Components Of Mycobacterial Cell Wall Research Articles

Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages.

The recognition of mycobacterial cell wall components causes macrophages to secrete tumor necrosis factor alpha (TNF-alpha) and other cytokines that are essential for the development of a protective inflammatory response. We show that toll-like receptors are required for the induction of TNF-alpha in macrophages by Mycobacterium tuberculosis. Expression of a dominant negative form of MyD88 (a signaling component required for toll-like receptor signaling) in a mouse macrophage cell line blocks TNF-alpha production induced by M. tuberculosis. We identify toll-like receptor-2 (TLR2) as the specific toll-like receptor required for this induction by showing that expression of an inhibitory TLR2 (TLR2-P681H) blocks TNF-alpha production induced by whole M. tuberculosis. Further, we show that TLR2-dependent signaling mediates responses to mycobacterial cell wall fractions enriched for lipoarrabinomannan, mycolylarabinogalactan-peptidoglycan complex, or M. tuberculosis total lipids. Thus, although many mycobacterial cell wall fractions are identified to be inflammatory, all require TLR2 for induction of TNF-alpha in macrophages. These data suggest that TLR2 is essential for the induction of a protective immune response to mycobacteria.

Human Toll-Like Receptors Mediate Cellular Activation by Mycobacterium tuberculosis

Abstract Recent studies have implicated a family of mammalian Toll-like receptors (TLR) in the activation of macrophages by Gram-negative and Gram-positive bacterial products. We have previously shown that different TLR proteins mediate cellular activation by the distinct CD14 ligands Gram-negative bacterial LPS and mycobacterial glycolipid lipoarabinomannan (LAM). Here we show that viable Mycobacterium tuberculosis bacilli activated both Chinese hamster ovary cells and murine macrophages that overexpressed either TLR2 or TLR4. This contrasted with Gram-positive bacteria and Mycobacterium avium, which activated cells via TLR2 but not TLR4. Both virulent and attenuated strains of M. tuberculosis could activate the cells in a TLR-dependent manner. Neither membrane-bound nor soluble CD14 was required for bacilli to activate cells in a TLR-dependent manner. We also assessed whether LAM was the mycobacterial cell wall component responsible for TLR-dependent cellular activation by M. tuberculosis. We found that TLR2, but not TLR4, could confer responsiveness to LAM isolated from rapidly growing mycobacteria. In contrast, LAM isolated from M. tuberculosis or Mycobacterium bovis bacillus Calmette-Guérin failed to induce TLR-dependent activation. Lastly, both soluble and cell wall-associated mycobacterial factors were capable of mediating activation via distinct TLR proteins. A soluble heat-stable and protease-resistant factor was found to mediate TLR2-dependent activation, whereas a heat-sensitive cell-associated mycobacterial factor mediated TLR4-dependent activation. Together, our data demonstrate that Toll-like receptors can mediate cellular activation by M. tuberculosis via CD14-independent ligands that are distinct from the mycobacterial cell wall glycolipid LAM.

Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells.

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 mannose-capped 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 CD4-expressing 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 devoid of ManLAM. The implication of protein tyrosine kinase(s), protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7. It was also determined, using electrophoresis mobility shift assays, that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-kappaB. M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type, but not the kappaB-mutated, HIV-1 LTR region. Finally, the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-alpha. The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection.

The mycobacterial cell wall: structure, biosynthesis and sites of drug action

Structural analysis has been successfully implemented recently to obtain valuable information on the mycobacterial cell wall components, many of which have formed the basis for biosynthesis and functional studies towards developing better drugs and possible vaccines. The highly complex and well organized structure unique to mycobacteria, represents the best target for novel antimycobacterial agents. Until recently, our knowledge of the enzymes responsible for the biogenesis of the cell wall components was almost negligible. The pathways are now being elucidated in several laboratories. Highlights of this review include significant advances in the structure and biochemistry of the major cell wall components and potenital targets for generation of new drugs.

