Mycobacterium tuberculosis, the causative agent for the disease Tuberculosis, is a serious public health problem that is responsible for 1.6 million deaths each year. The WHO’s recent report on Tuberculosis estimates that a third of the world’s population is latently infected with the bacteria, and, of those, 10% will progress to active disease. M. tuberculosis is a successful pathogen mainly due to its ability to adapt and survive in changing environments. It can survive a dormant state with limited metabolic activity during latent infection, while also being able to escape the macrophage and disseminate into active disease. Efforts to eradicate the disease must be based on understanding the biology of this organism, and the mechanisms it uses to infect, colonize, and evade the immune system. Understanding the behaviour of pathogenic mycobacteria in the macrophage is also important to the discovery of new drug targets. In this thesis, we employed state of the art mass spectrometry techniques, which allowed us to unpack the biology of this bacterium in different growth environments and expand our understanding of the mechanisms it employs to adapt and survive. We investigated protein regulation by the process of phosphorylation, through sensory kinases, which add a phosphate group to a protein of interest, thereby regulating its function. First, we interrogated the phosphoproteomic landscape between M. bovis BCG and M. smegmatis to explain how differential protein regulation results in the differences between slow and fast growth of mycobacteria. Second, we focused on Protein Kinase G (PknG), which plays an important role in bacterial survival by blocking phagosome/lysosome fusion. We identified the in vivo physiological substrates of this kinase in actively growing M.bovis BCG culture. Our results revealed that this kinase is a regulator of protein synthesis. We then examined the mechanisms of survival in murine RAW 246.7 macrophages mediated by PknG, using M. bovis BCG reference strain and PknG knock-out mutant. Our results indicated strong evidence that pathogenic mycobacteria disrupt the macrophagic cytoskeleton, through phosphorylation of proteins that are involved in cytoskeleton rearrangement. These results explain the strategies that pathogenic mycobacteria employ mediated by PknG to block phagosome-lysosome fusion and evade the host immune system and survive for prolonged periods in the macrophages. The findings of this thesis contribute to our understanding of the physiology of pathogenic mycobacteria and their interaction with the host.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Protective immunity against pathogenic mycobacteria depends principally on cell-mediated immunity executed by efficient anti-infectious functions of type 1 T helper (Th1) subset of CD4+ T cells. The polarization of Th1 responses is orchestrated by IL-12 secreted by antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs). A hallmark of Th1 type CD4+ T cells is the production of IFN-γ that activates plethora of innate cell-mediated immunity. It is well known that cytokines such as IFN-γ, IL-12 and TNF-α are required for control of mycobacterial infection in humans as well as in mice. However, it remains unclear that why the immune response controls mycobacteria, but does not eradicate infection suggesting critical roles for series of survival strategies employed by pathogenic mycobacteria. In general, these evasion strategies include blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, induced secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. that ultimately suppress the secretion of IL-12 and IFN-γ from APCs and T cells respectively, culminating in a skewed Th1/Th2 balance towards unprotective Th2 responses. Th2 cells secrete IL-4, IL-5, IL-9, IL-10 and IL-13 but are deficient in clearing intracellular infections including pathogenic mycobacteria. This eventually leads to inhibition of host’s immuno-protective responses with concomitant increase in the vulnerability to chronic mycobacterial infection. In this intricate process, modulation of cyclooxygenase-2 (COX-2) levels, a key enzyme catalyzing the rate-limiting step in the inducible production of prostaglandin E2 (PGE2), by mycobacteria like Mycobacterium bovis BCG assumes critical importance in influencing the overall host immune response. PGE2, an immunosuppressive member of prostaglandin family, is known to restrain production of IL-12, as well as reactive oxygen intermediates. PGE2-mediated inhibition of IL-12R, diminishes IL-12 responsiveness of macrophages and dendritic cells. PGE2 also inhibits the secretion of IFN-γ, which is important in activating T cells and macrophages. In contrast, PGE2 promotes IL-10 production by macrophages, dendritic cells and Th1-to-Th2 shift of acquired immune responses by inhibiting IL-2 and enhancing IL-4 production. Albeit, mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways are generally believed to be involved, little