the carbohydrate- and lipid-containing cell wall of mycobacteria, phenolic glycolipids: structure...

Microbiology

The Carbohydrate- and Lipid- Containing Cell Wall of Mycobacteria, Phenolic Glycolipids: Structure and Immunological Properties

Germain Puzo

ABSTRACT

Phenolic glycolipids were first discovered as cell-wall constituents of M . bovis. M . bovis BCG, M . rnarinum, and M . kansasii. Recently, such compounds were also isolated from M . leprae and have been shown to be specific-species serological markers. Moreover, they seem to be involved, in the case of lepromatous leprosy, in the stimulation of the suppressor T-cells. The functional activities of these phenolic glycolipids over the immune cells stimulation emphasized the role played by these molecules in the mycobacteria pathogenicity. Phenolic glycolipids have also been found in M . gasrri and M . tuberculosis strain Canefti. From a stmctural point of view, these glycolipids contain the same aglycon moiety mainly assigned to phenolphthiocerol diester while the sugar part structure confers to some of these glycolipids their antigenic specificity. The search of immunoreactive glycolipids and their function analysis remain a challenge for chemists and immu- nologists for the understanding of the mycobacteria pathogenicity.

1. INTRODUCTION

Certain members of the mycobacteria genus such as M . tuberculosis and M . leprae are the etiologic agents of important diseases in humans and animals. Tuberculosis and leprosy still remain major health problems especially in the developing countries.

The association of atypical pathogen mycobacteria with the human immunodeficiency virus (HW) in acquired immunodeficiency syndrome patients is changing the epidemiology picture of the tuberculosis in developed countries and thus might explain the recent increase in the Occurrence of tuberculosis. In spite of BCG vaccinations and multidrug chemotherapy, it now clearly appears that tuberculosis has not yet been eradicated in the developed countries. We believe that the structural identification of immunoreactive epitopes in- cluding specific probes and “protective antigens” could be in the future one key for the eradication of tuberculosis.

The purpose of this article is to focus on recent developments of the structural characterization and immunological properties of individual mycobacterial lipidic antigens components in an approach to understand the host immune response

to mycobacterial infection at the molecular level. Although this strategy is not new, the recent developments in purification and analytical (Mass Spectrometry, Nuclear Magnetic Reso- nance) and immunological techniques have allowed significant advances in the knowledge of the molecular entities involved in the host immune response to mycobacterial infections. For instance, the identification of species-specific B cell epitopes has led to important progress in the mycobacterial infection diagnosis. The challenge posed by the characterization of the molecular entities involved in T-cell epitopes and in resistance to the intramacrophagic environment has caused chemists, im- munologists, clinicians, and molecular biologists to show a revived interest in mycobacteria.

It can be assumed that molecular components on the mycobacterial cell surface play a key role in the host immune response. The mycobacterial cell wall is mainly composed of glycolipids, most of which are restricted to the genus mycobacteria. In the area of bacterial lipids, the term “glycolipids” designates the lipid fraction which contains carbohydrate and remains soluble in organic solvents. The chemical linkage between lipid and sugar can either be glycosidic, ester, or amido. Historically, phenolic glycolipids and peptidoglycolipids were first characterized by Smith et al.l-3 and defined as “glycolipids or glycolipid peptides limited in distribution to a single species of mycobacteria”. However, this definition is no longer re- strictive but these substances are not ubiquitous in the mycobacteria genus. Mycobacterial glycolipids have been recently reviewed in excellent monographs by B r e ~ a n , ~ . ’ Gaylord and Brennan,6 and Young.7

11. WORLD STATUS OF HUMAN MYCOBACTERIOSIS DISEASES

M . tuberculosi? and M . l e p r ~ e , ~ the etiologic agents of tuberculosis and leprosy, respectively, are the best known members of the mycobacterial genus. In 1882, the tubercle bacilli was discovered by Koch10 and that of leprosy was identified by Hansen in 1873.” People began to “dream” that recognition of the causative agent would very quickly lead to methods of the disease eradication. Later the discovery of ef- ficient mycobacterial drugs offered a similar promise for the eradication of mycobacteriosis. 12-“ However, after a century of investigations, tuberculosis and leprosy remain a major human health problem particularly in developing countries. It has been estimated by the World Health Organization that every

~~ ~

G. Puzo earned his Ph.D. at the University of Paul Sabatier. Toulousc. France. Presently, Dr. Puzo is Director of Research at the Center of Biochemical and Genetic Research, Centre National de la Recherche Scientifique, Toulouse, France.

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year, over the world, about 10 million people develop tuberculosis and at least 3 million die from the disease.I6.l7 The same organization estimated the presence of 10 to 12 million leprosy patients of whom about 1 to 3 million have physical

In 1986, Marius Jahely, a French leper in the Pondichery leper hospital testified the horror caused by leprosy: “Mon corps est casse, j’ai tout perdu. Je n’ai plus de doigts, je n’ai plus de jambe gauche et de pied droit, la maladie a ronge mon nez, mon poumon, et finalement mes yeux. Quand j’ai perdu mes doigts et ma vue, j’etais A penser beaucoup de tristesse. Ma malchance c’est de vivre comme un cadavre vi- vant dans ce monde.”

In developed countries, leprosy can be considered as being eradicated. However, it is disconcerting that tuberculosis remains a public health hazard (level in U . S . : 11.9 cases per 100,000 population per year in 1983).16 Moreover, the epidemiology of mycobacteriosis has recently changed consid- erably.” The incidence of pulmonary diseases caused by M . tuberculosis has steadly declined while atypical tuberculosis caused by mycobacteria other than tuberculosis (MOTT) also called nontuberculous mycobacteria (NTM) or atypical mycobacteria have become increasingly problematic. 20-23 Chronic pulmonary disease resembling tuberculosis associated with nontuberculous mycobacteria represents an important clinical problem (see Table 1). The M . aviurn complex (MAC) and M . kansasii are the most frequently encountered NTM etiologic agents of atypical tuberculosis. The other ones are M. xenopi, M . szulgai, M . simiae, M . forruiturn, and M . ~ k e l o n a e . ~ ~ Pa- tients who develop atypical tuberculosis are epidemiologically distinct from tuberculosis patients. They are older, more often white and often have underlying chronic lung disease such as bronchiectasis or silicosis. 15 This epidemiology picture is changing since now an important category of patients are im- munodepressed. These are kidney transplanted,26 cancer,27 and more recently AIDS patient^.^^-^'

The association of nontuberculous mycobacteria, mainly M . avium complex”.32 and some others such as M . kansasii or M . ~ e n o p i , ’ ~ . ~ ~ with the HIV constitutes a large proportion of atypical tuberculosis (39%).’9 In 1986, the Center for Dis- ease Control (CDC) in the U.S. reported that 2.2% of AIDS patients also had tuberculosis. Thus an association between AIDS and tuberculosis is now clearly recognized, and its mag- nitude varies with location and ethnic group. This might explain that the number of reported cases of tuberculosis for 1986 in the U . S . has gone up for the first time since 1953. This situation could in the future constitute a much greater public health risk. The clinical presentation of tuberculosis in AIDS is often different to tuberculosis without the HIV association since in the majority of cases it is extrapulmonary, increasing the difficulty of its diagnosis.

It can be assumed that it is the tropism of HIV for the CD4 + T helper cells which underlies the mechanism of reactivation or increased susceptibility to mycobacterial infection. This as-

Table 1 Incidence Rates of All Nontuberculous Lung Mycobacteriosis, Lung Disease Caused by Mycobacterium avium Complex and Lung Disease Caused by Mycobacterium kansasii per 100,000 Population Per Year

Population Case NTM Mycobacterial isolates

Japan (1983) 1.73’ 70 25 (1976) 1 99 0

U . S . (1983) 1.78b 62 24

Data from 1981 to 1984. number of cases per 1OO.ooO pnp- ulation per year. U.S. Centers for Disease Control, period 1981 to 1983

sumption is supported by the fact that the incidence of tuberculosis among AIDS patients appears to increase with the number of tuberculous patients recorded locally (association of M . tuberculosis in AIDS Haitian patient^^^.)^). Nevertheless the prominence of the M . avium complex in association with HIV compared to M . tuberculosis and M. kansasii suggests a hy- pothetical relationship between the M . avium complex and HIV.’* Investigation in this area will lead to a greater understanding of the immune defense against mycobacterial infections. This immune defense is composed of both cell-mediated and humoral immunities. This is best exemplified by the break- down of cell-mediated immunity in lepromatous leprosy and of humoral immunity in tuberculosis or tuberculoid leprosy.

The main aim in the investigation of mycobacteria is the total eradication of mycobacteriosis. It is now clear that the identification and characterization of T and B antigens is the key for the development of the following:

Immunodiagnosis, by the use of species-specific B-cell probes Preventive tools, by the design of molecular vaccines based on mycobacterial T cell epitopes important in protective cell-mediated immunity Immunopathology, by understanding the pathologic immune response to mycobacteria and their resistance to being killed by the immune system

In the first part of this review, we briefly summarize the structures of the glycolipids constitutive of the mycobacterial cell wall and their immunological properties. In the second part, we describe the species-specific glycolipids and particularly discuss the phenolic glycolipids in more depth.

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111. OVERVIEW OF STRUCTURE AND IMMUNOLOGICAL REACTIVITY OF SOME

MYCOBACTERIA CELL WALLS CARBOHYDRATE AND LIPID-CONTAINING

In the structural molecular analysis of mycobacterial cell walls, lipids and carbohydrates have fascinated research groups from 1950 diverting interest until recently from protein antigens. This particular interest for mycobacterial lipids results from early observations that M . tuberculosis virulence is notably attenuated by its delipidation suggesting a prominent role of the lipids in pathogenesis. Effectively, the Gram-positive mycobacterial cell walls contains up to 60% of lipids compared to some 20% for Gram-negative organisms.” Moreover, from experiments with isolated rnycobacterial cell walls, it is clear that many of the biological properties of mycobacteria such as adjuvancity , enhancement of nonspecific resistance mechanisms to infection and cutaneous tumors are due to the cell wall c o r n p ~ n e n t s ~ * - ~ (see Table 2 ) .

