Cord Factor Research Articles

Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide

Mycobacterium tuberculosis is a facultative intracellular pathogen that inhibits phagosome maturation in macrophages thereby securing survival and growth. Mycobacteria reside in an early endocytic compartment of near-neutral pH where they upregulate production of complex glycolipids such as trehalose dimycolate. Here, we report that trehalose dimycolate coated onto beads increased the bead retention in early phagosomes, i.e. at a similar stage as viable mycobacteria. Thus, a single mycobacterial lipid sufficed to divert phagosome maturation and likely contributes to mycobacterial survival in macrophages. Previous studies showed that activated macrophages promote maturation of mycobacterial phagosomes and eliminate mycobacteria through bactericidal effectors including nitric oxide generated by inducible nitric-oxide synthase. We show that deceleration of bead phagosome maturation by trehalose dimycolate was abolished in immune-activated wild type, but not in activated nitric-oxide synthase-deficient macrophages, nor when hydroxyl groups of trehalose dimycolate were chemically modified by reactive nitrogen intermediates. Thus, specific host defence effectors of activated macrophages directly target a specific virulence function of mycobacteria.

Antitumor effect of a synthetic cord factor, 6,6'-di-O-decanoyl-alpha,alpha-trehalose (SS554), in mice.

The antitumor effect on Meth-A fibrosarcoma in BALB/c mice of a synthetic cord factor, 6,6'-di-O-decanoyl-alpha,alpha-trehalose (designated as SS554), was examined. Only intratumoral injection had a curative effect; subcutaneous, per oral, or intravenous routes had no such effect. The co-presence of an oily vehicle has been shown to be necessary for antitumor activity of a natural cord factor. When SS554 was examined in suspensions of sesame oil, squalane, squalene or sesame oil and water emulsion, a 60% cure rate was achieved. However, no such effect was obtained with a suspension in Tween-PBS or a solution. It should also be noted that sequential but independent administrations of SS554 and oil were found to be as effective as the simultaneous administration of oil and SS554 in emulsion form. In the case of the emulsion, the amount of sesame oil necessary was over 10%, or 0.01 ml in absolute terms. Cures were obtained in a dose-dependent manner by injection of SS554 in amounts in excess of 1 mg. The effect of the time of administration was also examined; the best result was obtained when intratumoral injection was done on day 3 after tumor implantation. Mice cured by SS554 exhibited growth inhibition and rejection of rechallenged Meth-A cells. However, this immunity was specific; it did not extend to a rechallenge with RLmale-1 leukemia cells.

A rapid approach to lipid profiling of mycobacteria using 2D HSQC NMR maps

Mycobacteria, including Mycobacterium tuberculosis, are characterized by a unique cell wall rich in complex lipids, glycolipids, polyketides, and terpenoids. Many of these metabolites have been shown to play important roles in mycobacterial virulence and their inherent resistance to many antibiotics. Here, we report the development of a new simple method for global analysis of these metabolites using two-dimensional (1)H-(13)C heteronuclear single quantum coherence nuclear magnetic resonance. The major advantages of this method are as follows: the small amount of sample and the minimal sample manipulation required; a relatively short procedural time; and the ability to rapidly attain a qualitative and quantitative lipid profile of a mycobacterial sample in which the majority of the clinically relevant lipids can be observed simultaneously. The effectiveness of this method is demonstrated in four different areas of major concern to the mycobacterial research community: i) adaptive changes in cell wall lipids as a result of drug treatment; ii) analysis of gene function; iii) characterization of new mycobacterial species; and iv) analysis of the production of virulence factors in clinical isolates of M. tuberculosis. This method is complementary to mass spectrometry-based lipidomic technologies and provides an urgently needed tool to gain a better understanding of the role of lipids in mycobacteria pathogenesis.

