The PPE2 protein of Mycobacterium tuberculosis is secreted during infection and facilitates mycobacterial survival inside the host.

Mycobacterium tuberculosis secrets various effector proteins to evade host immune responses for facilitating its intracellular survival. The bacterial genome encodes several unique PE/PPE family proteins, which have been implicated to play important role in mycobacterial pathogenesis. A member of this family, PPE2 have been shown to contain a monopartite nuclear localization signal (NLS) and a DNA binding domain. In this study, we demonstrate that PPE2 protein is present in the sera of mice infected with either M. smegmatis expressing PPE2 or a clinical strain of M. tuberculosis (CDC1551). It was found that exogenously added PPE2 can permeate through the macrophage cell membrane and eventually translocate into the nucleus which requires the presence of NLS which showed considerable homology to HIV-tat like cell permeable peptides. Exogenously added PPE2 could inhibit NO production and decreased mycobacterial survival in macrophages. PPE2-null mutant of M. tuberculosis failed to inhibit NO production and had poor survival in macrophages which could be rescued by complementation with full-length PPE2. PPE2-null mutants also had poor survival in the lungs of infected mice indicating that PPE2 even when present in the bloodstream can confer a survival advantage to mycobacteria.

The cause-effect relation of tuberculosis on incidence of diabetes mellitus.

Tuberculosis (TB) is one of the oldest human diseases and is one of the major causes of mortality and morbidity across the Globe. Mycobacterium tuberculosis (Mtb), the causal agent of TB is one of the most successful pathogens known to mankind. Malnutrition, smoking, co-infection with other pathogens like human immunodeficiency virus (HIV), or conditions like diabetes further aggravate the tuberculosis pathogenesis. The association between type 2 diabetes mellitus (DM) and tuberculosis is well known and the immune-metabolic changes during diabetes are known to cause increased susceptibility to tuberculosis. Many epidemiological studies suggest the occurrence of hyperglycemia during active TB leading to impaired glucose tolerance and insulin resistance. However, the mechanisms underlying these effects is not well understood. In this review, we have described possible causal factors like inflammation, host metabolic changes triggered by tuberculosis that could contribute to the development of insulin resistance and type 2 diabetes. We have also discussed therapeutic management of type 2 diabetes during TB, which may help in designing future strategies to cope with TB-DM cases.

Vaccines against Tuberculosis: Where Are We Now?

Tuberculosis (TB) is among the top 10 leading causes of death in low-income countries. Statistically, TB kills more than 30,000 people each week and leads to more deaths than any other infectious disease, such as acquired immunodeficiency syndrome (AIDS) and malaria. TB treatment is largely dependent on BCG vaccination and impacted by the inefficacy of drugs, absence of advanced vaccines, misdiagnosis improper treatment, and social stigma. The BCG vaccine provides partial effectiveness in demographically distinct populations and the prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB incidences demands the design of novel TB vaccines. Various strategies have been employed to design vaccines against TB, such as: (a) The protein subunit vaccine; (b) The viral vector vaccine; (c) The inactivation of whole-cell vaccine, using related mycobacteria, (d) Recombinant BCG (rBCG) expressing Mycobacterium tuberculosis (M.tb) protein or some non-essential gene deleted BCG. There are, approximately, 19 vaccine candidates in different phases of clinical trials. In this article, we review the development of TB vaccines, their status and potential in the treatment of TB. Heterologous immune responses generated by advanced vaccines will contribute to long-lasting immunity and might protect us from both drug-sensitive and drug-resistant TB. Therefore, advanced vaccine candidates need to be identified and developed to boost the human immune system against TB.

Rab7l1 plays a role in regulating surface expression of toll like receptors and downstream signaling in activated macrophages.

