Structure of a novel sulfate-containing mycobacterial glycolipid.

We described previously the unusual structures of the two major C-mycoside glycopeptidolipids from Mycobacterium fortuitum biovar. peregrinum. More polar glycolipids, potentially more interesting in terms of antigenicity, were also present in the strains. A combination of FAB mass spectrometry, NMR, chemical analyses, and radiolabeling was successfully applied to these glycolipids to arrive at the unexpected and novel structure for the more polar compound. This consisted of the "orthodox" basic structure of the apolar C-mycosides, modified at the alaninol end by the presence of a sulfate group on position 2 of a 3,4-di-O-methylrhamnosyl residue. This novel and second class of sulfate-containing mycobacterial glycolipid may provide a chemical basis for the differentiation and classification of members of the M. fortuitum complex, the main group causing human diseases among the many fast-growing mycobacteria widely distributed in nature.     

Wild Animal Mycobacterial Isolates

Thirteen strains of mycobacteria isolated from deer and various species of wild birds were analysed by gas chromatography (GG) for cellular fatty acids and by thin-layer chromatography (TLG) for polar lipids. These strains were compared to reference strains of Mycobacterium avium, M. para tuberculosis and M. mal-moense. All the examined strains exhibited a generally similar fatty acid pattern characterized by relatively large amounts of hexadenca-noate (16:0), octadecenoate (18:1), octadecanoate (18:0) and 10-me-thyl-octadecanoate (tuberculostearic acid, 10-Me-18:0). Several additional acids were also generally present but in smaller amounts. By means of small but distinct differences in fatty acid composition, the wild animal isolates could be distinguished from both M. paratuber-culosis and M. malmoense but not from M. avium. The TLG polar lipid patterns on the other hand separated the wild animal isolates into 2 distinct groups of complex and simple polar lipid composition which corresponded to the morphologically smooth and rough types, respectively. The complex patterns of the smooth strains were comparable to those of the M. avium serovars whereas both the rough wild animal isolates and all the M. paratuber-culosis strains showed a simple pattern of polar lipids. Both fatty acid profiles and TLG polar lipid patterns support allocation of the wild animal isolates to the MAIS complex. Moreover, the 2 chemical techniques, particularly the GC procedure, are very useful for a more rapid and precise identification of the slow-growing wild animal mycobacterial isolates which have hitherto been characterized on basis of vague criteria.     

The presence of novel glucuronic acid-containing, type-specific glycolipid antigens within Mycobacterium spp. Revision of earlier structures

Previously, we had described the structures of the haptenic oligosaccharides of the surface glycopeptidolipid antigens from serotypes 9 and 25 of the Mycobacterium avium complex and had synthesized these units as putative antigenic probes. The lack of chemical concordance between the synthetic products and the haptens has prompted a re-examination of these structures utilizing the instrumental techniques not previously available of fast atom bombardment-mass spectrometry, Fourier transform infra-red, and high resolution NMR spectroscopy. With the additional information thus available, more extensive chemical fragmentations by base degradation, followed by alkylation, have furnished supportive evidence to allow formulation of revised and novel structures, all of which contain glucuronic acid: serotype 9, 2,3-di-O-Me-L-Fucp(alpha 1----4)-D-GlcAp(beta 1----4)-2,3-di-O-Me-L-Fucp(alpha 1----3)-L-Rhap(alpha 1----2)-6dTal; and serotype 25, 4-acetamido-4,6-dideoxy-2-O-Me-hexosyl(alpha 1----4)-D- GlcAp(beta 1----4)2-O-Me-L-Fucp(alpha 1----3)-L-Rhap(alpha 1----2)6dTal. Glucuronic acid, acetamido sugars, and other novel sugars appear to be widespread in the glycopeptidolipid antigens of Mycobacterium spp. The revised structures will allow renewed synthesis of artificial antigen probes and rational approaches to preparing monoclonal antibodies, both necessary for the new diagnostics required to trace the sources of widespread infections due to M. avium and Mycobacterium intracellulare.     

Function of phosphatidylinositol in mycobacteria

Phosphatidylinositol (PI) is an abundant phospholipid in the cytoplasmic membrane of mycobacteria and the precursor for more complex glycolipids, such as the PI mannosides (PIMs) and lipoarabinomannan (LAM). To investigate whether the large steady-state pools of PI and apolar PIMs are required for mycobacterial growth, we have generated a Mycobacterium smegmatis inositol auxotroph by disruption of the ino1 gene. The ino1 mutant displayed wild-type growth rates and steady-state levels of PI, PIM, and LAM when grown in the presence of 1 mM inositol. The non-dividing ino1 mutant was highly resistant to inositol starvation, reflecting the slow turnover of inositol lipids in this stage. In contrast, dilution of growing or stationary-phase ino1 mutant in inositol-free medium resulted in the rapid depletion of PI and apolar PIMs. Whereas depletion of these lipids was not associated with loss of viability, subsequent depletion of polar PIMs coincided with loss of major cell wall components and cell viability. Metabolic labeling experiments confirmed that the large pools of PI and apolar PIMs were used to sustain polar PIM and LAM biosynthesis during inositol limitation. They also showed that under non-limiting conditions, PI is catabolized via lyso-PI. These data suggest that large pools of PI and apolar PIMs are not essential for membrane integrity but are required to sustain polar PIM biosynthesis, which is essential for mycobacterial growth.     

