Structural features of the arabinan component of the lipoarabinomannan of Mycobacterium tuberculosis

The recent availability of pure lipoarabinomannan (LAM) from Mycobacterium spp. has resulted in its implication in host-parasite interaction, which events may be mediated by the presence of a phosphatidylinositol unit at the reducing end of LAM. Herein we address the structure of the antigenic, nonreducing end of the molecule. Through the process of 13C NMR analysis of the whole molecule and gas chromatography/mass spectrometry of alditol acetates derived from the differential per-O-alkylated lipopolysaccharide, the majority of the arabinosyl residues were recognized as furanosides. Second, through analysis of per-O-alkylated oligoarabinosyl arabinitol fragments of partially hydrolyzed LAM, it was established that the internal segments of the arabinan component consists of branched 3,5-linked alpha-D-arabinofuranosyl (Araf) units with stretches of linear 5-linked alpha-D-Araf residues attached at both branch positions, whereas the nonreducing terminal segments of LAM consist of either of the two arrangements, beta-D-Araf-(1----2)-alpha-D-Araf-(1----5)- alpha-D-Araf---- or [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2---- (3 and 5)-alpha-D-Araf----. Since this latter arrangement also characterizes the terminal segments of the peptidoglycan-bound arabinogalactan of Mycobacterium spp., we propose that mycobacteria elaborate unique terminal arabinan motifs in two distinct settings. In the case of the bound arabinogalactan, these motifs provide the nucleus for the esterified mycolic acids, entities which dominate the physicochemical features of mycobacteria and their peculiar pathogenesis. In the case of LAM, these motifs, non-mycolylated, are the dominant B-cell antigens responsible for the majority of the copious antibody response evident in most mycobacterial infections.     

Lipoarabinomannan of Mycobacterium tuberculosis. Capping with mannosyl residues in some strains.

Previously we had demonstrated that the termini of the arabinan component of mycobacterial cell wall arabinogalactan, the site of mycolic acid location, consists mostly of clusters of a pentaarabinofuranoside, [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2----(3 and 5)-alpha-D-Araf. Subsequently, the same arrangement was shown to dominate the non-reducing ends of lipoarabinomannan (LAM), a key component in the interaction of mycobacteria with host cell. Accordingly, we had proposed that mycobacteria universally elaborate the same Araf-containing motifs in two settings for different pathophysiological purposes. However, we now report that the termini of LAM from the virulent, Erdman, strain of Mycobacterium tuberculosis, unlike those from the attenuated H37Ra strain, are extensively capped with mannosyl (Manp) residues, either a single alpha-D-Manp, a dimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp), or a trimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp ). The use of monoclonal antibodies demonstrates a clear difference in the antigenicity of the basic and mannose-capped LAM. The possibility that these structures are a factor in the virulence of some strains of M. tuberculosis and represent an example of carbohydrate mimicry in mycobacterial infections is discussed.     

A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate,Lipoarabinomannan

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5–7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10–12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.     

Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species

Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules. In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis. A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain. In cell-free assays, extracts from M. smegmatis overexpressing the M. tuberculosis Rv2611c gene incorporated [14C]palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain. Cell-free extracts from the Rv2611c mutant of M. smegmatis were greatly impaired in these respects. This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme. We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria.     

Trehalose-containing lipooligosaccharide antigens of Mycobacterium sp.: presence of a mono-O-methyltri-O-acyltrehalose "core"