A novel application of affinity biosensor technology to detect antibodies to mycolic acid in tuberculosis patients

Tuberculosis has re-emerged as a global health problem due to co-infection with HIV and the emergence of drug-resistant strains of Mycobacterium tuberculosis. HIV co-infection introduced a 30% underestimation in TB diagnosis based on sputum analysis, calling for a reliable and fast serodiagnostic assay to assist in the management of TB in HIV-burdened populations. Serodiagnosis with mycobacterial lipid cell wall antigens gave promising results, in particular with LAM and cord factor. Free mycolic acids have also been considered because they are unique in structure to each species of Mycobacterium and can be economically extracted and purified. In a standard immunoassay such as ELISA, however, an unacceptable number of false positive and false negative test results were obtained. Here we report a much improved biosensor method to detect antibodies to mycolic acids in patient serum as surrogate markers of active tuberculosis. Mycolic acid (MA) liposomes were immobilized on a non-derivatized twin-celled biosensor cuvette and blocked with saponin. A high dilution of serum was used to calibrate the binding signal of the two cells, followed by contact with patient serum at a lesser dilution, but pre-incubated with either antigen-carrying, or empty liposomes. The serum, or the protein A purified IgG thereof, from sputum-positive tuberculosis patients could be inhibited from binding to the MA in the biosensor by prior incubation with MA-containing liposomes. The accuracy of the inhibition test was 84% if HIV-positive patients for whom a negative TB sputum analyses could not be relied upon to serve as a reference standard were excluded. If biosensor technology could be made suitable for high throughput screening, then it may provide the solution to the serodiagnosis of tuberculosis against a background of HIV.

Peripheral cell wall lipids of Mycobacterium tuberculosis are inhibitory to surfactant function

The transmission of Mycobacterium tuberculosis (TB) requires extensive damage to the lungs to facilitate bacterial release into the airways, and it is therefore likely that the microorganism has evolved mechanisms to exacerbate its local pathology. This study examines the inhibitory effects of lipids extracted and purified chromatographically from TB on the surface-active function of lavaged bovine lung surfactant (LS) and a clinically relevant calf lung surfactant extract (CLSE). Total lipids from TB greatly inhibited the surface activity of LS and CLSE on the pulsating bubble surfactometer at physical conditions applicable for respiration in vivo (37 degrees C, 20 cycles/min, 50% area compression). Minimum surface tensions for LS (0.5 mg/ml) and CLSE (1 mg/ml) were raised from <1 mN/m to 15.7+/-1.2 and 18.7+/-1.3 mN/m after 5 min of bubble pulsation in the presence of total TB lipids (0.15 mg/ml). TB mixed waxes (0.15 mg/ml) and TB trehalose monomycolates (TMMs, 0.15 mg/ml) also significantly inhibited the surface activity of LS and CLSE (minimum surface tensions of 10-16 mN/m after 5 min of bubble pulsation), as did purified trehalose 6,6'-dimycolate (TDM, cord factor). Phosphatidylinositol mannosides (PIMs, 0.15 mg/ml) from TB had no inhibitory effect on the surface activity of LS or CLSE. Concentration dependence studies showed that LS was also inhibited significantly by total TB lipids at 0.075 mg/ml, with a smaller activity decrease apparent even at 0.00375 mg/ml. These findings document that TB contains multiple lipids that can directly impair the biophysical function of endogenous and exogenous lung surfactants. Direct inhibition by TB lipids could worsen surfactant dysfunction caused by plasma proteins or other endogenous substances induced by inflammatory injury in the infected lungs. TB lipids could also inhibit the effectiveness of exogenous surfactants used to treat severe acute respiratory failure in TB patients meeting criteria for clinical acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS).

