Lipid domains of mycobacteria studied with fluorescent molecular probes (original) (raw)

Identification of the bound and unbound lipids on the cell envelopes of Mycobacterium bovis

2002

Sixteen Mycobacterium bovis isolates from bovine tissues and one reference strain M. bovis AN5 were investigated for different bound and unbound lipids. For comparison, other mycobacterial species included in the study were M. tuberculosis, M. bovis BCG and M. avium. The thin layer chromatographic (TLC) study indicated the absence of C-mycosides in all the M. bovis isolates while phosphatidyl inositol mannosides (PIMs) was present in all M. bovis isolates, M. bovis BCG and M. tuberculosis as shown by TLC with the solvent chloroform-methanol-water (60:30:6, v/v × 1). The polar glycolipids were present in all the M. bovis isolates and M. tuberculosis showing seven bands with Rf value 0.62, 0.55, 0.44, 0.40, 0.38, 0.31 and 0.09 with the solvent n-propanol-water-ammonia (sp. gr. 0.89) (75:22:3, v/v × 1). The unidimensional TLC mycolates patterns of all M. bovis isolates including AN5 showed one spot corresponding to alpha-mycolate (as in M. tuberculosis) and the other spot being more di...

Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2011

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.

Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid

2005

ever, the molecular components of biosynthetic machinery involved in the synthesis of these complex Mohd. Zeeshan Ansari, Rashmi Tickoo, lipids have not been elucidated. Vijayalakshmi Sridharan, Debasisa Mohanty, The tremendous efforts in recent years have demonand Rajesh S. Gokhale* strated that a number of mycobacterial lipids are National Institute of Immunology biosynthesized by the combined action of fatty acid Aruna Asaf Ali Marg synthases (FASs) and polyketide synthases (PKSs) New Delhi 110 067 (Camacho et al., 2001; Cox et al., 1999; Kolattukudy et India al., 1997; Sirakova et al., 2001). Whereas FASs are known to catalyze the biosynthesis of fatty acids, PKSs usually produce secondary metabolites that are a rich Summary source of commercially important therapeutic agents (Hopwood, 1997; Khosla, 2000). Traditionally, these Mycobacterium tuberculosis cell envelope is a treamultienzymatic assemblies have been studied indepensure house of biologically active lipids of fascinating dently, and their modes of interaction to produce hybrid molecular architecture. Although genetic studies have molecules of fatty acids and polyketides have not been alluded to an array of genes in biosynthesis of cominvestigated (Gokhale and Tuteja, 2001; Kolattukudy et plex lipids, their mechanistic, structural, and bioal., 1997; Minnikin et al., 2002). In mycobacteria, several chemical principles have not been investigated. Here, PKS disruption mutants display altered lipid profiles, we have dissected the molecular logic underlying the some of which also exhibit attenuation in their virulence biosynthesis of a virulence lipid phthiocerol dimycoproperties (Sirakova et al., 2001; Dubey et al., 2002). cerosate (PDIM). Cell-free reconstitution studies dem-Genetic studies have identified an array of PKS onstrate that polyketide synthases, which are usually genes that are required for biosynthesis of PDIMs and involved in the biosynthesis of secondary metaboglycosylated phenolphthiocerol esters, which are surlites, are responsible for generating complex lipids in face-exposed lipids unique to the virulent strains of mycobacteria. We show that PapA5 protein directly mycobacteria. The pks7, pks10, pks12, ppsA-E, and transfers the protein bound mycocerosic acid ana-pks15/1 mutant strains of M. tuberculosis are deficient logs on phthiocerol to catalyze the final esterification in phthiocerol derivatives and yield strains with attenustep. Based on precise identification of biological ated growth in the murine model (Kolattukudy et al., functions of proteins from Pps cluster, we have ratio-1997; Rousseau et al., 2003; Sirakova et al., 2003a, nally produced a nonmethylated variant of mycocero-2003b). Most of the genes proposed to be involved in sate esters. Apart from elucidating mechanisms that PDIM biosynthesis can be classified in two large clusgenerate chemical heterogeneity with PDIMs, this ters. The pps locus consists of three transcriptional study also presents an attractive approach to explore units with 15 open reading frames and encompasses host-pathogen interactions by altering mycobacterial w50 kbp of the genome. The enzymic activity for three surface coat. of these proteins from pps cluster has been elucidated. We recently characterized FadD26 protein as fatty acyl

