The Glycolipids of Mycobacteria - ACS Publications - American

Chapter 14

The Glycolipids of Mycobacteria

Downloaded by UNIV MASSACHUSETTS AMHERST on July 29, 2012 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0541.ch014

G. S. Besra and P. J. Brennan Department of Microbiology, Colorado State University, Fort Collins, CO 80523

Research over the past 15 years has shown that members of the Mycobacterium genus express on their surfaces large quantities of glycolipids of highly unusual structures. Members of the Mycobacterium avium complex, important opportunistic pathogens, are characterized by glycopeptidolipids, and serotypic variation in this complex is due to variations in the composition of the short oligosaccharide haptens of the glycopeptide lipid antigens. Other atypical mycobacteria (environmental; sometimes pathogenic) are marked by the lipooligosaccharides, i.e., oligosaccharides based on an acylated trehalose core. Another major group of glycolipids, found to best effect in Mycobacterium leprae, are the phenolic glycolipids based on a phenolphthiocerol. In this review, emphasis is placed on structure, especially glycosyl composition and sequence.

The resurgence of interest in mycobacteria and their constituents stems from the resurgence of tuberculosis itself (1) and from long-time fascination with leprosy. Similarly, the involvement of "atypical," nontuberculous environmental mycobacteria, such as the members of the Mycobacterium avium complex, as opportunistic pathogens in patients with underlying immune dysfunctions (2) has mitigated interest in the bacteria themselves and also in their peculiar immunopathogenesis. Most, if not all, of the nontuberculous mycobacterial species are endowed with large quantities of a variety of glycolipid antigens with remarkable structural features (J). These glycolipids, and the more complex lipopolysaccharides that accompany them, have been implicated in diverse aspects of disease pathogenesis, such as interaction of bacterium and macrophage and persistence of bacteria within the intracellular environment (4). The variable oligosaccharide constituents of these glycolipid antigens are usually of sufficient

0097-6156/94/0541-0203$08.50/0 © 1994 American Chemical Society In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

204

MASS SPECTROMETRY FOR CHARACTERIZATION OF MICROORGANISMS

antigenicity as to evoke corresponding specific antibodies and thereby allow serodiagnosis of individual mycobacterioses (3), and, in the case of leprosy, a good means for specific serodiagnosis (5). Three major classes of glycolipids with the generic structures shown in Figure 1 have been identified, the trehalosecontaining lipooligosaccharides (LOS) (6, 7), the glycopeptidolipids (GPL) (8), and the phenolic glycolipids (PGL) (3) (Figure 1).


The Characteristic Lipooligosaccharide Antigens of Mycobacterium spp. (Table I) The acyltrehalose-containing lipooligosaccharides (LOS) are the predominant surface immunogens of a host of environmental mycobacteria and represent a rare principle in carbohydrate chemistry, glycosidically linked trehalose. The acyltrehalose residue invariably occupies the putative reducing terminus, whereas the non-reducing end is variable, being responsible for immunological specificity, and is characteristic of each individual species. First members of this class were reported by Saadat and Ballou (P) who called substances in M. smegmatis pyruvylated glycolipids since they contain pyruvate ketal units. Initially, Saadat and Ballou were seeking biosynthetic precursors of the 6-0-methylglucose lipopolysaccharide and had isolated from cell extracts of M. smegmatis several acidic methylglucose-containing glycolipids. The acidic property was found to be due to the presence of a pyruvate group, rather than the glycerate that characterizes the methylglucose-containing lipopolysaccharide. More recently, a pyruvylated LOS glycolipid was isolated from Mycobacterium butyricum (10) which differed solely by the extent of acylation of the oligosaccharide core as compared to the acidic oligosaccharide A derived from the pyruvylated glycolipid of Saadat and Ballou (9) (Table I). Schaefer had reported that the surface antigens of Mycobacterium kansasii involved in specific agglutination were soluble in organic solvents, and their antigenic properties, unlike those from M. avium, were susceptible to mild alkali treatment (77). Hunter et al (6) demonstrated that the serologically active lipids of Af. kansasii, previously described by Schaefer, in fact, belonged to the new class of glycosylated acylated trehaloses. The core structure of eight of these glycolipids from M. kansasii (Figure 2) were revealed by a combination of acetolysis, partial acid cleavage, *H- and C - N M R spectroscopy, chemical ionization and ΕΙ/MS of permethylated products (6, 7, 12). They are composed of variable residues of xylose, 3-O-methylrhamnose, fucose, and a novel N-acylamino sugar linked to a common tetraglucose core, distinguished by the presence of a glycosidically linked α,α'-trehalose substituent. The novel N-acyl amino sugar was later shown to be 4,6-dideoxy-2-0-CH -3-CH -4-(2'methoxypropionamido)-a-L-mannohexopyranosyl (72), and when the terminal, non-reducing end N-acylkansoaminyl(l->3)fucosyl disaccharide appeared on the more polar glycolipids (LOS IV-VIII), antigenicity, and precise anti-M. kansasii antigenicity at that, was conferred on these molecules. Thus, this unit is the unique antigenic epitope of Af. kansasii, and the amino sugar N-acylkanosamine is the single most characteristic feature of the organism. Three molecules of 2,413