Avaliação do crescimento em cordas na identificação presuntiva do complexo Mycobacterium tuberculosis

Virulent strains of the Mycobacterium tuberculosis complex, under certain appropriate conditions, grow as characteristic ropes, bundles or serpentine cords known as cord factor or growth in cords. The objective of the present study was to evaluate cord factor detection as a method of achieving presumptive identification of the M. tuberculosis complex, comparing it to conventional typing tests. A total of 743 strains were analyzed from January of 2002 to December of 2005 in the Mycobacteria Sector of the Adolfo Lutz Institute, located in the city of Santos, Brazil. Samples were obtained from clinical specimens collected from patients with respiratory symptoms treated at basic health clinics in the greater metropolitan area of Santos. Ziehl-Neelsen-stained smears were prepared, 301 (40.5%) in MB/BacT broth and 442 (59.5%) on solid media, either Lowenstein-Jensen or Ogawa-Kudoh. The sensitivity, specificity, positive predictive value and negative predictive value obtained during the performance comparison of the two methods (cord factor detection and conventional typing) using both isolation media were, respectively, 98.5, 88, 97 and 93%. The method was more sensitive on solid medium (100%), and the difference in sensitivity between the two media types was only 2.7%. Taking into consideration the results obtained, we conclude that, in laboratories with a high incidence of M. tuberculosis complex isolation and limited economic resources, cord factor detection is a fast and valid criterion for identifying these mycobacteria using liquid or solid medium. It also enables subsequent conclusive identification tests, as well as additional sensitivity tests when necessary.

Mycobacterial glycolipid trehalose 6,6′-dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes

The granulomatous response is a characteristic histological feature of Mycobacterium tuberculosis infection responsible for organism containment. The development of cell-mediated immunity is essential for protection against disease, as well as being required for maintenance of the sequestering granulomatous response. Trehalose 6,6'-dimycolate (TDM; cord factor), a glycolipid associated with the cell wall of mycobacteria, is implicated as a key immunogenic component in M. tuberculosis infection. Models of TDM-induced hypersensitive granulomatous response have similar pathologies to that of active tuberculosis infection. Prior immunization (sensitization) of mice with TDM results in exacerbated histological damage, inflammation and lymphocytic infiltration upon subsequent TDM challenge. Adoptive transfer experiments were performed to ascertain the cell phenotype governing this response; CD4(+) cells were identified as critical for development of related pathology. Mice receiving CD4(+) cells from donor TDM-immunized mice demonstrated significantly increased production of Th1-type cytokines IFN-gamma and IL-12 within the lung upon subsequent TDM challenge. Control groups receiving naïve CD4(+) cells, or CD8(+) or CD19(+) cells isolated from TDM-immunized donors, did not exhibit an exacerbated response. The identified CD4(+) cells isolated from TDM-immunized mice produced significant amounts of IFN-gamma and IL-2 when exposed to TDM-pulsed macrophages in vitro. These experiments provide further evidence for involvement of a cell-mediated response in TDM-induced granuloma formation, which mimics pathological damage elicited during M. tuberculosis infection.

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

Sensitive profiling of chemically diverse bioactive lipids

Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. alpha-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria.

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6′-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both α- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.

Molecular and supra-molecular structure related differences in toxicity and granulomatogenic activity of mycobacterial cord factor in mice

To establish the structure biological activity relationship of cord factor (trehalose 6,6′-dimycolate, TDM), we compared the molecular or supra-molecular structure of TDM micelles with toxicity, thymic atrophy and granulomatogenicity in lungs and spleen of BALB/c mice. According to the difference in the mycolyl subclass composition, TDM was divided into two groups, one possessing α-, methoxy- and keto-mycolates in M. tuberculosis H 37Rv, M. bovis BCG and M. kansasii (group A) and the other having α-, keto- and wax ester-mycolates in M. avium serotype 4, M. phlei and M. flavescens (group B), although mycolic acid molecular species composition differed in each group considerably. Supra-molecular structure of TDM micelle differed species to species substantially and the micelle size of TDM from M. bovis BCG Connaught was the largest. The highest toxicity was shown with TDM from M. tuberculosis H 37Rv which possessed the highest amount of α- (47.3%) and methoxy-mycolates (40.8%), while TDM from M. phlei having the low amount of α-mycolate (11.6%) showed almost no toxicity with the given doses. The thymic atrophy was observed with TDM from group A, but not with TDM from group B. On the other hand, TDM from group B showed massive lung granulomatogenic activity based on the histological observations and organ indices. Taken together, group A TDM showed a wide variety of micelle sizes and specific surface areas, high to low toxicity and marked to moderate granulomatogenicity, while group B TDM showed smaller sizes of micelles and larger specific surface areas, lower toxicity but higher granulomatogenicity in lungs. Existence of higher amount of longer chain α-mycolates in TDM appeared to be essential for high toxicity and thymic apoptotic activity, whereas TDM possessing wax ester-mycolate with smaller sized micelles seemed to be less toxic, but more granulomatogenic in lungs in mice. Thus, the mycolic acid subclass and molecular species composition of TDM affect critically the micelle forms, toxicity and granulomatogenicity in mice, while the relative abundances and carbon chain length of α-mycolate affected the toxicity in mice.