is known about the signaling molecules playing significant roles upstream of MAPK and NF-κB pathways during mycobacteria triggered COX-2 expression. Further, information on early receptor proximal signaling mechanisms essential during mycobacteria mediated induction of COX-2 remains scanty. In this regard, signaling cascade triggered upon recognition of mycobacterial components by pattern recognition receptors (PRR) signify as critical event in overall regulation of cell fate decisions. PRR like Toll like receptor (TLR2) and nucleotide-binding oligomerization domain 2 (NOD2) are two nonredundant recognition mechanisms of pathogenic mycobacteria. Several components of mycobacteria have been identified as being responsible for TLR2-dependent activation including 19-kDa lipoprotein, lipomannan etc.; while NOD2 recognizes mycobacterial peptidoglycans through its interaction with muramyl dipeptide (MDP). Interestingly, although mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), trehalose 6,6′-dimycolate (TDM; cord factor), PE/PPE family proteins etc., are released and traffic out of the mycobacterial phagosome platform into endocytic compartments. Importantly, these antigens could gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represents a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. Further, PIM2 is a known TLR2 agonist and reported to activate NF-κB, AP-1, and MAPK suggesting that mycobacterial envelope antigen PIM2 could modulate the inflammatory responses similar to mycobacteria bacilli. In this context, we explored the signaling events modulated by M. bovis BCG, and role for TLR2 and NOD2 in this intricate process, to trigger the expression of COX-2 in macrophages. Our studies demonstrated that M. bovis BCG triggered TLR2-dependent signaling leads to COX-2 expression and PGE2 secretion in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or CSF of tuberculosis patients. Similarly, mycobacterial TLR2 agonist PIM2 and NOD2 ligand MDP triggered COX-2 expression in macrophages. The induced COX-2 expression in macrophages either by M. bovis BCG or PIM2 or MDP was dependent on NF-κB activation, which was in turn mediated by iNOS/NO and Wnt-β-Catenin dependent participation of the members of Notch1-PI3K signaling cascade. Importantly, loss of iNOS activity either in iNOS null macrophages or by pharmacological intervention in wild type macrophages severely abrogated M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD) as well as activation of PI3K signaling cascade. On contrary, treatment of macrophages with SIN-1, an NO donor, resulted in a rapid increase in generation of NICD, activation of PI3K pathway as well as the expression of COX-2. Interestingly, pharmacological inhibition as well as siRNA mediated knockdown of Wnt-β-Catenin signaling compromised ability of M. bovis BCG to induce activation of Notch1-PI3K signaling and drive COX-2 expression. Concomitantly, activation of Wnt-β-Catenin signaling by LiCl triggered activation of Notch1 and PI3K pathway as well as COX-2 expression. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbation experiments suggested involvement of the cross-talk of Notch1 with PI3K signaling cascade. In this perspective, we propose TLR2 and NOD2 as two major receptors involved in mycobacteria mediated activation of Notch1PI3K signaling, and the activation of iNOS/NO and Wnt-β-Catenin signaling axis as obligatory early receptor proximal signaling events during mycobacteria induced COX-2 expression in macrophages. Functional characterization of mycobacterial antigens that are potent modulators of host immune responses to pathogens by virtue of induced expression of COX-2 assumes critical importance for deciphering pathogenesis of mycobacterial diseases as well as to identify novel therapeutic targets to combat the disease. In this context, a group of novel antigens carried by M. tuberculosis that are expressed upon infection of macrophages belong to PE and PPE family of proteins. Ten percent of the coding capacity of M. tuberculosis genome is devoted to the PE and PPE gene family members, exemplified by the presence of Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many members of the PE family exhibit multiple copies of polymorphic guanine-cytosine– rich sequences (PGRS) at the C-terminal end, which are designated as the PE_PGRS family of proteins. A number of PE/PPE proteins associate with the cell wall and are known to induce strong T & B cell responses in humans. However information related to effects of PE/PPE antigens on the maturation and functions of human dendritic cells and eventual modulation of T cell responses as well as underlying signaling events remains obscure. Our results demonstrated that two cell wall associated/secretory PE_PGRS proteins PE_PGRS 17, PE_PGRS 11 and PPE family protein PPE 34 recognize TLR2, induce maturation and activation of human dendritic cells and enhance the ability of dendritic cells to stimulate CD4+ T cells. In