Kay-Hooi Khoo

The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis.

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizingd-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosisgenomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

Truncated Structural Variants of Lipoarabinomannan in Ethambutol Drug-resistant Strains of Mycobacterium smegmatis INHIBITION OF ARABINAN BIOSYNTHESIS BY ETHAMBUTOL

The anti-tuberculosis drug, ethambutol (Emb), was previously shown to inhibit the synthesis of arabinans of both the cell wall arabinogalactan (AG) and lipoarabinomannan (LAM) of Mycobacterium tuberculosis and other mycobacteria. However, an Emb-resistant mutant, isolated by consecutive passage of the Mycobacterium smegmatis parent strain in media containing increasing concentrations of Emb, while synthesizing a normal version of AG, produced truncated forms of LAM when maintained on 10 μg/ml Emb (Mikušová, K., Slayden, R. A., Besra, G. S., and Brennan, P. J. (1995) Antimicrob. Agents Chemother. 39, 2482-2489). We have now isolated and characterized the truncated LAMs made by both the resistant mutant and a recombinant strain transfected with a plasmid containing the emb region from Mycobacterium avium which encodes for Emb resistance. By chemical analysis, endoarabinanase digestion, high pH anion exchange chromatography, and mass spectrometry analyses, truncation was demonstrated as primarily a consequence of selective and partial inhibition of the synthesis of the linear arabinan terminal motif, which constitutes a substantial portion of the arabinan termini in LAM but not of AG. However, at higher concentrations, Emb also affected the general biosynthesis of arabinan destined for both AG and LAM, resulting in severely truncated LAM as well as AG with a reduced Ara:Gal ratio. The results suggested that Emb exerts its antimycobacterial effect by inhibiting an array of arabinosyltransferases involved in the biosynthesis of arabinans unique to the mycobacterial cell wall. It was further concluded that the uniquely branched terminal Ara6 motif common to both AG and LAM is an essential structural entity for a functional cell wall and, consequently, that the biosynthetic machinery responsible for its synthesis is the effective target of Emb in its role as a potent anti-tuberculosis drug.

Structural characterization of the N-glycans from Echinococcus granulosus hydatid cyst membrane and protoscoleces

Infection by the tapeworm Echinococcus granulosus in the intermediate host results in the development of a hydatid cyst which contains the protoscoleces within a fluid-filled cavity enclosed by the bilayered cyst membrane. N-glycans were enzymatically released from crude extracts of homogenates of hydatid cyst membranes and protoscoleces and their structures were defined by high sensitivity fast atom bombardment mass spectrometry in conjunction with sequential exoglycosidase digestions. The major N-glycans from the cyst membrane were found to be non-charged structures having complex-type antennae and core fucosylation. The antennae are either truncated at the first N-acetylglucosamine or are extended with beta-galactose to form N-acetyllactosamine (lacNAc). A significant proportion of the lacNAc backbones are capped by alpha-galactose. The resulting Gal alpha-Gal beta-terminal structures may account for the earlier observation that antibodies against the blood group P1 epitope recognise components of hydatid cyst extracts. The complex-type N-glycans identified in the protoscoleces extracts were the same as the neutral structures found in the cyst membrane but a small proportion of high mannose structures and truncated di- and trimannosyl core structures were also identified. Sialylated N-glycans were identified as minor constituents of the cyst membrane preparation but were not observed in protoscoleces extracts. Whether the sialylated glycans are host derived or endogenously synthesized by the parasite remains to be established. This is the first reported structural analysis of N-glycans from cestodes and provides new insights into protein glycosylation in helminths.

ALTERED EXPRESSION PROFILE OF THE SURFACE GLYCOPEPTIDOLIPIDS IN DRUG-RESISTANT CLINICAL ISOLATES OF MYCOBACTERIUM AVIUM COMPLEX

Members of the Mycobacterium aviumcomplex are the most frequently encountered opportunistic bacterial pathogens among patients in the advanced stage of AIDS. Two clinical isolates of the same strain, numbers 397 and 417, were obtained from an AIDS patient with disseminated M. avium complex infection before and after treatment with a regimen of clarithromycin and ethambutol. To identify the biochemical consequence of drug treatment, the expression and chemical composition of their major cell wall constituents, the arabinogalactan, lipoarabinomannan, and the surface glycopeptidolipids (GPL), were critically examined. Through thin layer chromatography, mass spectrometry, and chemical analysis, it was found that the GPL expression profiles differ significantly in that several apolar GPLs were overexpressed in the clinically resistant 417 isolate at the expense of the serotype 1 polar GPL, which was the single predominant band in the ethambutol-susceptible 397 isolate. Thus, instead of additional rhamnosylation on the 6-deoxytalose (6-dTal) appendage to give the serotype 1-specific disaccharide hapten, the accumulation of this nonextended apolar GPL probably provided more precursor substrate available for further nonsaccharide substitutions including a higher degree of O-methylation to give 3-O-Me-6-dTal and the unusual 4-O-sulfation on 6-dTal. Further data showed that this alteration effectively neutralized ethambutol, which is known to inhibit arabinan synthesis. Thus, in contrast with derived Emb-resistant mutants ofMycobacterium smegmatis or Mycobacterium tuberculosis, which are devoid of a surface GPL layer, the lipoarabinomannan from resistant 417 isolate grown in the presence of this drug was not apparently truncated.

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides.

Derivatisation/FABMS strategies applicable to the structure analysis of low microgram quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitrous acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.