EXPRESSION OF MONOCYTE CHEMOTACTIC PROTEIN-3 IN HUMAN MONOCYTES EXPOSED TO THE MYCOBACTERIAL CELL WALL COMPONENT LIPOARABINOMANNAN

Monocyte chemotactic protein-3 (MCP-3) is a C–C chemokine which interacts with the CCR1, CCR2 (MCP-1) and CCR3 receptors and has a distinct spectrum of action. The present study was designed to assess whether mycobacterial components were able to induce expression and production of MCP-3 in human monocytes. Mycobacterial lipoarabinomannan (LAM) induced expression of MCP-3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside (PLM). Among mononuclear cells, monocytes were responsible for LAM-induced MCP-3 mRNA expression. Whole mycobacteria ( Mycobacterium bovisBCG) strongly induced MCP-3 expression. Pretreatment with actinomycin D abolished LAM-induced MCP-3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced MCP-3 expression was associated with the production of immunoreactive PTX3. Interleukin 10 (IL-10) and IL-13 inhibited the induction of MCP-3 by LAM. Thus mycobacterial cell wall components induced expression of MCP-3 in human monocytes. MCP-3, a chemokine active on mononuclear phagocytes, NK cells, T cells and dendritic cells, may be relevant to the induction and expression of immunity against mycobacteria.

New horizons in the treatment of tuberculosis

The development of new chemotherapy for the treatment of tuberculosis has three major objectives: first, the development of faster-acting drugs to shorten the duration of treatment; second, the development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies; and, third, the development of chemotherapeutics that specifically target dormant bacilli to treat the one-third of the world's population latently infected with tubercle bacilli. Strategies based upon optimizing the inhibition of known targets require an extensive knowledge of the detailed mechanism of action of current antimycobacterial agents. For many agents such as isoniazid, ethambutol, rifampin, and pyrazinamide such knowledge is now available. Strategies based upon the identification of novel targets will necessitate the identification of biochemical pathways specific to mycobacteria and related organisms. Many unique metabolic processes occur during the biosynthesis of mycobacterial cell wall components, and some attractive new targets have emerged. The development of targets specific to latency will require a detailed picture of the metabolism and biochemical pathways occurring in dormant bacilli. Recent evidence suggests that anaerobic metabolic pathways may operate in dormant bacilli, and the enzymes involved in such pathways may also provide significant new targets for intervention. The combination of the mycobacterial genome sequence that is anticipated to become available this year with an improved understanding of the unique metabolic processes that define mycobacteria as a genus offers the greatest hope for the elimination of one of mankind's oldest enemies.

Interaction of human mannose-binding protein with Mycobacterium avium.

The interaction between human mannose-binding protein (MBP) and Mycobacterium avium was explored. By ELISA, calcium-dependent and mannan-inhibitable binding of human recombinant MBP (rMBP) to live M. avium was observed. Preincubation of M. avium with rMBP resulted in a 2-fold increase in uptake by human neutrophils. Mycobacterial cell wall components were assessed by ELISA for their ability to bind the carbohydrate recognition domain of rMBP. The best ligand was mannosyl-lipoarabinomannan, followed by lipomannan, phosphatidylinositol mannoside, arabinosyl-lipoarabinomannan, and dimycolated trehalose (cord factor). rMBP did not bind to partially purified lipid fractions containing glycopeptidolipids. These results are consistent with the known structural basis for rMBP ligand recognition. They suggest that MBP may play a role in host defense against M. avium by opsonizing both whole organisms and free cell wall components for internalization.

Expression of a long pentraxin, PTX3, by monocytes exposed to the mycobacterial cell wall component lipoarabinomannan.