addition, tuberculosis patients were found to have a high frequency of T cells specific to PE_PGRS and PPE antigens. We further found that PE/PPE proteins-mediated activation of dendritic cells involves participation of ERK1/2, p38 MAPK and NF-κB signaling pathways. While, PE_PGRS antigens-matured dendritic cells secreted high amounts of inflammatory cytokine IL-12, PPE 34 triggered maturation of dendritic cells was associated with secretion of high amounts of anti-inflammatory cytokine IL-10 but not the Th1-polarizing cytokine IL-12. Consistent with these results, PPE 34-matured dendritic cells favored secretion of IL-4, IL-5 and IL-10 from CD4+ T cells and contributed to Th2 skewed cytokine balance ex vivo in healthy individuals and in patients with pulmonary tuberculosis. Interestingly, PPE 34-skewed Th2 immune response involved induced expression of COX-2 in dendritic cells. Our results suggest that by inducing differential maturation and activation of human dendritic cells, PE/PPE proteins could potentially modulate the initiation of host immune responses against mycobacteria. Taken together, our observations clearly signify the potential role for TLR2 and NOD2 triggering by M. bovis BCG in activating receptor proximal Notch1-PI3K signaling during induced COX-2/PGE2 expression which represents a crucial immune subversion mechanism employed by mycobacteria in order to suppress or attenuate host immune responses. Further, differential maturation of human dendritic cells by PE_PGRS and PPE antigens as well as their ability to stimulate CD4+ T cells towards Th1 and Th2 phenotype respectively, improves our understanding about host-mycobacteria interactions and clearly paves a way towards the development of novel combinatorial therapeutics.

Mycobacterium are important human pathogens and their strength lies in establishing acute infections, latent infections as well as co-existing with other dreadful infectious agents like HIV. The success of mycobacterium infection often relies in its ability to evade immune-surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immune regulatory molecules. In this scenario, the role of pattern recognition receptors (PRRs) in orchestrating host immune responses assumes central importance. Of the PRRs, the Toll-like receptors (TLRs) or intracellular surveillance receptors such as retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) govern key immune-surveillance mechanisms in recognition as well as control of mycobacterial or viral infections. The first part of this study illustrates the role of SHH signaling in macrophage induced neutrophil recruitment during mycobacterial infections. The present investigation demonstrates that, in response to mycobacterium infection, macrophages displayed robust activation of TLR2 dependent SHH signaling. By utilizing the well-documented experimental air pouch model, we show that the ability of pathogenic mycobacterium infected macrophages to recruit polymorph nuclear leukocytes (PMNs) like neutrophils to the infected site was dependent on SHH signaling. The activated SHH signaling differentially regulated the expression of proteolytic enzymes, MMP-9 and MMP-12 that would contribute to PMN migration. Interestingly, SHH-responsive krüppel-like family (KLF) of transcription factors, KLF4 and KLF5 were found to modulate these chemokine effectors to regulate neutrophil recruitment. Subsequent chapters describe novel functions of SHH signaling during RIG-I mediated anti-viral immunity and RIG-I mediated modulation of TLR2 anti-inflammatory signature in mycobacteria infected macrophages. In this perspective, we demonstrate that RIG-I ligand robustly induces the activation of SHH signaling via the phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. Furthermore, we show that the sustained inhibition of PKA-GSK-3β-SUFU negative regulatory axis upon RIG-I engagement with 5'3pRNA is critical for the activation of SHH signaling. Gain or loss of function studies implicate the necessity of RIG-I triggered MAVS-TBK1 canonical axis in the inhibition of PKA-GSK-3β-SUFU negative regulatory axis that contributes to SHH signaling activation. The RIG-I activated SHH signaling drives the production of anti-viral type 1 interferons leading to the inhibition Japanese encephalitis virus (JEV) replication. Further, RIG-I-mediated anti-viral type 1 interferon production and subsequent control of viral replication suggested the involvement of two transcriptional factors, IRF3 and YY1 in the response along a SHH axis. Further, mounting evidence clearly depicts a significant cross talk among the molecular events initiated by given TLRs and RLRs like RIG-I. Clearly, these studies present an interesting challenge in delineating the events during polymicrobial infection of host immune cells like macrophages or DCs. Altogether, our results improve our understanding of mycobacteria associated confections’ and may add significantly to the current knowledge of the delicate balance that determines a successful mycobacterial infection.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