A. The Mycoloyl Arabinogalactan-Peptidoglycan Complex

This polymer constitutes the basal covalent layer which confers its rigidity to the bacterial cell wall. Its general structure was assigned to an arabinogalactan-mycolate complex cova- lently linked to the p e p t i d o g l y ~ a n ~ ~ ~ (Figure 1). More pre- cisely the peptidoglycan chains are formed by alternating N- acetyl-glucosamine and N-glycolylmuramic acids linked to a tetrapeptide hai in,^'.^^ connecting the glycan chains by inter- peptidic linkage. This structure appears common to the mycobacterial genus; however, the M . leprae peptidoglycan presents glycine instead of L-alanine at the amino terminal of the peptide chain.43

The arabinogalactans are esterified on the arabinose residues by mycolic acids, and are linked by the arabinose units, through a phosphodiester bridge, to the muramic acid residue .41.42 The arabinogalactans of various mycobacterial species

FIGURE 1 . Tentative structw of mycoloyl -arabinolactan- peptidoglycan. Myc: rnycolic acid csterifies the arabinogalactan probably at the arabinose side chain. DAP: diarninopimelic acid. (Adapted from Ledenr, E., Pure Appl . Chcm., 25. 135, 1971.)

have essentially similar structures defined as polymers of 5- linked arabinofuranose and either Clinked galactopyranose or 5-linked galactofuranose. The presence of P-galactofuranosyl residues was initially established by Vilkas et who isolated the disaccharide p-D-galf-(l - 6) -~ -ga l from the M. tuberculosis arabinogalactan. Recently, by an elegant and sophisticated chemical process, McNeil et al.46 have established that the M . leprae and M . tuberculosis arabinogalactans contain exclusively arabinofuranosyl and galactofuranosyl residues. Furthermore, they have determined that the galactofuranosyl residues are either 5-, 6-, or 5,6-linked while the arabinofuranosyl is only 5-linked.

This work suggests that some structural points must be clarified: the attachment of the arabinose- and galactose-containing regions and the phosphoric diester bridge. Since this complex probably constitutes the dominant mycobacterial antigen, its structure must be elucidated exactly.

The adjuvancity, which reflects the potency to unspecifically enhance the antibody response to antigens, represents a well-known property of mycobacterial cell walls which is mainly assigned to the arabinogalactan-peptidoglycan complex. Fur- ther analytical studies delineated N-glycosylmuramyl-L-alanyl-

Table 2 Immunological Properties of Some Mycobacterial Cell-Wall Glycolipids

Clycolipid type Immunological properties Assumed epitope

Lipoarabinornannan (LAM-B)’B

Liprnannan (LAM-A)”

Lipoarabinogalactan“ Peptidoglycan c o m p l ~ x ~ ~ Cord- factor”

sulfa tide^^ Mannophosphoinositidesa.@

Nonspecific immunosupprcs- sor,n 71 187 antigenic, high uter in sera of infected patients,79

a-( 1 + 5)-linked-D-araf

Nonanogenic

Antigenica2 Adjuvant” Muramyl dipcptide” (MDP) Immun~stirnulant~’ haptenic. induce

Haptcnic Antigenic" haptenic protecuve Undetermined

a-( 1 - S)-linkcd D-araf

granulomas toxicity in mice

~mmunity’~

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D-isoglutamine (muramyl dipeptide, MDP) (Figure 2) as the minimal common structure which confers the adjuvanP and antitumor properties to the peptidoglycan." Recent studies have shown that the reintroduction in MDP of branched fatty acids does restore all the immunological activity of the mycobacterial cell wall, such as. for instance, in the case of the granuloma induction in Guinea pigs."' The arabinogalactan is considered by Misaki et al.m as a common polysaccharide antigen of mycobacterial species. Its epitope contains the terminal 5-lir1ked arabinofuranose residue which is common to another mycobacterial cell wall polysaccharide: arabinomannan.sO

Hol

NHAC

I# D

I ( CH2),COOH

I CH3

I H3C- CH - C O - N H - C H - C O - N H - C H ~ C O ~ N H ~

0

FIGURE 2. L-danyt-D- isogtute .

Muramyt dipeptide (MDP) structure. N-acetyl-muramyl-

B. Lipoarabinomannan (LAM) The heteropolysaccharide (AM) formed by D-arabinofur-

anose and D-mannopyranose has been identified in earlier studies.s1-53 A tentative structure was proposed by Misaki et al.54 consisting of a backbone of a-( 1 + 6)-linked D-mannopyranose units with short side chains of a-(1 -P 2)-D-mannopyranose residues and a-( 1 ---+ 5)-linked D-arabinofuranose residues. Pal- mitic and tuberculostearic acids esterify the heteropolysac- har ride.^^"^ Weber and Gray demonstrated the presence, in the M . smegmatis LAM, beside the sugar residues Arafand Manp, of phosphate, succinyl, and lactate groups.57 Recently, Hunter et al.s8 have proposed a tentative structure for the LAM of M . tuberculosis and M . leprae (Figure 3 ) previously assigned to the 30- to 35-kDa glycoprotein of the leprosy bacillus. The key of its structural elucidation was its purification in the native form in detergent containing buffers by anion exchange and gel filtration chromatography. It appears homogeneous on PAGE and yields a broad diffuse band in the vicinity of proteins with molecular weight of 30 to 35 m a . This work supported the presence of lactate, succinate, and phosphate but also of inositol and glycerol. The most unexpected feature is the presence of glycosidically linked diacylglycerol residues which apparently arise in mannosylated phosphatidylinositols. Hunter et al.5* concluded that LAM may be an amphipatic multiglyco- sylated extension of the phosphomannoinositides. In spite of the sophisticated chemical methods and purification techniques

FIGURE 3. Tentative S ~ N C ~ U R of the M. leprae lipoarabinomannan

used, the exact structure of LAM has not yet been clearly determined since the tuberculostearic, palmitic, and succinic acids, which appear to be an integral part of LAM have not yet been located. This conclusion is supported by the absence of succinic acid and glycerol in the LAM isolated from M . paratuberculosis .lr9

Most mycobacteria contain, beside LAM, lipomannan 0 and AM. LAM are serologically active while the LM and AM are less or not active.s8 LAM appears as a dominant B-cell antigen of M . tuberculosis, M . leprae, and M . paratuberculosis. The M . leprae LAM reacts with antibodies of lepro- rnatous and tuberculoid leprosy patients. It is immunologically cross-reactive with related compounds from M . tuberculosis, but certain monoclonal antibodies raised against M . leprue bound the M . leprae LAM more efficiently than that of M . tuberculosis.'" Serological studies suggest that LAM is a mul- tiepitopic antigen whose immunodominant epitope can be assigned to the a-(I + 5)-linked D-arabinofuranosyl chain. Moreover, in a recent in vizro study M . leprae LAM appears to be a potent inhibitor of y-interferon-mediated activation of mouse macrophages .61 These observations suggest the conmbution of LAM to the defective activation of granuloma macro phage^.^^.^^

C. Phosphatidyl lnositol Mannosides, (PIMan) The early history of these substances was reviewed by

AsselineauU and Goren.M The structure of the least glycosy- lated (PIMan,) was established as 1-phosphatidyl-L-myo-inositol-2-0-a-~-mannopyranoside~~~' while (Figure 4) presents a second a-D-mannopyranosyl residue attached to the 6-hydroxyl group of the myo-inositol ring. PIMan, and PIMan, arise from PIMan, by the glycosylation of the mannose linked to the dhydroxyl group of the myo-inositol by one and two mannose residues in a linear a-(1 + 6 ) array. However, in PMan, (Figure 5 ) the mannotriose is terminated at the nonreducing end by a single a-( 1 --+ 2) linked mannose.- PIMan, was found esterified by two, three, and four molecules of palmitic and tuberculostearic acids, but the positions of the acyl groups which do not esterify the phosphatidic acids are still undetermined.

Mannophosphoinositides were found to induce both hu-


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OH

0 I ’r ‘OH

CH2 Q HC - 0 - C - 4

I

H26-O-Z-R, 0

R1 , R2 : mixture of palmitoyl and tuberculostearoyl fatty acids

FIGURE 4. PIMan, smcture

9

OH

p HC -O-C-Ra

H,C-O-C -R, I

0

R, , R 2 : mixture of palmitoyl and . tuberculostearoyl fatty acids

FIGURE 5. PlMan, smcture.

moral and cell mediated immune responses in mice when in- jected as a mannoside-methylated bovine serum albumin complex. Moreover, this complex induces protective immunity in rabbits against challenge with M . tuberculosis H,,Rv which is mediated by the cooperation of T- and B-~ells.~’ Antibodies against PIMan have also been detected in human i n f e c t i ~ n s . ~ ~ , ~ ~ Thus, these glycolipids are the basis of a useful enzyme-linked immunosorbent assay (ELISA) for the clinical diagnosis of mycobacterial in fe~t ions .~~

D. Cord Factor and Sulfatides The cord-factor structure (a-a-D-trehalose 6,6’-dimyco-

late) and its biological properties have been largely reviewed by Lede~er, ,~ by A~selineau,’~ by Goren and B~ennan,’~ and by Bekierkun~t.’~ The history of the sulfatide has been reviewed by Gored’ and by Goren and B~ennan.,~ As far as we know, at this date no decisive data concerning either the structure or immunological properties have been recently published. Thus, these topics would not be presented in this treatise.

