Lewinsohn D.M.
      • Reed S.G.
      • Dillon D.C.
      Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.
      ). Despite these important characteristics shown by EsxL, its role in pathogenesis is still unknown. Comparative genome analyses revealed that the M. bovis BCG genome contains Mb1230, an orthologue of M. tuberculosis esxL, whereas the M. smegmatis genome does not contain any esxL orthologue (Tuberculist database).
      Figure thumbnail gr1
      Figure 1Genetic organization, growth analysis, bacterial survival, and MtbΔesxL mutant construction. A, schematic representation of esxL in the M. tuberculosis H37Rv genome. RAW 264.7 (B) and THP-1 (C) were infected with M. smegmatis (Msm) pSMT3 and recombinant M. smegmatis esxL strains. The cells were lysed, and intracellular survival was determined 1, 8, and 24 h post-infection by a cfu assay. D, in vitro growth curve of the M. smegmatis WT, M. smegmatis pSMT3, and recombinant M. smegmatis esxL was determined by growing bacteria in 7H9 medium and measuring OD (O.D600 nm). E, extracellular expression of the esxL transcript was measured by qRT-PCR after growing M. smegmatis esxL in vitro for 4, 12, and 24 h. RNA was isolated at the respective time points. cDNA was synthesized, and the expression of esxL was determined using qRT-PCR. Transcript levels are represented relative to mRNA -fold change of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. F, intracellular expression of esxL transcript was measured by qRT-PCR. RNA was isolated from M. smegmatis esxL-infected macrophages at different time points. cDNA was synthesized, and the expression of esxL was determined using qRT PCR. Transcript levels are represented relative to mRNA level of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. G, schematic representation of construction of MtbΔesxL mutant by homologous recombination. The location of primers used for the confirmation of deletion mutant generation is depicted. H, confirmation of MtbΔesxL mutant generation. F1 and R2 primers were designed beyond the flanks, whereas R1 and F2 primers anneal to sacB-hygr cassette. PCR using F1 and R1 is expected to give no product with the M. tuberculosis (lane 1) and ∼1.3 kb with the MtbΔesxL (lane 2); F2-R2 primer sets were expected to give no product with M. tuberculosis and ∼1.5 kb in MtbΔesxL mutant. Amplification of udgB with gene-specific primers was performed as a control. The experiments were performed in triplicate (n = 3). Results are shown as mean ± S.D. (error bars). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns, not significant.
      Because EsxL was found to be a member of the ESAT-6 family proteins, a key virulence factor, we compared the intracellular bacillary persistence of M. smegmatis harboring pSMT3 vector (M. smegmatis pSMT3) and M. smegmatis expressing esxL (M. smegmatis esxL) strains in mouse macrophage RAW 264.7 and phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. The infected cells were lysed at different time points post-infection, and the bacterial survival was determined by colony-forming unit (cfu) enumeration. The bacterial input and time 0 (T0) counts were determined to calculate the intracellular bacterial survival. The recombinant M. smegmatis esxL strain showed significantly high bacterial burden in RAW 264.7 (p ≤ 0.001; Fig. 1B) and THP-1 cells (p ≤ 0.001; Fig. 1C) when compared with M. smegmatis pSMT3 strain after 24 h of infection. We did not observe any differences in the growth patterns of M. smegmatis wild-type (MsmWT), M. smegmatis pSMT3, and M. smegmatis esxL strains (Fig. 1D), suggesting that the observed increased bacterial survival was not due to differences in the growth kinetics of bacteria. The extracellular and intracellular expressions of esxL were determined by qRT-PCR at the 4-, 12-, and 24-h time points. As shown, esxL was expressed under both in vitro (Fig. 1E) and ex vivo (Fig. 1F) conditions. In conclusion, the above data showed that M. tuberculosis esxL has a role in increased bacterial persistence inside the macrophages. The generation of MtbΔesxL is shown in Fig. 1, G and H.

      EsxL induces NO production and iNOS expression in macrophages

      iNOS, which catalyzes formation of NO, has immunomodulatory activities that determine the outcome of M. tuberculosis infection (
      • Bose M.
      • Farnia P.
      • Sharma S.
      • Chattopadhya D.
      • Saha K.
      Nitric oxide dependent killing of Mycobacterium tuberculosis by human mononuclear phagocytes from patients with active tuberculosis.
      ). In addition to its antibacterial properties, NO is also known as a key regulator in the initiation and maintenance of anti-TB protective immunity (
      • Bogdan C.
      Nitric oxide and the immune response.
      ) and is known to modulate the cellular signaling pathways that can either support or inhibit the bacterial growth, depending upon the cytokine milieu (
      • Kanangat S.
      • Meduri G.U.
      • Tolley E.A.
      • Patterson D.R.
      • Meduri C.U.
      • Pak C.
      • Griffin J.P.
      • Bronze M.S.
      • Schaberg D.R.
      Effects of cytokines and endotoxin on the intracellular growth of bacteria.
      ). Previously, ESAT-6 was shown to induce NO production in macrophages (
      • Singh G.
      • Singh B.
      • Trajkovic V.
      • Sharma P.
      Mycobacterium tuberculosis 6 kDa early secreted antigenic target stimulates activation of J774 macrophages.
      ). We found significantly higher NO production in M. smegmatis esxL-infected RAW 264.7 cells as compared with M. smegmatis pSMT3-infected cells at the indicated time points (p ≤ 0.05 and p ≤ 0.01; Fig. 2A). Similarly, we observed an ∼18-fold increase in iNOS at transcriptional (p ≤ 0.001; Fig. 2B) and significant increase at translational levels (Fig. 2C) in M. smegmatis esxL-infected macrophages. Immunofluorescence studies using iNOS-specific antibody also showed significantly increased expression in M. smegmatis esxL-infected macrophages as compared with uninfected and M. smegmatis pSMT3-infected cells (Fig. 2D). On the contrary, decreased iNOS expression was observed in MtbΔesxL-infected THP-1 cells when compared with M. tuberculosis-infected cells (Fig. 2E). We did not observe any significant differences in the production of ROS in infected macrophages (data not shown), indicating that M. tuberculosis esxL specifically induces NO production in macrophages.
      Figure thumbnail gr2
      Figure 2Determination of NO production and iNOS expression in M. smegmatis pSMT3-, M. smegmatis esxL-, M. tuberculosis H37Rv-, and MtbΔesxL-infected cells. A, RAW 264.7 cells were infected with M. smegmatis (Msm) pSMT3 and recombinant M. smegmatis esxL for 2 h. The production of NO was quantified using Griess reagent 24 and 48 h after infection. B, transcript levels of iNOS in M. smegmatis pSMT3- and M. smegmatis esxL-infected macrophages were determined by qRT-PCR 24 h post-infection. GAPDH was taken as internal control. iNOS expression was checked by Western blotting (C) and fluorescence-microscopic analysis (D) using iNOS-specific antibody in M. smegmatis pSMT3- and M. smegmatis esxL-infected macrophages. E, THP-1 cells were infected with M. tuberculosis (Mtb) and MtbΔesxL mutant for 2 h. The level of iNOS was checked by Western blotting 24 h post-infection. The experiments were performed in triplicate (n = 3). Results are shown as mean ± S.D. (error bars). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