Studies in lipid histochemistry. XIII. The OPA (osmiumtetroxide-periodic acid-alpha-naphthylamine) method for the detection of apolar lipids.

A new procedure for the detection of apolar lipids is described. It is a modification of the OTAN method (Adams, 1959) using periodic acid which oxidatively removes lower osmium derivatives from polar sites only, leaving those in apolar lipids intact and demonstrable with alpha-naphthylamine. Control steps for the exclusion of the possible interference of some less polar complex lipids and of lipopigments are described. The described technic is superior to the conventionally used sudan dyes due partly to the fact that only aqueous solutions are employed thus excluding any extraction of lipids, partly to the more distinct coloration.     

Direct detection of Mycobacterium tuberculosis lipid antigens by thin-layer chromatography

Abstract Free lipids were extracted from Mycobacterium tuberculosis H37Rv, and their antigenicity was assessed directly on thin-layer chromatograms (TLC) by an immunostaining technique. A family of glycolipids, composed of trehalose acylated with multimethyl branched long-chain fatty acids, was investigated. The most polar of these glycolipids was identified as a possible specific surface antigen. A pair of novel polar glycolipids also showed positive antigenic reactions.     

Mycosides C: Behavior as Receptor Site Substance for Mycobacteriophage D4

Interpretation of an earlier published infrared spectrum of Mycobacterium smegmatis lipids with receptor site activity for D4 phage led us to the inference that the active substance is very likely a mycoside C. This hypothesis was confirmed: the well-characterized mycosides C(s) and C(1217) elaborated by the heterologous strains M. scrofulaceum and Mycobacterium species 1217, respectively, are essentially indistinguishable from the smegmatis lipids in their behavior toward D4. Minute quantities adsorb and extensively inactivate the phage on appropriate incubations. In accord with derivative expectations, Mycobacterium species 1217 is a permissive host, attacked and lysed by D4. However, our current strains of M. butyricum, M. avium, and M. scrofulaceum, which reputedly produce various related mycosides C, are neither lysed by nor do they significantly adsorb the phage. Implications of these observations are discussed.     

Peptidoglycolipid nature of the superficial cell wall sheath of smooth-colony-forming mycobacteria.

The most superficial cell wall layer present in smooth-colony-forming mycobacteria was isolated from serovar 20 of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) serocomplex and examined chemically and by electron microscopy. Most (70 to 80%) of the fibrillar material consisted of an array of serologically active, acetylated C-myosidic peptidoglycoplipids with the basic structure (formula, see text) but in which the location of acetyl groups and the arrangement of monosaccharides have not been defined. Apparently, all serovars within the MAIS complex are characterized by structurally related superficies in which the monoglycosyl-lipopeptide portion is invariable but the oligosaccharide attachment is peculiar to each serovar. These unique inert structures may be an important factor in shielding the pathogen within phagolysosomes from lysosomal enzymes.     

Serological Relationships of Salmonella A Bacteriophages Isolated from Salmonellae in Different Kauffmann- White Groups

Six different Salmonella group A phages from salmonellae in Kauffmann- White groups B, C(1), C(2), and D were examined serologically. Those phages which were specific for a particular somatic antigen were found to be serologically very similar. Antiserum against a phage with one specificity was able to neutralize a different phage with the same specificity but unable to neutralize, in the normal way, a phage with a different specificity. Phages mixed with heterologous phage antiserum responded with an "inhibition response" in which there appeared to be a neutralization of the phage infectivity for the first 10 min, followed by a reversal of the neutralization until, by 20 or 25 min, there was no apparent neutralization. This response was interpreted to indicate that the adsorption antigens, probably situated on the tail fibers, were different for phages with different specificities but sufficiently similar so that heterologous antibodies could react with the antigens; but the antigen-antibody complex was quickly disassociated, resulting in a modification of the antibody molecules but no change in the specificity sites of the antigen. A subgrouping of the Salmonella A phages based on their antigenic specificity is suggested.     

Electron-microscopic study of the serological affinity between the antigenic components of phages T4 and DDVI.

