Proteomics and Network Analyses Reveal Inhibition of Akt-mTOR Signaling in CD4(+) T Cells by Mycobacterium Tuberculosis Mannose-Capped Lipoarabinomannan (original) (raw)
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Cellular Immunology, 2012
Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust CD4 + T cell responses. We have shown previously that M. tuberculosis cell wall glycolipids, including mannose capped lipoarabinomannan (ManLAM), directly inhibit polyclonal murine CD4 + T cell activation by blocking ZAP-70 phosphorylation. We extended these studies to antigen-specific murine CD4 + T cells and primary human T cells and found that ManLAM inhibited them as well. Lck and LAT phosphorylation also were inhibited by ManLAM without affecting their localization to lipid rafts. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that ManLAM insertion into T cell membranes was required. Thus, M. tuberculosis ManLAM inhibits antigen-specific CD4 + T cell activation by interfering with very early events in TCR signaling through ManLAM's insertion in T cell membranes.
Infection and Immunity, 2009
Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust adaptive CD4 ؉ T-cell responses. We have previously shown that M. tuberculosis can indirectly inhibit CD4 ؉ T cells by suppressing the major histocompatibility complex class II antigen-presenting cell function of macrophages. This study was undertaken to determine if M. tuberculosis could directly inhibit CD4 ؉ T-cell activation. Murine CD4 ؉ T cells were purified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4 ؉ T cells were activated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence of M. tuberculosis and its subcellular fractions. CD4 ؉ T-cell activation was measured by interleukin 2 production, proliferation, and expression of activation markers, all of which were decreased in the presence of M. tuberculosis. Fractionation identified that M. tuberculosis cell wall glycolipids, specifically, phosphatidylinositol mannoside and mannosecapped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-like receptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin. ZAP-70 phosphorylation was decreased in the presence of M. tuberculosis glycolipids, indicating that M. tuberculosis glycolipids directly inhibited CD4 ؉ T-cell activation by interfering with proximal T-cell-receptor signaling.
European Journal of Immunology, 2008
We evaluated the role of regulatory T cells (CD4+ CD25+ Foxp3+ cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19-kDa protein and heat shock protein 65 had no effect. Anti-IL-10 and anti-TGF-β alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF-β and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4+ IFN-γ cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8+ cells decreased the frequency of IFN-γ+cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.
Frontiers in immunology, 2014
CD4(+) T cells are prominent effector cells in controlling Mycobacterium tuberculosis (Mtb) infection but may also contribute to immunopathology. Studies probing the CD4(+) T cell response from individuals latently infected with Mtb or patients with active tuberculosis using either small or proteome-wide antigen screens so far revealed a multi-antigenic, yet mostly invariable repertoire of immunogenic Mtb proteins. Recent developments in mass spectrometry-based proteomics have highlighted the occurrence of numerous types of post-translational modifications (PTMs) in proteomes of prokaryotes, including Mtb. The well-known PTMs in Mtb are glycosylation, lipidation, or phosphorylation, known regulators of protein function or compartmentalization. Other PTMs include methylation, acetylation, and pupylation, involved in protein stability. While all PTMs add variability to the Mtb proteome, relatively little is understood about their role in the anti-Mtb immune responses. Here, we review ...
European journal of immunology, 2016
Cells in homeostasis metabolise glucose mainly through the tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), whilst activated cells switch their basal metabolism to aerobic glycolysis. In thus study, we examined whether metabolic reprogramming towards aerobic glycolysis is important for the host response to Mycobacterium tuberculosis (Mtb). Through transcriptional and metabolite analysis we show that Mtb induces a switch in host cellular metabolism towards aerobic glycolysis in human peripheral blood mononuclear cells (PBMCs). The metabolic switch is TLR2-dependent but NOD2-independent, and is mediated in part through activation of the AKT-mTOR pathway. We show that pharmacological inhibition of the AKT/mTOR pathway inhibits cellular responses to Mtb both in vitro in human peripheral blood mononuclear cells, and in vivo in a model of murine tuberculosis. Our findings reveal a novel regulatory layer of host responses to Mtb which will aid understanding of host s...
2021
Objectives Identification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study. Methods Biochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome. Results Rv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likel...
Microbiology (Reading, England), 2017
Mycobacterium tuberculosis employs two-component systems (TCSs) for survival within its host. The TCS MtrAB is conserved among mycobacteria. The response regulator MtrA is essential in M. tuberculosis. The genome-wide chromatin immunoprecipitation (ChIP) sequencing performed in this study suggested that MtrA binds upstream of at least 45 genes of M. tuberculosis, including those involved in cell wall remodelling, stress responses, persistence and regulation of transcription. It binds to the promoter regions and regulates the peptidoglycan hydrolases rpfA and rpfC, which are required for resuscitation from dormancy. It also regulates the expression of whiB4, a critical regulator of the oxidative stress response, and relF, one-half of the toxin-antitoxin locus relFG. We have identified a new consensus 9 bp loose motif for MtrA binding. Mutational changes in the consensus sequence greatly reduced the binding of MtrA to its newly identified targets. Importantly, we observed that overexp...
Journal of General Virology, 1998
Tuberculosis has emerged as an epidemic, extended by the large number of individuals infected with human immunodeficiency virus type 1 (HIV-1). The major goal of this study was to determine whether the mycobacterial cell wall component mannosecapped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis (M. tuberculosis) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system. These experiments are of prime importance considering that CD4expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals. Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR, it was first found that culture protein filtrates (CFP) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts
Journal of Biological Chemistry, 2001
Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAMstimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (⌬p85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr 308 and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with ⌬p85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/ Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis.
Anergy via GRAIL T + Induces CD 4 Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan from
2015
Mycobacterium tuberculosis cell wall glycolipid, lipoarabinomannan, can inhibit CD4 + T cell activation by downregulating the phosphorylation of key proximal TCR signaling molecules: Lck, CD3z, ZAP70, and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an Ag encounter, yet remain viable and hyporesponsive. We tested whether mannose-capped lipoarabinomannan (LAM)-induced inhibition of CD4 + T cell activation resulted in CD4 + T cell anergy. The presence of LAM during primary stimulation of P25 TCR-transgenic murine CD4 + T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25 TCR-transgenic CD4 + T cells primed in the presence of LAM also exhibited decreased response upon restimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hyporesponsiveness to restimulation was not due to apoptosis, generation of Foxp3-positive regulatory T cells, or inhibitory cytokines. Acquisition of the anergic phenotype correlated with upregulation of gene related to anergy in lymphocytes (GRAIL) protein in CD4 + T cells. Inhibition of human CD4 + T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA-mediated knockdown of GRAIL before LAM treatment abrogated LAM-induced hyporesponsiveness. In addition, exogenous IL-2 reversed defective proliferation by downregulating GRAIL expression. These results demonstrate that LAM upregulates GRAIL to induce anergy in Ag-reactive CD4 + T cells. Induction of CD4 + T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.