      EsxL down-regulates MHC-II and CIITA in macrophages

      Pathogenic mycobacteria are known to down-regulate the surface expression of MHC-II molecules in macrophages (
      • Pai R.K.
      • Convery M.
      • Hamilton T.A.
      • Boom W.H.
      • Harding C.V.
      Inhibition of IFN-γ-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion.
      ). The MHC-II-dependent antigen presentation is tightly regulated by a key transcription factor, CIITA. Mice deficient for CIITA showed a marked reduction in MHC-II expression (
      • Chang C.H.
      • Guerder S.
      • Hong S.C.
      • van Ewijk W.
      • Flavell R.A.
      Mice lacking the MHC class II transactivator (CIITA) show tissue-specific impairment of MHC class II expression.
      ). It has been shown that M. bovis BCG infection inhibited MHC-II expression by inducing NO production in macrophages (
      • Ghorpade D.S.
      • Holla S.
      • Sinha A.Y.
      • Alagesan S.K.
      • Balaji K.N.
      Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress major histocompatibility complex II expression during Mycobacterium bovis bacillus Calmette-Guerin infection.
      ). In view of these reports, we checked the expression of MHC-II and CIITA in M. tuberculosis, MtbΔesxL, M. smegmatis pSMT3, and M. smegmatis esxL-infected macrophages. As shown, M. smegmatis esxL infection abrogated CIITA expression at both transcriptional (p ≤ 0.01; Fig. 3A) and translational levels (Fig. 3B). M. tuberculosis-infected THP-1 cells also showed a time-dependent decrease in CIITA expression (Fig. 3C), whereas increased CIITA expression was observed in MtbΔesxL-infected cells (Fig. 3D). We further confirmed the effect of CIITA on MHC-II expression. A significant decrease in MHC-II expression was observed at both transcriptional (p ≤ 0.01; Fig. 3E) and translational (Fig. 3F) levels in M. smegmatis esxL-infected RAW 264.7 macrophages when compared with M. smegmatis pSMT3-infected cells. Flow cytometry analysis also showed a significant decrease in MHC-II expression in M. smegmatis esxL-infected THP-1 (Fig. 3G) and RAW 264.7 macrophages (Fig. 3H). In agreement with previous reports, we also observed a time-dependent decrease in MHC-II expression in M. tuberculosis-infected THP-1 cells (Fig. 3I) when compared with MtbΔesxL-infected cells (Fig. 3J). Altogether, these data suggest that M. smegmatis esxL-, M. bovis BCG-, and M. tuberculosis-mediated MHC-II inhibition is due to down-regulation of CIITA.
      Figure thumbnail gr3
      Figure 3Expression of CIITA and MHC-II in M. smegmatis pSMT3-, M. smegmatis esxL, M. tuberculosis H37Rv-, and MtbΔesxL-infected macrophages. A, RAW 264.7 cells were infected with M. smegmatis (Msm) pSMT3 and recombinant M. smegmatis esxL for 2 h. The transcript level of CIITA was quantified using qRT-PCR 24 h post-infection. Uninfected cells were used as control. B, RAW 264.7 cells were infected with M. smegmatis pSMT3 and recombinant M. smegmatis esxL for 2 h. Cell lysates were prepared at the indicated time points (12, 24, and 48 h). CIITA expression was checked by Western blotting. C, the level of CIITA after M. tuberculosis (Mtb) infection was checked by Western blotting. Expression of CIITA protein was determined in M. tuberculosis H37Rv-infected THP-1 cells by Western blotting using an antibody specific to CIITA after 24, 48, and 72 h of infection. D, Western-blotting analysis to check expression of CIITA in THP-1 cells infected with M. tuberculosis and MtbΔesxL after 48 h of infection. The expression level of MHC-II was checked at both transcriptional (E) and translational (F) levels in RAW 264.7 cells infected with M. smegmatis pSMT3 and recombinant M. smegmatis esxL strains 24 h post-infection. THP-1 (G) and RAW 264.7 cells (H) were infected with M. smegmatis pSMT3 and recombinant M. smegmatis esxL strains for 2 h. Flow cytometry analysis of MHC-II expression was determined by using anti-MHCII antibody and analyzed through 10,000 gated cells 24 h post-infection. I, expression of MHCII protein was determined in M. tuberculosis H37Rv-infected THP-1 cells by Western blotting using an antibody specific to MHCII after 24, 48, and 72 h post-infection. J, the level of MHCII was checked in M. tuberculosis H37Rv- and MtbΔesxL-infected THP-1 cells by Western blotting after 24 and 48 h post-infection. For qRT-PCR, GAPDH was taken as an internal control. The experiments were performed in triplicate (n = 3). Results are shown as mean ± S.D. (error bars); **, p ≤ 0.01.

      M. smegmatis EsxL infection down-regulates IL-2 and IL-10 and up-regulates IL-6 and TNF-α production in macrophages

      It is known that inhibition of antigen presentation prevents T-cell activation (
      • Nelson B.H.
      IL-2, regulatory T cells, and tolerance.
      ). As mentioned above, IL-2 is a key cytokine involved in T-cell activation (
      • Boyman O.
      • Sprent J.
      The role of interleukin-2 during homeostasis and activation of the immune system.
      ,
      • Pennini M.E.
      • Liu Y.
      • Yang J.
      • Croniger C.M.
      • Boom W.H.
      • Harding C.V.
      CCAAT/enhancer-binding protein β and δ binding to CIITA promoters is associated with the inhibition of CIITA expression in response to Mycobacterium tuberculosis 19-kDa lipoprotein.
      ). Therefore, we checked IL-2 levels using the Bioplex cytokine analysis kit. For this, macrophages were first infected with M. smegmatis pSMT3 and M. smegmatis esxL strains, followed by co-culture with BALB/c mouse splenocytes. We found significant down-regulation of IL-2 (p ≤ 0.05; Fig. 4A) and IL-10 (p ≤ 0.05; Fig. 4B) cytokines in supernatant obtained from M. smegmatis esxL-infected cells as compared with M. smegmatis pSMT3 infection after 24 h. However, the presence of G9a inhibitor UNC0638 increased production of both of the cytokines, suggesting that M. smegmatis esxL infection suppressed T-cell activation. In contrast, TNF-α (p ≤ 0.01; Fig. 4C) and IL-6 (p ≤ 0.01; Fig. 4D) were up-regulated in M. smegmatis esxL-infected cells.
      Figure thumbnail gr4
      Figure 4Cytokine analysis. Analysis of IL-2, IL-6, IL-10, and TNF-α cytokines in RAW 264.7 (M) and splenocyte (S) co-cultured cells (M + S) infected with M. smegmatis (Msm) pSMT3 and recombinant M. smegmatis esxL strains. The level of IL-2 (A), IL-10 (B), TNF-α (C), and IL-6 (D) cytokines was determined by using the Bioplex cytokine analysis kit. Infected RAW 264.7 cells were co-cultured with splenocytes isolated from BALB/c mice in the presence and absence of G9a inhibitor (UNC0638), and cell supernatants were collected 24 h post-infection. The experiments were performed in triplicate (n = 3). Results are shown as mean ± S.D. (error bars); *, p ≤ 0.05; **, p ≤ 0.01.