IV. IMMUNOREACTIVE SPECIES-SPECIFIC G LY CO LI PI DS

The discovery of a new class of glycolipids called mycosides, their structural determination, and the elucidation of their immunological properties in the goal of identifying pathogenic mycobacteria by chemically defined antigens constitute a fascinating and exciting history (see reviews in References 4, 85, and 86).

It began in 1954 when Smith et al.’ associated the immunizing properties of fractions from tubercle bacilli with their infrared (IR) spectra. Later these fractions were shown to be specific lipids and their IR spectra led to the characterization of mycobacterial species.2 These lipids which, in 1960, were called mycosides3 were defined as “glycolipid or glycolipid peptides limited in distribution to a single species of mycobacteria”. However, these mycosides are now known to be produced by other species. The amino-acid devoid mycosides A, B, and G were isolated from M. kansasii, M . bovis,, and M . m ~ r i n u m , ~ ~ respectively, and belong to the phenolic glycolipid class. The glycolipid peptide or mycoside C was extracted from the M. avium complex, M . butyricurn, M . smegmaris, and other mycobacteria. At the present time, the immunoreactive species-specific glycolipids have been classified into three classes: the phenolic glycolipids, the C-mycoside glycopeptidolipids, or polar C-mycosides and the trehalose-containing lipooligosaccharides (LOS). Ln this section we briefly summarize the basic structures and biological properties of the glycopeptidolipids (GPL) or polar C-mycosides and the trehalose-containing lipooligosaccharides (LOS). The phenolic glycolipid antigens are reviewed with more em- phasis in the next section.

A. C-Mycoside Glycopeptidolipids (GPL) The historical background of the polar C-mycosides has

been carefully reviewed by Goren.88 By seroagglutination of whole cells, Schaefer subdivided the M . avium, intracellulare, scrofulaceum group (MAIS) into 31 distinct serotypes. Inde- pendently, Marks and Jenkins observed that MAIS serotypes are defined by specific g l y c o l i p i d ~ . ~ ~ ~ ~ Then, Brennan and Goren demonstrated that the Schaefer typing antigens and the Marks-Jenkins glycolipids were s y n o n y m ~ u s . ~ ’ . ~ These glycolipids were characterized by Brennan as polar C-mycosides.

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They assigned a specific structure to the polar C-mycosides of each serotype. These structures shared a common core which corresponds to C-mycosides (Figure 6), and they differ by their oligosaccharidic appendix, linked to the 6-deoxytalose, which is responsible for their antigenic subspecies specificity. These oligosaccharide appendage structures summarized in Table 3 present a common basal disaccharide a-r-Rhap-( 1 -+ 2)-6-d- Tal. Glycopeptidolipids are not exclusively confined to the MAIS group organisms; they are also observed in other mycobacterial species such as M . paratuberculosis, M. chelonae subsp. chelonae, M . fortuitum biovar peregrineurn, and M. simiae I , 11.

The GPL antigens represent major constituents of the out- ermost layer of mycobacteria in the M . aviurn complex suggesting that they play a role in the mycobacterial surviving within the host macrophage. By using GPL antigens radiola- beled either on the common core or in the oligosaccharide d e t e ~ m i n a n t , ~ . ~ ' it has been established that these compounds are relatively inert to macrophage degradation. Moreover, i t was found that GPLs inhibit the in vivo and in ritro proliferation of murine splenic mononuclear cells to nonspecific mitogens .06

It was established that macrophages are not a major contributor to the GPL immunosuppression property.

Both smooth and rough cultures of MAIS were observed.

OR 7 H2 y H3 7 H3 Q

I CH,-(CH,),-CH=CH --(CH~),-CH-CH~-CO-NH -CH-CO-NH-?H-CO -NH -CH-co -NH -CH-CH, -O-R2

CH-CH, I 0 I Ri

FIGURE 6. General swcture of C-mycosides R = CH,, H. n = 13. 14. 15. m = 7 lo 1 I . R , = 6-deoxytslosc or 3-0-Me-6-deoxytalose. R, = 3-4-di-0- Me-rhamnose or 3-0-Me-rhamnosc, and R , and R, can be partially 0-acetylated.

Table 3 Structure of the Oligosaccharide Appendices from the Glycopeptidolipids (GPL) of Mycobacteria

Mycobaderium strains Structure of the oligosaccharide Ref.

Avium complex 1 2 4 8 9

12

14

17

19

20 21 25

26 Paratuberculosis Chelonae bsp.

chelonae Fortuitum biovar

peregrineurn Sirniae 1. I1

a-L-Rhap( 1 -+ 2)-6-d-~-Tal 2.3-Di-O-Mc-a-~-Fucp-( 1 -+ 3)-~-Rhap-( I + 2)-6-d-~-Tal CO-Me-a-~.-Rhap-( I -. 4)-2-O-Me-a-~-Fucp-( 1 + 3)-a-~-Rhap-( I -+ 2)-6-d-~-TaI 4,&( 1 '-Carboxyethylidene)-3-O-Me-~-~-Glcp-( I -+ 3)-a-~-Rhap-( I -+ 2)-6-d-~-Tal 4-0-Ac-2.3-di-O-Me-a-~-Fucp-( I -+ 4)-P-D-GlcpA-( I -+ 4)-2.3-di-O-Me-a-~-Fucp-( I -+ 3)-a-~-Rhap-( I -+ 2)-6-d-

4-Lacrylamido-3-O-Me-4,6-dideoxy-P-Glc-( 1 -+ 3)-4-O-Me-a-~-Rhap-( 1 + 3)-a-~-Rhap-( 1-+3)-a-~-Rhap-( 1 -+ 2 ) -

4-Formamido-3,4,6-trideoxy-2-0-Me-3-C-Me-a-Man-( I -+ 3)-2-O-Me-a-~Rhap-( 1 -+ 3)-?-O-Mc-a-~-Fucp-( 1 -+

4-(2'-Mclhyl-3'-hydroxybutyramido)-4,6-dideoxy-Hex-( 1 ---t 3)-4-O-Me-a-~-Rhap-( I -+ 3)-a-~-Rhap-( 1 + 3 ) - a - ~ -

3,4-di-O-Me-B-~-GlcpA-( 1 -+ 3)-2,4-di-O-Me-3-C-Me-6-d-a-Hex-( I -+ 3)-a-~-Rhap-( 1 + 3)-a-~.-Rhap-( I + 1-)-6-

2-O-Me-a-pRhap( 1 -+ 3)-2-O-Me-a-Fucp-( 1 + 3)-a-~-Rhap-( 1 -+ 2)-6-d-~-Tal 4.6-( 1 '-Carboxycthylidcne)-~-pGlcp-( 1 ---L 3)-a-~-Rhap-( 1 -+ 2)-6-d-~-Tal 4-Acctamido-4.6-dideoxy-2-O-Mc-a-Hex-( I -+ 4)-P-~-GlcpA-( 1 -+ 4)-2-O-Me-a-~-Fucp-( 1 + 3)-a-~-Rhap-( 1 -+

2.4-di-O-Me-a-Fucp-( 1 -+ 4)-P-GlcpA-( 1 -* 4)-2-O-Me-a-~-Fucp-( 1 -+ 3)-a-~-Rhap-( 1 -+ 2)-6-d-~-Tal M. aviurn scrovar 2 structure 3.4-di-0-Me-Rhap - 6-d-L-Tal

L - T ~

6-d-~-Tal

3)-a-~-Rhap-( 1 + 2)-6-d-~-Tal

Rhap-( I -+ 2)-6-d-L-Tal

d-L-Tal

2)-&d-~-Tal

Undetermined

Undetermined

97 98 99 100 101

97

102

97

103

104,91 97 101

97 105 106

106

106

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Recent studies by Brennan et al. , 4 report the absence of GPL in rough strains, and their disappearance during the smooth to rough morphology transformation of M. srnegrnatis.

The immunochemical analysis of the GPL allowed the serotyping by ELISA of M . avium isolates and provided better tools for the identification of these infections. The association of some particular M. avium serotypes (4 and 8) with HIV has revived the interest for M. aviurn complex immunochemistry.

6. Trehalose-Containing Lipooligosaccharides (LOS)

In pioneering studies, Schaefer reported that the M. kansasii surface antigens involved in specific agglutination were soluble in organic solvents. lo' Their antigenic properties were abolished by mild alkali treatment. Independently, Szulga and Markslog observed that M. kansasii specific lipids give a black color in their reaction to orcinol-H,SO, while the glycopeptidolipids show a characteristic yellow color. Brennan et al. demonstrated that the serologically active Marks-Jenluns lipids of M. kansasii are synonymous to those of Schaefer. They belong to the trehalose-containing LOS class. The M. kansasii LOS structures were established by Brennan et al.4 They are composed of variable residues of xylose, 3-O-Me-Rhap and Fucp which are linked to a common tetraglucose core.'09 Only the LOSs which contained, on the opposite point of the core, the 4,6-dideoxy-2-0-Me-3-C-Me-4-(2'-methoxypropionam- ido)-a-L-manno-hexopyranosyl-( 1 + 3)-fucosyl disaccharidic unit are species-specific (Figure 7). Three molecules of 2,4-dimethyltetradecanoic acid esterified the hydroxyl groups at the C-3, C-4, and C-6 terminal glucosyl residue.'I2 However, the acetyl and formyl residues are still unlocated.

FIGURE 7. Structure of the disaccharidic epitope unit present in the M. knnsoii lipooligosaccharide (LOS) Kanh'acyl-( 1->3)-Fucp R = see Table 4.

