Trisaccharides of Phenolic Glycolipids Confer Advantages to Pathogenic Mycobacteria through Manipulation of Host-Cell Pattern-Recognition Receptors.

Despite mycobacterial pathogens continue to be a threat to public health, the mechanisms that allow them to persist by modulating the host immune response are poorly understood. Among the factors suspected to play a role are phenolic glycolipids (PGLs), produced notably by the major pathogenic species such as Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report an original strategy combining genetic reprogramming of the PGL pathway in Mycobacterium bovis BCG and chemical synthesis to examine whether sugar variations in the species-specific PGLs have an impact on pattern recognition receptors (PRRs) and the overall response of infected cells. We identified two distinct properties associated with the trisaccharide domains found in the PGLs from M. leprae and M. tuberculosis. First, the sugar moiety of PGL-1 from M. leprae is unique in its capacity to bind the lectin domain of complement receptor 3 (CR3) for efficient invasion of human macrophages. Second, the trisaccharide domain of the PGLs from M. tuberculosis and M. leprae share the capacity to inhibit Toll-like receptor 2 (TLR2)-triggered NF-κB activation, and thus the production of inflammatory cytokines. Consistently, PGL-1 was found to also bind isolated TLR2. By contrast, the simpler sugar domains of PGLs from M. bovis and Mycobacterium ulcerans did not exhibit such activities. In conclusion, the production of extended saccharide domains on PGLs dictates their recognition by host PRRs to enhance mycobacterial infectivity and subvert the host immune response.

Mycobacterial phenolic glycolipids with a simplified lipid aglycone modulate cytokine levels through Toll-like receptor 2.

Phenolic glycolipids (PGLs) are virulence factors present in the cell walls of many pathogenic mycobacteria. PGLs have been implicated in various aspects of mycobacterial disease, but there are limited structure-activity data available for these molecules. We report here the preparation of seven synthetic PGL analogues, differing from the native compounds in the replacement of the complex phenolic lipid moiety with a p-methoxyphenyl group. The ability of these compounds to stimulate or inhibit the production of cytokines (TNF-α, IL-1ß, IL-6, MCP-1) and nitric oxide (NO) was then evaluated by ELISA-based assays. None of the compounds stimulated the production of these biological signalling molecules. In contrast, they each displayed concentration-dependent inhibitory activity, related to the methylation pattern of the molecule and mediated by Toll-like receptor 2. Additional studies revealed that native PGL-I from Mycobacterium leprae and a synthetic PGL-I analogue containing a simplified lipid domain had enhanced inhibitory activities relative to the corresponding analogues containing the p-methoxyphenyl aglycone; however, the natural lipid phenolthiocerol was only weakly active. These studies reveal that synthetic molecules of this type can be used as probes for PGL function. Moreover, their ease of synthesis relative to the natural glycolipids, as well as their more favourable aqueous solubility, should allow for more thorough structure-activity relationship studies.

Phenolic-glycolipid-1 and lipoarabinomannan preferentially modulate TCR- and CD28-triggered proximal biochemical events, leading to T-cell unresponsiveness in mycobacterial diseases.

BACKGROUND: Advanced stages of leprosy show T cell unresponsiveness and lipids of mycobacterial origin are speculated to modulate immune responses in these patients. Present study elucidates the role of phenolicglycolipid (PGL-1) and Mannose-capped lipoarabinomannan (Man-LAM) on TCR- and TCR/CD28- mediated signalling. RESULTS: We observed that lipid antigens significantly inhibit proximal early signalling events like Zap-70 phosphorylation and calcium mobilization. Interestingly, these antigens preferentially curtailed TCR-triggered early downstream signalling events like p38 phosphorylation whereas potentiated that of Erk1/2. Further, at later stages inhibition of NFAT binding, IL-2 message, CD25 expression and T-cell blastogenesis by PGL-1 and Man-LAM was noted. CONCLUSION: Altogether, we report that Man-LAM and PGL-1 preferentially interfere with TCR/CD28-triggered upstream cell signalling events, leading to reduced IL-2 secretion and T-cell blastogenesis which potentially could lead to immunosupression and thus, disease exacerbation, as noted in disease spectrum.

Role of PGL-I of M. leprae in TNF-alpha production by in vitro whole blood assay.