3

3

In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

14. BESRA & BRENNAN


205


L I P O O L I G O S A C C H A R I D E S (LOS)

OLIGOSACCHARIDE^

GLYCOPEPTIDOLIPIDS (GPL) C H e

5

CH2

CH3

R - NH - C H - C O - NH - C H " C O " NH - CH -< 1 HC-CHo 1 OLIGOSACCHARIDE Ο 3

OCH

3

P H E N O L I C G L Y C O L I P I D S (PGL)

OLIGOSACCHARIDE

O — ^

^ — ( C H

2

)

1

8

OR

OR

ι

ι

- C H - C H

2

R = L O N G CHAIN FATTY

OCH3

- C H - (CH ) 2

ι

-CH-CH

4

- C

2

ACYL H

Figure 1. Generic structures of the three major classes of "new glycolipids of Mycobacterium spp. In the case of the lipooligosaccharide class, the point of attachment of the oligosaccharide unit may also be the 4- or the 6-OH group of the acyltrehalose unit.


H

5

In Mass Spectrometry for the Characterization of Microorganisms; Fenselau, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993. ND ND

ND

p-O-Xy\p-(1^4)-3-0-Me-a-L-ltoap-(l-+3yp-O-G\cp(1— 3)- β - D -Glcρ -(1— 4)-a- D-Glcp -(l«-*l)-a-D -Glcp

( β-D-Xylp ) -(l—4)-3-0-Me-a- L -Rhap -(1—3)- β -D -Glcp (l-*3)^-D-Glcp-(l— 4)-a-D-Glcp -(Wl)-a-D-Glcp

Kantfacyl-(1— 3)-Fuc-(l—4)-( β - D -Xylp ) -3-O-Me- a- L -Rhaρ (1— 3)- β - D-Glcp -(1— 3)- β - D-Glcp -(1-* 4)-a-D -Glcp (Wl)-a-D-Glcp

LOS I

LOS II, III

LOS IV, V, VI

LOS VII, VIII

LOS II

LOS-I

M. kansasii

M. kansasii

M. kansasii

M. kansasii

M. malmoense

M. szulgai

3-,4-, and 6-hydroxyls of terminal 2-0-Me-Glcp unit of terminal monoO-Me-trehalose

a-L-2-0-Me-Fucp-(l-*3)-a-L-Rhap-(l—3)-β-L-Rhap-(1—3)$-O-G\cp-(1^6)-a-O-G\cp-(l*+l)-a-O-2-0-Me-G\cp

13

14

3-,4-, and 6-hydroxyls of terminal Glcp unit of terminal trehalose

a - D-Manp -(l-*3)-a-D -Manp -(1-* 2)-a- L-Rhap -(l-*2)- [a- L 3-0-Me-Rhap-(l -*2)] - α - L-Rhap -(l-^3)-a-D-Glcp -(1 —l)-aD-Glcp 2

6,7,12

6

3-,4-, and 6-hydroxyls of terminal Glcp unit of terminal trehalose

Ref.