Related LOSs were identified as species-specific antigens of M . szulgai,"' M . malrnoen~e ,"~ and a smooth variant of a Mycobacteriurn sp. (M. linda) isolated from patients with Crohn's disease. The LOS antigen species-specific structures are summarized in Table 4. Whatever their mycobacterial source, all the LOS contain a triacylated a-a-D-trehdose unit except for that of M. szulgai which is based on a novel core containing 2-O-Me-methyltrehalose.

In the goal of finding biosynthetic precursors of the 6-0- methylglucose lipopolisaccharides in M. smegrnaris, Saadat and Ballou"6 have found related structures called pyruvylated glycolipids based on a trehalose core. Fatty acid analysis revealed the presence of C,,, C,,, and C,, fatty acids located on each of the glycosyl units of the a-a-trehalose core."'

V. PHENOLIC GLYCOLIPIDS

A. Historical Introduction Phenolic glycolipid history began in 1954 with the pi-

oneering task of Smith's group which characterized, by IR spectra, the immunizing fractions from tubercle bacilli. These IR spectra indicated an aromatic group contained by the sub- stance named "glycolipid G". ' They were found in restricted species of the Mycobacterium genera. M. kansasii, and M . bovis and called G, and G,, respectively. It was shown that G, is composed of three kinds of 6-deoxyhexoses (2,4-di-0- Me-rhamnose, 2-O-Me-rhamnose, and 2-O-Me-fucose) while exclusively 2-O-Me-rhamnose was found for G,. 118-170 These substances were further renamed mycoside A and rnycoside B3 and are now classified as members of the phenolic glycolipid family.

From 1970 to 1980, these substances were neglected and a revival of interest arose from the discovery, in 1981 by Brennan and Hunter, that the M. leprae-specific antigen belongs to the phenolic glycolipid class. To date phenolic glycolipids have been described in M . kansasii, M. gasrri, M . bovis BCG, M. marinurn. M . leprae, and M . tuberculosis strain Caneni.

6. Phenolic Glycolipids from M. bovis BCG 1. Retrospective Structural Studies (7953 to 1970)

The M. bovis phenolic glycolipid (mycoside B) was discovered by Randall and Smith.I2' MacLennan and Randall characterized the mycoside B carbohydrate part as 2-0-Me- rhamnose while No11 and Smith assigned the partial structure of the aglycon to an aromatic alcohol esterified by mycocerosic acids.z,122 Later, using new analytical tools such as gas liquid chromatography, 'H-NMR, and EI-mass spectrometry, the mycoside B structure was investigated by Demarteau-Ginsburg et al.123 and Gastambide-Odier et al.124.125 By UV spectroscopy (two maxima at 225 and 279 nm) and IR analysis (absorption bands at 1610, 1595, 1510, and 825 cm-I), the aromatic alcohol was assigned to a phenolic nucleus with its para position linked to an aliphatic chain. After saponification and hydrolysis, the phenolic nucleus was specifically methylated yielding mono-methyl phenol glycol. This product, in the presence of acetone yields an isopropylidene derivative which is in agreement with the hydroxyl groups being either in the a or p position. The difference 6v = 87 cm- ' between the IR absorption frequencies of the free hydroxyl and the hydrogen bonded one supported a p-glycol structure. From these data

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Table 4 Structures of the Major lmmunoreactive Trehalose-Containing Lipooligosaccharides

Mycobadend strain Otigosaccharides structure Fatty acids structure Ref.

kamasii KanNAcyl-( 1 ~ ) - F u c ~ - ( 1 --* ~)-[P-D- ' Xylp-( I -* 4)]&-L-3-0- Me-Rhv-( 1 -* 3 ) - p - ~ Glcp-( 1 -* 3)-P-~Glcp-( 1 -* 4)-a- &lcp-(l - l)-a-D-Glcp

s:ulgai a-~-2-O-Me-Fucp-( 1 -* 3)-a-~-Rhap- (1 -+ 3)-a-~-Rhap-( 1 4 3bP-D- Glcp-( 1 + 6)-a-&lcp-( 1 - l)-a- ~-2-O-MeGlcp

maltnoenre a-D-Manp-( 1 -+ 3 ) - a - ~ - Manp( 1 42)-a-~-Rhap-( I 4 2)- [a-~-3-O-Me-Rhap( 1 + 2)],-a-~- map-( 1 + 3 ) - a - ~ G l c p ( 1 - I )-O-D

Glcp

~ G l c p - ( 1 - I)-a-~-Glcp linda P-DGlcp-( 1 + 3)-a-~-Rhap-( 1 -3)-a-

2.4di-Mc-C,, 109 110.111

2.4.6-ei-Me-Cn 113 2.4-di-Me-Cm 2-MC ,3-OH-C I4

2-Me-C,, c* 1 I4

2.4-di-Me-C, 2-Me-Cm

2,4-di-Me-C,, I I5

and those of EI-mass spectrometry, Demarteau-Ginsburg et al. proposed the following partial structure for the phenol glycol core in which the exact position of the glycol group was not determined.

The paraphenolic structure of mycoside B was also supported by 'H-NMR analysis (two resonance doublets at 6 = 6.95 and 7.14 ppm) and the glycol unit was localized by Gas- tambide-Odier et al. 124.125 according to the procedure described in Scheme 1.

OCH] I u.o*-cq-(cq]. - c n - c q - c n - i c q ~ ~ - c n - c n - c ~ - c n l

OH OH CHa

. Oxydativr drgradrlion

. Diaiomclhrnr rslerincaiion

ocnl I

I Idenlificilion of Ihr sub-producu by CC and EI-MS rndysis :

nlcooc -(c%jr-cn-cn -c%-cn, I CH,

SCHEME 1 . Structural elucidation of the mycoside B phcnolphthioccrol core by Gastambide-Odicr et al."'.'"

The phenolphthiocerol Structure of mycoside B shown in Scheme 1 was confirmed by the presence of characteristic fragment ions on their EI-mass spectrum, resulting from the cleavage of the bonds located a- to the glycol unit. From this careful study the following phenolphthiocerol structure was proposed by Gastambide-Odier et al. ' 2 4 , ' 3 for the phenolglycol core of mycoside B.

gcosidm B phenolphthiocerol structure

The phenolphthiocerol structure shown next was found to be esterified by palmitic and C-29 mycocerosic acids. This latter compound belongs to a family of levorotatory polymethyl branched fatty acids with the following structure:

Finally, from optical rotatory value of mycoside B monosaccharide, the exact structure of the mycoside B carbohydrate was assigned by Demarteau-Ginsburg et al. 123 to a 2-O-Me-P- D-rhamnopyranosyl.

C. Recent Developments 7. Structure of the Mycoside 6 and New-Found Phenolic Glycolipids

Recently, the mycoside B structure was reinvestigated by


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three groups: Laneelle,’28 Brennan,lZ9 and Puzo. I 3 O . l 3 I The mycoside B molecular weight determined by desorption-chemical- ionizationimass spectrometry (DCVMS) using ammonia as reagent gas (Figure 8) are in agreement with the presence of two CZ6, C,,, CZ9, or C,, mycocerosic acids instead of one palmitic and one mycocerosic acid esterifying the phenolphthiocerol. 130.131 Mycoside B is formed by at least eight molecular species which only differ by the aglycon chain length.

Moreover, by gas liquid chromatography of the mmethylsilyl-2-~-butyl-glycosides, the 2-O-Me-Rhap was found to belong to the “L” series instead of the “D” series. Also, from the mycoside B coupling ’T-NMR spectrum the value of J,.,, of 164 Hz leads to the “a” anomeric configuration. This assignment was supported by two-dimensional proton- NMR Nuclear Overhauser Effect Spectroscopy (NOESY) analysis of mycoside B.I3, So, from these recent data, the exact structure summarized in Figure 9 can be proposed for mycoside B.

Beside mycoside B (called Phe GI B), three other quan- titatively minor phenolic glycolipids were purified by gradient direct-phase high performance liquid Chromatography (Figure 10). Using DCVMS and two-dimensional ’H-NMR homonu- clear Correlated Spectroscopy (COSY) and NOESY experiments, the structures of these new phenolic glycolipids, called B- 1, B-2, and B-3 according to their retention times in HPLC (Figure lo), were established and are summarized in Table 5 . 1 3 , Two-dimensional NMR studies allow both sugar and anomeric assignment and also the interglycosidic linkage type determination. Figures l l and 12 show the COSY and NOESY B-3 spectra used for the B-3 carbohydrate structural elucidation as the disaccharide : Rhap-a-(1 + 3)-2-O-Me-Rhap (Figure 13).

2. Serological Studies By ELISA technique, we have demonstrated that mycoside

[I, +un.]* 1412.

B and the minor phenolic glycolipids react against anti-M. boris BCG rabbit polyclonal antibodies. 1 3 * Moreover, the native Phe GI B , the phenolic glycolipid B-3 are immunogenic and rabbit polyclonal antibodies against these two antigens were obtained.

Brennan et al. have synthesized a neoantigen containing 2 - 0 - M e - a - ~ - R h a p . I ~ ~ The 9-carbon spacer arrn pioneered by Lemieux et al.1’3 is summarized in Scheme 2 , was selected to link the sugar epitope to the protein carrier. Serum from a cow experimentally infected by M . bovis reacts in ELISA with mycoside B and the neoantigen. The highest rabbit antibody titers against native and synthetic antigens were obtained when the rabbit was inoculated with the complex mycoside B - methylated BSA. From this last study, the neoantigen appears to be M . bovis specific since, using a battery of rabbit antisera against various other pathogenic mycobacteria, no cross-re- actions were observed in ELISA when the neoantigen was coated. Thus these authors concluded that rnycoside B may play a role as serological probe for the screening of M . bovis infections. However, mycoside B is not observed in most M . bovis strains and consequently cannot be considered as a re- liable marker of M . bovis.