Pathogenic Mycobacteria are among the most unrelenting pathogens known to mankind as one-third of the world population is latently infected with Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis. Despite many species of mycobacteria elicits robust host T cell responses as well as production of cytokines like interferon-γ (IFN- γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. One potential mechanism by which mycobacteria may achieve a state of long-term persistence amid a robust host immune response is by modulating the signaling cascades leading to macrophage activation. Activation of proinflammatory responses by the host macrophages upon infection with mycobacteria requires the involvement of a variety of signaling events. Studies have indicated that macrophages infected with pathogenic mycobacteria produce significantly less tumor necrosis factor (TNF)-α and other proinflammatory molecules compared with infection with nonpathogenic mycobacteria, which likely play a role in enhancing mycobacterial survival in vivo. Furthermore, macrophages infected with mycobacteria become refractory to many cytokines including IFN-γ and modulation of host cell signaling responses is critical for the suppression of a generalized inflammatory response which might influence the persistence of mycobacteria within the host. In this context, Suppressor of cytokine signaling (SOCS) 3, a member of SOCS family function as negative regulators of multiple cytokine and toll like receptor induced signaling. The SOCS3 has been shown to specifically inhibit signaling by IFN-γ, IL-6 family of cytokines and can act as a negative regulator of inflammatory responses. In this regard, many species of mycobacteria including M. bovis BCG triggers the inducible expression of SOCS3. Further, it has been suggested that M. bovis BCG triggered SOCS3 and SOCS1 proteins leads to the inhibition of IFN- γ stimulated JAK/STAT signaling in macrophages. Albeit JAK/STAT signaling pathway is generally believed to be involved, STAT-independent signals are suggested to take part in the induction of SOCS proteins in many systems signifying the involvement of multiple signal pathways in regulation of SOCS expression. Further little is known about the early, receptor proximal signaling mechanisms underlying mycobacteria-mediated induction of SOCS3. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, many cell wall antigens like LAM, PIM, TDM, PE family antigens etc are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extra cellular environment in the form of exocytosed vesicles. In this context, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. PIM are suggested to be the common anchor of LM and LAM as PIM, LM, and LAM originate from identical biosynthetic pathway. PIM are present in virulent M. tuberculosis H37Rv as well as in M. bovis BCG and a number of biological functions have been recently credited to PIM2. PIM2 is suggested to trigger the activation of cells via Toll like receptor (TLR)-2 and stimulation resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. PIM2 induces proinflammatory stimuli such as TNF-α and IL-12 in murine and human macrophages in a TLR2 dependent manner. PIM exhibited pulmonary granuloma-forming activities as well as was shown to be responsible for the recruitment of NKT cells to granulomas. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether M. bovis BCG or novel cell surface antigens like PIM2 or Rv0978c, a PE-PGRS protein with unknown function can contribute to M. bovis BCG triggered molecular signaling events leading to SOCS3 expression in macrophages. Our studies clearly demonstrated that M. bovis BCG can trigger SOCS3 expression in macrophages. The inception of signaling by M. bovis BCG is TLR2-MyD88 dependent, but not TLR4 dependent. The perturbation of TLR2 signaling and the downregulation of MyD88 resulted in significant decrease in SOCS3 expression implicating the role of TLR2-MyD88 axis in M. bovis BCG triggered signaling. Experiments with cycloheximide and neutralizing antibodies to IL-10 evinced that M. bovis BCG triggered SOCS3 expression is a primary response and requires direct activation of signaling cascades. In the current study, we show for the first time that infection of macrophages with M. bovis BCG activates NOTCH1 signaling events, which leads to expression of SOCS3. The perturbation of NOTCH signaling in infected macrophages either by siRNA mediated down regulation of NOTCH1 or RBP-Jk or by inhibition with pharmacological inhibitor gamma secretase-I, resulted in the marked reduction in the expression of SOCS3. Further, the enforced expression of the NOTCH1 intracellular domain (NICD) in RAW264.7 macrophages induces the expression of SOCS3, which can be further potentiated by M. bovis BCG. Furthermore, the inhibition of TLR2 signaling by a TLR2 dominant-negative construct resulted in