Lipomannan and Lipoarabinomannan from a Clinical Isolate of Mycobacterium kansasii: NOVEL STRUCTURAL FEATURES AND APOPTOSIS-INDUCING PROPERTIES (original) (raw)
Related papers
Journal of Biological Chemistry, 2002
Lipomannan (LM) and lipoarabinomannan (LAM) are major glycolipids present in the mycobacterial cell wall that are able to modulate the host immune response. In this study, we have undertaken the structural determination of these important modulins in Mycobacterium chelonae, a fast growing pathogenic mycobacterial species. One-dimensional and two-dimensional NMR spectra were used to demonstrate that LM and LAM from M. chelonae, designated CheLM and CheLAM, respectively, possess structures that differ from the ones reported earlier in other mycobacterial species. Analysis by gas chromatography/mass spectrometry of the phosphatidyl-myo-inositol anchor, which is thought to play a role in the biological functions of these lipoglycans, pointed to a high degree of heterogeneity based on numerous combinations of acyl groups on the C-1 and C-2 positions of the glycerol moiety. Characterization of the mannan core of CheLM and CheLAM revealed the presence of novel ␣1,3-mannopyranosyl side chains. This motif, which reacted specifically with the lectin from Galanthus nivalis, was found to be unique among a panel of nine mycobacterial species. Then, CheLM and CheLAM were found to be devoid of both the mannooligosaccharide cap present in Mycobacterium tuberculosis and the inositol phosphate cap present in Mycobacterium smegmatis and other fast growing species. Tumor necrosis factor-␣ and interleukin-8 production were assessed from human macrophages with LAM preparations from different species. Our results suggest that the inositol phosphate capping may represent the major cytokineinducing component of LAMs. This work not only underlines the diversity of LAM structures among various mycobacterial species but also provides new structures that could be useful to dissect the structure-function relationships of these complex molecules.
mBio, 2013
Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to -lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to -lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis.
Genetics of Mycobacterial Arabinogalactan and Lipoarabinomannan Assembly
Microbiology Spectrum, 2014
The cell wall of Mycobacterium tuberculosis is unique in that it differs significantly from those of both Gram-negative and Gram-positive bacteria. The thick, carbohydrate- and lipid-rich cell wall with distinct lipoglycans enables mycobacteria to survive under hostile conditions such as shortage of nutrients and antimicrobial exposure. The key features of this highly complex cell wall are the mycolyl-arabinogalactan-peptidoglycan (mAGP)–based and phosphatidyl- myo -inositol–based macromolecular structures, with the latter possessing potent immunomodulatory properties. These structures are crucial for the growth, viability, and virulence of M. tuberculosis and therefore are often the targets of effective chemotherapeutic agents against tuberculosis. Over the past decade, sophisticated genomic and molecular tools have advanced our understanding of the primary structure and biosynthesis of these macromolecules. The availability of the full genome sequences of various mycobacterial spe...
Journal of The American Chemical Society, 2006
Mycobacteria produce a cell-surface glycoconjugate, lipoarabinomannan (LAM), which has been shown to be a potent modulator of the immune response that arises from infection by these organisms. Recently, LAM from the human pathogens Mycobacterium tuberculosis and M. kansasii has been shown to contain an unusual 5-deoxy-5-methylthio-xylofuranose (MTX) residue as well as its corresponding oxidized counterpart, 5-deoxy-5-methylsulfoxy-xylofuranose (MSX). To date, the absolute configuration of these residues and their linkage position to the polysaccharide are unknown, as is their biological role. Through the combined use of chemical synthesis and NMR spectroscopy, we have established that the MTX/MSX residues in these glycoconjugates are of the D-configuration and that they are linked R-(1f4) to a mannopyranose residue in the mannan portion of the glycan. Conformational analysis of the MTX/MSX residue using NMR spectroscopy showed differences in ring conformation and as well as in the rotamer populations about the C-4-C-5 bond, as compared to the parent compound, methyl R-D-xylofuranoside. Two of the synthesized disaccharides, 3 and 34, were tested in cytokine induction assays, and neither led to the production of TNF-R or IL-12p70. In contrast, both demonstrated modest inhibitory properties when these same cytokines were induced using a preparation of Interferon-γ and Staphylococcus aureus Cowan strain (SAC/IFN-γ). These latter observations suggest that this motif may play a role in the immune response arising from mycobacterial infection.