PTX3 is a prototypic long pentraxin composed of a C-terminal domain similar to those of classical pentraxins (e.g., C reactive protein) and an unrelated N-terminal portion. PTX3 is expressed in a variety of cell types, notably mononuclear phagocytes and endothelial cells, after exposure to the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The present study was designed to assess whether mycobacterial components were able to induce expression and production of PTX3. Mycobacterial lipoarabinomannan (LAM) induced expression of PTX3 mRNA in human peripheral blood mononuclear cells. The non-mannose-capped version of lipoarabinomannan (AraLAM) was considerably more potent than the mannose-capped version ManLAM or the simpler version phosphatidylinositol mannoside. Among mononuclear cells, monocytes were responsible for LAM-induced PTX3 mRNA expression. Whole mycobacteria (Mycobacterium bovis BCG) strongly induced PTX3 expression. Pretreatment with actinomycin D abolished LAM-induced PTX3 expression, whereas cycloheximide only partially reduced the expression. LAM-induced PTX3 expression was associated with the production of immunoreactive PTX3. IL-10 and IL-13 did not inhibit the induction of PTX3 by LAM. Under the same conditions, these anti-inflammatory cytokines inhibited MCP-1 expression. In contrast, gamma interferon inhibited LAM-induced PTX3 expression. Thus, in addition to IL-1, TNF, and lipopolysaccharide, mycobacterial cell wall components also induce expression and production of the long pentraxin PTX3. The significance of PTX3 in the immunobiology of mycobacterial infection and its relevance in relation to clinical involvement remain to be determined.

Prospects for new interventions in the treatment and prevention of mycobacterial disease.

Mycobacterium tuberculosis claims more lives each year than any other single human pathogen. Despite the availability of effective drugs, the incidence of tuberculosis is increasing in much of the developing world and has recently reemerged as a public health problem in industrialized countries. In the first section of this chapter, current understanding of the fundamental biology of mycobacterial infection is reviewed from the perspective of development of new tools for disease control. A second section describes strategies for identification of novel antimycobacterial agents, with particular emphasis on recent progress in defining biosynthetic pathways for unique mycobacterial cell wall components. The third section focuses on current approaches to the development of new vaccine candidates consisting of live attenuated bacteria or individual antigenic subunits.

Ex vivo induction of TNF-α and IL-6 mRNA in bovine whole blood by Mycobacterium paratuberculosis and mycobacterial cell wall components

Johne's disease is a chronic enteritis of cattle and other ruminant species that is of worldwide economic importance. The cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Using reverse transcriptase polymerase chain reaction (RT-PCR) and specific bovine oligonucleotide cytokine primers and probes for bovine TNF-α and IL-6, we examined theex vivoexpression of mRNA for these inflammatory cytokines in whole blood from healthy cattle. Cytokine mRNA levels increased after a brief incubation of bovine whole blood withMycobacterium paratuberculosisor its lipoarabinomannan (LAM). Muramyl dipeptide (MDP) andEscherichia coliLPS also stimulated TNF-α and IL-6 mRNA expression. Several strains ofM. paratuberculosiswere tested and found to have similar abilities to stimulate TNF-α and IL-6 mRNA expression. Several strains of the closely relatedMycobacterium avium, and the unrelated saprophyte,Mycobacterium phlei, had somewhat less ability to stimulate TNF-α and IL-6 mRNA expression.

Mycobacterial cell wall components induce the production of TNF-α, IL-1, and IL-6 by bovine monocytes and the murine macrophage cell line RAW 264.7

Johne's disease is characterized by a chronic enteritis that results in granulomatous inflammation, cachexia, and eventual death of cattle infected with Mycobacterium paratuberculosis. The cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) have been associated with granuloma formation and wasting in other disease syndromes. The potential role of these cytokines in the development and progression of Johne's disease has not been investigated. Freshly isolated bovine peripheral blood monocytes and the murine macrophage cell line RAW 264.7 were examined for their ability to release inflammatory cytokines in response to mycobacterial cell wall components. Bovine monocytes and RAW 264.7 cells incubated with M. paratuberculosis lipoarabinomannan (LAM), muramyl dipeptide (MDP), or lipopolysaccharide (LPS) released TNF-α, IL-1β, and IL-6 as detected by appropriate bioassays. Using the RAW 264.7 cells, cytokine mRNA levels were elevated after in vitro incubation with live M. paratuberculosis or LPS as determined using a reverse-transcriptase polymerase chain reaction procedure.

Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis.