inhibition of NOTCH1 activation. Additionally, our results demonstrates for the first time that physical association of TLR2 with both Phosphoinositide-3 Kinase (PI3K) and NOTCH1, which suggest the significant role of TLR2 triggering by of M. bovis BCG in the activation of PI3K and NOTCH1. More importantly, signaling perturbations data suggest the involvement of cross-talk among the members of PI3K and MAPK cascades with NOTCH1 signaling in SOCS3 expression. In addition, SOCS3 expression requires the NOTCH1 mediated recruitment of CSL/RBP-Jk and Nuclear Factor-B (NF-B) to the SOCS3 promoter. A number of biological functions triggered by mycobacteria are often attributed to many of the cell wall antigens. As part of our current investigation, we explored whether two novel cell wall associated antigens namely PIM2 and a PE-PGRS antigen, Rv0978c could play as significant or crucial cell wall ingredients which imparts ability to M. bovis BCG to trigger activation of NOTCH signaling leading to SOCS3 expression. Akin to M. bovis BCG, PIM2 activates NOTCH1 signaling resulting NICD formation which leads to the expression of SOCS3 in a TLR2-MyD88 dependent manner. PIM2 mediated NOTCH1 activation, both directly influences the SOCS3 expression by serving as coactivator in RBP-Jk complex and indirectly triggers SOCS3 expression by activating PI3K-MAPK-NF-κB cascade. One important outcome of the genome sequencing project of M. tuberculosis was the discovery of two new multigene families designated PE and PPE, named for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. Many PE and PPE proteins are composed only of PE or PPE homologous domains. However, in other proteins, the PE domain is often linked to a unique domain of various lengths that is rich in alanine and glycine amino acids, termed the PGRS domain (PE-PGRS subfamily). PE family genes were suggested to play roles in the virulence of the pathogen and many members of PE family proteins are reported be localized on the surface of M. tuberculosis bacilli. Some of the PE proteins may play a role in immune evasion and antigenic variation or may be linked to virulence. Additionally, it has been suggested that the PE-PGRS subfamily of PE genes is enriched in genes with a high probability of being essential for M. tuberculosis. The uniqueness of the PE genes is further illustrated by the fact that these genes are restricted to mycobacteria. However, despite their abundance in mycobacteria, very little is known regarding the expression or the functions of PE family genes. In this context, we have chosen to study Rv0978c as a typical member of PE-PGRS family based on the following observations. Rv0978c was upregulated in TB bacilli upon infection of macrophages. Rv0978c was demonstrated to be a member of a group of genes called in vivo-expressed genomic island, which were shown to be upregulated in M. tuberculosis bacilli during infection of mice. Rv0978c was also shown to be upregulated, at least eightfold, in human brain microvascular endothelial cell-associated M. tuberculosis infection, suggesting a role for endothelial cell invasion and intracellular survival. In the current investigation, we have demonstrated that Rv0978c is hypoxia responsive gene based on promoter analysis and upregulated in M. tuberculosis during the infection of macrophages. Further, Rv0978c is associated with cell wall and is exposed outside the surface of the bacterium suggesting the possible access to intracellular compartments of the infected macrophages. In this perspective, our results clearly demonstrate that Rv0978c triggers SOCS3 expression by activating PI3K-ERK1/2-NF-B cascade in mouse macrophages. Additionally, Rv0978c elicited humoral antibody reactivities in a panel of human sera or in cerebrospinal fluid samples obtained from different clinical categories of tuberculosis patients. DNA immunizations experiments in mice clearly suggested that Rv0978c is an immunodominant antigen demonstrating significant T cell and humoral reactivites. These observations clearly advocate that Rv0978c protein is expressed in vivo during active infection with M. tuberculosis and that the Rv0978c is immunogenic. These results clearly describe the cross-talk of NOTCH1 signaling with signaling pathways like PI3K and MAPK pathways during infection of macrophages with M. bovis BCG eventually resulting in regulation of specific gene expressions, such as SOCS3. These observations lead to a possibility of differential effects of NOTCH1 signaling activated upon infection by an intracellular bacillus, which could be involved in modulation of macrophage functions depending on a local immunological milieu. Taken together, our findings suggest that, induction of Suppressors of Cytokine Signaling 3 molecule by M. bovis BCG or by its cell wall antigens represents a crucial immune subversion mechanism in order to suppress or attenuate host responses to cytokines to generate the conditions that favor survival of the mycobacteria.