Phenolic glycolipid-I (PGL-I) is known to be a major antigen of Mycobacterium leprae. We have studied the influence of PGL-I on the production of Tumour Necrosis Factor alpha (TNF-alpha) using the in vitro whole blood assay. Armadillo-derived M. leprae (ADML) are thought to be depleted of PGL-I during the purification process. M. leprae obtained from mouse foot pad material (MFPML) has been subjected to a less rigorous purification process; their PGL-I coating is therefore believed to be more intact than that of ADML. PGL-I or ADML alone induced the secretion of minimal levels of TNF-alpha in whole blood assay; when added in combination, higher levels of this cytokine were observed. The highest TNF-alpha response was seen following stimulation with MFPML. MFP material not infected with ML did not elicit any response. The difference in TNF-alpha response shown by ADML and MFPML was postulated to be largely due to the presence of higher levels of PGL-I in MFPML. This increase in TNF-alpha production suggests that PGL-I may play a significant role in the induction of TNF-alpha during natural infection.

Effect of unique Mycobacterium leprae phenolic glycolipid-I (PGL-I) on tumour necrosis factor production by human mononuclear cells.

Mycobacterium leprae cell wall-associated components are found in large amounts in the tissues of leprosy patients, particularly those at the lepromatous pole. Among these molecules, the phenolic glycolipid-I (PGL-I), unique to M. leprae, has been involved in the selective anergy observed in the lepromatous patients. Armadillo-derived M. leprae retains only a small proportion of the total PGL-I found in infected tissues. Therefore, the addition of PGL-I to M. leprae in vitro is important for a better understanding of M. leprae effects in vivo. We have studied the influence of PGL-I on TNF production by normal human peripheral blood mononuclear cells (PBMC) and by a human monocytic leukaemia cell line (THP-1) following stimulation with killed M. leprae. PGL-I alone did not induce TNF secretion by PBMC, but when associated with a sub-optimal dose of armadillo-derived M. leprae increased the release of this cytokine. In agreement with these results, M. leprae-exposed THP-1 cells did not secrete detectable levels of TNF unless PGL-I was simultaneously added to the culture. This increase in TNF production suggests that PGL-I plays a role in the induction of TNF during the natural infection. In addition, the modulatory effect of PGL-I on TNF release by THP-1 cells reinforces that monocytes are one of the possible targets of this molecule.

Production of transforming growth factor-beta 1 (TGF-beta1) by blood monocytes from patients with different clinical forms of leprosy.

In the present study, the concentration of TGF-beta1 secreted by adherent cells isolated from human peripheral blood mononuclear cells (PBMC) and either stimulated with PGL-1 or lipopolysaccharide (LPS) or left unstimulated was determined by ELISA. The cells were isolated from untreated patients with different clinical forms of leprosy and healthy individuals. The adherent cells exhibited spontaneous release of TGF-beta1 in all clinical forms of leprosy and in healthy individuals; however, lepromatous leprosy/borderline leprosy (LL/BL) patients presenting erythema nodosum leprosum (ENL) displayed significantly higher concentrations of TGF-beta1 than either the other patients studied or the controls. These high TGF-beta1 levels were consistently observed when LL/BL ENL cells were stimulated with phenolic glycolipid (PGL-1) or LPS, and even in the absence of a stimulus (P < 0.01). The most significant differences in TGF-beta1 levels were observed when comparing the results in the presence of PGL-1 from ENL with, in order of significance: tuberculoid leprosy (TT) patients (P < 0.001), LL/BL patients without ENL (P < 0.01), healthy individuals (P < 0.01) and borderline-borderline/borderline-tuberculoid (BB/BT) patients with reversal reaction (RR) (P < 0.01). The BB/BT patients produced equivalent levels of TGF-beta1 compared with LL/BL patients without ENL, for all types of stimuli (P > 0.05). In contrast, TT patients produced the lowest levels of TGF-beta1 among all the subjects studied (both patients and healthy controls), especially following PGL-1 stimulation (P < 0.001, and P < 0.05, respectively). In conjunction with our previous data regarding TGF-beta1 expression in dermal lesions, it appears that TGF-beta1 probably plays different roles in leprosy: (i) to mediate a suppressive action locally, associated with the presence of PGL-1, and (ii) to induce proinflammatory effects when secreted systemically by monocytes, thereby acting as a modulatory cytokine in the acute inflammatory reactions of ENL and associated with the Th2 immune response in multibacillary forms of leprosy.

Alterations in early biochemical events following T cell activation in leprosy patients.

