Mycobacterial Lipids Research Articles

In Vivo Activity of Released Cell Wall Lipids ofMycobacterium bovisBacillus Calmette-Guérin Is Due Principally to Trehalose Mycolates

The hallmark of Mycobacterium-induced pathology is granulomatous inflammation at the site of infection. Mycobacterial lipids are potent immunomodulators that contribute to the granulomatous response and are released in appreciable quantities by intracellular bacilli. Previously we investigated the granulomagenic nature of the peripheral cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin (BCG) by coating the lipids onto 90-microm diameter microspheres that were mixed into Matrigel matrix with syngeneic bone marrow-derived macrophages and injected i.p. into mice. These studies demonstrated that BCG lipids elicit proinflammatory cytokines and recruit leukocytes. In the current study we determined the lipids responsible for this proinflammatory effect. BCG-derived cell wall lipids were fractionated and purified by liquid chromatography and preparative TLC. The isolated fractions including phosphatidylinositol dimannosides, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, trehalose monomycolate, trehalose dimycolate, and mycoside B. Trehalose dimycolate, when delivered to bone marrow-derived murine macrophages, induced the greatest secretion of IL-1beta, IL-6, and TNF-alpha in vitro. Trehalose dimycolate similarly induced the greatest secretion of these proinflammatory cytokines in ex vivo matrices over the course of 12 days. Trehalose monomycolate and dimycolate also induced profound neutrophil recruitment in vivo. Experiments with TLR2 or TLR4 gene-deficient mice revealed no defects in responses to trehalose mycolates, although MyD88-deficient mice manifested significantly reduced cell recruitment and cytokine production. These results demonstrate that the trehalose mycolates, particularly trehalose dimycolate, are the most bioactive lipids in the BCG extract, inducing a proinflammatory cascade that influences granuloma formation.

Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.

Dissecting the Mechanism and Assembly of a Complex Virulence Mycobacterial Lipid

Mycobacterium tuberculosis cell envelope is a treasure house of biologically active lipids of fascinating molecular architecture. Although genetic studies have alluded to an array of genes in biosynthesis of complex lipids, their mechanistic, structural, and biochemical principles have not been investigated. Here, we have dissected the molecular logic underlying the biosynthesis of a virulence lipid phthiocerol dimycocerosate (PDIM). Cell-free reconstitution studies demonstrate that polyketide synthases, which are usually involved in the biosynthesis of secondary metabolites, are responsible for generating complex lipids in mycobacteria. We show that PapA5 protein directly transfers the protein bound mycocerosic acid analogs on phthiocerol to catalyze the final esterification step. Based on precise identification of biological functions of proteins from Pps cluster, we have rationally produced a nonmethylated variant of mycocerosate esters. Apart from elucidating mechanisms that generate chemical heterogeneity with PDIMs, this study also presents an attractive approach to explore host-pathogen interactions by altering mycobacterial surface coat.

The Human CD1-Restricted T Cell Repertoire Is Limited to Cross-Reactive Antigens: Implications for Host Responses against Immunologically Related Pathogens

The repertoires of CD1- and MHC-restricted T cells are complementary, permitting the immune recognition of both lipid and peptide Ags, respectively. To compare the breadth of the CD1-restricted and MHC-restricted T cell repertoires, we evaluated T cell responses against lipid and peptide Ags of mycobacteria in leprosy, comparing tuberculoid patients, who are able to restrict the pathogen, and lepromatous patients, who have disseminated infection. The striking finding was that in lepromatous leprosy, T cells did not efficiently recognize lipid Ags from the leprosy pathogen, Mycobacterium leprae, or the related species, Mycobacterium tuberculosis, yet were able to efficiently recognize peptide Ags from M. tuberculosis, but not M. leprae. To identify a mechanism for T cell unresponsiveness against mycobacterial lipid Ags in lepromatous patients, we used T cell clones to probe the species specificity of the Ags recognized. We found that the majority of M. leprae-reactive CD1-restricted T cell clones (92%) were cross-reactive for multiple mycobacterial species, whereas the majority of M. leprae-reactive MHC-restricted T cells were species specific (66%), with a limited number of T cell clones cross-reactive (34%) with M. tuberculosis. In comparison with the MHC class II-restricted T cell repertoire, the CD1-restricted T cell repertoire is limited to recognition of cross-reactive Ags, imparting a distinct role in the host response to immunologically related pathogens.