In an investigation of the antigenic fine structure of phages T4 and DDVI with the use of the neutralization reaction and electron-microscopic observation of the phage-antibody complexes, it has been possible to establish that the head of phage T4 consists of proteins which have antigenic determinants of two types: The first type is identical to the antigens of the head of phage DDVI, and the second type is apparently absent in phage DDVI. The phage DDVI head contains mostly determinants which are common to the phage T4 head, since it was not possible to detect antigenically specific components in the phage DDVI head. The tail sheaths of phage T4 and DDVI appear to be identical in the antigenic respect. A difference has been observed in the fibers and the base plates of the phages investigated. The presence of the following three types of antigens has been established: 1) common to phages T2, T4, and DDVI, 2) common to phages T4 and DDVI, and 3) specific for each phage investigated.     

Recognition of novel glycolipid antigens from smooth variants of Mycobacterium tuberculosis

A major polar and three minor slightly less polar glycolipids were identified in extracts of two smooth (Canetti) strains of Mycobacterium tuberculosis. Immunostaining on thin-layer chromatograms and enzyme-linked immunosorbent assay (ELISA) of purified lipids demonstrated that the major and the two most polar of the minor glycolipids are potent antigens, reacting with homologous antisera and also with that raised against the type strain (H37Rv).     

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies

Phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties since conventional hybridoma technologies have yielded mostly low-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties in immobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for protein antigens cannot be readily applied. We adapted phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterization of phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. We first constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) using Man3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences were isolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residues at nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Furthermore, first round characterization of scFvs expressed from respective phages indicated good affinity and specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness of this strategy in constructing human scFv against various carbohydrate antigens. Further studies on the purification and characterization of these scFvs are presented in an accompanying paper in this issue.     

Antigenic glycolipids of Mycobacterium fortuitum based on trehalose acylated with 2-methyloctadec-2-enoic acid

Three characteristic glycolipids were identified in extracts of 10 representative strains of Mycobacterium fortuitum. The two most polar major lipids were shown to be antigens with strong reactions against homologous antiserum and sera raised against Mycobacterium tuberculosis H37Rv. Both of these lipids were shown to be acyl trehaloses, containing substantial amounts of 2-methyloctadec-2-enoic acid in addition to straight-chain and monounsaturated acids. These lipids correspond to 'mycoside F', lipids previously identified by infra-red spectroscopy in extracts of Myco. fortuitum.     

Membranes of Tetrahymena. IV. Isolation and characterization of temperature-responsive smooth and rough microsomal subfractions.

Temperature-responsive microsomes of the ciliate protozoan Tetrahymena have been originally fractionated by step centrifugation on two-layered, Mg2+-containing sucrose gradients. Three fractions have been obtained, which are termed smooth I, smooth II and rough according to the appearance of the membrane vesicles upon electron-microscopy. Smooth I, smooth II, and rough microsomes exhibit RNA/protein ratios of 0.09, 0.20, and 0.34; their phospholipid/protein ratios and their neutral lipid/phospholipid ratios were 0.52, 0.43 and 0.25, and 0.17, 0.18 and 0.13, respectively. All three fractions contain equivalent, low succinic dehydrogenase and 5'-nucleotidase activities. Glucose-6-phosphatase and acid phosphatase are more concentrated in smooth I membranes than in rough membranes. The reverse is true for ATPase. The smooth II membranes occupy an intermediate position except that their ATPase activity is the lowest of the three fractions. The specific activities of these enzymes of the three microsomal fractions are compared to those of homogenates of whole cells. Thin-layer chromatography reveals a very similar polar and nonpolar lipid pattern of the three microsomal fractions. The major phospholipid compounds are phosphatidlethanolamine, glycerideaminoethylphosphonate and phosphatidylcholine, while diglycerides, an unknown NL-compound, and triglycerides are the major apolar lipids. Gas liquid chromatography shows that the fatty acids are mainly even-numbered ranging between C12 and C18. The smooth I, smooth II and rough membranes contain 65.2, 69.3 and 72.7% unsaturated fatty acids in their polar lipids, whereas only 52.7, 49.7 and 48.3% unsaturated acids are found in their apolar lipids, respectively. The fatty acids are more unevenly distributed among the individual polar lipids than in the apolar ones.     

Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1.

We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase.     