Rab GTPases are known for controlling intracellular membrane traffic in a GTP-dependent manner. Rab7l1, belonging to family of Rab GTPases, is important for both endosomal sorting and retrograde transport. In our previous study, we identified a novel role of Rab7l1 in phagosome maturation. However, its role in regulating macrophage innate-effector signaling and cytokine response is not clearly understood. In this study, we have demonstrated that upon treatment of Rab7l1-knocked-down (Rab7l1-KD) THP-1 macrophages with lipopolysaccharide (LPS) and Pam3CSK4 has led to higher induction levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) as compared to the control cells that received scrambled shRNA. Similar results were observed in Rab7l1-KD RAW 264.7 and Balb/c peritoneal macrophages. The phospho-ERK 1/2 (extracellular signal-regulated kinase 1/2) and phospho-p38 MAPK (mitogen-activated protein kinase) levels, known to be responsible for higher induction of TNF-α and IL-10 respectively, were higher in Rab7l1-KD THP-1 macrophages which also displayed higher nuclear translocation of p50/p65 nuclear factor kappa B (NF-κB) upon stimulation with LPS. Surface expression levels of toll-like receptor 2 (TLR2), TLR4 and CD14 receptors were higher in Rab7l1-KD THP-1 macrophages as compared to the control cells. However, intracellular levels of these receptors were lower in Rab7l1-KD THP-1 macrophages as compared to the control group. Together, our study suggests that Rab7l1 has a role in regulating MAPK signaling and cytokine effector responses in macrophages by regulating the surface expression of membrane receptors.

Therapeutic application of PPE2 protein of Mycobacterium tuberculosis in inhibiting tissue inflammation.

There is an increasing need to develop biological anti-inflammatory agents that are more targeted, effective, and with lesser side effects as compared to conventional chemical drugs. In the present study, we found that Mycobacterium tuberculosis protein PPE2 and a synthetic derivative peptide can suppress the mast cell population and inhibit several vasoactive and fibrogenic mediators and pro-inflammatory cytokines induced by mast cells in formalin-induced tissue injury. PPE2 was found to inhibit transcription from the promoter of stem cell factor, important for mast cell maintenance and migration. Thus, PPE2/peptide can be used as a potent nonsteroidal therapeutic agent for the treatment of inflammation and tissue injury.

Secretory proteins of Mycobacterium tuberculosis and their roles in modulation of host immune responses: focus on therapeutic targets.

Mycobacterium tuberculosis, the bacterium responsible for tuberculosis, is one of the most successful pathogens in human history. An extremely resilient cell wall and highly evolved and coordinated strategies for immune evasion have made it a very formidable pathogen. The secretory proteins of M. tuberculosis play a crucial role in its virulence and immune evasion. The secretory proteins are secreted through tightly regulated secretion systems and modulate the host immune responses through a plethora of strategies, including epigenetic reprogramming of infected cells, targeting antigen presentation, inhibition of phagosomal maturation, modulation of cytokine production, apoptosis and redox regulation, etc. Upon infection, the secretory proteins become localized into various cellular organelles, such as nucleus, cytoplasm, phagosomes and Golgi, bodies and hijack the host machineries through their wide gamut of functions, including kinase, phosphatase, methyl transferase activities and interaction with several host partners. In this review, we discuss the secretion systems, the functions of various secretory proteins of M. tuberculosis and their roles in modulating immune responses of the host. We also discuss the feasibility of their use as possible therapeutic targets. This information is likely to improve our understanding of the host-pathogen interaction and help in the design of effective anti-tuberculosis therapeutics.

Phagosome maturation and modulation of macrophage effector function by intracellular pathogens: target for therapeutics.

Macrophages are important cells that regulate various innate functions. Macrophages after engulfment of pathogens proceed for phagosome maturation and finally fuse with lysosomes to kill pathogens. Although pathogen degradation is one of the important functions of phagosomes, various immune-effector functions of macrophages are also dependent on the phagosome maturation process. This review discusses signaling processes regulating phagosome maturation as well as various effector functions of macrophages such as apoptosis, antigen presentation, autophagy and inflammasome that are dependent on the phagosome maturation process. It also discusses strategies adopted by various intracellular pathogens to counteract these functions to evade intracellular destruction mechanisms. These studies may give direction for the development of new therapeutics to control various intracellular infections.