Etiology of Crohn’s disease: Do certain food additives cause intestinal inflammation by molecular mimicry of mycobacterial lipids?

Crohn's disease is a chronic granulomatous inflammation of the gastrointestinal tract which was first described in the beginning of the 20th century. The histological similarity with intestinal tuberculosis has led to the assumption of an involvement of mycobacteria and mycobacterial antigens, respectively, in the etiology. A major defense mechanism against mycobacterial lipid antigens is the CD1 system which includes CD1 molecules for antigen presentation and natural killer T cells for recognition and subsequent production of cytokines like interferon-gamma and tumour necrosis factor-alpha. These cytokines promote granulomatous transformation. Various food additives, especially emulsifiants, thickeners, surface-finishing agents and contaminants like plasticizers share structural domains with mycobacterial lipids. It is therefore hypothesized, that these compounds are able to stimulate by molecular mimicry the CD1 system in the gastrointestinal mucosa and to trigger the pro-inflammatory cytokine cascade. The understanding of Crohn's disease as a CD1-mediated delayed-type hypersensitivity to certain food additives would lead to strong emphasis on a dietary treatment. Related aspects of pathology, physiology and epidemiology of Crohn's disease are presented.

Clinical evaluation of serodiagnosis of active tuberculosis by multiple-antigen ELISA using lipids from Mycobacterium bovis BCG Tokyo 172

The humoral immune responses of 69 active tuberculosis (TB) patients against three major mycobacterial lipid antigens, monoacyl phosphatidylinositol dimannoside (Ac-PIM2), trehalose 6,6'-dimycolate (TDM-T) and trehalose 6-monomycolate (TMM-T) from Mycobacterium bovis BCG Tokyo 172, were examined by ELISA. IgG antibodies from active TB patients were reactive against each of the three lipid antigens (Ac-PIM2, TDM-T and TMM-T), giving positive results of 42.0-59.4%. If tests were combined and an overall positive was scored when any of the three tests was positive, sensitivity was 71.0%, showing better discrimination between patients and normal subjects. Although this value is not satisfactory for the clinical diagnosis of active TB, it is still higher than values for Determinar TBGL (56.5%) and MycoDot (31.9%) test results, both of which are commercially available. IgG antibody responses to particular lipid antigens in an individual patient differed diversely between patients. Positive IgG antibody rates and IgG antibody levels to lipid antigens were mostly paralleled by the amount of mycobacteria excreted and by the severity of pathological lesions (size and cavity formation). Serologically positive responsiveness was shown in 60.0% of tuberculin skin test (TST)-negative TB patients. Furthermore, seropositivity for multiple-antigen ELISA in active TB patients was demonstrated in other possible immune-suppressed cases, such as senile, diabetes mellitus and fulminant TB patients. Therefore, in contrast to tests based on cellular immune responses such as the TST, the humoral immune responses of TB patients against mycobacterial lipid antigens were disease-specific and were shown to be useful for the early diagnosis of active TB disease in conjunction with smear and cultivation tests, even if cellular immune responses are decreased.

Mycobacterium tuberculosis pks12 Produces a Novel Polyketide Presented by CD1c to T Cells

CD1c-mediated T cells are activated by a mycobacterial phospholipid antigen whose carbohydrate structure precisely corresponds to mammalian mannosyl β-1-phosphodolichol (MPD), but contains an unusual lipid moiety. Here, we show that this T cell antigen is a member of a family of branched, alkane lipids that vary in length (C30-34) and are produced by medically important mycobacteria such as M. tuberculosis and M. bovis Bacille-Calmette-Guerin. The alkane moiety distinguished these mycobacterial lipid antigens from mammalian MPDs and was necessary for activation of CD1c-restricted T cells, but could not be accounted for by any known lipid biosynthetic pathway. Metabolic labeling and mass spectrometric analyses suggested a mechanism for elongating lipids using alternating C2 and C3 units, rather than C5 isopentenyl pyrophosphate. Inspection of the M. tuberculosis genome identified one candidate gene, pks12, which was predicted to encode the largest protein in M. tuberculosis, consisting of 12 catalytic domains that correspond to key steps in the proposed pathway. Genetic deletion and complementation showed that Pks12 was necessary for antigen production, but did not affect synthesis of true isoprenols. These studies establish the genetic and enzymatic basis for a previously unknown type of polyketide, designated mycoketide, which contains a lipidic pathogen-associated molecular pattern.