D. Phenolic Glycolipids from M. kansasii 1. Retrospective Structural Studies (1957 to 1970)

Mycoside A was discovered by Smith et al. in 19572 in photochromogenic mycobacteria notably M . kansasii. IR (ab- sorptions 1615 cm-’. 1515 cm-I) and UV analyses (absorption 223, 276, and 283 nm) indicated the presence of an aromatic grou~.~.’~ Macknnan et al. in 1961’19 established the absence of nitrogen and phosphorus atoms, and N011”~ has found that rnycocerosic acids esterdy the aromatic alcohol. Mycoside B and mycoside A were identified by thin layer chromatography on silicic acid (mycoside A Rf = 0.78, rnycoside B Rf = 0.7 ; eluent CHCI,/CH,OH, 9317 vlv.)

The structure of the aglycon assigned to a diacylphenol-

I45m

FIGURE 8. Phc GI B (mycoside B) partial DCI mass-spectrum. mlr 1482, M,:MW = 1464 Da, phenolphthioccrol. C, + C, major homolog. x = IS; mycocerosic acids, d z 1524. M,:MW = 1506 Da. phcnolphthiocerol. C,, + CZ9; major homolog, x = IS; mycoccrosic acids.

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FH, -CH -CH* -CH-(CH,). -CH - c H - c H , - C ~

I ocn, I

HoM+-&+** oa ' OR

f 2-0-Me-a-L-Rhap n . n , c - ( c n , ~ , - C n - c ~ ~ n - c ~ - c n - c o - I OM. cn, cn,

FIGURE 9. 1464 Da, C, H,,, O,,,.

Phe GI B (mycoside B) major homolog stn~cturc. M,, MW =

' 0 ° 1

1 . 1 ~ ' I ~ " ' ' ~ ' ' ' , 10 20

R t T E W T l O N TIME (rnin)

Y .. I 0 L .. Y - 0 c u Y e Y 0

FIGURE 10. Characterization of the phenolic glycolipids B, B-I, B-2. and B-3 by their retention times in direct-phase HPLC. Linear gradient of solvent B [CHCIJCH,OH (8:2 vlv) ] in solvent A [CHCI,] during the first 9 min: 0 to 20% of B then 20 to 100% of B over a period of 16 min at a flow rate of I ml/min.

x - 17, g. 19. 20 - cl2* Cl4, CIS, c18 and c27. Czg, Q~,Q a d C32 mycocrrosic aclds.

phthiocerol was established by Gastambide-Odier et al. 124.125

using the procedure described in Scheme 1. In 1961, Macknnan and Randall demonstrated by paper

chromatography that three different kinds of monosaccharides in equal amounts composed mycoside A: 2-0-methyl-rhamnose, 2-O-methyl-fucose, and 2,4-di-O-methyl-rhamnose.

However, these authors concluded: "the question remains whether or not mycoside A is a single glycolipid containing three sugars or a mixture of glycolipids each containing one or more of the three sugars. " I I s

The monosaccharide composition of mycoside .4 was reex- ahined in 1970 by Gastambide-Odier who developed new analytical approaches for the structural analysis of the carbohydrate.l' Methanolysis of the mycoside A was selected instead of hydrolysis to yield methyl glycosides which were purified on dry florisil column chromatography and further identified as trimethylsilyl or acetyl derivatives by gas liquid chromatography, 'H-RMN and EI-Mass spectrometry. Beside the monosaccharides described by MacLennan and Randall,"* four other kinds of glycosides were identified, 2- and 3-0- methyl-rhamnofuranosides and 2- and 3-0-methyl-fucofuran- osides. At that time, these data strongly supported, as suggested by MacLennan and Randall, the heterogeneity of mycoside A. However, the methanolysis conditions used (38 h) could explain the formation of furanose forms from deoxyhexopyra- noses. In summary, in 1970, although the structure of the mycoside A aglycon was well identified, the structure of the carbohydrate moiety remained unclear.

2. New Developments The major developments in the structure and properties of

the phenolic glycolipids from M. kansasii arise from our group. First, a revised structure was proposed for mycoside A renamed Phe G1 K-I; second, minor phenolic glycolipids were purified and the structure of one of them called Phe GI K-I1 was established. Finally, the antigenicity and the immunogenicity of Phe GI K-I were demonstrated as well as its haprenic smcture.

The abundance of new data on the structure of M . kansasii phenolic glycolipids resulted from the development of new purification and structural strategies based on the use of modem analytical tools. In the goal of obtaining structural information on the native glycolipid, fast atom bombardment-mass-spectrometry (FAB-MS), 135.136 tandem mass-spectometry (MS/ ,,),I3' two-dimensional COSY and NOESY '38 'H-NMR were successfully applied.

3. Purification Procedures The purification to homogeneity of the phenolic glycolipids

is a difficult and tedious task mainly due to the mixture com- plexity and the amphipatic nature of these molecules, but it still remains a key step prior to any structural elucidation. The homogeneity on silicic acid TLC of mycoside A renamed Phe G1 K-I was obtained by standard column chromatography on florisil and silicic acid followed by flash chromatography and gradient HPLC equipped with a semipreparative direct phase column 5p-~pherisorb.'~~.'"O Figure 14 shows the -gradient HPLC profile of Phe G1 K-I and Phe GI K-11.


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I I I I 5 4 3 2 1

ppm

FIGURE 11. Two dimensional 'H-NMR COSY spectrum of the peracety- lated Phe GI B-3. Proton assignment of the distal Rhap mycoside monosaccharide H'-1 (6 = 4.96 ppm, doublet J, . ,* . = 1.8 Hz), H'-2 (quadret, 6 = 5.15 ppm, J,.,,. = 1.8 Hz, Jz.,,. = 3.3Hz: H'-2eq., H'-3ax.), H'-3 (quadret, 6 = 5.28 ppm, J , .,,, = 3.3 Hz, J ,.,, . = 10 Hz: H'-3ax.. H'-4ax.), HI-4 (triplet. 6 = 5.09 ppm. I,.,. = J,.,,. = 9.8 Hz), H'-5 (multiplct. 6 = 4.12 ppm). C- 6H, (6 = 1.25 ppm). Proton assignment of 2-0-Me-Rhap H-1 (doublet, 6 = 1.8 Hz), H-2 (quadrct, 6 = 3.73 ppm), H-3 (quadret, J,,, = 9.8 Hz. J2,, = 3.2 Hz: H-2eq.. H-3ax.. H4ax.) H-4 (triplet, 5.16 ppm J,,, = J,,: H 4 a x . . H-Sax.), H-5 (6 = 3.75 ppm). C-6H, (6 = 1.17 pprn).

4. Revised Structure of the Mycoside A (Phe GI K-I) For the fmt time, the molecular weight of Phe GI K-I was

unambiguously established by FAB-mass spectrometry using the cationization t e ~ h n i q u e . ~ ~ ' . ' ~ ~ The data obtained with this technique were the starting point of a reinvestigation of the mycoside A structure. A new kind of matrix, rnonobutyltrie- thylene glycol (MBTG) was employed instead of the standard glycerol and thioglycerol matrices for the FAB-MS analysis of these amphipatic molecules. The glycerol and thioglycerol matrices have been successfully used in FAB-mass spectrometry analysis of hydrosoluble biomolecules. However, the liposoluble Phe GI K-I tends to aggregate into micellar systems in these matrices, hindering their FAB ionization. The use of

7 6 5 4 3 PPm

FIGURE 12. Two dimensional 'H-NMR NOESY spectrum of the perace- tylated Phe GI B-3. H- I ' correlated to H-3 indicates a I + 3 intaglycosidic linkage. H-1 comlatcd to H-2, to the methoxyl group(0-CH,) and to the orrho aromatic protons (doublet, 6 = 6.95 ppm) indicates an a-anomeric configuration.

an adequate matrix, the MBTG, enabled the FAB-MS analysis of chloroform soluble glycolipids.

This study indicates that Phe GI K-I is composed at least of four kinds of molecular species of molecular weight 2152, 2136, 2124, and 2108 Da (Figure 15). The molecular species of molecular weight 2152 and 2124 Da are the most abundant. The latter was greater, by 186 Da, than the calculated value based on the postulated rnycoside A trisaccharide structure. 'VH-Nh4R spectroscopy 139.144 indicated a tetrasaccharide structure (four H-1 resonances) and also the presence of an acetoxyl residue (singlet at 2.15 ppm) on the carbohydrate moiety. in summary, prior to any chemical degradation of Phe GI K-I, these data allowed the 186 Da to be assigned to an acetoxyl group (42 Da) and to an anhydro-mono-0-Me-dideoxyhexose residue of 144 Da. By FAB-tandem rnass-spectrometry (FAB-MS/MS)139 (Figure 16) of the deacylafed Pbe GI K-I (d-Phe GI K-I) this extra sugar residue was localized

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FIGURE 13. BCG .

Structure of the phenolic glycolipid B-3 isolated from M. bovis

s-O-(CH2)8-c02 He 8 -me thoxylcarbonyloctyl

S - carbohydrate epitopa I

S-O-(CH2)8-CONH-~2 8-hydrazino carbonyloctyl

1 S-O-(CH2)g-CON3

I carrier protein

neoantigen.

SCHEME 2. Synthetic pathway proposed by Lemieux et al.") for neoantigen synthesis.

at the oligosaccharide nonreducing end. The fragmentation pathway leads us to propose the following partial tetrasaccharide sequence for Phe GI K-I: mono-0-Me-dideoxyhexosyl + mono-0-Me-deoxyhexosyl + mono-0-Me-deoxyhexosyl + 2,4-di-O-Me-Rhap.