KanWacyl-(l—3)-Fucp -(l-*4)-[p-L-Xylp -(1—4)] -a- L-3-0Me-Rhap-(l-O)- $-O-G\cp-(l-+ 3)-β-D-Glcp (1—4)-a-DGlcp -(R*l)- a- D-Glcp

4

ND

3-0-Me-a- L -Rhap -(1— 3)- β-D -Glcp -(1— 3)- β -D -Glcp (l-*4)-a-D-Glcp -(Wl)-a-D-Glcp

LOS I'

Af. kansasii

2

ND

4,6-(l' -carboxyethylidene)-3-0-Me- β-D-Glcp -(l-*3)- β -D Glcp -(l-*4)- β - D-Glcp -(1-*6)-α- D-Glcp -(1—1)- a- D-Glcp

(Acidic oligosaccharide B2)

M. smegmatis

4 and 6-hydroxyls of terminal trehalose

Positions of Acvl Residues

ND"

(Acidic oligosaccharide Β ι )

Af. smegmatis

4,6-(l' -carboxyethylidene)-3-0-Me- β -D -Glcp -(1-* 3)4,6-(l'-(»rboxyethylidene)-β-D-Glcp -(1-* 4)-β-D-Glcp (1-* 6)-a - D-Glcp -(1*-* l)-a-D-Glcp

Structure of Oligosaccharide

4,6-(l' -carboxyethylidene)- β -D -Glcp -(l-*4)- β -D -Glcp (1^6)-a-D-Glcp-(l«*l)-ct-D-Glcp

(Acidic oligosaccharide A)

Af. smegmatis

Trivial Name

Structures of Major Trehalose Containing Lipooligosaccharides of Mycobacterium

Species

Tabic I



LOS-]

Af. gordonae 990

2

2,3,4 and 6-hydroxyls of terminal trehalose

N-acy l-4-amino-4,6-dideoxy-2,3-0-Me- α -Galρ -(1 —3)-2- O-Me-40-Ac-a-L-Fucp-(l— 3)^-D-Glcp-(l—S^-O-Me-a-LRhap-(1—3)-[β-ο-Χγ1ρ-(1—2)]-a-L-Rhap-0^ (1—3)-a-L -Rhap-(1— 3)-6-0-Me-a-D-Glcp -(l+*l)-a-D-Glcp

LOS-I

Af. gordonae 989

ND = Not determined

fl

2 and 3 hydroxyls of terminal trehalose

a-D-Glcp-(l-*l)-a-D-Glcp

Af. tuberculosis ΌΑΎ , DAT H37Rv

Χ

2' and 2,3,6-hydroxyls of terminal trehalose

β-D-Glcp -(l-*6)-Q-n-Glcp -(!«·-* I )-a-D-Glcp

Af. fortuitum biovar fortuitum

a - L -Rhap -(1— 2)-3-0-Me-a-L -Rhap -(1—3)-[β-θ-Χγ1ρ -(1—2)] - 2,3,4 and 6-hydroxyls of α - L -Rhap -(1-*3)-β -D -Glcp -(1—3)- β-D-Glcp -(1—3)-a- L -Rhap - terminal trehalose (1— 3)-6-0-Me-a-D -Glcp -(Wl)-a -D -Glcp

2,3,6 and 3,4,6-hydroxyls of terminal trehalose in the proportions of 2:3

2' and 3,4-hydroxyls of 4,6-(methyl l'-carboxyethylideneJ-S-O-Me-p-D-Glcp-il—3)-4,6(methy 1 l' -carboxyelhy 1 idcne)- β - D -G lc/> -( 1 — 4)- β - D -G lcp -( 1—6) terminal trehalose -a-D-Glcp-(l«-*l)-a-D-Glcp