We have described the surface antigens of Mycobacterium kansasii as trehalose-containing lipooligosaccharides (LOS) which at the nonreducing "epitope" end bear a unique amino sugar containing diglycosyl unit, whereas the putative reducing end consists of an acylated alpha, alpha-trehalose-containing tetraglucosyl "core" [Hunter, S. W., Jardine, I., Yanagihara, D. L., & Brennan, P. J. (1985) Biochemistry 24, 2798-2805]. The presence of a new variation on this core, in Mycobacterium szulgai, is now reported, ----3)beta-D-Glcp-(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O- Me-alpha-D-Glcp, representing the first example of an O-methyltrehalose unit in nature. The simplest of the LOS class of glycolipids in M. szulgai was defined as alpha-L-2-O-Me-Fucp(1----3)alpha-L-Rhap(1----3)alpha-L-Rh ap(1----3) beta-D-Glcp(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O-Me-alpha-D-G lcp. Further glycosylation of this nonantigen, by an incompletely defined 6-deoxyhexosyl residue, confers specific antigenicity on the organism. Thus, these extraordinary structures, in a manner analogous to the better known lipopolysaccharides from rough variants of Enterobactericiae, are highly amphipathic and display variability not only in the immunogenic, distal region but also in the "invariant" lipophilic core. The contribution of these glycolipids to the hydrophobic barrier, the pseudo outer membrane of mycobacteria, is discussed.     

Structural studies on O-specific polysaccharides of lipopolysaccharides from Yersinia enterocolitica serovars O:1,2a,3, O:2a,2b,3 and O:3

Lipopolysaccharides from Yersinia enterocolitica serovars O:1,2a,3, O:2a,2b,3 and O:3 have been isolated and characterized. 6-Deoxy-L-altrose residues were shown to be the main constituents of lipopolysaccharides isolated in addition to residues of L-rhamnose, D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid being minor components of sugar chains. Mild hydrolysis of lipopolysaccharides with acetic acid furnished O-specific polysaccharides, which are composed of 6-deoxy-L-altrose. Using 13C-NMR spectroscopy and methylation data, the structural features of backbones have been elucidated as follows: ----2)-6d-L-Altp(beta 1----2)-6d-L-Altp(beta 1----3)-6d-L-Altp)(beta 1----for serovars O:1,2a,3 and O:2a,2b,3;----2)-6d-L-Altp(beta 1----for serovar O:3. In addition, O-polysaccharide of serovar O:2a,2b,3 was found to contain an O-acetyl group at the C-3 position of some 1,2-linked sugar residues.     

Structural study of lipomannan and lipoarabinomannan from Mycobacterium chelonae. Presence of unusual components with alpha 1,3-mannopyranose side chains

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 alpha1,3-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-alpha 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 cytokine-inducing 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.     

Use of 1H NMR ROESY for structural determination of O-glycosylated amino acids from a serine-containing glycopeptidolipid antigen.

A serine-containing glycopeptidolipid antigen isolated from Mycobacterium xenopi typified a new class of mycobacterial glycopeptidolipid antigens devoid of the C-mycoside core structure [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063]. The lipopeptide core assigned to C12-Ser-Ser-Phe-alloThr-OCH3 exhibits three potential sites of glycosylation. The carbohydrate parts are composed of 3-O-methyl-6-deoxy-alpha-L-talopyranosyl and 2,3,4-tri-O-methyl-L- rhamnopyranosyl(alpha 1----3)-2-O-lauroyl-L-rhamnopyranosyl(alpha 1----3)-L- rhamnopyranosyl(alpha 1----3)-2,4-di-O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendages. In the present work, the carbohydrate attachment sites were successfully determined by ROESY experiments on the native glycopeptidolipid using chloroform as solvent. From the NOE contacts, we unambiguously established that the acylated serine is glycosylated by the 3-O-methyl-6-deoxy-alpha-L-talopyranosyl appendage while the 2,3,4-tri-O-methyl-L-rhamnopyranosyl(alpha 1----3)-2-O- lauroyl-L-rhamnopyranosyl(alpha 1----3)-L-rhamnopyranosyl(alpha 1----3)-2,4-di- O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendage is bound to the C-terminal alloThr-OCH3. From these data, the acetyl and lauroyl residues on the C-2 and C-4 of the basal monosaccharide unit were successfully localized. Furthermore, the "L" absolute configuration for the serines and the phenylalanine residues and the "D" configuration for the allothreonine were established. The primary structure of this novel type of mycobacterial antigen, a serine-containing glycopeptidolipid, has now been fully established.     