Rabaptin5 acts as a key regulator for Rab7l1-mediated phagosome maturation process.

Phagosome maturation is an important innate defence mechanism of macrophages against pathogen infections. Phagosome-lysosome (P-L) fusion is a highly regulated process. Different RabGTPases are involved in P-L fusion. Rab7l1 is shown to regulate P-L fusion process. In this study, we demonstrate that Rabaptin5 is a guanine nucleotide exchange factor (GEF) for Rab7l1. We reveal that Rabaptin5 interacts with Rab7l1-GTP form and promotes its recruitment to phagosome. In the absence of Rabaptin5, localization of P-L markers like EEA1, Rab7, LAMP1 and LAMP2 was found to be poorer. Thus, our data suggest that Rabaptin5 works upstream to Rab7l1 and triggers Rab7l1 activation for further recruitment of P-L markers and downstream regulation of phagosomal maturation process.

Moonlighting by PPE2 Protein: Focus on Mycobacterial Virulence.

In Mycobacterium tuberculosis, ∼10% of its genome encodes the proline-glutamic acid and proline-proline-glutamic acid (PPE) family of proteins, some of which were recently established to be key players in mycobacterial virulence. PPE2 (Rv0256c) is one among these proteins that we found to have pleiotropic effects during mycobacterial infection. PPE2 weakens the innate immune system by disturbing NO and reactive oxygen species production and myeloid hematopoiesis. We showed that PPE2 is unique for having nuclear localization signal, DNA binding domain, and SRC homology 3 (PXXP) binding domain, which enable it to interfere with the host immune system. Interestingly, PPE2 is a secretary protein, expressed during active tuberculosis (TB) infection, and is involved in facilitating survival of M. tuberculosis Thus, PPE2 could be a valuable drug target for developing effective therapeutics against TB. In this article, we describe possible roles of PPE2 in TB pathogenesis and the importance of PPE2 as a novel therapeutic target against TB.

Aptamers: An Emerging Tool for Diagnosis and Therapeutics in Tuberculosis.

Tuberculosis (TB) has been plaguing human civilization for centuries, and currently around one-third of the global population is affected with TB. Development of novel intervention tools for early diagnosis and therapeutics against Mycobacterium tuberculosis (M.tb) is the main thrust area in today's scenario. In this direction global efforts were made to use aptamers, the chemical antibodies as tool for TB diagnostics and therapeutics. This review describes the various aptamers introduced for targeting M.tb and highlights the need for development of novel aptamers to selectively target virulent proteins of M.tb for vaccine and anti-TB drugs. The objective of this review is to highlight the diagnostic and therapeutic application of aptamers used for tuberculosis. The discovery of aptamers, SELEX technology, different types of SELEX development processes, DNA and RNA aptamers reported for diseases and pathogenic agents as well have also been described in detail. But the emphasis of this review is on the development of aptamers which can block the function of virulent mycobacterial components for developing newer TB vaccine candidates and/or drug targets. Aptamers designed to target M.tb cell wall proteins, virulent factors, secretory proteins, or combination could orchestrate advanced diagnosis and therapeutic measures for tuberculosis.

Mycobacterium tuberculosis protein PPE2 binds to DNA region containing promoter activity.

ppe2 gene is predicted to be present in operon with non pe/ppe genes, cobq1 and cobu as ppe2-cobq1-cobu. Thus, we were interested to investigate the role of ppe2 in operonic organization. We performed microscale thermophoresis (MST) experiment which revealed that PPE2 protein could bind to upstream DNA segments of ppe2-cobq1-cobu operon. Upstream region of ppe2 had shown promoter activity in ß-gal assay. In this study, for the first time, a physical interaction between PPE2 protein and DNA fragment was reported, suggesting that PPE2 protein plays a role in the regulation of the putative ppe2-cobq1-cobu operon, via unknown mechanism.

