Mtb Lipid Research Articles

Biocompatible chitosan nanoparticles as an efficient delivery vehicle for Mycobacterium tuberculosis lipids to induce potent cytokines and antibody response through activation of γδ T cells in mice

The activation of cell-mediated and humoral immune responses to Mycobacterium tuberculosis (Mtb) is critical for protection against the pathogen and nanoparticle-mediated delivery of antigens is a more potent way to induce different immune responses. Herein, we show that mice immunized with Mtb lipid-bound chitosan nanoparticles (NPs) induce secretion of prominent type-1 T-helper (Th-1) and type-2 T-helper (Th-2) cytokines in lymph node and spleen cells, and also induces significantly higher levels of IgG, IgG1, IgG2 and IgM in comparison to control mice. Furthermore, significantly enhanced γδ-T-cell activation was observed in lymph node cells isolated from mice immunized with Mtb lipid-coated chitosan NPs as compared to mice immunized with chitosan NPs alone or Mtb lipid liposomes. In comparison to CD8+ cells, significantly higher numbers of CD4+ cells were present in both the lymph node and spleen cells isolated from mice immunized with Mtb lipid-coated chitosan NPs. In conclusion, this study represents a promising new strategy for the efficient delivery of Mtb lipids using chitosan NPs to trigger an enhanced cell-mediated and antibody response against Mtb lipids.

Selection of phage-displayed human antibody fragments specific for CD1b presenting the Mycobacterium tuberculosis glycolipid Ac2SGL

Objective/backgroundThe development of new tools capable of targeting Mycobacterium tuberculosis (Mtb)-infected cells have potential applications in diagnosis, treatment, and prevention of tuberculosis. In Mtb-infected cells, CD1b molecules present Mtb lipids to the immune system (Mtb lipid–CD1b complexes). Because of the lack of CD1b polymorphism, specific Mtb lipid–CD1b complexes could be considered as universal Mtb infection markers. 2-Stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL) is specific for Mtb, and is not present in other mycobacterial species. The CD1b–Ac2SGL complexes are expressed on the surface of human cells infected with Mtb. The aim of this study was to generate ligands capable of binding these CD1b–Ac2SGL complexes. MethodsA synthetic human scFv phage antibody library was used to select phage-displayed antibody fragments that recognized CD1b–Ac2SGL using CD1b-transfected THP-1 cells loaded with Ac2SGL. ResultsOne clone, D11—a single, light-variable domain (kappa) antibody (dAbκ11)—showed high relative binding to the Ac2SGL–CD1b complex. ConclusionA ligand recognizing the Ac2SGL–CD1b complex was obtained, which is a potential candidate to be further tested for diagnostic and therapeutic applications.

Mycobacterial lipid antigen-specific innate T cell responses in TB-naïve macaques (INC4P.328)

Abstract Current tuberculosis (TB) vaccine strategies are targeted mainly towards conventional T cell responses to mycobacterial protein antigens. However, there is growing evidence for the role of mycobacterial lipid specific CD1-restricted T cell responses in anti-mycobacterial immunity. Understanding how the CD1-restricted T cells contribute to TB may provide critical insights into innate mechanisms of resistance to TB and aid the development of new therapies against TB. In this pilot study, we utilized cynomolgus macaques (CM) to characterize innate T cell responses to mycobacterial lipid antigens. PBMC from naïve CM were stimulated with purified mycobacterial lipid antigens, including Lipoarabinomannan (LAM), Phosphatidylinositol mannoside (PIM-1, 2, & 6) and total cellular lipids of MTb H37Rv, presented on CD1-a, -b, -c and -d transfectant C1R B cell lines. T cell responses were measured by IFN-γ, TNF-a and IL-2 ELISA in culture supernatants, and by intracellular cytokine staining for IFN-g, TNF-a, and IL-13. Robust TNF-a and IFN-g production was observed in response to CD1d/PIM and CD1b/LAM in all of the eight CM following 16 hours stimulation. Mainly CD8 positive and CD4/CD8 double negative T cells contributed to the dominant CD1-restricted Th1 cytokine response. The innate Th1-type response of T cells to MTb lipid antigens, particularly LAM and PIM, may be a significant component of the innate immune response in TB and thus promising targets for improved TB vaccines.