      M. smegmatis EsxL induces histone modification (H3K9 hypermethylation) in macrophages

      A few studies have shown that pathogenic mycobacteria and its antigens induce epigenetic changes to evade host immune responses (
      • Kumar P.
      • Agarwal R.
      • Siddiqui I.
      • Vora H.
      • Das G.
      • Sharma P.
      ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner.
      ,
      • Pennini M.E.
      • Pai R.K.
      • Schultz D.C.
      • Boom W.H.
      • Harding C.V.
      Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling.
      ,
      • Pennini M.E.
      • Liu Y.
      • Yang J.
      • Croniger C.M.
      • Boom W.H.
      • Harding C.V.
      CCAAT/enhancer-binding protein β and δ binding to CIITA promoters is associated with the inhibition of CIITA expression in response to Mycobacterium tuberculosis 19-kDa lipoprotein.
      ). We hypothesized that EsxL might render repressive epigenetic modifications at the CIITA promoter that subsequently inhibit MHC-II-dependent antigen presentation. H3K9me2/3 is involved in transcriptional repression (
      • Bannister A.J.
      • Zegerman P.
      • Partridge J.F.
      • Miska E.A.
      • Thomas J.O.
      • Allshire R.C.
      • Kouzarides T.
      Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.
      ). Therefore, we analyzed the status of H3K9me2/3 in infected macrophages. Indeed, immunoblotting (Fig. 5A) and immunofluorescence (Fig. 5B) analysis showed significantly elevated levels of H3K9me2/3 in M. smegmatis esxL-infected macrophages as compared with control cells. A significant increase in H3K9me2/3 puncta was observed in M. smegmatis esxL-infected cells. We did not observe any significant differences in H3K4me3 and total H3 in M. smegmatis pSMT3- and M. smegmatis esxL-infected macrophages (Fig. 5C), indicating that esxL induces H3K9me2/3 in macrophages.
      Figure thumbnail gr5
      Figure 5Expression of H3K9me2/3 in macrophages. RAW 264.7 cells were infected with M. smegmatis (Msm) pSMT3 and recombinant M. smegmatis esxL for 2 h. The expression of H3K9me2/3 was determined by Western blotting (A) and immunofluorescence microscopy (B) 24 h post-infection. C, Western-blotting analysis of H3K4me3 and total H3 was performed in M. smegmatis pSMT3 and recombinant M. smegmatis esxL-infected macrophages 24 h post-infection. D, expression of H3K9me2/3 was determined in M. bovis BCG-infected RAW 264.7 cells in the presence and absence of G9a inhibitor (UNC0638) 24 h post-infection. E, the level of H3K9me2/3 was determined in M. tuberculosis (Mtb) H37Rv-infected THP-1 by Western blotting using an antibody specific to H3K9me2/3 24, 48, and 72 h post-infection. F, differentiated THP-1 cells were infected with M. tuberculosis and MtbΔesxL for 4 h. The level of H3K9me2/3 was checked by Western blotting 24 and 48 h post-infection. The level of G9a expression was checked in M. smegmatis pSMT3- and recombinant M. smegmatis esxL-infected RAW 264.7 cells (G) and M. tuberculosis- and MtbΔesxL-infected THP-1 cells (H) by qRT-PCR. I, RAW 264.7 cells were treated with UNC0638 (G9a inhibitor), followed by infection with M. smegmatis pSMT3 and recombinant M. smegmatis esxL. Expressions of CIITA and H3K9me2/3 were checked by Western blotting 24 h post-infection. A ChIP assay was performed to check the H3K9me2/3 enrichment at the CIITA promoter (pIV) after infecting RAW 264.7 cells with M. smegmatis pSMT3 and recombinant M. smegmatis esxL without treatment (J) and after treatment with G9a inhibitor (K) 24 h post-infection. Quantification of the data was done by qRT-PCR using specific ChIP primers. The GAPDH promoter was taken as an additional negative control for ChIP qRT-PCR. For qRT-PCR, GAPDH was taken as an internal control. The experiments were performed in triplicate (n = 3). Results are shown as mean ± S.D. (error bars); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns, not significant.

      M. bovis BCG and M. tuberculosis infection induces H3K9me2/3 in macrophages

      To further confirm the role of EsxL in inducing repressive histone modification, we analyzed the expression of H3K9me2/3 in M. bovis BCG-, M. tuberculosis-, and MtbΔesxL-infected macrophages. Concordantly, Western-blotting analysis showed increased levels of H3K9me2/3 in M. bovis BCG (Fig. 5D)- and M. tuberculosis (Fig. 5E)-infected macrophages, suggesting that M. tuberculosis and BCG may down-regulate CIITA expression by inducing H3K9me2/3 in macrophages. In contrast, decreased H3K9me2/3 expression was observed in MtbΔesxL-infected THP-1 cells when compared with M. tuberculosis-infected cells (Fig. 5F).

      M. smegmatis esxL and M. bovis BCG induce H3K9me2/3 modification by up-regulating EHMT2 methyltransferase activity

      To further investigate the mechanism of H3K9me2/3 induction, we studied the activity of methyltransferases in infected macrophages. Several H3K9-specific lysine methyltransferases, such as Eset, KMT1E, and G9a, are involved in H3K9 methylation (
      • Fritsch L.
      • Robin P.
      • Mathieu J.R.
      • Souidi M.
      • Hinaux H.
      • Rougeulle C.
      • Harel-Bellan A.
      • Ameyar-Zazoua M.
      • Ait-Si-Ali S.
      A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex.
      ). Among them, G9a, also known as EHMT2, is a dominant histone methyl transferase responsible for methylation of H3K9 (
      • Tachibana M.
      • Sugimoto K.
      • Nozaki M.
      • Ueda J.
      • Ohta T.
      • Ohki M.
      • Fukuda M.
      • Takeda N.
      • Niida H.
      • Kato H.
      • Shinkai Y.
      G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis.
      ). Transcriptional analysis showed a significant increase in G9a level in M. smegmatis esxL (p ≤ 0.01; Fig. 5G)- and M. tuberculosis (p ≤ 0.001; Fig. 5H)-infected THP-1 cells, whereas MtbΔesxL infection down-regulated G9a expression (p ≤ 0.001; Fig. 5H). Otherwise, treatment with G9a inhibitor (UNC0638) subdued the expression of H3K9me2/3 in M. smegmatis esxL-infected macrophages when compared with untreated cells (Fig. 5I). Similar results were obtained with M. bovis BCG infection, where BCG infection increased the level of H3K9me2/3, whereas treatment with G9a inhibitor reduced H3K9me2/3 level (Fig. 5D). Collectively, these results indicate that M. smegmatis esxL, M. tuberculosis, and M. bovis BCG induce H3K9me2/3 via G9a methyltransferase.
      To assess whether CIITA down-regulation during M. smegmatis esxL infection is dependent on G9a-mediated H3K9me2/3, we checked the expression of CIITA in untreated and G9a inhibitor-treated macrophages. Immunoblotting analysis showed that pretreatment with G9a inhibitor severely reduced the capacity of M. smegmatis esxL to inhibit CIITA expression in macrophages (Fig. 5I), indicating that observed CIITA down-regulation was due to G9a-mediated induction of H3K9me2/3 in infected macrophages.