Mycobacteria are multifaceted pathogens capable of causing both acute disease as well as an asymptomatic latent infection. Host immune responses during mycobacterial infection involve potent cell effector functions including that of CD4+, CD8+ and γδT cells, macrophages and dendritic cells (DCs). Further, the critical regulators of protective immunity to mycobacterial infection include IFN-γ, IL-12, IL-23, TNF-α, lymphotoxins, CD40, nitric oxide and reactive oxygen species. However, the success of mycobacterial infection often relies in its ability to evade immune surveillance mechanisms mediated by sentinels of host immunity by modulating host signal transduction pathways and expression of immunoregulatory molecules. Therefore, the key to control mycobacterial growth and limit pathogenesis lies in the understanding the interactions between Mycobacterium and primary responders like macrophages and DCs. In this scenario, the role of pattern recognition receptors (PPRs) in orchestrating host immune responses assumes central importance. The cell surface receptors play crucial role in influencing overall immune responses. Of the PRRs, the Toll-like receptors (TLRs) form key immune surveillance mechanisms in recognition as well as control of mycobacterial infection. Among them, TLR2 is the primary interacting receptor on antigen presenting cells that recognize the invading mycobacteria. Mycobacterial cell wall constituents such as LAM, LM, PIM and 19-kDa protein have been shown to activate TLR2 signaling leading to proinflammatory responses. Recent reports have suggested that PE_PGRS antigens of M. tuberculosis interact with TLR2. For example, RV0754, Rv0978c, RV1917c have been implicated in modulation of human DCs. The 19-kDa lipoprotein, LpqH (Rv3763) and LprG (Rv1411c) utilize TLR2 signaling to inhibit macrophage responsiveness to IFN-γ triggered MHC class II expression and mycobacterial antigen presentation. Interestingly, recognition and amplification of pathogenic-specific signaling events play important roles in not only discriminating the invading microbes, but also in regulating explicit immune responses. In this context, integration of key signaling centers, which modulate host immunity to pathogenic mycobacterial infections, remains unexplored. In accordance to above observations, signal transduction pathways downstream to TLRs play a critical role in modulation of battery of host cells genes in terms of expression and production of immune modulatory cytokines and chemokines, recruitment of cellular machineries to site of infections etc. This suggests the decisive role for TLRs in modulation of host cell fate decisions. However, during the ensuing immunity to invading pathogens, beside TLR signaling pathways, various other signaling molecules are thought to execute specific functions in divergent cellular contexts. Recent studies from our laboratory have clearly demarcated a novel cross talk of TLR2-NOTCH1 and TLR2-Wnt signaling pathways during mycobacterial infections. The current study primary focuses on the broad range of cross talk of TLR2 and Sonic hedgehog (SHH) signaling pathways and its functional significance. The present investigation demonstrates that M. bovis BCG, a vaccine strain, triggers a robust activation of SHH signaling in macrophages compared to infection with diverse Gram-positive or Gram-negative microbes. This observation was further evidenced by the heightened SHH signaling signatures during in vivo scenario in cells /tissues from pulmonary tuberculosis (TB) individuals as well as tuberculous meningitis (TBM) patients. Furthermore, we show that the sustained TNF-α secretion by macrophages upon infection with M. bovis BCG is a critical necessity for SHH activation. Significantly, perturbation studies implicate a vital role for M. bovis BCG stimulated TLR2/PI3K/PKC/MAPK/NF-κB axis to induce TNF-α, that contributes to enhance SHH signaling. The TNF-α driven SHH signaling downregulates M. bovis BCG induced TLR2 signaling events leading to modulation of battery of genes that regulate various functions of macrophages genes like Vegf-a, Socs-3, Cox-2, Mmp-9 and M1/M2 genes. Importantly, utilizing whole-genome microRNA (miRNA) profiling, roles for specific miRNAs were identified as the molecular regulators that bring about the negative-feedback loop comprising TLR2-SHH signaling events. Thus, the current study illustrates how SHH signaling tightly regulates the kinetics and strengths of M. bovis BCG specific TLR2 responses, emphasizing a novel role for SHH signaling in host immune responses to mycobacterial infections. As described, variety of host factors contributes for