Since dideoxyhexoses are acido-labile, mild Phe GI K-I hydrolysis conditions were used, and mono-0-Me-dideoxyhexose was recovered and purified on a C,, Sep-pack cartridge. Its molecular weight of 162 Da established by CI-MS using ammonia as reagent gas confims the 0-Me-dideoxyhexose structure. Moreover, its derivatization into alditol acetate followed by ELMS analysis allows the characterization of a 2,6- dideoxy-4-0-Me-hexopyranose. 145 Finally, from the optical rotatory value and 'H-NMR analysis of the methyl glycoside derivative, we propose the following structure: methyl 2,6- dideoxy-4-O-Me-arabino-a-~-hexopyranoside which corresponds to a new found sugar in nature.i46

The tetrasaccharide composition and the type of interglycosidic linkage were established by a new alternative approach to the alditol acetate method. It is based on the identification of the trimethylsilyl derivatives of partially methylated methyl glycosides by gas chromatography/mass-spectometry (GC/ MS). 14'

Beside the dideoxyhexose, which is chemically decom- posed when standard oligosaccharide hydrolysis or methanolysis conditions are used, the monosaccharides described by

FIGURE 14. Charactenzaoon of the M kansarir phenolic glycolipids K-I (mycoside A) and K-U by their retention omes in HPLC equiped with a 5-k direct phase column Linear gradient of solvent B [CHCIJCH,OH (8 2 V N ) ] in solvent A [CHCI,] D u n g the first 6 min 0 to 208 of B then 20 to 100% of B over a penod of 19 nun at a flow rate of 1 d m i n

2100 2100

FIGURE 15. Phe GI K-I partial FAB-mass spectra in positive mode with monobutylaiethyleneglycol as matrix. (A) Without inorganic salt addition. (B) In the presence of cesium iodide; the signal shift allows the assignment of the signals observed in spectrum A to protonated molecular ions.

Macknnan and Randall,ii8.119 2-O-Me-a-fucopyranose, 2-0- Me-a-rhamnopyranose, and 2,4-di-O-Me-a-rhamnopyranose were identified. However, their furanose forms mentioned by Gastambide-OdierlY were not observed. The Phe G1 K-I interglycosidic linkage type (1 + 3) was determined after Phe G1 K-I permethylation, methanolysis, trimethylsilylation, and analysis of the subproducts by GC/MS in €1 mode. Finally, determination of the Phe GI K-I sequence order of 2-0-Me- Rhap and 2-0-Me-Fucp required, from partial Phe GI K-I hydrolysis, the purification of the diglycosylated phenolphtiocerol residue followed by its 'H-NMR analysis. Thus, from the Ji .* coupling constant values of the anomeric protons and from the previous data, the following Phe GI K-I tetrasaccharide sequence is proposed: 2,6-dideoxy40-Me-a-~-arabino-hexopyranosyl-( 1 --* 3)-2-O-Me-a-~-Fucp-( 1 + 3)-2-0-Me-a-~- map-( 1 + 3)-2,4-di-O-Me-a-~-Rhap.

The acetoxyl group revealed by Phe GL K-I 'H-NMR


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100,

> I-

VI I W c z

- - 50, w > I-

-I W

- < a

1 0 7 1 I m l r : 1 2 1 5

FIGURE 16. FAB-tandem mass specroometry of the precursor ion m/z 1215 (M + H’) arising from the deacylated major phenolic glycolipid. The rnono- saccharides are eliminated in the anhydro form.

analysis was located on the penultimate monosaccharide residue by pyrolysis EI-mass spectrometry of perdeuteroacetylated Phe GI K-I (Figure 17).i39 This assignment was supported by two-dimensional COSY ‘H-NMR analysis of the native Phe GI K-I which unambiguously indicated that the acetoxyl group is on the C-4 fucosyl residue (unpublished results).

The aglycon moiety structure was reinvestigated and was found responsible for the Phe GI K-I heterogeneity observed by FAB-MS analysis. The major phenol glycol core homolog corresponds to a phenolphthiocerol esterified only by C,,, C,, and C,, mycocerosic acids and not by CI6, C,, fatty acids as previously described. The presence of phenolphthiodolone, although in small amounts, was identified and along with the

variable chain length of the mycocerosic acids support the Phe GI K-I heterogeneity revealed by FAB-MS analysis. The Phe GI K-I major hornolog structure is represented in Figure 18.

The structure of Phe GI K-I1 (Figure 191, a minor phenolic glycolipid, was established using the same analytical strategy employed for Phe GI K-I. Phe G1 K-I and Phe GI K-I1 present a common core assigned to mono-acetyl-tnglycosyl-diacyl- phenolphthiocerol. They differ by the distal monosaccharide residue: 2,4-di-O-Me-a-~-mannopyranosyl, for Phe GI K-I1 and 2,6-dideoxy-4-0-Me-ar-~-arabino-hexopyranosy1 for Phe GI K-I.

5. Phe GI K-l and Phe GI K-ll Serological Reactivities

Phe GI K-I and Phe GI K-I1 react against polyclonal antibodies arising from rabbits inoculated with killed M. kansasii.145 Moreover, high titer rabbit polyclonal antibodies against Phe GI K-I were obtained when the rabbits were infected by the native Phe GI K-I.”9

In the aim of determining the Phe GI K-I epitope, the “L” and “D” enantiomers of methyl 2,6-dideoxy-4-0-Me-a-uru- bino-hexopyranoside were synthesized. Is’ Also, the deacetylated di-, and triglycosylated Phe GI K-I were purified from Phe GI K-I partial hydrolysis products.’+’ By the ELISA inhibition technique the “D” enantiorner and the mono-acetyl- triglycosyl-Phe GI K-I inhibit the linkage reaction of Phe GI K-I to the Phe GI K-I antibodies. The other di-, h-iglycosyl- Phe GI K-I and the deacetylated glycolipid are not recognized by the Phe G1 K-I antibodies. Thus, the Phe K-I epitope was delineated as the distal monoacetylated disaccharide of the Phe GI K-I tetrasaccharide moiety. It is the first example of an alkal-labile phenolic glycolipid epitope. This property was previously used for the LOS antigens characterization.

We have begun the synthesis of this epitope by the syn-

I

L, .-

. Z r‘

i *i’ I

! 1 9 0 d

FIGURE 17. oxonium ions allowing tetrasaccharide sequencing and acetoxyl location on the fucopyranosyl residue.

Pyrolysis EI-mass spectrum of the perdeuteroacetylated Phe GI K-I derivative. The main fragment ions result from glycosidic cleavage yielding

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lli C K

FIGURE 18. from M. kansasii (mycoside A renamed Phe GI K-I).

Structure of the major phenolic glycolipid homolog isolated

I S

S

A ,

K - I %-*

on. '

2,4-di-O-He-a-D-Hanp

FIGURE 19. kansasii (Phe GI K-11).

Structure of a minor phenolic glycolipid isolated from M .

thesis of methyl 2,6-dideoxy-4-0-Me-a-~-arabinohexopyran- oside in a five-step sequence, summarized in Scheme 3.'" An alternative synthetic pathway has been proposed by Gujar et al.156

By ELISA technique using polyclonal antibodies against Phe GI K-I we have shown that this antigen is common to different M. kansasii isolates but restricted to M. kansasii and M. gasrri species.'49.1so The phenolic glycolipids from M. gastri were purified, and it was demonstrated that the major phenolic glycolipid called Phe GI G-I has the same structure as Phe GI K-1.Is'

Beside the Phe GI K-I antigen, M. kansasii cell wall also contains a distinct class of surface species-specific glycolipidic antigens: the trehalose-containing Iipooligosaccharides. Both antigens were observed in smooth colony variants whereas all rough variants were devoid of LOS."* These data and those of Collins and CunninghamIs3 have established that the rough M. kansusii variants are persistent in animals while the smooth variants are rapidly cleared. Belisle and Brennan assume that

........... R I u90Ac OCW,

6.deoxy-D-glucal mechyl 2-acecoxymercur:o-2.6 dldeoxy-a-D-mannopyranoslde

............ .......... ' Ph-S: -w ocn,

'R

mechyl 2.6-didaoxy-a-D- methyl 3-3-c.rt-bucy:dlphenyl arablnohexopyranoslda sllyl-2.6-dideoxy-a-D-

arabino-hexopycanocide

mechyl 2.6-dldeoxy-h-O-!b. a.D-arabinohexopyranosido

SCHEME 3. Synthesis of the Phe GI K-I immunodormnant rnonosacchande. '"

the absence of LOS allow a better exposure of the phenolic glycolipids and lipoarabinomannan, both of which inhibit in v i m T-cell proliferation. However, Rastogi and David have shown that M. gusrri, a nonpathogenic mycobacteria and M. Kasnsasii which share the same phenolic glycolipids, have different specific behavior inside the macrophages. M . kansasii multiplies intracellularly whereas M . gasrri does not. Is4

E. Phenolic Glycolipids from M. Leprae

Brennan.IJ7 This topic has been reviewed in a pertinent treatise by

1. Structural Elucidation The investigations into the immunoreactive entities of M .

leprae were hampered by the lack of in v i m cultures of this microorganism. Until recently, the only sources of M . leprae were from infected human tissues and mouse foot tissues. From the qualitative and quantitative analysis of the lipids of M . leprae in skin biopsy samples from lepromatous patients, Young demonstrates the presence of 6-deoxyhexose-containing glycolipid related to the glycolipid mycosides A and B from M . kansasii and M . bovis.158 However, the amount of material available from such clinical specimens was insufficient for the exact elucidation of this M . leprae glycolipid. Independently, in a deliberate search for a specific M . leprae antigen, Brennan and Barrow found evidence for a lipidic compound with such properties. 165

In 1981, the availability of large amounts of M . leprae from infected armadillo tissues provided the opportunity of structural studies of serological M . leprae markers. Then, Hunter and Brennan assigned this lipidic antigen to a phenolic gly-


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colipid structure (PGL-I). Independently, Draper found evidence for a phenolic glycolipid antigen in M . feprue.'* The first structure of this PGL-I antigen was determined by Bren- nan's group. 16' By partial hydrolysis, 'H-NMR experiments and by GCiMS analysis of the alditol acetate derivatives, the PGL-I carbohydrate structure was assigned to the following trisaccharide: 3,6-di-O-Me-P-~-GIcp-( 1 + 4)-2,3-di-O-Me-a- ~-Rhap-( 1 + 2)-3-O-Me-cr-~-Rhap.