Mycobacterium tuberculosis PPE18 protein inhibits MHC class II antigen presentation and B cell response in mice.

PPE18 protein belongs to PE/PPE family of Mycobacterium tuberculosis. We reported earlier that PPE18 protein provides survival advantage to M. tuberculosis during infection. In the current study, we found that PPE18 inhibits MHC class II-mediated antigen presentation by macrophages in a dose-dependent manner without affecting the surface level of MHC class II or co-stimulatory molecules. PPE18 does not affect antigen uptake or presentation of preprocessed peptide by macrophages. Antigen degradation was found to be inhibited by PPE18 protein due to perturbation in phagolysosomal acidification. PPE18-mediated inhibition of MHC class II antigen presentation caused poorer activation of CD4 T cells. Mice infected with M. smegmatis expressing PPE18 exhibited reduced maturation and activation of B cells and had decreased Mycobacteria-specific antibody titers. Thus M. tuberculosis probably utilizes PPE18 to inhibit MHC class II antigen presentation causing poorer activation of adaptive immune responses. This study may be useful in understanding host-pathogen interaction and open up directions of designing novel therapeutics targeting PPE18 to tackle this nefarious pathogen.

PPE2 protein of Mycobacterium tuberculosis affects myeloid hematopoiesis in mice.

Irregularity in hematopoiesis is noted in humans during tuberculosis. However, influence of mycobacterial protein(s) on bone marrow hematopoiesis is not fully understood. In this study, we have demonstrated the role of a mycobacterial protein, PPE2 (Rv0256c) in suppressing hematopoiesis during infection. PPE2 belongs to PPE (proline-proline-glutamine) family of mycobacterial proteins which are well known for hijacking host machineries for better survival inside host. In the present study, we have shown that mice infected with Mycobacterium smegmatis expressing PPE2 (M. smeg-PPE2) had a marked reduction in cells of myeloid lineage in bone marrow and peripheral blood along with altered bone marrow phenotype. Bone marrow of M. smeg-PPE2-infected mice showed an overall hypo-cellularity with an increase in population of immature cells, along with reduction in mature cell population. Higher number of M. smeg-PPE2 bacilli was observed in bone-marrow, lung, liver and spleen of mice as compared to the control mycobacteria (M. smeg-pVV16). M. smeg-PPE2-infected mice also showed higher expression of IFN-γ than those infected with M. smeg-pVV16. We conclude that PPE2 affects bone-marrow hematopoiesis of myeloid cells, probably by increasing IFN-γ levels, both locally and systemically, thus favoring the bacilli to establish a positive infection.

ESAT-6 Protein of Mycobacterium tuberculosis Increases Holotransferrin-Mediated Iron Uptake in Macrophages by Downregulating Surface Hemochromatosis Protein HFE.

Iron is an essential element for Mycobacterium tuberculosis; it has at least 40 enzymes that require iron as a cofactor. Accessibility of iron at the phagosomal surface inside macrophage is crucial for survival and virulence of M. tuberculosis ESAT-6, a 6-kDa-secreted protein of region of difference 1, is known to play a crucial role in virulence and pathogenesis of M. tuberculosis In our earlier study, we demonstrated that ESAT-6 protein interacts with ß-2-microglobulin (ß2M) and affects class I Ag presentation through sequestration of ß2M inside endoplasmic reticulum, which contributes toward inhibition of MHC class I:ß2M:peptide complex formation. The 6 aa at C-terminal region of ESAT-6 are essential for ESAT6:ß2M interaction. ß2M is essential for proper folding of HFE, CD1, and MHC class I and their surface expression. It is known that M. tuberculosis recruit holotransferrin at the surface of the phagosome. But the upstream mechanism by which it modulates holotransferrin-mediated iron uptake at the surface of macrophage is not well understood. In the current study, we report that interaction of the ESAT-6 protein with ß2M causes downregulation of surface HFE, a protein regulating iron homeostasis via interacting with transferrin receptor 1 (TFR1). We found that ESAT-6:ß2M interaction leads to sequestration of HFE in endoplasmic reticulum, causing poorer surface expression of HFE and HFE:TFR1 complex (nonfunctional TFR1) in peritoneal macrophages from C57BL/6 mice, resulting in increased holotransferrin-mediated iron uptake in these macrophages. These studies suggest that M. tuberculosis probably targets the ESAT-6 protein to increase iron uptake.