A rapid method of diagnosing pulmonary tuberculosis using stir bar sorptive extraction–thermal desorption–gas chromatography–mass spectrometry

A fast method for detection of tuberculostearic acid (TBSA) in sputum samples is described. The samples, obtained from patients with known or suspected pulmonary tuberculosis, were decontaminated and concentrated before being analyzed by stir bar sorptive extraction–thermal desorption–gas chromatography–mass spectrometry (SBSE–TD–GC–MS). Prior to extraction, the mycobacterial lipids were hydrolyzed and then derivatized with ethyl chloroformate to increase the sorption of the compounds by the polydimethylsiloxane (PDMS) stir bar coating. The limit of detection (LOD) is 0.2 ng ml −1. Four sputum samples that were classified by direct microscopy as smear-positive or negative were analyzed by GC–MS. TBSA was detected at concentrations ranging from 0.47 to 2.3 ng ml −1. The method is sufficiently sensitive to detect TBSA directly in clinical samples without the need to culture the organisms.

High‐Polarity Mycobacterium avium‐Derived Lipids Interact with Murine Macrophage Lipid Rafts

Cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are widely recognized as portals for pathogenic micro-organisms. A growing body of evidence demonstrates mobilization of host plasma cell membrane lipid rafts towards the site of contact with several pathogens as well as a strict dependence on cholesterol for appropriate internalization. The fate of lipid rafts once the pathogen has been internalized and the nature of the pathogen components that interact with them is however less understood. To address both these issues, infection of the J774 murine cell line with Mycobacterium avium was used as a model. After demonstrating that M. avium induces lipid raft mobilization and that M. avium infects J774 by a cholesterol-dependent mechanism, it is shown here that mycobacterial phagosomes harbour lipid rafts, which are, at least in part, of plasma cell membrane origin. On the other hand, by using latex microbeads coated with any of the three fractions of M. avium-derived lipids of different polarity, we provide evidence that high-polarity, in contrast to low-polarity and intermediate-polarity, mycobacterial lipids or uncoated latex beads have a strong capacity to induce lipid raft mobilization. These results suggest that high-polarity mycobacterial lipid(s) interact with host cell cholesterol-enriched microdomains which may in turn influence the course of infection.

Microbial type I fatty acid synthases (FAS): major players in a network of cellular FAS systems.

The present review focuses on microbial type I fatty acid synthases (FASs), demonstrating their structural and functional diversity. Depending on their origin and biochemical function, multifunctional type I FAS proteins form dimers or hexamers with characteristic organization of their catalytic domains. A single polypeptide may contain one or more sets of the eight FAS component functions. Alternatively, these functions may split up into two different and mutually complementing subunits. Targeted inactivation of the individual yeast FAS acylation sites allowed us to define their roles during the overall catalytic process. In particular, their pronounced negative cooperativity is presumed to coordinate the FAS initiation and chain elongation reactions. Expression of the unlinked genes, FAS1 and FAS2, is in part constitutive and in part subject to repression by the phospholipid precursors inositol and choline. The interplay of the involved regulatory proteins, Rap1, Reb1, Abf1, Ino2/Ino4, Opi1, Sin3 and TFIIB, has been elucidated in considerable detail. Balanced levels of subunits alpha and beta are ensured by an autoregulatory effect of FAS1 on FAS2 expression and by posttranslational degradation of excess FAS subunits. The functional specificity of type I FAS multienzymes usually requires the presence of multiple FAS systems within the same cell. De novo synthesis of long-chain fatty acids, mitochondrial fatty acid synthesis, acylation of certain secondary metabolites and coenzymes, fatty acid elongation, and the vast diversity of mycobacterial lipids each result from specific FAS activities. The microcompartmentalization of FAS activities in type I multienzymes may thus allow for both the controlled and concerted action of multiple FAS systems within the same cell.