TNF and IFN-gamma are thought to be involved in the immune response to mycobacterial infection because they exhibit antimycobacterial effects in vitro. To investigate the roles of these cytokines in vivo at the site of disease activity in human tuberculosis, we evaluated local cytokine production in patients with tuberculous pleuritis. Both TNF and IFN-gamma were selectively concentrated 5- to 30-fold in pleural fluid, compared to blood of the same patients. Messenger RNA for both cytokines was detected in pleural tissue by in situ hybridization, suggesting that selective cytokine concentration is due to local cytokine production. Two Mycobacterium tuberculosis cell wall components, the protein-peptidoglycan complex and lipoarabinomannan, caused dose-dependent release of TNF by pleural fluid mononuclear cells and may constitute the stimuli for TNF production in the pleural space. In contrast to results obtained for TNF release, the protein-peptidoglycan complex, but not lipoarabinomannan, stimulated IFN-gamma release by pleural fluid mononuclear cells. The clinical manifestations of tuberculous pleuritis, such as fever, exudative pleural effusion, and tissue necrosis, may be due to the effects of elevated local TNF concentrations, produced in response to mycobacterial cell wall components.

Regulation of in vivo IgE Biosynthesis in Mice with Complete Freund’s Adjuvant

Complete Freund's adjuvant (CFA) administered before sensitization dampened the normal and cyclophosphamide-enhanced response of high and moderate IgE responder phenotype mice (CAF1 and C57B1/6J, respectively). CFA-induced suppression of IgE biosynthesis was effective in reducing anaphylactic histamine release from approximately 2,900 ng histamine per milliliter to background levels (less than 100 ng/ml). CFA-induced ascites fluid was able to reduce the cyclophosphamide-enhanced IgE response of low-responder phenotype SJL mice from 1:320 to less than 1:5 as determined by passive cutaneous anaphylaxis. Muramyl dipeptide, a mycobacterial cell wall component capable of eliciting effects similar to those seen with CFA, was shown to induce suppression of IgE production if incorporated in incomplete Freund's adjuvant. Muramyl dipeptide administered in saline was ineffective, while incomplete Freund's adjuvant alone had some immunoregulatory properties. Ongoing IgE responses were less susceptible to regulation. CFA administered to sensitized C57B1/6J mice was ineffective in inducing IgE suppression when animals were challenged with antigen.

In vitro effects of lipopolysaccharides and mycobacterial cell wall components on swine alveolar macrophages

The possible activation of swine alveolar macrophages (AM) by lipopolysaccharides (LPS) and mycobacterial cell wall components such as muramyl dipeptide (MDP) and interphase material (IPM) was investigated. Swine AM were harvested by post mortem lung washings and the following functions were assayed: adherence and spreading in cultures; phagocytosis of 51Cr-labelled chicken red cells; cytostatic activity against xenogeneic tumour cells (P815 mastocytoma cells); monokine synthesis; interferon and LAF (lymphocyte activating factor or inter-Ieukin-1). Incubation of swine AM with either MDP, LPS or IPM (0·1 μg to 100 μg/ml) for 24 hours did not affect the cell viability but increased their adherence and spreading slightly. Phagocytosis was not markedly modified. Under the same experimental conditions, the unstimulated AM cultures exhibited a strong cytostatic activity which was not modified by these components. No interferon synthesis could be observed in the normal or stimulated AM cultures. In contrast, LAF activity was consistently increased after 48 hours of incubation with LPS, whereas MDP produced this effect only in some experiments.

Arthritogenic activity of a synthetic immunoadjuvant, muramyl dipeptide.

A synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine, dissolved in saline only and applied subcutaneously to rats of the Lewis inbred strain, produced arthritis, clinically manifest by hind feet paresis but without apparent paw swelling in most cases. Histologically, the disease was characterized by edema and hyperemia of connective tissues, joint synovias, and tendon sheaths, with massive accumulation of inflammatory cell infiltrates composed mainly of lymphoplasmocytes and partly of neutrophil leukocytes. Fibrin exudation and fibrinoid necrosis in connective tissues were observed in the most severe cases. Synovial layers of the talocrural joint, especially on their villi, exhibited marked swelling or cell desquamation of the inner zone. Clinical symptoms of the disease disappeared spontaneously within 5 days after cessation of the treatment; also, histological examinations showed that the effects were reversible. Our results prove that (i) muramyl dipeptide is the principal substance involved in the production of arthritis, (ii) there is no necessity for the presence of additional mycobacterial cell wall components, and (iii) the involvement of the oil moiety is not requisite for the production of arthritis.