Uncovering Structural and Molecular Dynamics of ESAT-6:ß2M Interaction: Asp53 of Human ß2-Microglobulin Is Critical for the ESAT-6:ß2M Complexation.

ESAT-6 is a small secreted protein of Mycobacterium tuberculosis involved in the ESAT-6 secretion system (ESX-1)-mediated virulence and pathogenesis. The protein interacts with ß2M, causing downregulation of MHC class I Ag presentation, which could be one of the mechanisms by which it favors increased survival of the bacilli inside the host. In an earlier study, we have shown that the C-terminal region of ESAT-6 is crucial for its interaction with ß2M. However, the interface of ß2M involved in interaction with ESAT-6 and detailed physicochemical changes associated with ESAT-6:ß2M complexation are not fully defined. In this study, using computational and site-directed mutagenesis studies, we demonstrate the presence of strong noncovalent hydrophobic interactions between ESAT-6 and ß2M in addition to the vital hydrogen bonding between the aspartate residue (Asp53) of ß2M and methionine (Met93) of ESAT-6. Docking-based high-throughput virtual screening followed by 16-point screening on microscale thermophoresis resulted in the identification of two potent inhibitors (SM09 and SM15) that mask the critical Met93 residue of ESAT-6 that is required for ESAT-6:ß2M interaction and could rescue cell surface expression of ß2M and HLA in human macrophages as well as MHC class I Ag presentation suppressed by ESAT-6 in peritoneal macrophages isolated from C57BL/6 mice. Both SM09 and SM15 significantly inhibited intracellular survival of M. tuberculosis in human macrophages. Further, we characterized the physicochemical properties involved in the ESAT-6:ß2M complexation, which may help in understanding host-pathogen interactions.

Mycobacterium tuberculosis PPE2 Protein Interacts with p67phox and Inhibits Reactive Oxygen Species Production.

Mycobacterium tuberculosis employs defense mechanisms to protect itself from reactive oxygen species (ROS)-mediated cytotoxicity inside macrophages. In the current study, we found that a secretory protein of M. tuberculosis PPE2 disrupted the assembly of NADPH oxidase complex. PPE2 inhibited NADPH oxidase-mediated ROS generation in RAW 264.7 macrophages and peritoneal macrophages from BALB/c mice. PPE2 interacted with the cytosolic subunit of NADPH oxidase, p67phox, and prevented translocation of p67phox and p47phox to the membrane, resulting in decreased NADPH oxidase activity. Trp236 residue present in the SH3-like domain of PPE2 was found to be critical for its interaction with p67phox Trp236Ala mutant of PPE2 did not interact with p67phox and thereby did not affect ROS generation. M. tuberculosis expressing PPE2 and PPE2-null mutants complemented with PPE2 survived better than PPE2-null mutants in infected RAW 264.7 macrophages. Altogether, this study suggests that PPE2 inhibits NADPH oxidase-mediated ROS production to favor M. tuberculosis survival in macrophages. The findings that M. tuberculosis PPE2 protein is involved in the modulation of oxidative response in macrophages will help us in improving our knowledge of host-pathogen interactions and the application of better therapeutics against tuberculosis.

Dribbling through the host defence: targeting the TLRs by pathogens.