      ChIP analysis shows that H3K9 hypermethylation occurs at the promoter IV region of CIITA

      A ChIP assay was performed to check H3K9me2/3 in the CIITA promoter. Sequence analysis revealed the presence of three promoter regions (CIITApI, CIITApIII, and CIITApIV) in CIITA (
      • Kumar P.
      • Agarwal R.
      • Siddiqui I.
      • Vora H.
      • Das G.
      • Sharma P.
      ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner.
      ). As shown, M. smegmatis esxL down-regulated CIITA expression by inducing H3K9me2/3 at the promoter IV region of CIITA (p ≤ 0.001; Fig. 5J), whereas no such modification was observed at CIITApI and CIITApIII promoters (data not shown). Moreover, we did not observe any H3K9me2/3 enrichment in M. smegmatis pSMT3-infected cells. Importantly, inhibition of G9a significantly decreased H3K9 hypermethylation at CIITA promoter IV in M. smegmatis esxL-infected macrophages (p ≤ 0.01; Fig. 5K). These results clearly indicate that M. smegmatis esxL down-regulates G9a-dependent CIITA expression by promoting H3K9me2/3 in the promoter IV region of CIITA.

      M. smegmatis esxL triggers H3K9me2/3-mediated CIITA inhibition by inducing the MAPK-signaling pathway

      NO acts as a key intermediate in regulation of cell-fate decisions by modulating several signaling pathways in the host cells (
      • Kapoor N.
      • Narayana Y.
      • Patil S.A.
      • Balaji K.N.
      Nitric oxide is involved in Mycobacterium bovis bacillus Calmette-Guerin-activated Jagged1 and Notch1 signaling.
      ,
      • Bansal K.
      • Balaji K.N.
      Intracellular pathogen sensor NOD2 programs macrophages to trigger Notch1 activation.
      ). Hence, we postulated that signaling cascades that regulate NO production could act as a focal point in M. smegmatis esxL infection-triggered histone modification that subsequently leads to inhibition of MHC-II or CIITA. MAPK pathways are known to regulate eukaryotic gene expression by modulating the chromatin structure of regulatory elements (
      • Vermeulen L.
      • Vanden Berghe W.
      • Beck I.M.
      • De Bosscher K.
      • Haegeman G.
      The versatile role of MSKs in transcriptional regulation.
      ). Increased MAPK leads to recruitment of histone deacetylases (HDACs), leading to gene repression (
      • Yang S.H.
      • Vickers E.
      • Brehm A.
      • Kouzarides T.
      • Sharrocks A.D.
      Temporal recruitment of the mSin3A-histone deacetylase corepressor complex to the ETS domain transcription factor Elk-1.
      ). Mycobacteria, in addition to selective antigens, are known to induce MAPK signaling in macrophages (
      • Schorey J.S.
      • Cooper A.M.
      Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way.
      ). In this context, we addressed the role of the MAPK-signaling pathway in the regulation of H3K9me2/3 and CIITA expression. We found that M. smegmatis esxL infection triggered the activation of pp38 (Fig. 6A) and pERK (Fig. 6B) when compared with control conditions. Similarly, M. tuberculosis infection also induced pp38 expression when compared with MtbΔesxL-infected THP-1 cells (Fig. 6C). Importantly, pretreatment with p38 inhibitor (SB203580) abrogated the M. smegmatis esxL-induced inhibition of CIITA (Fig. 6D, top). Similarly, treatment with SB203580 down-regulated the expression of H3K9me2/3 in M. smegmatis esxL-infected macrophages (Fig. 6D, middle). On the other hand, inhibition of pERK by the pharmacological inhibitor U0126 did not show any effect on the expression of either CIITA or H3K9me2/3 during M. smegmatis esxL infection when compared with untreated macrophages (Fig. 6E). These results clearly suggest that the M. smegmatis esxL-triggered p38 MAPK-signaling pathway holds the capacity to modulate H3K9me2/3 expression to regulate CIITA/MHC-II expression.
      Figure thumbnail gr6
      Figure 6Role of p38, ERK, and iNOS in CIITA and H3K9me3 expression. RAW 264.7 cells were infected with M. smegmatis (Msm) pSMT3 and recombinant M. smegmatis esxL for 2 h. The expression of pp38 (A) and pERK (B) was estimated by Western blotting 24 h post-infection. C, THP-1 cells were infected with M. tuberculosis (Mtb) and MtbΔesxL for 2 h, and the level of pp38 was determined by Western blotting 24 h post-infection. The expression of CIITA and H3K9me3 were checked in M. smegmatis pSMT3- and recombinant M. smegmatis esxL-infected cells after treatment with SB203580 (p38 inhibitor) (D) and U0126 (ERK inhibitor) (E) by Western blotting 24 h post-infection. F, the expression of CIITA, H3K9me3, and iNOS were checked in M. smegmatis pSMT3- and recombinant M. smegmatis esxL-infected RAW 264.7 cells after treatment with 1400W (iNOS inhibitor). The experiments were performed in triplicate (n = 3).

      M. smegmatis esxL-induced NO production regulates induction of H3K9me2/3 and inhibition of CIITA expression

      Next, we assessed the role of iNOS/NO during M. smegmatis esxL infection in modulating the expression of H3K9me3 and CIITA. For this, macrophages were infected with M. smegmatis pSMT3 and M. smegmatis esxL strains and then treated with an iNOS inhibitor, 1400W. Treatment with the 1400W inhibitor severely down-regulated the expression of H3K9me2/3 in M. smegmatis esxL-infected macrophages (Fig. 6F). On the other hand, inhibition of iNOS led to increased expression of CIITA when compared with untreated conditions (Fig. 6F). These results confirm the crucial role of NO in repression of CIITA by increasing hypermethylation of H3K9.