The early events of activation and cytokine profiles (IL-2, 4, and 6) were studied in lymphocytes of paucibacillary (TT/BT) and multibacillary (BL/LL) leprosy patients after stimulation with PMA/A23187 and Mycobacterium leprae antigen (PGL-1). Lymphocytes from BT/TT patients showed proliferation in response to both PMA/A23187 and PGL-1 compared to BL/LL. The levels of early activation signaling molecules such as IP3, calcium, and protein kinase C (PKC) in the particulate fraction were found to be elevated in BT/TT and BL/LL patients and showed a further significant increase after stimulation with PMA/A23187 in BT/TT patients. PGL-1 marginally increased the IP3 levels in BT/TT patients, whereas in BL/LL patients, it had no effect. The levels of IL-2 were enhanced in lymphocytes of BT/TT leprosy patients and were further augmented by PPD and PGL-1, while the levels of IL-4 and IL-6 were increased in LL/BL lymphocytes and further augmented by PGL-1. Thus PGL-1 seems to be a major culprit in inducing the TH2-type cytokine response observed in lepromatous leprosy patients.

Phospholipase activity of Mycobacterium leprae harvested from experimentally infected armadillo tissue.

Three types of phospholipase activity--phospholipase A1, A2, and lysophospholipase--were detected in Mycobacterium leprae harvested from armadillo tissue at about 25% of the specific activity found in a slowly growing mycobacterium, Mycobacterium microti, which was grown in medium to optimize its phospholipase activity. The highest activity found was lysophospholipase, which released fatty acid from 2-lyso-phosphatidylcholine. Phospholipase activity was detected by using phosphatidylcholine and phosphatidylethanolamine. Differences in relative activities with these three types of substrate distinguished phospholipase activity in M. leprae extracts from armadillo liver extracts. Furthermore, retention of activity in M. leprae after NaOH treatment showed that the activity associated with M. leprae was not host derived. The specific activity of phospholipase was 20 times higher in extracts of M. leprae than in intact M. leprae organisms. Diazotization, a treatment which abolishes activities of surface enzymes exposed to the environment by the formation of covalent azide bonds with exposed amino groups, did not affect M. leprae's phospholipase activity, with one exception: release of arachidonic acid from phosphatidylcholine, which was partially inhibited. Phenolic glycolipid I, the major excreted amphipathic lipid of M. leprae, inhibited phospholipase activity, including release of arachidonic acid, for both M. leprae- and armadillo-derived activity.

Interleukin-1 beta production by monocytes from leprosy patients.

The cause responsible for the lack of an efficient cell-mediated immunity or a delayed type hypersensitivity to M. leprae in lepromatous patients is poorly understood. But the resistance to M. leprae infection in humans is likely mediated by the activated macrophages to present M. leprae antigen to T cells for cell-mediated immunity. Phenolic glycolipid-I (PGL-I) is a M. leprae-specific antigen and is supposed to play a significant role in the long lasting unresponsiveness in lepromatous leprosy. In this study, IL-1 activities were tested among leprosy patients to evaluate monocyte function and the role of IL-1 in the immunosuppression in leprosy. We found that peripheral blood mononuclear cells (PBMCs) from tuberculoid patients were strongly reactive to M. leprae (mean cpm; 28,853 +/- 28,916), but the proliferative responses of PBMCs from lepromatous patients (mean cpm; 6,051 +/- 803) were significantly lower. IL-1 concentration in culture supernatant of monocytes from lepromatous patients was similar to that from tuberculoid patients with stimulation of M. leprae (lepromatous: 1,014 +/- 637 pg/ml, tuberculoid: 1,012 +/- 167 pg/ml) or lipopolysaccharides (IPS) (lepromatous: 3,479 +/- 2,188 pg/ml, tuberculoid: 4,246 +/- 2,432 pg/ml). The IL-1 concentration is sera from lepromatous patients (42 +/- 30 pg/ml) tended to be higher than those from tuberculoid patients (28 +/- 69 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the human complement system by phenolic glycolipid 1 of Mycobacterium leprae.

The activation of the complement system by phenolic glycolipid 1 (PGL) from Mycobacterium leprae was studied. It was found that PGL consumed haemolytic complement through both the classical and the alternative pathways. This was further studied at the level of C3. Although the activation was independent of anti-PGL antibodies present in normal human serum, the addition of antibody augmented the activation of complement by PGL. The uptake of C3 through the classical pathway was enhanced predominantly by IgM antibody whereas, IgG antibody against PGL was responsible for the augmentation of the alternative pathway activation. Furthermore, it was found that both the disaccharide and trisaccharide components of PGL were able to activate the complement system.

Phenolic glycolipid-1 from M. leprae inhibits oxygen free radical production by human mononuclear cells.

We studied the effect of PGL1, a phenolic glycolipid unique to Mycobacterium leprae, on the activation of the phagocyte oxidative respiratory burst, by measuring the chemiluminescence (CL) generated by normal mononuclear cells. PGL1 induced a decrease in oxygen free radical production stimulated by mycobacteria (M. leprae, BCG and M. kansasii) or by phorbol myristate acetate, but did not prevent the binding or ingestion of fluorescein-conjugated mycobacteria. In contrast, mycoside A from M. kansasii, a structurally related compound, did not alter the CL response. In addition, treatment of M. leprae with anti-PGL1 antibodies failed to restore the response to this microorganism. PGL1 could act as an oxygen species scavenger and protect M. leprae from killing by toxic oxygen metabolites.