The immune system is well-equipped with sensors that detect invading pathogens and dictate subsequent immune responses for clearing the infections. One such class of sensor is the toll-like receptor (TLR), that can sense diverse molecules of pathogen origin such as proteins, lipids, carbohydrate, DNA, RNA, and trigger suitable immune responses to prevent infections. However, successful pathogens have evolved strategies to bypass the TLR-driven host immune responses to enable their survival inside the host. In this review, we have discussed about the recent advances in TLR biology and strategies adopted by various pathogens (bacteria, virus, and parasites) to subvert the TLR-signalling for evading host-immune attack. Further, we have discussed how TLRs are linked in augmenting infection burden and disease severity in host during co-infection. This information is likely to be helpful to design TLR-based immunotherapeutics to control various infections and pathophysiological disorders.

Calcium Signaling Commands Phagosome Maturation Process.

Phagosome-lysosome (P-L) fusion is one of the central immune-effector responses of host. It is known that phagosome maturation process is associated with numerous signaling cascades and among these, important role of calcium (Ca2+) signaling has been realized recently. Ca2+ plays key roles in actin rearrangement, activation of NADPH oxidase and protein kinase C (PKC). Involvement of Ca2+ in these cellular processes directs phagosomal maturation process. Some of the intracellular pathogens have acquired the strategies to modulate Ca2+ associated pathways to block P-L fusion process. In this review we have described the mechanism of Ca2+ signals that influence P-L fusion by controlling ROS, actin and PKC signaling cascades. We have also discussed the strategies implemented by the intracellular pathogens to manipulate Ca2+ signaling to consequently subvert P-L fusion. A detail study of factors associated in manipulating Ca2+ signaling may provide new insights for the development of therapeutic tools for more effective treatment options against infectious diseases.

PPE65 of M. tuberculosis regulate pro-inflammatory signalling through LRR domains of Toll like receptor-2.

Our understanding of the PE/PPE family of proteins in M. tuberculosis (Mtb) pathogenesis is still evolving and their critical roles in the host immunomodulation are still in the discovery process. Earlier studies from our group have shown that TLR2-LRR domain plays an important role in regulating cytokine signalling by PPE proteins. The importance of TLR2-LRR domain 16-20 in the regulation of PPE17-induced pro-inflammatory signalling has been established recently. However, it is yet to find whether other PPE protein also targets the TLR2-LRR 16-20 domain for induction of pro-inflammatory responses. In the current study, we have explored the structural parameters and possible role of PPE65 in generating pro-inflammatory signalling molecules mediated through IRAK3 downstream of TLR2-LRR domain 16-20. This study conceptualizes the functional characteristics of PPE65 in infection condition and might possibly provide valuable information in exploring this protein as an immunomodulator in Mtb infection.

PPE17 (Rv1168c) protein of Mycobacterium tuberculosis detects individuals with latent TB infection.

Latent tuberculosis infection (LTBI) is a clinically distinct category of Mycobacterium tuberculosis (Mtb) infection that needs to be diagnosed at the initial stage. We have reported earlier that one of the Mtb proline-proline-glutamic acid (PPE) proteins, PPE17 (Rv1168c) is associated with stronger B-cell and T-cell responses and could be used to diagnose different clinical categories of active TB patients with higher specificity and sensitivity than PPD and ESAT-6. Based on these observations we further tested the potential of PPE17 for the diagnosis of LTBI. We tested 198 sera samples collected from LTBI individuals (n = 61), QFT-negative (n = 58) and active TB patients (n = 79). Individuals were defined as LTBI by QuantiFERON-TB Gold In-Tube test (QFT-GIT) positive results, while active TB patients were confirmed based on the guidelines of the Revised National TB Control Programme of India. The antibody responses against PPE17, ESAT-6:CFP-10 and PPD were compared in these subjects by enzyme-linked immunosorbent assay. We observed that LTBI individuals show a higher sero-reactivity to PPE17 as compared to currently used latent TB diagnostic antigens like ESAT-6, CFP-10 and PPD. The LTBI and active TB patients display almost similar sensitivity. Interestingly, PPE17 could discriminate LTBI positive subjects from the QFT-negative subjects (P < 0.001). Our study hints that PPE17 may be used as a novel serodiagnostic marker to screen the latently infected subjects and may also be used as a complimentary tool to the QFT-GIT.