Effect of Mycobacterium leprae lipids on BCG- and carrageenan-induced cellular recruitment in mouse pleurisy

Pathogenic mycobacteria survive inside macrophages and deactivate these cells, using a mechanism that is still poorly understood. Mycobacterial cell wall lipids constitute the first contact with the host cell. Although Mycobaterium leprae and M. bovis BCG share common antigens, they induce opposite inflammatory responses. Apolar M. leprae lipids have been shown to be anti-inflammatory by down-regulating macrophage activation and T-cell functions. We wonder if these lipids would influence cellular migration to BCG or to other inflammatory agent. We investigated the effect of M. leprae, its lipids or delipidated bacteria on acute and chronic BCG- or carrageenan-induced pleurisy. Previous injection of intact or delipidated M. leprae did not alter either the BCG- or carrageenan-induced pleural inflammatory reaction. However, M. leprae lipids enhanced carrageenan-induced acute cellular migration without impairing BCG inflow; moreover, they reduced BCG chronic response. Together these data suggest distinct mechanisms for intracellular deactivation and pleural cell recruitment exerted by mycobacterial structures.

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.

Role for malonyl coenzyme A:acyl carrier protein transacylase (MCAT) in the growth-inhibitory effect of the calmodulin antagonist trifluoperazine in Mycobacterium bovis BCG.

To determine whether the fatty acid synthesis enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT) is involved in the growth-inhibitory effect of trifluoperazine in the tubercle bacillus Mycobacterium bovis BCG. BCG was grown in liquid culture with various concentrations of trifluoperazine and growth was monitored by OD measurement. To determine the effect of trifluoperazine on MCAT protein level, total protein was extracted from BCG cultures and was analysed by 2D gel electrophoresis and western blot. To confirm trifluoperazine-dependent reduction in the MCAT protein level, two BCG strains overexpressing MCAT at a low and high constitutive level were similarly tested. The synergic effect of trifluoperazine and isoniazid was tested at sub-MIC levels in liquid cultures. Trifluoperazine inhibition of growth correlates with reduction in the steady-state level of MCAT protein. Overexpression of MCAT confers resistance to trifluoperazine. Trifluoperazine acts synergically (albeit weakly) with isoniazid and no resistance towards isoniazid alone was observed due to overexpression of MCAT. This suggests MCAT to be a specific target of trifluoperazine. These results indicate MCAT as a target of trifluoperazine and provide an explanation for the inhibitory effect of trifluoperazine on mycobacterial lipid synthesis observed earlier. This makes MCAT a potential target for new antimycobacterials.

Diacylated Sulfoglycolipids Are Novel Mycobacterial Antigens Stimulating CD1-restricted T Cells during Infection with Mycobacterium tuberculosis

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent on previous contact with M. tuberculosis, but independent from the development of clinically overt disease. These properties identify Ac2SGL as a promising candidate to be tested in novel vaccines against tuberculosis.

Decolorization of malachite green and crystal violet by waterborne pathogenic mycobacteria.

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite green and its reduced, decolorized product were detected in the lipid fraction of M. avium strain A5 cells grown in the presence of malachite green, suggesting that a minor component of resistance could be due to sequestering the dyes in the extensive mycobacterial cell surface lipid. The membrane fraction of M. avium strain A5 had at least a fivefold-higher specific decolorization rate than did the crude extract, suggesting that the decolorization activity is membrane associated. The malachite green-decolorizing activity of the membrane fraction of M. avium strain A5 was abolished by either boiling or proteinase exposure, suggesting that the decolorizing activity was due to a protein. Decolorization activity of membrane fractions was stimulated by ferrous ion and inhibited by dinitrophenol and metyrapone.

T-Cell Responses to CD1-Presented Lipid Antigens in Humans with Mycobacterium tuberculosis Infection

CD1-restricted presentation of lipid or glycolipid antigens derived from Mycobacterium tuberculosis has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with M. tuberculosis documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with Mycobacterium bovis BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1(+) immature dendritic cells revealed that T cells from asymptomatic M. tuberculosis-infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to M. tuberculosis, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of M. tuberculosis-reactive T cells in healthy individuals with previous exposure to M. tuberculosis.

Attenuation of Mycobacterium tuberculosis by disruption of a mas-like gene or a chalcone synthase-like gene, which causes deficiency in dimycocerosyl phthiocerol synthesis.