Structure of the sidechain of lipopolysaccharide from Pseudomonas syringae pv. morsprunorum C28

The sidechain of the lipopolysaccharide from the phytopathogen Pseudomonas syringae pv. morsprunorum C28 was shown to be composed of D-rhamnose. Using 1H and 13C-NMR spectroscopy, methylation analysis, Smith degradation and optical rotation data, the repeat unit was found to have the structure: ----3)-D-Rhap-(alpha 1----3)-D-Rhap-(alpha 1----2)-D-Rhap-(alpha 1---- and a degree of polymerization of approximately 70. Attention is drawn to the possible prevalence of D-6-deoxyhexoses in the lipopolysaccharides of plant pathogenic bacteria.     

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.     

Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans

Lipoarabinomannans (LAM) and lipomannans (LM) are integral parts of the mycobacterial cell wall recognized by cells involved in the innate immune response and have been found to modulate the cytokine response. Typically, mannosylated LAM from pathogenic mycobacteria have been reported to be anti-inflammatory, whereas phosphoinositol-substituted LAM from nonpathogenic species are proinflammatory molecules. In this study, we show that LM from several mycobacterial species, including Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis bacillus Calmette-Guérin, display a dual function by stimulating or inhibiting proinflammatory cytokine synthesis through different pathways in murine primary macrophages. LM, but none of the corresponding LAM, induce macrophage activation characterized by cell surface expression of CD40 and CD86 and by TNF and NO secretion. This activation is dependent on the presence of Toll-like receptor (TLR) 2 and mediated through the adaptor protein myeloid differentiation factor 88 (MyD88), but independent of either TLR4 or TLR6 recognition. Surprisingly, LM exerted also a potent inhibitory effect on TNF, IL-12p40, and NO production by LPS-activated macrophages. This TLR2-, TLR6-, and MyD88-independent inhibitory effect is also mediated by LAM from M. bovis bacillus Calmette-Guérin but not by LAM derived from M. chelonae and M. kansasii. This study provides evidence that mycobacterial LM bear structural motifs susceptible to interact with different pattern recognition receptors with pro- or anti-inflammatory effects. Thus, the ultimate response of the host may therefore depend on the prevailing LM or LAM in the mycobacterial envelope and the local host cell receptor availability.     

Identification of a novel protein with a role in lipoarabinomannan biosynthesis in mycobacteria

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.     

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan

Based on chemical analysis, we have previously concluded thatthe biologically important lipoarabinomannan (LAM) and lipomannan(LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositolmannosides (PIMs), the characteristic cell envelope mannophosphoinositidesof mycobacteria Using definitive analytical techniques, we havenow re-examined the reported multiacylated nature of PIMs inorder to gain a better insight into their possible roles asbiosynthethic precursors of LM and LAM. High-sensitivity fastatom bombardment-mass spectrometry analyses of the perdeuteroacetyland permethyl derivatives of PIMs from Mycobacterium tuberculosisand Mycobacterium leprae enabled us to define the exact fattyacyl compositions of the multiacylated, heterogeneous PIM families,notably the dimannoside (PIM₂) and the hexamannoside (PIM₆).Specifically, in conjunction with other chemical and gas chromatography-massspectrometry (GCMS) analyses, the additional C16 fatty acylsubstituent on PIM₂ and its lyso form were defined as attachedto the C6 position of mannose. We also present evidence fortriacylated mannophosphoinositide as a common lipid anchor forboth LM and LAM, and further postulate that acylation of PIM₂may constitute a key regulatory step in their biosynthesis. FAB-MS lipoarabinomannan lipomannan Mycobacterium tuberculosis phosphatidylinositol mannosides     

Structural studies on O-specific polysaccharides of lipopolysaccharides from Yersinia enterocolitica serovars O:5 and O:5,27

Lipopolysaccharides of Yersinia enterocolitica serovars O:5 and O:5,27 were shown to have a similar sugar composition, consisting of L-rhamnose, D-glucose, D-galactose, D- and L-glycero-D-manno-heptose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 3-deoxy-D-manno-octulosonate and D-threo-pent-2-ulose (D-xylulose). Partial hydrolysis of lipopolysaccharides with acetic acid produced rhamnans with the following repeating unit: ----3)-L-Rha rho(alpha 1----3)-L-Rha rho(alpha 1----3)-L-Rha rho(beta 1----. 13C-NMR and methylation studies of the lipopolysaccharides gave the following structure for the repeating unit of the two O-specific polysaccharides: ----3)-L-Rha rho(alpha 1----3)-L-Rha rho(alpha 1----3)-L-Rha rho(beta 1----. (formula; see text)     