The Cell-Wall Core of Mycobacterium tuberculosis in the Context of Drug Discovery.

Present-day understanding of the architecture of the entire cell-wall of Mycobacterium tuberculosis amounts to a "core" template comprised of peptidoglycan with phosphodiester linkage, via a linker disaccharide, to a linear D-galactofuran, to which, in turn, are attached several strands of a highly branched D-arabinofuran. The cell-wall mycolic acids are linked via an ester to the majority of the non-reducing termini of the D-arabinan. The mycolic acids are oriented perpendicular to the plane of the membrane and provide a truly special lipid barrier responsible for many of the physiological and disease-inducing aspects of M. tuberculosis. Intercalated within this environment are the phthiocerol dimycocerosates, cord factor or dimycolyltrehalose, sulfolipids, phosphatidylinositol mannosides and the related lipomannan and lipoarabinomannan, etc., agents responsible for much of the pathogenesis of tuberculosis. Interest in the biosynthesis of the cell-wall core, regarded, unlike the ancillary lipids, as essential to bacterial viability and integrity, is now driven by the pressing need for alternative drugs to counteract drug-resistant tuberculosis. In a manner analogous to the roles of lipid I and II in peptidglycan formation, synthesis of the entire arabinogalactan is initiated by transferring activated sugars to decaprenyl-phosphate, giving rise to the linker disaccharide, followed by stepwise elongation of the galactan, and the arabinan, apparently one sugar at a time. The genetics and enzymology of these polymerization events have not been well defined, nor have the final steps, namely the attachment of mycolic acids and ligation to peptidoglycan. However, what is known of the earlier events in cell-wall core synthesis has attracted interest in terms of new anti-tuberculosis drug development.

A novel trehalase from Mycobacterium smegmatis − purification, properties, requirements

Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.

Efficient Syntheses of a Series of Trehalose Dimycolate (TDM)/Trehalose Dicorynomycolate (TDCM) Analogues and Their Interleukin-6 Level Enhancement Activity in Mice Sera

We found an IL-6 level-enhancing compound during our synthetic study of trehalose-6,6'-dimycolate (1, TDM, formerly called cord factor) analogues. TDM is a glycolipid distributed in the cell wall of Mycobacterium tuberculosis and shows significant antitumor activity based on an immunoadjuvant activity. However, due to its significant toxicity, TDM is not yet applicable for practical use. In 1993, Datta and Takayama reported the purification of trehalose-6,6'-dicorynomycolate (2c, TDCM) from Corynebacterium spp. We have previously reported the synthesis of four diastereomeric TDCMs and showed that the synthetic (2R,3R,2'R,3'R)-TDCM (2c, hereafter abbreviated RRRR-TDCM-C14) is identical to natural TDCM; we also demonstrated that 2c and SSSS-TDCM-C14 (3c) showed significant antitumor activity as well as inhibitory activity in experimental lung metastasis based on the immunoadjuvant activity. Furthermore, we found that the significant lethal toxicity in mice by TDM (1) was no longer observed with the shorter-chain analogues of TDCMs. Therefore, we have elucidated that the 2,3-antistereochemistry (RR or SS) of the fatty acid residue is promising for biological activities. The chain length of the fatty acid residue should also be important for the biological activity, and thus, we designed a general synthetic procedure for trehalose diesters with 2,3-antistereochemistry and a series of chain lengths by using Noyori's asymmetric reduction of beta,beta-ketoesters followed by antiselective alkylation according to Frater to give beta,beta-hydroxy alcohols as the key steps. Thus, we prepared trehalose diesters (TDCM) 2a-d, 3a-d, and 4a-d as well as monoesters (TMCM) 5a-d and 6a-d. Immunological activities of TDCMs and TMCMs were evaluated by determining IL-6 level enhancement in mouse serum, and we found that RRRR-TDCM-C14 (2c) and RRSS-TDCM-C14 (4c) showed significant IL-6 level enhancement activities.

Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen

There is a clinical need for a more effective vaccine against hepatitis B, and in particular vaccines that may be suitable for therapeutic administration. This study assesses the potential of cationic surfactant vesicle based formulations using two agents; the cationic amine containing [ N-( N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or dimethyl dioctadecylammonium bromide (DDA) with hepatitis B surface antigen (HBsAg). Synthetic mycobacterial cord factor, trehalose 6,6′-dibehenate (TDB) has been used as an adjuvant and the addition of 1-monopalmitoyl glycerol (C16:0) (MP) and cholesterol (Chol) to DDA–TDB is assessed for its potential to facilitate formation of dehydration–rehydration vesicles (DRV) at room temperature, and the effect of this on immune responses. A DRV formulation is directly compared to an adsorbed formulation of the same composition and preparation protocol (MP:dioleoyl phosphoethanolamine (DOPE):Chol:DC-Chol) and the direct substitution of MP with phosphatidylcholine (PC) is also compared in DRV antigen-entrapped formulations. MP and Chol were shown to facilitate the use of DDA–TDB in DRV formulations prepared at room temperature, whilst there was marginal alteration of immunogenicity (a reduction in HBsAg-specific IL-2). The HBsAg adsorbed DRV formulation was not significantly different from the HBsAg entrapped DRV formulation. Overall, DDA formulations incorporating TDB showed markedly increased antigen specific splenocyte proliferation and elicited cytokine production concomitant with a strong T cell driven response, delineating formulations that may be useful for further evaluation of their clinical potential.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

CD3+ cells transfer the hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6′-dimycolate in mice

The granulomatous response is the characteristic histological feature of Mycobacterium tuberculosis infection that is essential for organism containment. Trehalose 6,6-dimycolate (TDM), a cell-wall glycolipid present on most mycobacterial species, has been implicated in the pathogenesis of M. tuberculosis infection. TDM has potent immunoregulatory and inflammatory properties, and can be used to model granulomatous reactions that mimic, in part, pathology caused during active infection. This study examined the hypersensitive granulomatous response, focusing on cellular responses specific to TDM. Lungs from mice immunized with TDM emulsion demonstrated exacerbated histological damage, inflammation, and lymphocytic infiltration upon subsequent challenge with TDM. Splenocytes recovered from these mice demonstrated significant interferon (IFN)-gamma production during recall response to TDM, as well as increased production of proinflammatory mediators (tumour necrosis factor-alpha, interleukin-6 and macrophage inflammatory protein-1alpha). The exacerbated response could be adoptively transferred to naïve mice. Administration of non-adherent lymphocytes or purified CD3(+) cells from TDM-immunized mice led to increased inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon challenge with TDM. Recipient mice that received immunized CD3(+) lymphocytes demonstrated significant increases in Th1-type cytokines and proinflammatory mediators in lung tissue following TDM challenge. When CD1d(-/-) mice were immunized with TDM, they failed to generate a specific IFN-gamma response, suggesting a role for this molecule in the generation of hypersensitivity. These experiments provide further evidence for the involvement of TDM-specific CD3(+) T cells in pathological damage elicited during M. tuberculosis infection.