      Discussion

      M. tuberculosis adopts various strategies to evade the host defense mechanisms to facilitate its survival. One such mechanism involves epigenetic modifications in the host proteins to dampen antibacterial effector functions of host cells. In this context, various M. tuberculosis proteins, including ESAT-6, CFP-10, lipoproteins, and PE/PPE (proline-glutamic acid/proline-proline-glutamic acid) proteins, are known to be involved in the establishment of the infection process (
      • de Jonge M.I.
      • Pehau-Arnaudet G.
      • Fretz M.M.
      • Romain F.
      • Bottai D.
      • Brodin P.
      • Honoré N.
      • Marchal G.
      • Jiskoot W.
      • England P.
      • Cole S.T.
      • Brosch R.
      ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity.
      ,
      • Renshaw P.S.
      • Lightbody K.L.
      • Veverka V.
      • Muskett F.W.
      • Kelly G.
      • Frenkiel T.A.
      • Gordon S.V.
      • Hewinson R.G.
      • Burke B.
      • Norman J.
      • Williamson R.A.
      • Carr M.D.
      Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6.
      ,
      • Derrick S.C.
      • Morris S.L.
      The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression.
      ,
      • Choi H.H.
      • Shin D.M.
      • Kang G.
      • Kim K.H.
      • Park J.B.
      • Hur G.M.
      • Lee H.M.
      • Lim Y.J.
      • Park J.K.
      • Jo E.K.
      • Song C.H.
      Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis.
      ,
      • De Leon J.
      • Jiang G.
      • Ma Y.
      • Rubin E.
      • Fortune S.
      • Sun J.
      Mycobacterium tuberculosis ESAT-6 exhibits a unique membrane-interacting activity that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis.
      ,
      • Marongiu L.
      • Donini M.
      • Toffali L.
      • Zenaro E.
      • Dusi S.
      ESAT-6 and HspX improve the effectiveness of BCG to induce human dendritic cells-dependent Th1 and NK cells activation.
      ,
      • Fishbein S.
      • van Wyk N.
      • Warren R.M.
      • Sampson S.L.
      Phylogeny to function: PE/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity.
      ,
      • Chumduri C.
      • Gurumurthy R.K.
      • Zadora P.K.
      • Mi Y.
      • Meyer T.F.
      Chlamydia infection promotes host DNA damage and proliferation but impairs the DNA damage response.
      ). Herein, we reported that M. tuberculosis esxL represses CIITA/MHC-II expression by inducing H3K9me2/3 in the CIITA promoter. Previously, we and several others have used M. smegmatis as a surrogate model to elucidate the function of M. tuberculosis proteins in pathogenesis. For example, expression of M. tuberculosis Mce4A protein in non-pathogenic Escherichia coli increased invasion in HeLa cells (
      • Saini N.K.
      • Sharma M.
      • Chandolia A.
      • Pasricha R.
      • Brahmachari V.
      • Bose M.
      Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival.
      ), whereas expression of M. tuberculosis PE proteins in M. smegmatis increased its virulence properties (
      • Tiwari B.M.
      • Kannan N.
      • Vemu L.
      • Raghunand T.R.
      The Mycobacterium tuberculosis PE proteins Rv0285 and Rv1386 modulate innate immunity and mediate bacillary survival in macrophages.
      ). Based on this evidence, we expressed M. tuberculosis esxL in an M. smegmatis strain and also deleted esxL from the M. tuberculosis genome (MtbΔesxL) and proved its function using a macrophage infection model.
      The M. smegmatis genome does not contain esxL orthologue; therefore, the observed phenotypes can be attributed to the ectopic expression of esxL in M. smegmatis. Using human and mice macrophage infection models, we showed that recombinant M. smegmatis esxL strain survives more as compared with control strains, indicating that EsxL is involved in bacillary persistence in macrophages. It is well established that pathogenic M. tuberculosis facilitates its survival by modulating ROS and NO production (
      • Kumar A.
      • Farhana A.
      • Guidry L.
      • Saini V.
      • Hondalus M.
      • Steyn A.J.
      Redox homeostasis in mycobacteria: the key to tuberculosis control?.
      ,
      • Voskuil M.I.
      • Bartek I.L.
      • Visconti K.
      • Schoolnik G.K.
      The response of Mycobacterium tuberculosis to reactive oxygen and nitrogen species.
      ,
      • Lamichhane G.
      Mycobacterium tuberculosis response to stress from reactive oxygen and nitrogen species.
      ,
      • Firmani M.A.
      • Riley L.W.
      Reactive nitrogen intermediates have a bacteriostatic effect on Mycobacterium tuberculosis in vitro.
      ). We observed that M. smegmatis esxL strain increased NO and iNOS production in infected macrophages, whereas inhibition of iNOS decreased the intracellular survival of M. smegmatis esxL. On the contrary, MtbΔesxL reduced iNOS expression. The role of NO as an antimicrobial agent has been extensively studied in the context of host defense mechanisms. Nevertheless, a previous study (
      • Harding C.V.
      • Boom W.H.
      Regulation of antigen presentation by Mycobacterium tuberculosis: a role for Toll-like receptors.
      ) has provided evidence that reactive nitrogen intermediate helps in proliferation of M. tuberculosis, suggesting a bacteriostatic effect of reactive nitrogen intermediate on M. tuberculosis. In addition to antimicrobial properties, NO/iNOS are also known to modulate several signaling cascades that regulate cell fate decisions of host cells (
      • Tachibana M.
      • Sugimoto K.
      • Nozaki M.
      • Ueda J.
      • Ohta T.
      • Ohki M.
      • Fukuda M.
      • Takeda N.
      • Niida H.
      • Kato H.
      • Shinkai Y.
      G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis.
      ,
      • Bansal K.
      • Balaji K.N.
      Intracellular pathogen sensor NOD2 programs macrophages to trigger Notch1 activation.
      ). Previous studies have shown that M. tuberculosis down-regulates CIITA, thus altering antigen presentation (
      • Pattenden S.G.
      • Klose R.
      • Karaskov E.
      • Bremner R.
      Interferon-induced chromatin remodeling at the CIITA locus is BRG1 dependent.
      ). However, the specific M. tuberculosis protein responsible for observed down-regulation is not known. Our study has provided sufficient data that EsxL could be responsible for CIITA down-regulation. Studies have shown that epigenetic modifications are involved in regulating CIITA expression (
      • Pai R.K.
      • Convery M.
      • Hamilton T.A.
      • Boom W.H.
      • Harding C.V.
      Inhibition of IFN-γ-induced class II transactivator expression by a 19-kDa lipoprotein from Mycobacterium tuberculosis: a potential mechanism for immune evasion.
      ,
      • Zika E.
      • Greer S.F.
      • Zhu X.S.
      • Ting J.P.
      Histone deacetylase 1/mSin3A disrupts interferon-induced CIITA function and major histocompatibility complex class II enhanceosome formation.
      ,
      • Jung S.B.
      • Yang C.S.
      • Lee J.S.
      • Shin A.R.
      • Jung S.S.
      • Son J.W.
      • Harding C.V.
      • Kim H.J.
      • Park J.K.
      • Paik T.H.
      • Song C.H.
      • Jo E.K.
      The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes.
      ). Studies also showed that M. tuberculosis proteins like LpqH and ESAT-6 cause CIITA inhibition by decreasing histone H3K4 methylation level and acetylation levels (
      • Kumar P.
      • Agarwal R.
      • Siddiqui I.
      • Vora H.
      • Das G.
      • Sharma P.
      ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner.
      ,
      • Pennini M.E.
      • Pai R.K.
      • Schultz D.C.
      • Boom W.H.
      • Harding C.V.
      Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling.
      ). Our study has unambiguously shown that M. tuberculosis esxL induced H3K9me2/3 in host cells. H3K9me1/2/3 is known for transcriptional repression of the gene, which undergoes modification at its promoter region (
      • Pandey H.
      • Tripathi S.
      • Srivastava K.
      • Tripathi D.K.
      • Srivastava M.
      • Kant S.
      • Srivastava K.K.
      • Arora A.
      Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv.
      ,
      • Bannister A.J.
      • Zegerman P.
      • Partridge J.F.
      • Miska E.A.
      • Thomas J.O.
      • Allshire R.C.
      • Kouzarides T.
      Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.
      ). We have further proved that induction of H3K9me2/3 by EsxL is mediated via G9a, which is a known histone methyltransferase responsible for H3K9 methylation. Inhibition of G9a down-regulated H3K9me2/3 and simultaneously resulted in up-regulation of CIITA, indicating that G9a methyltransferase is responsible for H3K9 hypermethylation in the CIITA promoter. Indeed, a ChIP assay showed that M. tuberculosis esxL promoted H3K9me2/3 at the promoter IV region of the CIITA gene, which led to the transcriptional repression of CIITA that subsequently perturbed antigen presentation and T-cell activation. The level of H3K9me2/3 was also found to be increased in M. bovis BCG- and M. tuberculosis-infected macrophages, whereas levels of H3K9me2/3, CIITA, MHC-II, iNOS, and p38 were down-regulated in MtbΔesxL-infected macrophages. M. bovis BCG contains the esxL orthologue Mb1230. Therefore, M. bovis BCG-induced H3K9me2/3 could be attributed to this protein.
      The MAPK-signaling pathway plays a crucial role in mycobacterial infection. M. tuberculosis 38-kDa protein was shown to induce TNF-α and IL-6 through the MAPK pathway to facilitate mycobacterial infection (
      • Pandey H.
      • Tripathi S.
      • Srivastava K.
      • Tripathi D.K.
      • Srivastava M.
      • Kant S.
      • Srivastava K.K.
      • Arora A.
      Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv.
      ). As shown before, MAPK components ERK and p38 are known to alter gene transcription by altering chromatin structure (
      • Vermeulen L.
      • Vanden Berghe W.
      • Beck I.M.
      • De Bosscher K.
      • Haegeman G.
      The versatile role of MSKs in transcriptional regulation.
      ). The recruitment of HDACs at the promoter site of the gene is known to be facilitated by MAPK (
      • Yang S.H.
      • Vickers E.
      • Brehm A.
      • Kouzarides T.
      • Sharrocks A.D.
      Temporal recruitment of the mSin3A-histone deacetylase corepressor complex to the ETS domain transcription factor Elk-1.
      ). In this study, we found that M. smegmatis esxL infection up-regulated ERK and p38 levels in macrophages. However, inhibition of p38 down-regulated the expression of H3K9me2/3 and CIITA, indicating that EsxL-induced H3K9me2/3 is mediated via p38 signaling pathway. Previous studies have shown the involvement of p38 in repressive epigenetic modification. M. tuberculosis LpqH activates p38, which in turn facilitates the recruitment of HDAC to the promoter of CIITA, thus repressing gene transcription (
      • Pennini M.E.
      • Pai R.K.
      • Schultz D.C.
      • Boom W.H.
      • Harding C.V.
      Mycobacterium tuberculosis 19-kDa lipoprotein inhibits IFN-induced chromatin remodeling of MHC2TA by TLR2 and MAPK signaling.
      ). We further showed that EsxL-induced H3K9me2/3 is dependent on NO production. There are reports suggesting the involvement of NO/iNOS in down-regulation of CIITA. A study has shown that BCG infection increased NO production that up-regulated the expression of KLF4 transcription factor. KLF4 acts as a regulatory switch and inhibits CIITA expression (
      • Ghorpade D.S.
      • Holla S.
      • Sinha A.Y.
      • Alagesan S.K.
      • Balaji K.N.
      Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress major histocompatibility complex II expression during Mycobacterium bovis bacillus Calmette-Guerin infection.
      ).
      In summary, we have studied a mechanism in detail that leads to repression of CIITA/MHC-II during M. tuberculosis infection. Fig. 7 shows a schematic representation of a mechanism that leads to repression in antigen presentation and T-cell activation by inducing H3K9me2/3 in promoter IV region of CIITA via NO, p38 MAPK, and G9a during infection.
      Figure thumbnail gr7
      Figure 7Schematic representation showing the role of M. tuberculosis esxL in induction of hypermethylation of H3K9, which down-regulates the expression of CIITA, the major co-activator of MHCII. Thus, H3K9 hypermethylation results in down-regulation of MHCII expression and reduced production of IL-2.