Tuberculosis is one of the leading preventable causes of death. Emergence of drug-resistant tuberculosis makes the discovery of new targets for antimycobacterial drugs critical. The unique mycobacterial cell wall lipids are known to play an important role in pathogenesis, and therefore the genes responsible for their biosynthesis offer potential new targets. To assess the possible role of some of the genes potentially involved in cell wall lipid synthesis, we disrupted a mas-like gene, msl7, and a chalcone synthase-like gene, pks10, with phage-mediated delivery of the disruption construct, in which the target gene was disrupted by replacement of an internal segment with the hygromycin resistance gene (hyg). Gene disruption by allelic exchange in the case of each disruptant was confirmed by PCR and Southern blot analyses. Neither msl7 nor pks10 mutants could produce dimycocerosyl phthiocerol, although both could produce mycocerosic acids. Thus, it is concluded that these gene products are involved in the biosynthesis of phthiocerol. Both mutants were found to be attenuated in a murine model, supporting the hypothesis that dimycocerosyl phthiocerol is a virulence factor and thus the many steps involved in its biosynthesis offer potential novel targets for antimycobacterial therapy.

Identification and macrophage-activating activity of glycolipids released from intracellular Mycobacterium bovis BCG.

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow-derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14C-labelled BCG. Released BCG lipids were resolved by thin-layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2), mono- and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono- and dimycolates and the phenolic glycolipid, mycoside B. Culture-derived BCG lipids that co-migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat-killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid-coated microspheres were delivered to interferon gamma-primed macrophages (BMMØ or thioglycollate-elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor-alpha and monocyte chemoattractant protein-1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage-activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.

How Far Have We Reached in Tuberculosis Vaccine Development?

Tuberculosis, a bacterial disease prevalent since ancient times, continues to cause the most deaths globally compared with all other diseases. The causative agent Mycobacterium tuberculosis is responsible for different types of tuberculosis in humans; however, pulmonary tuberculosis is the most common and causes the most deaths. Mycobacterium tuberculosis is an intracellular pathogenic bacterium, which has developed sophisticated mechanisms to survive inside host mononuclear phagocytes and thus evade the host immune system. This is attributed primarily to an inadequate immune response toward infecting bacteria, which results in temporary growth inhibition rather than death and subsequently allows the bacteria to multiply immensely, leading to full-blown disease in an individual. This disease has become a challenge due to poor diagnosis, a low-efficiency tuberculosis vaccine (Mycobacterium bovis Bacillus Calmette-Guèrin [BCG]), a long-term antibacterial chemotherapy regimen (approximately 6 months), and an emergence of multiple drug resistant strains of Mycobacterium tuberculosis especially in people with human immune deficiency virus (HIV) infection, for whom researchers worldwide must develop effective short-term chemotherapy and an effective vaccine. In this review different aspects of vaccines in tuberculosis are discussed, and these include the traditional BCG vaccine, the modern auxotrophic vaccine, the subunit or acellular vaccine; and a DNA vaccine. We discuss also the potential of mycobacterial lipids as a vaccine or as an adjuvant in the future. Since complete genome information of Mycobacterium tuberculosis H37Rv and bioinformatics tools are available, it is possible to develop new strategies for a better and effective tuberculosis vaccine, which can replace the traditional BCG vaccine.

Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum.

Although most mycobacterial lipids are thought to be associated with the cell envelope, the authors previously observed substantial deposits of intracellular lipophilic material. A Nile-red-based cytological assay was used to determine factors which affect the presence and natural history of intracellular lipophilic inclusions (ILIs) in Mycobacterium smegmatis. Development of ILIs was associated with stationary-phase cultures in broth and with aged (6 days) colonies on agar. Using variants of Youmans' defined medium, the frequency and size of ILIs was observed to be minimal in carbon-poor medium. ILIs were observed to form within 15 min after provision of fatty acids to the medium and after a period of several days in nitrogen-poor medium. Analysis of the non-polar lipid extracts of ILI-rich and -poor preparations indicated that the triacylglycerols (TAGs) were a major component of the inclusions. The acyl substituents of the TAGs varied according to whether they were formed in Middlebrook 7H9 broth, in low-nitrogen Youmans' broth or rapidly after oleic acid supplementation of Youmans' broth. These studies support a storage function for TAGs in mycobacteria in addition to their previously suggested occurrence as components of the cell envelope. To assess a possible role for ILIs in Mycobacterium tuberculosis, a combined acid-fast (Auramine)/Nile red assay was applied to heavily positive sputum samples from patients with tuberculosis. Strong intracellular Nile red signals were obtained from acid-fast cells, indicating that ILI occur in M. tuberculosis in vivo. This may reflect a distinct physiological state of these cells, which it has not been possible to reproduce in vitro. These findings indicate that the uptake of long-chain fatty acids and TAG biosynthetic and degradative pathways are important aspects of mycobacterial lipid metabolism, meriting further investigation.