FIGURE 20. from M. leprne (PGL- 1).

Structure of the major phenolic glycolipid homolog isolated

The phenolglycol core belongs to the phenolphthiocerol class and was found to be esterified by a mixture of C,, C,,, and C, mycocerosic acids. Figure 20 represents the structure of the M . leprae phenolic glycolipid. Tarelli et al.16* confirmed, mainly by 'H-NMR and I3C-NMR experiments, the PGL-I carbohydrate structure and the anomeric configurations. The molecular weight of PGL-I established by FAB-MS using the MBTG matrix confirmed the proposed structure and indicated that the heterogeneity of PGL-I is due to the variable aglycon chain lengths.i43 More recendy, positive and negative ion PDMS technique was successfully applied for the PGL-I analysis. 163

Positive mass spectrum supported the fact that PGL-I is a complex mixture due to the fatty acids chain length. PGL-I molecular weights were determined from the cationized molecular ions (M + Na) + , and the partial sequence of the PGL- I trisaccharidic part can be established from the fragment ions at m/z 191, 365, and 526.

In addition to the PGL-I, small amounts of two other phenolic glycolipids PGL-II and PGL-I11 were isolated from infected liver (about 60 Fg/g of infected tissue).lM Their structure, established by Brennan's group, differ from that of PGL-I by the absence of a methoxyl group either at the C-2 of the penultimate rhamnose (EL-11) or at the C-3 of the distal glucose residue ( P G L - I ) (Figure 21).

FIGURE 21. Structures of the minor phenolic glycolipids PGL-I1 and FGL- 111 from M. leprae. EL-11: R , = -H. R, = CH,. PGL-HI: R , = C H , . R, = -H

2. Serological Studies and Neoantigen Synthesis Due to their apolar structure and thus their low solubility

in aqueous buffers, the antigenicity of PGL-I was a moot point for a period. Pnor to the discovery of PGL-I, Brennan et al.'65 suggested the presence of a species-specific lipidic antigen in the M . feprue cell wall. In 1982, Payne et al.160 incorporated PGL-I into liposomes and demonstrated the PGL-I reactivity against undiluted sera from lepromatous leprosy patients. In- dependently, three groups: Brennan et al.,IM Young and Buch- a r ~ a n , ' ~ ~ and Brett et al.I6' developed ELISA conditions, with dilute sera, adapted to the solubility of the apolar structure of PGL-I. Brennan et al. proposed a sonicated emulsion of PGL- I , Brett et al. used a detergent to aid in the initial step of solubilization, while Young and Buchanan proposed deacy- lation to increase its polarity. All these studies converged to- wards the application of PGL-I as a serological marker for the diagnosis of lepromatous leprosy patients. Once purified, the PGL-I was found to be M. leprae specific and over 90% of clinically diagnosed lepromatous patients demonstrated high antiglycolipid IgM immunoglobulin titers. 166 However, for tuberculoid leprosy, which is more difficult to diagnose, the assays were insensitive. Thus, the LAM combined with the specific PGL-I allows greater detection of both multibacillary and pau- cibacillary diseases (70%) than examining only IgM antibodies

The success of PGL-I as a specific probe for lepromatous leprosy patient diagnosis has triggered a challenge for the PGL- I epitope synthesis. The PGL-I major epitope was assumed to be its distal disaccharide using sera from human lepromatous leprosy patients and from rabbit inoculated with native PGL- I with phenolic glycolipids arising from other mycobacterial species.17o By an elegant study, Fujiwara et al.I7' defined the PGL-I epitope. They synthesized the PGL-I mono-, di-,172 and trisaccharide and demonstrated, by the use of ELISA inhibition techniques, that the distal monosaccharide unit is immunodominant, but its inhibiting efficiency is higher when the terminal PGL-I disaccharide is used. These results were supported by Young et al. who generated nine monoclonal antibodies against PGL-I, five of which selectively recognized the distal glucose.'73 Independently, Gigg et al. synthesized, in an alternative way, the PGL-I di- and t r i ~ a ~ ~ h a r i d e ~ ' ~ ~ ~ ' ~ ' and recently Verez-Bencomo's group proposed a new strategy for the PGL-I trisaccharide s y n t h e s i ~ . " ~ . ~ ' ~ In the first synthesis

to PGL-I (50%).

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of the PGL-I trisaccharide reported by Fujiwara et al.,17'.1'8 the glycosylation reaction yields a mixture of a- and p-an- omers. To overcome this problem, Verez-Bencomo et al. proposed a Lewis acid-catalyzed glycosylation which proceeds via an SN, mechanism. The coupling reaction of partially methylated syntons was carried out in the presence of triflate (boron trifluoride etherate).

Neoantigens were prepared by coupling either the PGL-I disaccharide or trisaccharide unit to a BSA carrier protein. In the first procedure, the penultimate sugar was degraded since the synthetic disaccharide was reductively aminated to the E-

amino group of the lysine.I7' In order to preserve the epitope and to increase sensitivity, the Lemieux strategy'33 (see Scheme 2) was applied to link the distal disaccharide of PGL-I as its 8-(methoxycarbonyl) octyl-glycoside derivative to the carrier protein. '79.180 The resulting neoantigen was called natural Jis- accharidic-octyl BSA (ND-0-BSA). More recently, Brennan et al. have again synthesized, using a similar approach to that of Bencomo's group, the PGL-I trisaccharide and prepared the NT-0-BSA neoantigen.

Fujiwara et al. have also synthesized, by the Lemieux synthetic pathway, BSA-conjugated PGL-I disaccharide and trisaccharide carrying the p-(2-methoxycarbonylethyl) phenyl group as a linker arm'82 namely ND-P-BSA and NT-P-BSA, respectively. Gigg et al. have synthesized PGL-I disaccharide involving the allyl linker arm and Brett et al.IS3 have described its coupling to BSA. The sensitivity and specificity of the neoantigens ND-0-BSA, NT-0-BSA, and NT-P-BSA tested against sera from leprosy patients by ELISA were found similar.'" These results support the PGL-I epitope assignment and c o n f m that the innermost monosaccharide residue did not contribute significantly to sensitivity or specificity. Compared to the native PGL-I antigen, these neoantigens do not present greater sensitivity or specficity . Nevertheless, their advantages of water solubility and low cost have led to the replacement of the native PGL-I by neoantigen in the serodiagnosis of lepromatous leprosy patients.

Another approach has been developed by Bencomo's group to prepare neoantigens according to the method described by Kotchekov.IU These neoantigens are copolymers of PGL-I trisaccharide with the allyl arm and acrylamide. They react in ELISA against IgM from pooled sera of leprosy patients. The comparison of these two kinds of neoantigens (glycoconjugates and copolymers) indicates that sensitivity was similar while spectficity was greater with glycoconjugates. 17) However, like the native PGL-I antigen, both neoantigens are inefficient for the diagnosis of tuberculoid leprosy.

Since in lepromatous leprosy large amounts of PGL-I have been found in host tissues, Brennan et al., aiming at the diagnosis of tuberculoid leprosy have developed chemical and immunological procedures for PGL-I quantitation in tissues. Another advantage of the quantitation of PGL-I rather than its corresponding antibodies in patients is the fact that amounts of the PGL-I decrease faster than the corresponding antibodies

Critical Reviews In

during chemotherapy, allowing a better monitoring of the disease. PGL-I was extracted from lepromatous leprosy sera by a chloroform-methanol solvent mixture. This extract was purified by column chromatography and identified by thin layer chromatography with a detection limit of about 0.5 to 1 pg. High performance liquid chromatography of untreated lepromatous sera gave from 0.8 to 3.7 pg PGL-L'ml. Immunological assay by ELISA inhibition technique with polyclonal antibodies against PGL-I corroborated the results obtained by HPLC. Sen- sitivity is similar for both quantitation methods, nevertheless DOT-ELISA on nitrocellulose with polyclonal and monoclonal IgG antibodies gave both greater sensitivity (500 pg) and semi- quantitative evaluation. Independently, Young et al.Is6 proposed the combination of chromatography and immunoblotting on a polysulfone membrane for PGL-I identification and quantitation.

3. Immunological Properties of M. leprae G l y ~ ~ l i p i d ~ ~ ~ ' ~ * ' * ~

The identification of T-cell epitopes is a key step in the understanding of immunity to mycobacterial infection and thereby in the design of molecular vaccines. In order to increase the rnacrophagic microbicidal activity, we can assume that the target of the antigens involved in protective immunity are T- cell: inducers of y-interferon production. However, the screening of such T-cell epitopes is hindered by the unavailability of adequately characterized T-cell subpopulations or clones. Moreover, cment dogma states that T-cells only recognize peptides associated with the class I1 major histocompatibility complex on antigen-presenting cells. So, the reported ability of T e l l s to specifically recognize carbohydrate must be clearly demonstrated.