      Experimental procedures

      Chemicals, reagents, and cell culture conditions

      Mycobacterium smegmatis mc2155 was grown in Middlebrook’s 7H9 broth medium (Difco) containing 0.05% Tween 80, 0.5% glucose, and 0.5% albumin at 37 °C on a shaker at 120 rpm. M. tuberculosis H37Rv and M. bovis BCG were grown in Middlebrook’s 7H9 broth medium (Difco) containing 0.05% Tween 80, 0.5% glucose, 0.5% albumin, and oleic albumin dextrose catalase at 37 °C on a shaker at 120 rpm. E. coli XL-10 Gold (Stratagene) was grown in Luria-Bertani (LB) broth supplemented with 20 μg/ml tetracycline. pSMT3 vector was a kind gift from Dr. Rakesh Sharma (Institute of Genomics and Integrative Biology, Delhi, India). The murine RAW 264.7 macrophage cell line was cultured in DMEM (HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% l-glutamine. THP-1 cells (
      • Jena P.
      • Mohanty S.
      • Mohanty T.
      • Kallert S.
      • Morgelin M.
      • Lindstrøm T.
      • Borregaard N.
      • Stenger S.
      • Sonawane A.
      • Sørensen O.E.
      Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.
      ) were grown in RPMI 1640 (Gibco) supplemented with 10% FBS, 10 mm HEPES, 1 mm sodium pyruvate, and penicillin-streptomycin solution. The cells were seeded onto 24-well culture dishes at a density of 2 × 105 cells/ml and treated overnight with 20 nm PMA (Sigma) for 24 h. Cells were then washed three times with PBS and incubated for one more day before performing the experiment. Anti-iNOS, anti-H3K9me3, anti-phospho-p38, anti-phospho-ERK1/2, anti- histone H3, anti-β-actin, anti-GAPDH, and secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Cell Signaling Technologies. Anti-CIITA antibody was purchased from Abcam (Cambridge, UK). Anti-MHC-II and anti-mouse IgG were procured from Santa Cruz Biotechnology, Inc. FITC-labeled anti-human MHCII antibody was purchased from Invitrogen. Secondary antibodies including goat anti-mouse IgG, Alexa Fluor 633, goat anti-rabbit IgG, and Alexa Fluor 488 were purchased from Thermo Fisher Scientific. Mounting solution with DAPI was purchased from DAKO. All of the pharmacological inhibitors were purchased from Sigma and Calbiochem and reconstituted in DMSO (Himedia, Mumbai, India) or sterile H2O at the following concentrations: 1400W (100 μm), U0126 (10 μm), SB203580 (10 μm), and UNC0638 hydrate (5 μm).