Fluidity of the lipid domain of cell wall from Mycobacterium chelonae

Proceedings of the National Academy of Sciences, 1995

The mycobacterial cell wall contains large amounts of unusual lipids, including mycolic acids that are covalently linked to the underlying arabinogalactanpeptidoglycan complex. Hydrocarbon chains of much of these lipids have been shown to be packed in a direction perpendicular to the plane of the cell surface. In this study we examined the dynamic properties of the organized lipid domains in the cell wall isolated from Mycobacterium chelonae grown at 30°C. Differential scanning calorimetry showed that much of the lipids underwent major thermal transitions between 30°C and 65°C, that is at temperatures above the growth temperature, a result suggesting that a significant portion of the lipids existed in a structure of extremely low fluidity in the growing cells. Spin-labeled fatty acid probes were successfully inserted into the more fluid part of the cell wall. Our model of the cell wall suggests that this domain corresponds to the outermost leaflet, a conclusion reinforced by the observation that labeling of intact cells produced electron spin resonance spectra similar to those of the isolated cell wall. Use of stearate labeled at different positions showed that the fluidity within the outer leaflet increased only slightly as the nitroxide group was placed farther away from the surface. These results are consistent with the model of mycobacterial cell wall containing an asymmetric lipid bilayer, with an internal, less fluid mycolic acid leaflet and an external, more fluid leaflet composed of lipids containing shorter chain fatty acids. The presence of the low-fluidity layer will lower the permeability of the cell wall to lipophilic antibiotics and chemotherapeutic agents and may contribute to the well-known intrinsic resistance of mycobacteria to such compounds.

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

Journal of Bacteriology, 2008

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

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 onl...

Spreading of a virulence lipid into host membranes promotes mycobacterial pathogenesis

Several lipids of the pathogen Mycobacterium tuberculosis are known to promote virulence at various stages of disease. However, the inability to probe these lipids during in vivo infection makes elucidation of their pathogenic mechanisms difficult. Using chemical extraction and reconstitution methods, we were able to define the lipid composition of the outer mycomembrane of Mycobacterium marinum prior to infection. Combining this approach with the synthesis of clickable, semi-synthetic lipids, we introduced a chemically tractable, biologically active variant of the virulence lipid phthiocerol dimycocerosate (PDIM) into the mycomembrane. We find that following infection of zebrafish larvae, PDIM spreads away from bacterial surfaces into the membranes of both macrophage and epithelial cells that it contacts. This spreading facilitates PDIM′s role in preventing bacterium-detrimental immune activation at the site of infection.

Trafficking and Release of Mycobacterial Lipids from Infected Macrophages

Traffic, 2000

Analysis of infected macrophages revealed that lipidcontaining moieties of the mycobacterial cell wall are actively trafficked out of the mycobacterial vacuole. To facilitate the analysis of vesicular trafficking from mycobacteria-containing phagosomes, surface-exposed carbohydrates were labeled with hydrazide-tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyzed by immunoelectron microscopy and by fluorescence microscopy of live cells. The released mycobacterial constituents were associated with several intracellular organelles and were enriched strikingly in tubular endocytic compartments. Subcellular fractionation of infected macrophages by density gradient electrophoresis showed temporal movement of labeled bacterial constituents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed five dominant bacteria-derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be observed in un-infected 'bystander' cells. Their transfer to bystander cells could expand the bacteria's sphere of influence beyond the immediate confines of the host cell.

Association of Mycobacterium Proteins with Lipid Droplets

Journal of Bacteriology

Mycobacterium tuberculosis is a global pathogen of significant medical importance. A key aspect of its life cycle is the ability to enter into an altered physiological state of nonreplicating persistence during latency and resist elimination by the host immune system. One mechanism by which M. tuberculosis facilitates its survival during latency is by producing and metabolizing intracytoplasmic lipid droplets (LDs). LDs are quasi-organelles consisting of a neutral lipid core such as triacylglycerol surrounded by a phospholipid monolayer and proteins. We previously reported that PspA (phage shock protein A) associates with LDs produced in Mycobacterium. In particular, the loss or overproduction of PspA alters LD homeostasis in Mycobacterium smegmatis and attenuates the survival of M. tuberculosis during nonreplicating persistence. Here, M. tuberculosis PspA (PspAMtb) and a ΔpspA M. smegmatis mutant were used as model systems to investigate the mechanism by which PspA associates with ...