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae.

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.

Inflammation induced by a glycolipid fraction from Mycobacterium leprae.

1. The inflammatory properties of a glycolipid fraction isolated from human recovered Mycobacterium leprae were investigated. The inflammatory reaction induced in mouse lung by the inoculation of the glycolipid fraction adsorbed to charcoal particles was characterized by a large influx of macrophages at various stages of maturation and of epithelioid cells around the particles. 2. When injected as an aqueous emulsion into the footpad of mice, the same fraction evoked a dose-dependent massive influx of mononuclear (MN) cells. The inflammatory reaction reached a peak at 6 days. The minimal effective dose of glycolipid was 0.1 micrograms. 3. The kinetics of inflammatory cell migration was studied by total and differential counts of leucocytes that migrated to the peritoneal cavity of mice inoculated intraperitoneally with the glycolipid fraction. This fraction initially induced intense polymorphonuclear (PMN) migration, which was later reduced, with a simultaneous increase in MN cells. 4. Adherent peritoneal cells (APC) incubated with glycolipid released one or more soluble factor(s) which induce active PMN and MN cell chemotaxis in vivo as well as in vitro. Thus, the MN cells may be attracted to the site of glycolipid inoculation by factor(s) released through the interaction of macrophages with the glycolipid fraction. 5. The present results demonstrate that a glycolipid containing trehalose and mycolic acid isolated from M. leprae reproduces some aspects of the fundamental lesion of leprosy.

Relationships between structure and biological activity of the mycolic acid-containing glycolipids from Nocardia asteroides "sensu stricto" and related species.

To reveal the taxonomical situation of Nocardia asteroides "sensu stricto", we compared the mycolic acid and mycolic acid-containing glycolipid composition and their granulomagenic activities in mice. The major glycolipids were glucose mono- and dimycolate, trehalose mono- and dimycolate and several unknown glycolipids, commonly, although the relative amount differed from strain to strains. On the other hand, molecular species composition of mycolic acids differed distinctively among the three closely related species: N. asteroides "sensu strico", N. farcinica and N. nova. GC/MS analysis showed the most abundant species of mycolic acids were C50(52) in N. asteroides, C54(52) in N. farcinica and C58(56) in N. nova, respectively with a different alpha-alkyl branch. Glucose mycolate and trehalose dimycolate possessing C50 mycolic acid showed a strong activity for granuloma formation in mice.

The effect of phenolic glycolipid-1 from Mycobacterium leprae on the antimicrobial activity of human macrophages.

Purified PGL-1 and dPGL from M. leprae can prevent bacterial killing by intact phagocytes and cell-free antimicrobial systems. Both glycolipids completely abolished the antimicrobial effect of the acetaldehyde-XO-Fe2+ system. Because the cytotoxicity of this system is inhibited by catalase, SOD, mannitol, and ethanol, but not by heated SOD or catalase, these data suggest that toxicity is due to OH. generated by the Haber-Weiss reaction. That the antimicrobial killing in the XO system is completely blocked by the addition of PGL-1 or dPGL suggests that these glycolipids can act as OH. scavengers. A modest protective effect against the cytotoxicity of the MPO-H2O2-halide system by both PGL-1 and dPGL was also observed. The antimicrobial activity of the MPO system was abolished with chloride, but not iodide, as the halide. The effect of the M. leprae-derived glycolipid on bacterial killing by intact phagocytes was examined. Two linking antibodies were used to bind the dPGL to a rapidly growing test organism, S. aureus, a murine IgM mAb specific for the terminal glycoside of PGL-1, and a rabbit IgG anti-mouse IgM which bound the staphylococcal protein A via its Fc region. Examination by transmission EM of human monocyte-derived macrophages which had ingested staphylococci either coated with both antibodies and dPGL, or coated only with the IgG and IgM antibodies, demonstrated the presence of bacteria in phagosomes of control and IFN-gamma-activated macrophages. Activation of the macrophage monolayers by pretreatment with IFN-gamma markedly increased their staphylocidal activity. When dPGL coated staphylococci were ingested, killing by both control and IFN-gamma-activated macrophages was completely blocked. These results, suggesting that PGL-1 can scavenge reactive oxygen species and prevent microbial death within the phagosome, may in part explain the intracellular survival of M. leprae in certain cell types.