The specific T-cell unresponsiveness in lepromatous leprosy was explained by the proliferation of the M. leprae specific suppressor T-cell of the T8 (CD8') phenotype activated in vim0 by lepromin.'88.'m'92 Mehra et al.'93 assigned this effect to PGL-I. They provided in vim0 evidence that T-cells recognize the E L - I trisaccharide since PGL-I is capable of sup- pressing the concanavalin A-triggered proliferation of lepromatous lymphocytes but not those from tuberculoid leprosy patients.

These results suggest a specific T-suppressor epitope in the PGL-I trisaccharide. However, Nath's group'94 provides evidence that PGL-I induces a general mitogenic suppression response unrelated to the leprosy type. Thus the specificity binding to PGL-I by T8 (CD8 +) cells is not yet clearly verified. Furthermore, the activation of T-suppressor cells by E L - I was not observed by Brett et al.195 in mice, and the two suppressor T-cell clones derived from leprosy patients are not addressed against PGL-I.'% The controversy concerning the PGL-I suppressor activity is not yet closed. Only the emergence of a T-cell clone that is reactive to the phenolic glycolipid PGL-I and to related neoantigens (ND-0-BSA) could clarify

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this point linlung the M. leprae specific antigen with the specific T-cell anergy.

Recently, Fournie et al.'97 have shown that purified phenolic glycolipids (Phe GI K-I, PGL-I, Phe GI B) inhibited in vitro proliferation of human mononuclear cells from healthy BCG vaccines. They demonstrated that this phenomenon: ( 1 ) is a general property of the Phe GIs and cannot be assigned to the carbohydrate moieties which express specific B-cell epitopes and (2) is independent of the stimulus (mitogens or mycobacterial antigens) and of the presence of APC or CD8' T- cells. However, according to the phenolic glycolipid structure, a dose effect was observed, suggesting that the presence of large amounts of phenolic glycolipids within the leprosy lesions could compromise effective clearance of mycobacteria.

LAM is also an inhibitor of in vino proliferation of peripheral blood mononuclear cells from healthy subjects or patients with tuberculosis when stimulated by mycobacterial or other antigens.77 Further studies have found that LAMS either from M . tuberculosis or M . leprae suppress the peripheral blood cells proliferation in both lepromatous and tuberculoid leprosy patients as well as in healthy subject^.'^ More recently, Moreno et al. have found that in v i m PBMC proliferation induced by IL-2 and PHA mitogens is not inhibited by the LAM while suppression was observed when mycobacterial antigens (MTSE, PPD) are used. Using a T-cell clone reactive to influenza virus, the comparative effect of LAM on assays with the T-cell epitope peptide and with the intact virus show that LAM inhibited the proliferative response to intact virus while no marked effect on the response to the peptide was observed. These data indicate that this effect is mediated at the level of the antigen presenting cell population and more particularly during the processing of the antigen by the macrophage.

So, from these in vitro experiments, we can assume that in vivo the phenolic glycolipids and the LAM epitopes which are exposed on the cell wall surface, unspecifically suppressed in synergy the T-cell proliferation thereby affecting mycobacterial clearance by the immune system.

It has also been suggested that PGL-I may function as part of a lipid capsule protecting the organism from the host environment. Neil1 and Klebanoff found that PGL-I is a weak scavenger of the toxic products of the peroxidase -H,O,- halide system and that it prevents bacterial killing by OH. generated by the xanthine oxidase and by IFN-y-activated macrophages. '9~

F. Phenolic Glycolipids from M. tuberculosis The discovery that nontuberculous mycobacteria contain

species-specific antigenic glycolipids raised the hope that M . ruberculosis cell walls would be endowed with related antigens. Reggiardo et al. isolated from M . bovis BCG three different classes of serologically active glycolipids used in passive hemagglutination and ELISA for the clinical diagnosis of tuber- c ~ l o s i s . ~ ~ . ' ~ However, these glycolipid fractions composed of PIMan and undefined compounds are ubiquitous in the M y - cobacrerium genus. In spite of the efforts of Brennan's group,

S1"l. C,..lti Y.0

FIGURE 22. isolated from M. ruberculosis strain Canett.

Strucfurc of the major phenolic glycolipid (PGT-I) homolog

no specific glycolipid antigens could be found in M . ruberculosis.2w Recently, phenolic glycolipids were identified by DafX et al. on M . tuberculosis strain Caneni.20t The structure of the major one, PGT-I, presented in Figure 22, was assigned to a triglycosyl-dimycocerosyl-phenolphthiocerol. 201 The trisaccharide moiety is composed of 1 --+ 3 linked tri-0-Me- Fucp, Rhap and 2-0-Me-Rhap while the aglycon moiety corresponds to the well-known dimycocerosyl-phenolphthiocerol structure. Besides this phenolic glycolipid, small amounts of a monoglycosylated phenolic glycolipid with the same structure as that of Phe GI B (mycoside B) were identified. Apparently, these phenolic glycolipids are restricted to the Canetri strain and were not observed by thin layer chromatography analysis of lipidic extracts from other M. ruberculosis strains.

Some structural analogy appears between the phenolic glycolipids of M . tuberculosis and those of M . bovis BCG. Both species contain Phe GI B (mycoside 8) and Phe GI B-2 (unpublished results) and PGT-1, cons ided as a glycolipid marker of the M . tuberculosis strain Canetti, differs only from Phe GI B-3 by the distal tri-0-Me-Fucp residue.

Preliminary evaluations of the PGT-I as a serological marker for tuberculosis diagnosis suggest that antibodies reacting with this antigen occur in the sera of most tuberculous patients investigated (97.5%).202 However, it is well established that only low amounts of antibodies to mycobacteria are present in human tuberculous sera. Thus, misleading data can arise from- considering as positive ELISA reaction ratios "tuberculosis to healthy" equal to or above two.

G. Phenolic Glycolipids from M. marinurn The M . rnarinum (strain balnei) phenolic glycolipid called

mycoside G was discovered in early works by Smith et al.,87 but was not observed in the M . marinurn type strain (ATCC 927). The mycoside G structure was established by Gastam- bide-Odier et al. 203.204 as a monoglycosylated dimycocerosyl phenolphthiocerol (Figure 23). The monosaccharide residue was identified as 3-O-Me-a-~-1hamnopyosyl and differs from the Phe G1 B (mycoside B) sugar residue by the location of the methoxyl group. The major aglycon is composed by phenolphthiocerol esterified by C24, C27, and C,, mycocerosic acids. Some minor homologs with methyl branches in the phenolphthiocerol (CH,), portion and also small amounts of phenolphthiodolone were identified. Beside mycoside G, a minor

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FIGURE 23. olog isolated from M. marinurn (M. balnei).

Structure of the major phenolic glycolipid (mycoside G) hom-

phenolic glycolipid called mycoside G’ was purified. Its structure2o5 differs from that of mycoside G by the presence of a mycoloyl group acylating the OH-2 of the 3-0-Me-rhamnopyranose. However, the presence of mycolic acids esterifying mycoside G’ is still doubtful and could result from structural analysis of a contaminated mycoside G’ fraction. Recently, with the purpose of developing an immunodiagnostic procedure, the antigenicity of mycoside G was examined. It was shown by ELISA that mycoside G is not antigenic.ZW

VI. SUMMARY

To date phenolic glycolipids have only been found in six mycobacteria species: M . leprae, M . kansasii, M . gastri. M . bovis BCG, M . marinurn, and M . tuberculosis strain Canerti. All the isolates M . leprae and M . kansasii, and M . gastri strains analyzed contain the phenolic glycolipids PGL-I and Phe GI K-I, respectively. This feature is not observed in M . bovis or M . marinurn since the phenolic glycolipids Phe GI B and mycoside G, respectively, are restrained to few strains. Finally, in the case of the M . tuberculosis spp. only the Canetri strain contains phenolic glycolipids.

The phenolic glycolipid structures are either species or subspecies specific markers except for Phe GI K-I which is common to two mycobacterial species: M . kansasii and M . gastri. The major structural difference between these glycolipids results from the peculiar structure of the carbohydrate moiety which confers to these antigens their species specificity. These major phenolic glycolipids are not single molecular species: their heterogeneity results from the aglycon structures. Beside these major phenolic glycolipids, small amounts of phenolic glycolipids called “minor” were identified and found to differ from the major ones by the carbohydrate structure. They differ by the degree of methoxylation in M . leprae, by the distal monosaccharide structure in M . kansasii and by the degree of glycosylation in M . bovis BCG and M . tuberculosis.

All these phenolic glycolipids are antigens and immuno- gens in rabbit and thus might be considered, with their related neoantigens, as potential serological probes for the screening of human mycobacterial infections. However, to date, only the PGL-I and its neoantigens have been successfully used for the diagnosis of lepromatous leprosy. For ELISA-based tuberculoid leprosy diagnosis, PGL-I appears to be inefficient and

preliminary results on untreated patients infected with M . kansasii show the presence in their sera of nonsignificant amounts of anti-Phe GI K-I antibodies. Thus, it seems that the presence of antibodies against PGT-I in tuberculous human sera, allowing serodiagnosis of the disease, must be confmed for instance by’the use of neoantigens.

In the same way, the PGL-I specific suppressive activity capable of inducing the proliferation of suppressor cells in lepromatous patients now appears dubious. It is rather likely that, due to their amphipatic structure, these glycolipids act in T-cell proliferation suppression by nonspecific mechanisms, unrelated to the leprosy type, which could result from lymphocyte membrane alteration.

ACKNOWLEDGMENTS

I wish to acknowledge Drs. J. J. Fournie and M. Riviere and the students A. Vercellone, M. Gilleron, and A. Venisse for their contribution to the results obtained in my own group in the area of mycobacterial phenolic glycolipids. Also, the author is particularly indebted to J. J. Fournie and M. Riviere for helpful and precious criticisms concerning the form of the review and Dr. P. Winterton for his help in correcting the English. I thank Huguette Dumoulin for typing the manuscript.

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