      Cloning and expression of esxL

      M. tuberculosis esxL was PCR-amplified using gene-specific primers (Table 1) and M. tuberculosis genomic DNA as template. The PCR-amplified products were gel-purified, double-digested with PstI and HindIII, and cloned into pSMT3 shuttle vector. The recombinant constructs were transformed into competent E. coli XL-10 gold. The positive clones were selected on LB agar plates supplemented with 20 μg/ml tetracycline and 50 μg/ml hygromycin. The positive clones were confirmed by colony PCR and sequencing using gene-specific primers. Finally, the recombinant constructs were transformed into electrocompetent M. smegmatis. The positive colonies were selected on 7H9 medium containing 50 μg/ml hygromycin B. The positive transformants were confirmed by colony PCR and sequencing using gene-specific primers.
      Table 1Oligonucleotides used in this study
      Serial no.Primer nameSequence (5′–3′)
      1esxL FPGTCCCTGCAGGATGACCATCAACTATC
      2esxL RPGTCCAAGCTTTCAGGCCCAGCTGGAG
      3iNOS FPTTC CAA GAG CCT TGC TGT TT
      4iNOS RPGTA GGT AAG GGC GTT GGT CA
      5CIITA FPACGCTTTCTGGCTGGATTAGT
      6CIITA RPTCAACGCCAGTCTGACGAAGG
      7MHCII FPTGGGCACCATCTTCATCATTC
      8MHCII RPGGTCACCCAGCACACCACTT
      9GAPDH FPGAGAGGCCCTATCCCAACTC
      10GAPDH RPTTCACCTCCCCATACACACC
      11esxL qRT FPGTTGACCGCGAGTGACTTTT
      12esxL qRT RPGGTTTGCGCCATGTTGTT
      13SigA FPCCAAGGGCTACAAGTTCTCG
      14SigA RPTGGATCTCCAGCACCTTCTC

      Generation of M. tuberculosis esxL mutant

      A temperature-sensitive phage-based transduction methodology was used for the generation of M. tuberculosis esxL deletion mutant (Fig. 1, G and H). Upstream (814-bp) and downstream (812-bp) flank regions were PCR-amplified, and the amplicons were digested with PflMI restriction enzyme. Flanks were ligated with the compatible sacB+hygr and oriEcos fragments from pYUB1471 to generate allelic exchange substrate. Allelic exchange substrate was packaged into phAE159 phasmid (a kind gift from Dr. William Jacob’s laboratory), and high-titer phages were generated and transduced into M. tuberculosis H37Rv harboring pNit-ET plasmid as described earlier (
      • Jain P.
      • Hsu T.
      • Arai M.
      • Biermann K.
      • Thaler D.S.
      • Nguyen A.
      • González P.A.
      • Tufariello J.M.
      • Kriakov J.
      • Chen B.
      • Larsen M.H.
      • Jacobs Jr., W.R.
      Specialized transduction designed for precise high-throughput unmarked deletions in Mycobacterium tuberculosis.
      ).

      Intracellular bacterial survival assay

      M. smegmatis harboring plasmid pSMT3 (M. smegmatis pSMT3) and recombinant M. smegmatis expressing M. tuberculosis esxL (M. smegmatis esxL) strains were grown to mid-exponential phase. Then bacterial cultures were pelleted, washed in 1× PBS, and resuspended in DMEM to a final A600 of 0.1. Bacterial clumps were broken by ultrasonication for 5 min, followed by a low-speed centrifugation for 2 min. RAW 264.7 macrophages (2 × 105 cells/well) were seeded on 24-well tissue culture plates with medium containing no antibiotic solution and grown for 18–20 h. Cells were infected with M. smegmatis pSMT3 and M. smegmatis esxL strains at a multiplicity of infection of 10, and intracellular survival was determined by lysing the infected macrophages with 0.5% Triton X-100 at different time points. Bacterial survival was determined by plating the serially diluted samples onto 7H9 plates. The equal input and time 0 (T0) counts of infecting bacilli were determined to calculate the percentage survival, % survival = cfu at required time/cfu of bacteria added × 100. For the THP-1 infection assay, cells were first treated with 20 nm PMA in RPMI medium, and the infection assay was performed as described above.

      Extracellular expression of esxL

      M. smegmatis esxL strain was grown in vitro for 24 h in 7H9 broth supplemented with 0.05% Tween 80 under shaking conditions. Cell pellets were harvested, followed by RNA isolation and cDNA synthesis. qRT-PCR was performed using gene-specific primers and cDNA as template. sigA was used as an internal control.

      Intracellular expression of esxL

      RAW 264.7 macrophages were infected with M. smegmatis esxL, and RNA was isolated at the 4-, 12-, and 24-h time points, followed by cDNA synthesis. qRT-PCR was performed using the cDNA as templates using gene-specific primers (Table 1). sigA was used as an internal control.

      Infection with M. tuberculosis H37Rv and MtbΔesxL

      THP1 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS and differentiated using PMA. The infection experiment with M. tuberculosis H37Rv and MtbΔesxL strains was performed as described earlier (
      • Soni V.
      • Upadhayay S.
      • Suryadevara P.
      • Samla G.
      • Singh A.
      • Yogeeswari P.
      • Sriram D.
      • Nandicoori V.K.
      Depletion of M. tuberculosis GlmU from infected murine lungs effects the clearance of the pathogen.
      ). For the lysate preparation, 8 × 106 cells were seeded in 10-cm cell culture dishes, and the infection was performed at a 1:5 multiplicity of infection. At different time points, cells were washed with PBS and lysed by using 600 μl of RIPA buffer, vortexed for 30 s, and kept in ice. The procedure was repeated three times, and the cell lysates were clarified at 13,000 rpm (Sigma 3-30K, 12154) at 4 °C.

      Free NO estimation

      RAW 264.7 cells (2 × 105 cells/well) were seeded on 24-well plates. Next day, the cells were infected with M. smegmatis pSMT3 and M. smegmatis esxL strains for 24 h. The accumulation of nitrite was measured by mixing 100 μl of culture supernatants with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride in 5% concentrated H3PO4) in 96-well plates. The plates were incubated for 10 min at room temperature, and absorbance was measured at 550 nm in a microtiter plate reader (EPOCH, BioTek). The nitrite concentrations (in μmol/sample) were determined by a least-square linear regression analysis using sodium nitrite as a standard (5–100 μm range). The values were averaged from three independent experiments.

      RNA isolation and quantitative real-time RT-PCR

      Total RNA was isolated from infected or uninfected macrophages using TRIzol reagent (Invitrogen) as per the manufacturer’s protocol. The cDNA synthesis kit (Thermo Fisher Scientific) was used for reverse transcription according to the manufacturer’s protocol. Quantitative real-time RT-PCR amplification using the SYBR Green PCR mixture (KAPA Biosystems) was performed for quantification of target gene expression in a Real Plex master cycler (Eppendorf, Hamburg, Germany) with initial denaturation at 95 °C for 10 min, final denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s to generate 200-bp amplicons. All reactions were repeated at least three times independently to ensure reproducibility of the result. The mRNA levels were normalized to the transcript levels of GAPDH, and the relative -fold changes were calculated.