Analysis of a new mannosyltransferase required for the synthesis of phosphatidylinositol mannosides and lipoarbinomannan reveals two lipomannan pools in corynebacterineae

The cell walls of the Corynebacterineae, which includes the important human pathogen Mycobacterium tuberculosis, contain two major lipopolysaccharides, lipoarabinomannan (LAM) and lipomannan (LM). LAM is assembled on a subpool of phosphatidylinositol mannosides (PIMs), whereas the identity of the LM lipid anchor is less well characterized. In this study we have identified a new gene (Rv2188c in M. tuberculosis and NCgl2106 in Corynebacterium glutamicum) that encodes a mannosyltransferase involved in the synthesis of the early dimannosylated PIM species, acyl-PIM2, and LAM. Disruption of the C. glutamicum NCgl2106 gene resulted in loss of synthesis of AcPIM2 and accumulation of the monomannosylated precursor, AcPIM1. The synthesis of a structurally unrelated mannolipid, Gl-X, was unaffected. The synthesis of AcPIM2 in C. glutamicum DeltaNCgl2106 was restored by complementation with M. tuberculosis Rv2188c. In vivo labeling of the mutant with [3H]Man and in vitro labeling of membranes with GDP-[3H]Man confirmed that NCgl2106/Rv2188c catalyzed the second mannose addition in PIM biosynthesis, a function previously ascribed to PimB/Rv0557. The C. glutamicum Delta NCgl2106 mutant lacked mature LAM but unexpectedly still synthesized the major pool of LM. Biochemical analyses of the LM core indicated that this lipopolysaccharide was assembled on Gl-X. These data suggest that NCgl2106/Rv2188c and the previously studied PimB/Rv0557 transfer mannose residues to distinct mannoglycolipids that act as precursors for LAM and LM, respectively.     

Carolinoside: a phytosteroidal glycoside from Solanum carolinense.

The glycoside of a new class of phytosteroids has been isolated from Solanum carolinense. The steroidal aglycone (carolinone) is alkylated at C-3 and is identified as C-[(2,4,5-trideoxy-3-keto-4,5-dehydro)-pentulopyranosyl]-(5----3)- (13,14- seco-14 beta,17 alpha-dihydroxy) estrogen. The hydrolytic labile glycosyl moiety is identified as O-(beta-D-glucopyranosyl) (1----1)-[L-(2,6-dideoxy-3-C-methyl)- arabinopyranose]. The linkage of this disaccharide in the steroidal glycoside (carolinoside) is shown to be O-(alpha-pentulopyranosyl)- (1----4)-O-(beta-L-arabinopyranosyl)-(1----1)-D-glucopyranose. Carolinoside occurs at concentrations of 10(-7)-10(-6) M in leaf tissue and was shown to be the host plant specific feeding induction factor for Manduca sexta.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.     

A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability.

A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.     

O-specific polysaccharide of Hafnia alvei lipopolysaccharide isolated from strain 1211. Structural study using chemical methods, gas-liquid chromatography/mass spectrometry and NMR spectroscopy.

The Hafnia alvei strain 1211 O-specific polysaccharide is composed of 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D-galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and D-glucose (1:1:2:2). On the basis of sugar and methylation analyses, Smith degradation, and one- and two-dimensional 1H- and 13C-NMR spectroscopy, the polysaccharide was shown to be an O-acetylated polymer of the repeating hexasaccharide unit, ----2D(4-OAc)Fucp3NAcyl beta 1----6DGlcpNAc alpha 1---- (DGlcp beta 1----3)4DGalpNAc alpha 1----3DGlcpNAc beta 1----2DGlcp beta 1----, where DFucp3NAcyl = 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D- galactopyranose. The O-specific polysaccharide showed some microheterogeneity due to incomplete substitution by terminal glucose.