New pyruvylated, glycosylated acyltrehaloses from Mycobacterium smegmatis strains, and their implications for phage resistance in mycobacteria

Phage resistance and apparent lysogenization of Mycobacterium smegmatis due to infection with mycobacteriophage D29 results in the emergence of new variations of the pyruvylated, acylated trehaloses described by Saadat and Ballou, J. Biol. Chem. 258 (1983) 1813-1818. Thin-layer chromatography of the glycolipids from two strains of phage-resistant M. smegmatis (mc(2)22 and mc(2)11) and comparison with those from phage-sensitive strains revealed a new, more mobile glycolipid in each case. The structures of these acyltrehalose-containing lipooligosaccharides were elucidated by a combination of gas-liquid chromatography-mass spectrometry, methylation analysis, 1H and 13C NMR spectroscopy, and fast-atom bombardment mass spectrometry. The glycolipid from M. smegmatis mc(2)22 is beta-D-Glcp-(1-->3)-4,6-O-(1-methoxycarbonylethylidene)-beta-D-Glc p-(1-->4)- beta-D-Glcp-(1-->6)-2-O-acyl-alpha-D-Glcp-(<==>1)-3,4-O-acyl-alpha-D-Glc p and that from M. smegmatis mc(2)11 is 4,6-O-(1-methoxycarbonylethylidene)-3-O-Me-beta-D-Glcp-(1-->3)-4,6 -O- (1-methoxycarbonylethylidene)-beta-D-Glcp-(1-->4)-beta-D-Glcp-(1-- >6)-2-O-acyl- alpha-D-Glcp-(1<==>1)-3,4-di-O-acyl-alpha-D-Glcp. These differ from the original pyruvylated glycolipids of Saadat and Ballou in the extent of their O-acylation and O-methylation. The findings are the first example of the definition of a chemical basis for phage resistance and presumed lysogeny in mycobacteria, and show parallels to related changes in gram-negative enteric bacteria.

Characterization of the specific antigenicity of representatives of M.senegalense and related bacteria

Representative strains of M. senegalense and an unusual strain, labelled M. farcinogenes (M280) were examined by thin-layer chromatography for the presence of characteristic surface glycolipids. In the case of M. farcinogenes M280 and M. senegalense M264, the glycolipids were of the alkali-labile acyltrehalose lipooligosaccharide (LOS) class of antigens, whereas M. senegalense M263 was found to contain the alkali-stable glycopeptidolipids (GPL). Through a combination of 1H-NMR, methylation analysis, FAB/MS, and other analytical techniques, the structures of these glycolipids were deduced. The LOS glycolipids were found to be similar in structure to the characteristic glucosyltrehalose-based glycolipids isolated previously from clinical isolates of M. fortuitum, but distinct from the diacyltrehaloses characteristic of the type strain of M fortuitum. The glycopeptidolipids from M. senegalense M263 were closely similar to those characterized previously from M.

Covalent structure determination of glycopolymers

Abstract The concerted application of high-resolution chromatographic procedures, together with a variety of powerful structural methods including chemical and enzymic degradation, mass spectrometry and NMR spectroscopy, has enabled the characterization of a wide variety of novel glycans. Structural research reviewed herein includes investigations of glycopolymers isolated from bacteria, insects, helminths, protozoa and higher animals.

S19.6 FAB-MS sequencing of mycobacterial glycolipid antigens

spectrometry to establish basic features of the LOS populations, such as the number of components present, their molecular weights, and their structural homology. Mild acid hydrolysis of the Hib A2 LOS mixture, which contained eleven components, produced free oligosaccharides which were analyzed by chemical methods, liquid secondary ion and tandem mass spectrometry, and 2D NMR spectroscopy. These studies established that the oligosaccharides were triantennary structures containing a heptose trisaccharide core with primarily glucose residues as branch sugars. Structures with nonreducing terminal Gall~4GlcNAc were minor components in the oligosaccharide fraction which were found to be quantitatively sialylated in the O-deacylated LOS mixture. These data support the view that presenting a complex profile of LOS surface structures offers Hib added flexibility in evading host defense mechanisms. This work was supported by grants from NIAID (AI21620 and AI24616) and NIH (RRO1614).

S9.14 Precise structural determination of unique highly branched multiantennaryN-glycan units present in fish egg hyosophorin

Electrospray mass spectrometry (ES-MS) has recently emerged as a new powerful technique for analysis of high molecular weight biopolymers (>100.000 Dal) at a subnanomolar level. The multiple charged molecules can be structurally attributed according to their state of charge. We used ES-MS for structural studies on glycopeptides. A superiority over well established FAB-MS methods was found in higher sensitivity as well as in ability to induce specific fragmentation of the carbohydrate portion. This allowed assignment of carbohydrate sequence and the binding site to the peptide portion. Using this approach, it is possible to avoid chemical derivatizations, which may influence alkali-labile groups, found frequently on carbohydrate chains. The positive and negative ion ES-MS was applied to analyse sialic acid containing O-glycopeptides from urine of patients suffering from inherited deficiency of lysosomal a-N-acetylgalactosaminidase. Beside the major species, Neu5Aca2-3Gal/31-(NeuAca2-6)3GalNAc-Ser(Thr), a number of mono-, diand trisialylglycans were structurally characterized in fractions, obtained after several chromatographic steps, including gel permeation and anion exchange chromatography. After appropriate Nand/or O-derivatizations, the samples were also analysed by FAB-MS for sequencing of sugar chains and by methylation analysis for linkage sites.