Journal of Biological Chemistry, 2021
In Mycobacterium tuberculosis (Mtb), surface-exposed Lipoarabinomannan (LAM) is a key determinant of immunogenicity, yet its intrinsic heterogeneity confounds typical structure-function analysis. Recently, LAM gained a strong foothold as a validated marker for active tuberculosis (TB) infection and has shown great potential in new diagnostic efforts. However, no efforts have yet been made to model or evaluate the impact of mixed polyclonal Mtb infections (infection with multiple strains) on TB diagnostic procedures other than antibiotic susceptibility testing. Here, we selected three TB clinical isolates (HN878, EAI, and IO) and purified LAM from these strains to present an integrated analytical approach of one-dimensional and two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy, as well as enzymatic digestion and site-specific mass spectrometry (MS) to probe LAM structure and behavior at multiple levels. Overall, we found that the glycan was similar in all LAM preparations, albeit with subtle variations. Succinates, lactates, hydroxybutyrate, acetate, and the hallmark of Mtb LAMmethylthioxylose (MTX), adorned the nonreducing terminal arabinan of these LAM species. Newly identified acetoxy/ hydroxybutyrate was present only in LAM from EAI and IO Mtb strains. Notably, detailed LC/MS-MS unambiguously showed that all acyl modifications and the lactyl ether in LAM are at the 3-OH position of the 2-linked arabinofuranose adjacent to the terminal β-arabinofuranose. Finally, after sequential enzymatic deglycosylation of LAM, the residual glycan that has 50% of α−arabinofuranose-(1→5) linked did not bind to monoclonal antibody CS35. These data clearly indicate the importance of the arabinan termini arrangements for the antigenicity of LAM. Lipoarabinomannan (LAM) is a heterogeneous lipoglycan, a major component of the cell wall of mycobacteria (1, 2). It is characterized by three distinct structural domains: (i) a phosphatidylinositol anchor, (ii) a mannan backbone, and (iii) several arabinan antennas emanating from the mannan backbone. The nonreducing end of the arabinan component of
Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane
Scientific Reports, 2017
The mycobacterial envelope is unique, containing the so-called mycomembrane (MM) composed of very-long chain fatty acids, mycolic acids (MA). Presently, the molecular composition of the MM remains unproven, due to the diversity of methods used for determining its composition. The plasma membranes (PM) and the native MM-containing cell walls (MMCW) of two rapid-growing mycobacterial species, Mycobacterium aurum and M. smegmatis, were isolated from their cell lysates by differential ultracentrifugation. Transmission electron microscopy and biochemical analyses demonstrated that the two membranes were virtually pure. Bottom-up quantitative proteomics study indicated a different distribution of more than 2,100 proteins between the PM and MMCW. Among these, the mannosyltransferase PimB, galactofuranosyltransferase GlfT2, Cytochrome p450 and ABC transporter YjfF, were most abundant in the PM, which also contain lipoglycans, phospholipids, including phosphatidylinositol mannosides, and only a tiny amount of other glycolipids. Antigen85 complex proteins, porins and the putative transporters MCE protein family were mostly found in MMCW fraction that contains MA esterifying arabinogalactan, constituting the inner leaflet of MM. Glycolipids, phospholipids and lipoglycans, together with proteins, presumably composed the outer leaflet of the MM, a lipid composition that differs from that deduced from the widely used extraction method of mycobacterial cells with dioctylsulfosuccinate sodium. Mycobacteria are probably the most successful microorganisms to parasite animals and humans. Among the 187 valid species described to date in the genus Mycobacterium, only three are strict pathogens for human: Mycobacterium tuberculosis (Mtu), M. leprae and M. lepromatosis 1. Tuberculosis still represents a major public health problem worldwide, remaining one of the world leading causes of death from an infectious agent, about one third of the world population being infected by the Koch bacillus and susceptible to develop the disease. In addition, two-thirds of mycobacteria species are opportunistic pathogens for human, and with large enough inoculum, all mycobacteria produce granulomatous lesions in experimental animals 2. The cell envelope is critical for the mycobacterial physiology, primarily because many crucial processes are located in this compartment. These include the protection of the bacterial cell from hostile environments, mechanical resistance of the cells, transport of solutes and proteins, adhesion to receptors. The hallmark of mycobacteria is their unique abundance in lipid, constituting up to 40% of the dry weight of the tubercle bacillus 3,4. The mycobacterial cell wall contains up to 60% of lipids, as compared with some 20% for the lipid-rich cell walls of Gram-negative microorganisms 4. These lipids include the exceptionally-long chain fatty acids (mycolic acids, MA) covalently linked to the cell wall polysaccharide arabinogalacatan (AG) and whose esterifying trehalose, as well as the numerous classes of exotic compounds typifying the Mycobacterium genus. To these lipids have been attributed many of the biological properties of mycobacteria 3,4. These include the very high resistance of the majority of mycobacterial species to most of the broad-spectrum antibiotics, except for instance streptomycin and rifamycins 2 and their recognized impermeability to nutrients, up to 100-to 1,000-fold less permeable than the most resistant Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa 5 .