Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

Human alveolar T lymphocyte responses to Mycobacterium tuberculosis antigens: role for CD4+ and CD8+ cytotoxic T cells and relative resistance of alveolar macrophages to lysis.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios of > or = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios of > or = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

CD4+ alpha beta T cell and gamma delta T cell responses to Mycobacterium tuberculosis. Similarities and differences in Ag recognition, cytotoxic effector function, and cytokine production.

CD4+ and gamma delta T cells are activated readily by Mycobacterium tuberculosis. To examine their role in the human immune response to M. tuberculosis, CD4+ and gamma delta T cells from healthy tuberculin-positive donor were studied for patterns of Ag recognition, cytotoxicity, and cytokine production in response to M. tuberculosis-infected mononuclear phagocytes. Both T cell subsets responded to intact M. tuberculosis and its cytosolic Ags. However, CD4+ and gamma delta T cells differed in the range of cytosolic Ags recognized: reactivity to a wide m.w. range of Ags for CD4+ T cells, and a restricted pattern for gamma delta T cells, with dominance of Ags of 10 to 15 kDa. Both T cell subsets were equally cytotoxic for M. tuberculosis-infected monocytes. Furthermore, both CD4+ and gamma delta T cells produced large amounts of IFN-gamma: mean pg/ml of IFN-gamma in supernatants was 2458 +/- 213 for CD4+ and 2349 +/- 245 for gamma delta T cells. By filter-spot ELISA (ELISPOT), the frequency of IFN-gamma-secreting gamma delta T cells was one-half of that of CD4+ T cells in response to M. tuberculosis, suggesting that gamma delta T cells on a per cell basis were more efficient producers of IFN-gamma than CD4+ T cells. In contrast, CD4+ T cells produced more IL-2 than gamma delta T cells, which correlated with diminished T cell proliferation of gamma delta T cells compared with CD4+ T cells. These results indicate that CD4+ and gamma delta T cell subsets have similar effector functions (cytotoxicity, IFN-gamma production) in response to M. tuberculosis-infected macrophages, despite differences in the Ags recognized, IL-2 production, and efficiency of IFN-gamma production.

Factors associated with medication adherence among tuberculosis patients in Timor-Leste

This thesis investigated medication adherence among people living with tuberculosis in Timor Leste. Suboptimal adherence was commonly reported, and was influenced by service inaccessibility, family poverty, patients' absence of disease symptoms and misperception of recovery, untreated depression and a popular cultural belief that luck or chance determines health outcomes. The study has implications for improving health literacy and counselling programs to achieve effective adherence to medication and good health outcomes.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M.tuberculosis and, in particular, the proline-glutamic acid-polymorphicguanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosismand host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid-polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

How pantothenol intervenes in Coenzyme-A biosynthesis of Mycobacterium tuberculosis

Coenzyme A is an indispensable cofactor for all organisms and holds a central position in a number of pathways. Prokaryotic enzymes involved in the synthesis of CoA are quite different from their mammalian counterparts; hence, they are good targets for the development of antimicrobials to treat many diseases. There are antimicrobials that act by inhibiting CoA biosynthesis. It has been suggested that pantothenol exhibits antibacterial activity by competitively inhibiting pantothenate kinase, a key regulatory enzyme for CoA synthesis. Contrary to these suggestions, in this paper, we demonstrate that pantothenol acts as a substrate for Mycobacterium tuberculosis and Escherichia coli pantothenate kinases. The product, 4'-phosphopantothenol, thus formed inhibits competitively the utilization of 4'-phosphopantothenate by CoaBC. Thus, it is the failure of CoaBC to utilize 4'-phosphopantothenol as a substrate that accounts for the bactericidal activity of pantothenol. (C) 2007 Elsevier Inc. All rights reserved.

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diamino-pelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1 s(-1). The K-m values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 mu M and 308.28 mu M, respectively whereas the V-max and k(cat) values for the enzyme are 0.02074 mu moles/min/ml and 0.003 s(-1), respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Inhibitory activity of Image -asparaginase from Mycobcacterium tuberculosis on Yoshida ascites sarcoma in rats

The antitumor activity of Image -asparagine amidohydrolases (EC 3.5.1.1) from Mycobacterium tuberculosis H37Rv and H37Ra strains has been tested on Yoshida ascites sarcoma in rats. The enzyme specific to M. tuberculosis H37Ra but not to H37Rv has proved to be effective in inhibiting the growth of the sarcoma. Comparative studies on the activity of this enzyme with that of similar enzyme from Escherichia coli B, has shown that at the same levels the former is more effective than the latter. Long-lived immunity to this tumor in A/IISc Wistar rats following treatment of tumor bearing animals with M. tuberculosis H37Ra, pH 9.6 Image -asparaginase has been observed. Immunity in these rats was demonstrated by tumor rejection and detection of humoral antibodies in the sera to the antigen of the cell-free extract of the tumor. The enzyme was ineffective in inhibiting fibrosarcoma in mice at the dose levels tested.

Structural and Biochemical Bases for the Redox Sensitivity of Mycobacterium tuberculosis RslA

An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis a factor sigma(L) regulates the expression of genes involved in cell-wall and polyketide syntheses. sigma(L) interacts with the cytosolic anti-sigma domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn2+ and can sequester sigma(L) in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn2+. This results in a substantial rearrangement of the sigma(L)/RslA complex, leading to an 8-fold decrease in the affinity of RslA for sigma(L). The crystal structure of the -35-element recognition domain of sigma(L), sigma(L)(4), bound to RslA reveals that RslA inactivates sigma(L) by sterically occluding promoter DNA and RNpolymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn2+ in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of sigma(L)/RslA interactions provide a template for understanding how variations in the rate of Zn2+ release and associated conformational changes could regulate the activity of a Zn2+-associated anti-sigma factor. (C) 2010 Elsevier Ltd. All rights reserved.

Image -asparaginases from Mycobacterium tuberculosis strains H37Rv and H37Ra

M. tuberculosis H37Ra possesses two Image -asparaginases while the H37Rv strain possesses only a single enzyme. These enzymes have been purified and their properties studied. The two Image -asparaginases in H37Ra strain differ from each other in pH optima, heat inactivation, Michaelis constant and effects of inhibitors, while one of them resembles the single Image -asparaginase present in the H37Rv strain. Image -Cysteine inhibits both Image -asparaginases in an allosteric manner probably because it is one of the end-products in Image -asparagine metabolism. This is the first time that a qualitative difference has been reported in the enzyme pattern between the avirulent and virulent strains of M. tuberculosis.