Inactivation of lsr2 results in a hypermotile phenotype in Mycobacterium smegmatis

Mycobacterial species are characterized by the presence of lipid-rich, hydrophobic cell envelopes. These cell envelopes contribute to properties such as roughness of colonies, aggregation of cells in liquid culture without detergent, and biofilm formation. We describe here a mutant strain of Mycobacterium smegmatis, called DL1215, which demonstrates marked deviations from the above-mentioned phenotypes. DL1215 arose spontaneously from a strain deficient for the stringent response (M. smegmatis Delta rel(Msm) strain) and is not a reversion to a wild-type phenotype. The nature of the spontaneous mutation was a single base-pair deletion in the lsr2 gene, leading to the formation of a truncated protein product. The DL1215 strain was complicated by having both inactivated rel(Msm) and lsr2 genes, and so a single lsr2 mutant was created to analyze the gene's function. The lsr2 gene was inactivated in the wild-type M. smegmatis mc(2)155 strain by allelic replacement to create strain DL2008. Strain DL2008 shows characteristics unique from those of both the wild-type and Delta rel(Msm) strains, some of which include a greatly enhanced ability to slide over agar surfaces (referred to here as "hypermotility"), greater resistance to phage infection and to the antibiotic kanamycin, and an inability to form biofilms. Complementation of the DL2008 mutant with a plasmid containing lsr2 (pLSR2) reverts the strain to the mc(2)155 phenotype. Although these phenotypic differences allude to changes in cell surface lipids, no difference is observed in glycopeptidolipids, polar lipids, apolar lipids, or mycolic acids of the cell wall.     

Detection of microbial biomass by intact polar membrane lipid analysis in the water column and surface sediments of the Black Sea

The stratified water column of the Black Sea produces a vertical succession of redox zones, stimulating microbial activity at the interfaces. Our study of intact polar membrane lipids (IPLs) in suspended particulate matter and sediments highlights their potential as biomarkers for assessing the taxonomic composition of live microbial biomass. Intact polar membrane lipids in oxic waters above the chemocline represent contributions of bacterial and eukaryotic photosynthetic algae, while anoxygenic phototrophic bacteria and sulfate-reducing bacteria comprise a substantial amount of microbial biomass in deeper suboxic and anoxic layers. Intact polar membrane lipids such as betaine lipids and glycosidic ceramides suggest unspecified anaerobic bacteria in the anoxic zone. Distributions of polar head groups and core lipids show planktonic archaea below the oxic zone; methanotrophic archaea are only a minor fraction of archaeal biomass in the anoxic zone, contrasting previous observations based on the apolar derivatives of archaeal lipids. Sediments contain algal and bacterial IPLs from the water column, but transport to the sediment is selective; bacterial and archaeal IPLs are also produced within the sediments. Intact polar membrane lipid distributions in the Black Sea are stratified in accordance with geochemical profiles and provide information on vertical successions of major microbial groups contributing to suspended biomass. This study vastly extends our knowledge of the distribution of complex microbial lipids in the ocean.     

Mycolipenates and mycolipanolates of trehalose from mycobacterium tuberculosis

Analysis of the lipids of Mycobacterium tuberculosis, by thin-layer chromatography, revealed the presence of two families of novel glycolipids each having two closely-related members but differing widely in polarity. The least and most polar families of lipids were characterized from M. tuberculosis strains C and H37Rv, respectively; all were based on trehalose, the least polar pair of glycolipids having more long-chain substituents than the more polar pair. The acyl substituents of the least polar of the four glycolipids were mainly straight-chain C16 and C18 acids and 2,4,6-trimethyltetracos-2-enoic (C27-mycolipenic) acid, and the second least polar glycolipid contained major amounts of 3-hydroxy-2,4,6-trimethyltetracosanoic (C27-mycolipanolic) acid in addition to these non-hydroxylated acids. The relatively polar pair of glycolipids were analysed together and released mainly straight-chain C16 and C18 acids, C27-mycolipanolic acid, minor amounts of C25- and C27-mycolipenic acids and major proportions of an acid having the chromatographic properties of 2,4-dimethyldocosanoic acid. The most polar pair of glycolipids co-chromatographed with glycolipid antigens previously detected in Mycobacterium bovis BCG.     

Theoretical model of human apolipoprotein B100 tertiary structure

Low density lipoprotein (LDL) particles are the main cholesterol carriers in human plasma. The organization of the particle, composed of apolar lipids and phospholipid monolayer stabilized by apolipoprotein B100 (apoB), is highly complex and still unknown. ApoB is an extremely large protein (4563 amino acids) and very little is known about its structure. A 3D model of the N-terminal region has been recently proposed and has provided interesting insights about the physico-chemical properties of the protein and putative interaction zones with lipids. In the present article, we propose the first tentative 3D modelling for most remaining residues. All predicted features emerging from the models are confronted with agreement to experimental data available. Using different up-to-date prediction methods, we decomposed the protein into eight domains and predicted 3D structure for each of them. The analysis of hydrophobic patches, polar regions, coupled with functional predictions based on the 3D models, gives new clues to understanding of the functional role of apoB. We suggest precise regions putatively involved in the lipid interactions, and discuss the position of apoB on the LDL particle. Finally, we propose relative organization of the domains, providing a shape quite compatible with the low resolution electron microscopy map.     

Compartmentalization of lipid biosynthesis in mycobacteria

The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.