      Western-blotting analysis

      RAW 264.7 cells were infected with M. smegmatis pSMT3 and M. smegmatis esxL. After 24 h of infection, protein samples were prepared by cell lysis using RIPA buffer (HiMedia) containing 5 mm EDTA, 5 mm EGTA, 1 mm PMSF, protease inhibitor mixture, 50 mm NaF, 1 mm DTT, 1 mm sodium orthovanadate. Proteins were electrophoresed in 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (GE Healthcare) overnight at 28 V. Then the blots were blocked with 5% BSA or skimmed milk in TBST (20 mm Tris-HCl, pH 7.4, 137 mm NaCl, and 0.1% Tween 20) for 60 min. Then blots were incubated with primary antibodies (1:1000) overnight at 4 °C and then with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies in 5% BSA or skimmed milk (1:1000) for 2 h at room temperature. The membrane was washed using 1× TBST, and X-ray film was developed using standard chemiluminescent solvent. β-Actin and GAPDH were used as loading controls.

      Immunofluorescence

      For immunofluorescence studies, RAW 264.7 macrophages (5 × 104) were seeded on coverslips. After infecting the cells with M. smegmatis pSMT3 and M. smegmatis esxL, cells were fixed with acetone/methanol (1:1) for 20 min at −20 °C and then blocked with 5% BSA for 1 h at room temperature and stained with primary anti-iNOS and anti-H3K9me3 antibodies overnight in the dark. Then the coverslips were stained with secondary antibodies for 2 h at room temperature. Finally, the cells were mounted in mounting solution with DAPI, and the images were analyzed using a BX61 Olympus fluorescence microscope and Cytovision software version 7.2.

      Flow cytometry analysis

      For flow cytometry analysis, THP-1 (2 × 105) and RAW 264.7 (2 × 105) cells were seeded onto 24-well cell culture plates. Cells were infected with M. smegmatis pSMT3 and M. smegmatis esxL for 24 h. The cells were then harvested and blocked with 0.1% BSA for 15 min on ice. The cells were then centrifuged at 2500 rpm (Sigma 3-30K, 12154) for 5 min, followed by staining with primary FITC-labeled anti-human MHC-II (FITC) and anti-mouse MHC-II (APC) antibodies for 30 min on ice and then with secondary antibodies. Uninfected cells were taken as negative control. Flow cytometry was performed by analyzing 10,000 gated cells using a FACS Canto II flow cytometer and FACS Diva software.

      ChIP assay

      For the ChIP assay, RAW 264.7 (1 × 107) cells were seeded onto 100-mm tissue culture disks. Cells were infected with M. smegmatis pSMT3 and M. smegmatis esxL. For the ChIP assay with G9a inhibition, RAW 264.7 cells were infected with M. smegmatis esxL and then treated with G9a inhibitor, UNC0638. After 24 h of infection, cells were washed twice with 1× PBS and then cross-linked with 11% formaldehyde solution for 15 min, followed by 2.5 m glycine treatment for quenching formaldehyde solution. The cells were then washed with ice-cold 1× PBS twice. The cells were then harvested by scrapping using ice-cold 1× PBS and centrifuged at 2500 rpm (Sigma 3-30K, 12154) for 5 min at 4 °C, followed by washing with 1× PBS. The pellets were then resuspended with 1 ml of ice-cold Farnham buffer and then centrifuged at 2000 rpm (Sigma 3-30K, 12154) for 5 min at 4 °C. The pellet was resuspended with 300 μl of RIPA buffer and then kept on ice for 10 min, followed by sonication in a Bioruptor at the high setting for a total time of 40 min, 30 s on, 30 s off at 4 °C. The chromatin length was then verified, and processed for further steps. The sonicated mixture was centrifuged at 14,000 rpm (Sigma 3−30K, 12154) for 15 min at 4 °C. The supernatant was collected and quantified, and the volume was adjusted with RIPA buffer so that each reaction has 150 μg/ml chromatin. The suspension was then incubated with previously prepared Protein A-Sepharose beads for 1 h at 4 °C in a rotator. After centrifugation at 1500 rpm (Sigma 3−30K, 12154) for 2 min at 4 °C, the supernatant was taken and incubated overnight with 6 μg of antibodies against H3K9me2/3 and mouse IgG per immunoprecipitation at 4 °C for overnight rotation. The next day, the suspensions were again incubated with Protein A-Sepharose beads at 4 °C for 2 h in a rotator and then centrifuged at 2000 rpm (Sigma 3−30K, 12154) for 1 min. The pellets were then washed using LiCl wash buffer (7–8 times) and TE buffer (once). The pellet was then dissolved in immunoprecipitation elution buffer for 30 min at room temperature, and then the supernatants were left at 65 °C overnight for reverse cross-linking. The next day, the RNA and protein were digested with RNase and Proteinase K to obtain purified DNA. The DNA (150 μg) isolated from 1 × 107 cells was further processed for quantitative PCR using specific primers for CIITApI, CIITApIII, CIITApIV, and GAPDH promoter. The quantitative PCR data were then normalized to input DNA. Primers for GAPDH promoter and antibody against mouse IgG both were used as negative controls (Table 2).
      Table 2Oligonucleotides used in ChIP assay
      Serial no.Primer nameSequence (5′–3′)
      1CIITApI FPGCATAGCAGATGCAAAACCA
      2CIITApI RPGGGCAGATTATTACAGATTAGTTGC
      3CIITApIII FPACGTCCAGAGAAACTCAATGC
      4CIITApIII RPAGAGCTGTTAGGGACATGGTG
      5CIITApIV FPCTACTGGCTCAAATCTGTCGTC
      6CIITApIV RPCAGGCAGATCTCACTTAGACCA
      7GAPDHpromoter FPGGATAGAATGTAGCCCTGGACTT
      8GAPDHpromoter RPTGTGCATGTATCTTTATTGGCTCT

      Cytokine profiling

      RAW 264.7 cells (2 × 105) were seeded onto a 24-well tissue culture plate and infected with M. smegmatis pSMT3 and M. smegmatis esxL. After 2 h of infection, 20 mg/ml gentamycin containing DMEM was added to kill the extracellular bacteria. After 1 h, the cells were then co-cultured with splenocytes isolated from BALB/c mice. After 24 h, the supernatant was harvested, and cytokine levels were estimated using the Bioplex kit assay (Bio-Rad).

      Statistical analysis

      All experiments were performed at least three times (n = 3). Statistical analyses were performed using the Mann-Whitney U test (two-tailed, equal variances). Significance is shown as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

      Author contributions

      S. S. planned the experimental setup, performed the experiments, analyzed the data, and wrote the manuscript. I. D., A. P., S. K. N., G. G., and K. P. P. analyzed the experiments and provided technical assistance. S. N. constructed the esxL knockout (MtbΔesxL) mutant and performed the M. tuberculosis infection assay. V. K. N. supervised the construction of the knock-out mutant and provided the BSL3 laboratory facility. A. A. contributed in performing the ChIP assay experiments and analysis of data. S. K. R. supervised the ChIP assay experiments and contributed in analyzing the data. A. S. planned the experimental setup and data analysis, wrote the manuscript, and provided all of the necessary resources and support for the completion of the study. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank members of the Sonawane laboratory for fruitful discussions and critical reading of the manuscript.
      • Department of Biotechnology, Ministry of Science and Technology (BT/PR5790/MED/29/602/2012) to
      • Indian Council of Medical Research (AMR/44/2011-ECD-I) to

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