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.

More human lives have been lost to tuberculosis than to any other disease and despite the availability of effective short course chemotherapy (DOTS) as well as the Bacilli Calmette Guerin (BCG) vaccine, tuberculosis continues to claim more than a million lives annually. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is one of the most successful and scientifically challenging pathogens of all time. However in the last two decades, the ability to perform molecular genetic analysis of M. tuberculosis has resulted in powerful new research tools, while the availability of the complete genome sequence has provided us with a wealth of new information and understanding of the biology of this major pathogen. One of the major challenges, however, is to analyze the properties and functions of those genes that are unique to M. tuberculosis genome. The identification and characterization of such genes which impart various survival strategies employed by M. tuberculosis for successful infection will be of particular significance. One of the important outcomes from the complete genome sequence of M. tuberculosis is the discovery of two multigene families designated PE (99 members) and PPE (69 members) named respectively for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. In addition to these motifs, proteins of the PE family possess highly homologous N-terminal domains of approximately 100 amino acids (PE domain), whereas the PPE proteins possess a highly homologous N-terminal domain of about 180 amino acids (PPE domain). Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. The current study focuses on Rv3097c gene of M. tuberculosis, a PE family gene that was bioinformatically predicted to be a triacylglycerol hydrolase (lipase). In order to decipher the role of the PE domain, we have carried out functional characterization of the Rv3097c gene (also named lipY) as it was, initially, the only known PE protein for which an enzymatic function (i.e. lipase activity) had been predicted. Further, to understand the function of PE family proteins, an important question that needs to be answered is; whether the PE domain of different PE family proteins has similar or different functions? In this context, our studies were focused on studying the functional role of the PE domain in LipY, as outlined below. In general, the in vivo function and subcellular localization of any protein are integrally connected. PE domain has been reported to be essential for cell wall localization of PE_PGRS33, another PE family protein. Therefore we investigated the subcellular localization of LipY and the influence of the PE domain on subcellular localization of LipY. LipY and a truncated form of LipY lacking the PE domain [LipY(ΔPE)] were expressed in mycobacteria(M. smegmatis and M. bovis BCG). Subcellular fractionation and western blot demonstrated that both LipY and LipY(ΔPE) were predominantly detected in the cell wall fraction, indicating that LipY is localized to the cell wall and the PE domain of LipY was not required for translocation of LipY to cell wall. This result is in contrast to the findings for PE_PGRS33, where the absence of the PE domain caused the cell wall associated protein to localize to the cytosol. Furthermore, immuno-electron microscopy of M. bovis BCG expressing LipY(ΔPE) clearly showed a cell surface localization of LipY(ΔPE). These results signify that the function of the PE domain might not always be similar amongst different PE family proteins. In order to further investigate the role of the PE domain in LipY, we studied the lipase activity of LipY and the influence of the PE domain on lipase activity. Bioinformatic analysis confirmed the presence of a lipase domain containing a GDSAG active site motif characteristic of lipases. Overexpression of LipY in mycobacteria (M. smegmatis and M. bovis BCG) resulted in a significant reduction in the pool of triacylglycerols (TAG), consistent with the lipase activity of this enzyme. Interestingly, this reduction was more pronounced in mycobacteria overexpressing LipY(ΔPE), suggesting that the presence of the PE domain diminishes the lipase activity of LipY. In vitro lipase assays also confirmed LipY(ΔPE) as a more efficient lipase compared to the wild-type LipY. Together these results suggest that the PE domain of LipY might be involved in the modulation of lipase activity. Surprisingly, M. marinum, another pathogenic mycobacteria, possesses a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain. The overexpression of LipYmar in M. smegmatis significantly reduced the TAG pool suggesting that it is a triacylglycerol hydrolase/lipase. Interestingly, similar to the removal of the PE domain of LipY, this reduction in the TAG pool was further pronounced when the PPE domain of LipYmar was removed. This suggests that PE and PPE domains might share similar functional roles in modulating the enzymatic activities of these lipase homologs. In order to assess the in vivo relevance of LipY expression during M. tuberculosis infection, we examined the humoral immune responses against LipY in sera derived from various clinical categories of tuberculosis patients. The presence of specific antibodies against any protein is suggestive of expression of the protein during infection and could potentially be used to differentiate between healthy individuals and infected patients (serodiagnosis of tuberculosis). The cell wall localization suggested that LipY may be accessible for interaction with the host immune system during infection. Moreover, humoral responses were observed against LipY in mice immunized with DNA constructs expressing LipY, indicating that LipY could be an effective B-cell antigen. Accordingly, a strong humoral response against LipY and LipY(ΔPE) was observed in tuberculosis patients compared to healthy individuals, suggesting that LipY is expressed during infection by clinical strains of M. tuberculosis and might represent an immunodominant antigen of M. tuberculosis with potential use in serodiagnosis of tuberculosis.