3,4, and 6-hydroxyls of terminal Glcp unit of terminal trehalose

JV-acyl-4-amino-4,6-dideoxy-Galp -(l-*4)-2-0-Me-a-L -Fucp (1—3)- β-ϋ-01ορ-(1—3)-2-0-Me-a- L-Rhap-(1—3)-2-0-Mea-L-Rhap-(l—3)-β-D-Glcp-(l->3)-4-0-Me-a-L-Rhap-(1—3)6-O-Me-a-D -Glcp -(WI)- a-D -Glcp

LOS-I

Af. butyricum

P-D-Glcp-(1—3)-a-L-Rhap-(l—3)-a-D-Glcp-(K* l)-a-D-Glcp

Af. tuberculosis LOS-I Canetti

LOS

Af. "linda"


MASS SPECTROMETRY FOR CHARACTERIZATION OF MICROORGANISMS


208

Figure 2. Thin-layer chromatogram of the individual purified LOS I-VIII from M. kansasii. Solvent, C H C 1 - C H 0 H - H 0 (10:5:1). A l l plates were sprayed with 10% H S 0 and heated. 3

2

3

2

4



14. BESRA & BRENNAN


209

dimethyltetradecanoic acid esterified the hydroxyl groups at C-3, C-4, and C-6 of the terminal glucosyl residue (Table I). The species-specific glycolipids of M. szulgai which were also recognized as members of the LOS class required special hydrolytic conditions, combined with GC/MS for full structural elucidation, and this allowed the recognition of a 2-0-methyl-a-D-glucopyranosyl unit, which, in turn, led to the recognition of the presence of glycosidically linked 2-0-methyltrehalose (13). The complete structure of the oligosaccharide from the simplest member of the LOS family in M. szulgai has been elucidated (Table I). M. malmoense, an atypical mycobacterium implicated in pulmonary infections, was also found to contain species-specific LOS glycolipids (14). De0-acylation, followed by partial acidic cleavage to release the glycosidicallylinked trehalose, α-mannosidase, 0-deuteriomethylation, partial acid hydrolysis, reduction with NaB H4, and Oethylation, combined with ^ - N M R , G C / M S and F A B / M S , revealed the structure of the major oligosaccharide as a-D-Manp-(l->3)a-D-Man/Kl[2)-a-L-Rhap-(-W^ which two of the 2-a-L-Rhap residues are O-methylated at C-3 (Table I). The distinct chromatographic mobility of the major LOS allowed one to distinguish M. malmoense sensu stricto from other closely, related, clinically significant mycobacteria. Mycobacterium tuberculosis (strain Canetti) was found to be characterized by the presence of two novel LOS glycolipids (15). The structure of the major glycolipid was established (Table I) and was found to be unique in that the trehalose substituent was methylated at the opposition. In addition, unlike other well characterized members of the LOS family, which are invariably acylated at the 3,4, and 6 (or 4' and 6) positions, the product from Af. tuberculosis Canetti consists of a mixture of 2,3,6- and 3,4,6-tri-O-acylated oligosaccharides. Two clinical isolates of Mycobacterium gordonae were also found to contain a novel series of glycolipids based on the alkali-labile LOS. Inherent features of interest included a novel branching of the oligosaccharide backbone in the form of a single residue of terminal (t)-j8-r>Xylp, (t)-j8-D-3-0-CH,-Xylp, or (t)-a-D-Ara£ and an incompletely defined N-acyl derivative of 4- amino-4,6dideoxy-2,3-0-CH -galactopyranose residue in some members. Also of interest was a novel mono-6'-0-CH -2,3,4,6-tetra-0-acyl-glucose unit, representing the first example of such a unit in the LOS family of glycolipids from mycobacteria (i3)-α- L-Rhap -(1-*·3)-α- L-Rhap -(l-»-2)-6deoxy- L-Tal

3,4-di-O-Me-a- L-Rhap -(l-*2)-3,4-di-0-Me-a- L-Rhap or

M. senegalense M263

a- L-Rhap-(l-*3>-a- L-Rhap 4-0-Ac-2^-di-0-Me-a- L-Fucp -

The Glycolipids of Mycobacteria - ACS Publications - American

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