ensuing effective host defenses and modulation of host cell fate decisions. Interestingly, avirulent pathogenic mycobacteria, including the vaccine strain M. bovis BCG, unlike virulent M. tuberculosis, cause extensive apoptosis of infected macrophages, which suggests a significant contribution of the apoptosis process to the initiation and subsequent amplification of innate as well as adaptive immune responses. Among various cues that could lead to apoptosis of host cells, the initiation of the apoptotic machinery by posttranscriptional mechanisms assumes significant importance. Among posttranscriptional control mechanisms, miRNAs are suggested to regulate several biological processes including immune responses. Various effectors of host immunity are known to be regulated by several miRNAs, and a prominent one among them, miRNA-155 (miR-155), often exhibits crucial roles during innate or adaptive immune responses. In this perspective, we identified a novel role of miR-155 during M. bovis BCG induced apoptosis of macrophages. The genetic and signaling perturbations data suggested that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in induced expression of the apoptotic genes as well as Caspase-3 cleavage and Cytochrome c translocation. Thus, augmented PKA signaling by M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, emphasizing a novel role for miR-155 in host immunity to mycobacterial infections. In perspective of these studies, important directives are often comprised of sequential and coordinated activation of TLR and NLR-driven signal transduction pathways, thus exhibiting foremost influence in determining the overall strength of the innate immune responses. As described, TLR2 exhibits dominant role in sensing various agonists including pathogen-associated molecular patterns (PAMPs) of microbes at the cell surface and generally considered as major effectuator of proinflammatory responses. Interestingly, NLRs like NOD1 or NOD2 often act in contrary, thus regulating anti-inflammatory responses as well as polarization of T cells towards skewed Th2 phenotype. This presents an interesting conundrum to functionality of DCs or macrophages in terms of effector functions during rapidly evolving immunological processes including effects originating from immunosuppressive effectors such as CTLA-4 or TGF-. DCs like macrophages are important sentinels of innate immunity, possesses array of PRRs that include TLRs and NOD-like receptors (NLRs). Signaling events associated with innate sensors like TLRs and NLRs often act as regulatory circuits that modulate the overall functions of DCs in terms of maturation process, cytokine or chemokine production, receptor expression, migration to secondary lymphoid organs for antigen presentation for effectuating Th polarization. TLR2, while acting as sensors for extracellular cues or endocytic network, drives signaling events in response to recognition of PAMPs including mycobacterial antigens like ESAT-6, PE_PGRS antigens, while NOD1 and NOD2 operate as cytosolic sensors initiating signaling pathways upon recognition of diaminopimelic acid (DAP) and muramyl dipeptide (MDP), components of bacterial peptidoglycan. Thus, TLRs or NOD receptors could trigger similar or contrasting immune responses by cooperative or non-cooperative sensing, consequently exhibiting immense complexity during combinatorial triggering of host DCs-PRR repertoire. In view of these observations, our current investigation comprehensively demonstrated that maturation process of human DCs were cooperatively regulated by signaling cascades initiated by engagements of TLR2, NOD1 and NOD2 receptors. Importantly, combined triggering of TLR2 and NOD receptors abolished the TGF-β or CTLA-4-mediated impairment of human DCs maturation, which required critical participation of NOTCH1-PI3K signaling cohorts. Thus, our data delineated the novel insights in modulation of macrophages and DCs effector functions by mycobacterial TLR2 or NOD agonists and broaden our understanding on the signal dynamics and integration of multiple signals from PRRs during mycobacterial infections. Altogether, our findings establish the understanding of conceptual frame work in fine tuning of TLR2 responses by SHH signaling as well as potential co-operativity among TLRs and NODs to modulate NOTCH1 dependent DCs maturation. Importantly, our study provides mechanistic and functional insights into various molecular regulators of macrophage cell fate decisions like miR-31. miR-150 and miR-155, which can fuel the search for attractive and effective drug targets and novel therapeutics to combat diseases of the hour like tuberculosis.