Immunotherapy of cancer: tumor suppression and regression by cell walls of Mycobacterium phlei attached to oil droplets.

Components of mycobacterial cell wall(s) (CW) attached to oil droplets were evaluated for their ability 1) to inhibit the growth of line-10 tumor transplants in the skin of syngeneic guinea pigs when inoculated together with 10(6) tumor cells (suppression experiments) and 2) to regress established 7-day-old intradermal tumors and eradicate microscopic lymph node metastases upon injection into the tumors (regression experiments). CW and cell-wall skeleton (CWS) preparations from Mycobacterium phlei, a fast-growing saprophyte of group IV of the atypical mycobacteria, suppressed tumor growth in essentially all animals when 37.5-mug doses were administered; at a dose of 300 mug, they cured 50-60% of the animals in regression tests. The addition of 300 mug of a purified trehalose mycolate, isolated from M. tuberculosis strain Aoyama B, to 300 mug M. phlei CW or CWS preparations significantly increased their tumor regressive potency to provide cure rates to about 90%. Because M. phlei can be propagated more readily, it can be used advantageously in place of BCG to prepare stable, non-living immunologic adjuvants of defined composition and consistently high potency to meet the need for standards with minimal residual malignant disease.

Biologically active components from mycobacterial cell walls: V. Granuloma formation in mouse lungs and guinea pig skin

The ability of certain mycobacterial cell wall components, when attached to minute droplets of oil, to induce granulomatous inflammation in mouse lung and guinea pig skin has been studied for clues to the mechanisms by which these components protect mice against aerosol challenge with virulent tubercle bacilli and are useful in immunotherapy of dermal tumors of guinea pigs. Fractions examined were the following: the cell wall skeleton (CWS-I), which consisted of a polymeric peptidoglycolipid; wax D, which contained mono- or oligomeric peptidoglycolipid; and trehalose mycolates (P3). A combination of CWS-I and P3 induced more granuloma in mouse lungs than either of these two entities alone. Similarly, when wax D was separated into a chloroform-methanol-insoluble portion (CMI), which contained the mono- or oligomeric peptidoglycolipid, and the soluble portion (CMS), which contained P3, it was found that both moieties were required to produce maximal granulomatous inflammation. The degree of granuloma induced by these nonliving vaccines, as measured by an increase in lung weight following intravenous inoculation, could be correlated with the degree of protection produced in mice against airborne infection with M. tuberculosis H37Rv and with the degree of regression of skin tumors in guinea pigs upon intralesional inoculation. Even though quantitation of the granulomatous response in the skin of guinea pigs is difficult, it would appear that intradermal inoculation of cell walls, CWS-I, or CMI produced comparable granuloma, whereas CMS or P3 alone was essentially inactive.

Preparation of an Adjuvant-Active, Tuberculin-Free Peptidoglycolipid from Human Tubercle Bacilli

Preparation of an Adjuvant-Active, Tuberculin-Free Peptidoglycolipid from Human Tubercle Bacilli

Immunization of mice by combinations of inactive fractions of Mycobacterium bovis strain BCG.

In an attempt to identify the mycobacterial cell wall components which enhance resistance in mice to airborne infection with Mycobacterium tuberculosis H37Rv, fractions of BCG were prepared by organic solvent extraction and by alkali or lipase treatment. Although certain of these fractions, when tested individually, were impotent in our protection test, vaccines containing combinations of solvent-extracted, alkali-treated, or lipase-treated cell walls and of an inactive chloroform or methyl alcohol extract were significantly effective. The wax D fraction, but not the wax B or C fractions, of the chloroform extract in combination with “inactive” cell walls was highly protective. Since combinations of either the “inactive” BCG cell wall residue or the BCG chloroform extract with heterologous substances failed to enhance resistance of mice, two or more specific and distinct mycobacterial components may be required in an effective vaccine.