Mycobacterial envelope lipids fingerprint from direct MALDI-TOF MS analysis of intact bacilli

Mycobacterium tuberculosis (Mtb) lipids including glycolipids and lipoglycans play a crucial role in the modulation of the host immune response by targeting the innate receptors C-type lectins, TLRs and the CD1 proteins of class 1. Glycolipids have been shown to be biomarkers of M. tuberculosis strains and also of opportunistic mycobacteria called non-tuberculous mycobacteria. Most of the structural and functional work of the Mtb lipids has been done using lipids arising from M. tuberculosis cell growth in vitro. However it is likely that lipid structures can change during infection or among the M. tuberculosis or opportunistic clinical strains. Here we describe a new, rapid and sensitive analysis of lipids directly on whole mycobacteria which can be done in few minutes and on less than 1000 mycobacteria by direct matrix-assisted laser desorption/ionization mass spectrometry using an unusual solvent matrix. By this new methodology, which does not require extraction or purification steps, we are able to discriminate mycobacteria belonging to the Mtb complex as well as opportunistic and non-pathogenic mycobacteria. This method was also found to be successful for identification of an envelope lipid mutant. This work opens a new analytical route for in vivo analysis of mycobacterial lipids.

The mycobacterial cell envelope-lipids.

Mycobacterium tuberculosis (Mtb) lipids are indelibly imprinted in just about every key aspect of tuberculosis (TB) basic and translational research. Although the interest in these compounds originally stemmed from their abundance, structural diversity, and antigenicity, continued research in this field has been driven by their important contribution to TB pathogenesis and their interest from the perspective of drug, vaccine, diagnostic, and biomarker development. This article summarizes what is known of the roles of lipids in the physiology and pathogenicity of Mtb and the exciting developments that have occurred in recent years in identifying new lead compounds targeting their biogenesis.

Molecular analysis of the Mycobacterium tuberculosis lux-like mel2 operon

Using a high throughput genetic strategy, designated Random Inducible Controlled Expression (RICE), we identified the six gene mel2 locus in Mtb and M. marinum. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, mel2 facilitates detoxification of reactive oxygen species (ROS). Through the use of thin layer chromatography (TLC) we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate (PDIM), in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol (TAG) in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis.

CD1-restricted adaptive immune responses to Mycobacteria in human group 1 CD1 transgenic mice

Group 1 CD1 (CD1a, CD1b, and CD1c)–restricted T cells recognize mycobacterial lipid antigens and are found at higher frequencies in Mycobacterium tuberculosis (Mtb)–infected individuals. However, their role and dynamics during infection remain unknown because of the lack of a suitable small animal model. We have generated human group 1 CD1 transgenic (hCD1Tg) mice that express all three human group 1 CD1 isoforms and support the development of group 1 CD1–restricted T cells with diverse T cell receptor usage. Both mycobacterial infection and immunization with Mtb lipids elicit group 1 CD1–restricted Mtb lipid–specific T cell responses in hCD1Tg mice. In contrast to CD1d-restricted NKT cells, which rapidly respond to initial stimulation but exhibit anergy upon reexposure, group 1 CD1–restricted T cells exhibit delayed primary responses and more rapid secondary responses, similar to conventional T cells. Collectively, our data demonstrate that group 1 CD1–restricted T cells participate in adaptive immune responses upon mycobacterial infection and could serve as targets for the development of novel Mtb vaccines.

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.

A study of mycobacterial lipid antigen-specific responses in a novel model of human group 1 CD1 (45.31)

Abstract Group 1 CD1 presents mycobacterial lipid antigens to various T cell subsets. Although group 1 CD1-restricted responses have been implicated in anti-mycobacterial immunity in humans, little is known about their function, as mice lack group 1 CD1. To study group 1 CD1-restricted immune responses in vivo, we have generated human group 1 CD1-transgenic (hCD1Tg) mice. These mice express group 1 CD1 in a pattern similar to humans and support the development of group 1 CD1-restricted T cells, which can be induced both through infection with Mycobacterium tuberculosis (Mtb) and immunization with Mtb lipid antigens. hCD1Tg mice respond to Mtb epitopes recognized by human group 1 CD1-restricted T cells. The kinetics of these group 1 CD1-restricted responses resembles those of conventional T cells and is more rapid upon secondary exposure to antigen, but is distinct from those of group 2 CD1-restricted NKT cells. Furthermore, we find that the number of group 1 CD1-expressing antigen-presenting cells is altered over the course of Mtb infection, and correlates with the kinetics of group 1 CD1-restricted T cells. Taken together, our data demonstrate the viability of hCD1Tg mice as a model for the study of group 1 CD1-restricted T cells in anti-mycobacterial immunity. Such studies could lead to the development of new vaccines against Mtb. This work is supported by NIH grant NIH-A157460.