Trans-cyclopropanation of mycolic acids on trehalose dimycolate suppresses Mycobacterium tuberculosis-induced inflammation and virulence

Recent studies have shown that fine structural modifications of Mycobacterium tuberculosis cell envelope lipids mediate host cell immune activation during infection. One such alteration in lipid structure is cis-cyclopropane modification of the mycolic acids on trehalose dimycolate (TDM) mediated by proximal cyclopropane synthase of alpha mycolates (pcaA), a proinflammatory lipid modification during early infection. Here we examine the pathogenetic role and immunomodulatory function of mycolic acid cyclopropane stereochemistry by characterizing an M. tuberculosis cyclopropane-mycolic acid synthase 2 (cmaA2) null mutant (Delta cmaA2) that lacks trans-cyclopropanation of mycolic acids. Although titers of WT and Delta cmaA2 organisms were identical during mouse infection, Delta cmaA2 bacteria were hypervirulent while inducing larger granulomas than WT M. tuberculosis. The hypervirulence of the Delta cmaA2 strain depended on host TNF-alpha and IFN-gamma. Loss of trans-cyclopropanation enhanced M. tuberculosis-induced macrophage inflammatory responses, a phenotype that was transferable with petroleum ether extractable lipids. Finally, purified TDM lacking trans-cyclopropane rings was 5-fold more potent in stimulating macrophages. These results establish cmaA2-dependent trans-cyclopropanation of TDM as a suppressor of M. tuberculosis-induced inflammation and virulence. In addition, cyclopropane stereochemistries on mycolic acids interact directly with host cells to both positively and negatively influence host innate immune activation.

Of mice, men, and elephants: Mycobacterium tuberculosis cell envelope lipids and pathogenesis

Mycolic acids and structures attached to them constitute a major part of the protective envelope of Mycobacterium tuberculosis, and for this reason, their role in tuberculosis pathogenesis has been extensively studied. In this issue of the JCI, Rao et al. examine the effect of trans-cyclopropanation of oxygenated mycolic acids attached to trehalose dimycolate (TDM) on the murine immune response to infection (see the related article beginning on page 1660). Surprisingly, they found that an M. tuberculosis mutant lacking trans-cyclopropane rings was hypervirulent in mice. The recent recognition of a hypervirulence phenotype in mice associated with laboratory and clinical M. tuberculosis strains with altered cell wall components has provided new insights into how M. tuberculosis may establish persistent infection. However, to date, characterization of these bioactive products in pathogenesis has been largely reductionistic; the relationship of their effects observed in mice to the persistent infection and tuberculosis caused by M. tuberculosis observed in humans remains obscure.

Mycobacterial sulfolipid shows a virulence by inhibiting cord factor induced granuloma formation and TNF-α release

Virulence mechanism of infection with Mycobacterium tuberculosis is currently focused to be clarified in the context of cell surface lipid molecule. Comparing two mycobacterial glycolipids, we observed toxicity and prominent granulomatogenic activity of trehalose 6,6′-dimycolate (TDM) injection in mice, evident by delayed body weight gain and histological observations, whereas 2,3,6,6′-tetraacyl trehalose 2′-sulfate (SL) was non-toxic and non-granulomatogenic. Likewise, TDM but not SL caused temporarily, but marked increase of lung indices, indicative of massive granuloma formation. Interestingly, co-administration of TDM and SL prevented these symptoms distinctively and SL inhibited TDM-induced release of tumor necrosis factor alpha (TNF-α) in a dose-dependent manner. Histological findings and organ index changes also showed marked inhibition of TDM induced granuloma formation by co-administration of SL. Simultaneous injection of SL together with TDM was highly effective for this protection, as neither injection 1 h before nor after TDM injection showed highly inhibitory. In parallel studies on a cellular level, TDM elicited strong TNF-α release from alveolar but not from peritoneal macrophages in vitro. This effect was blocked when alveolar macrophages were incubated in wells simultaneously coated with TDM and SL, indicating that SL suppresses TDM-induced TNF-α release from macrophages. Our results suggest a novel mechanism by which SL could contribute to virulence at early stage of mycobacterial infection or stimulation with the glycolipids by counteracting the immunopotentiating effect of TDM.