1721

The Role of Mycobacterium leprae Phenolic

Glycolipid I (PGL-I) in Serodiagnosis and in

the Pathogenesis of Leprosy

JOHN S. SPENCER & PATRICK J. BRENNAN

Department of Microbiology, Immunology & Pathology, Colorado

State University, Fort Collins, CO 80523-1682, USA

Accepted for publication 21 September 2011

Summary PGL-I (phenolic glycolipid I) emerged in the early 1980s on the one hand

as part of intensive efforts to dene the typing antigens of a host of Mycobacterium

spp. and also from characterisation of the lipids of skin biopsies from highly bacillary

positive lepromatous leprosy patients. PGL-I, despite its extreme lipophilicity due to

its inherent phthiocerol dimycocerosyl component, is highly antigenic evoking high

titre IgM antibodies in lepromatous leprosy patients, attributable largely to the unique

3,6-di-O-methyl-b-D-glucosyl entity at the non-reducing terminus of its trisacchar-

ide. PGL-I itself or in the form of semisynthetic neoglycoproteins containing the

synthetic terminal disaccharide or the whole trisaccharide chemically conjugated to

such as bovine or human serum albumin, has found its greatest utility in the serolo-

gical diagnosis, conrmation and management of lepromatous leprosy. PGL-I has

also been implicated in the tropism of M. leprae for Schwann cells, through specic

binding to laminin, and to play an important role in downregulation of the inam-

matory immune response and inhibition of dendritic cell maturation and activation,

thereby facilitating the persistence of M. leprae/leprosy.

Introduction

Among the most signicant achievements in leprosy research over the past 50 years was

the discovery in the early 1970s that Mycobacterium leprae could be grown to high

numbers in a living mammalian host, the nine-banded armadillo, Dasypus novemcinctus.

1

This development allowed for the rst time a reliable source of bacilli which could then be

used for lipidomic, proteomic, genomic, and metabolomic studies that eventually resulted in

major advances in understanding the basic biology of this human pathogen. The discovery of

a phenolic glycolipid (PGL-I) specic for M. leprae was rst reported in 1980,

2

with

subsequent reports that it was highly antigenic and capable of inducing high antibody titers

against the unique sugar epitopes of this molecule.

36

The history of how native PGL-I

Correspondence to: John S. Spencer (e-mail: [email protected]) or Patrick J. Brennan (e-mail: patrick.

[email protected])

Lepr Rev (2011) 82, 344357

344 0305-7518/11/064053+14 $1.00 q Lepra

was rst discovered, puried, chemically characterised, the sugar epitopes identied and

chemically synthesised and used as neoglycoproteins for the serodiagnosis of leprosy, and the

importance of PGL-I in aspects of the pathogenesis of leprosy, is now told.

DISCOVERY OF M. LEPRAE PGL- I

The recognition of an M. leprae-specic antigen, a glycolipid, came from two separate

approaches. Brennan and colleagues in the late 1970s/early 1980s had reported considerable

success in dening the surface/cell wall species and serovar/serotype-specic antigens of

a host of atypical/non-tuberculous mycobacteria (NTM), notably members of the, then

named, Mycobacterium avium/M. intracellulare/M. scrofulaceum (MAIS) complex, and

many others such as M. kansasii, M. szulgai, M. malmoense, M. gordonae, M. fortutium,

M. smegmatis, etc.

7

These were invariably members of but two precise chemical structural

entities: the so-called glycopeptidolipid (GPL) class, very characteristic of members of the

M. avium complex;

79

and the lipooligosaccharide (LOS) grouping, such as in M. kansasii,

M. malmoense, M. smegmatis, etc.

10

An additional class of glycolipids, the phenolic

glycolipids (PGLs), had been previously or subsequently recognised in M. bovis,

11

M. tuberculosis smooth morphology (e.g. M. tuberculosis strain Canetti),

12,13

but also in

such asM. kansasii

14

(in conjunction with theM. kansasii specic LOS (for a review, see [7]).

On the heels of the discovery that M. leprae could readily replicate in the armadillo

1

and

thereby generate a plentiful supply of the bacterium, the National Institute of Allergy and

Infectious Diseases (NIAID) of the NIH in 1978 issued an RFP (request for proposals) to

identify, characterise and provide to the research community, M. leprae-specic antigens.

Two contracts were awarded, one to National Jewish Hospital in Denver, CO (the Principal

Investigator, Patrick J. Brennan) based on the hypothesis thatM. leprae, as for the majority of

mycobacteria, is endowed with its own particular glycolipid, and like the majority of them,

such as those of the M. avium complex, should be highly antigenic and thus suitable for the

specic serodiagnosis of leprosy. A second contract was awarded to Pacic Medical Center

in Seattle, WA (the Principal Investigator, Thomas M. Buchanan), to pursue the identication

of M. leprae specic proteins.

15

Subsequently only one contract was awarded, to Colorado

State University (Brennan had moved there in 1980); indeed this contract was renewed

through many funding cycles until 2010 allowing a thorough exposition of the value of PGL-I

in various aspects of the disease of leprosy, particularly serodiagnosis.

Brennan and Barrow in 1980

2

rst reported evidence of a major glycolipid associated

with armadillo derived M. leprae, prepared according to the Draper 1979 gentle method.

16

Lyophilised M. leprae pooled from processed M. leprae infected armadillo livers was

extracted with acetone which favours removal of apolar lipids, followed by CHCl

3

-CH

3

OH to

extract residual soluble lipids. Column chromatography followed by serology using a version

of the classical Ouchterlony agar gel immunodiffusion technique

17

demonstrated the

presence of reactive lipid(s) in some of the early eluates off the column, reactive only with

antiserum from a lepromatous leprosy patient and an experimentally infected armadillo,

which did not extend to serum from patients with tuberculosis or an M. avium infection.

Secondly, it was established that the lipid was alkali stable and on acid hydrolysis yielded two

major sugars, tentatively identied by comparative gas chromatography (GC) as the alditol

acetates of the 6-deoxyhexoses, 3,4-di-O-methylrhamnose and 2,3-di-O-methylfucose. Both

designations (and the designation of a third minor sugar as 6-deoxytalose) proved to be wrong

and taught us a lesson in the fallacy of assigning sugar identity based only on comparative

Mycobacterium leprae Phenolic Glycolipid I 345

GC retention times. However, these authors cautiously warned: At this time we can merely

state that there is indirect evidence implicating 6-deoxyhexose-containing lipids with this

serological activity. A further prescient note was made: Currently we are looking for

this lipid antigen in liver fractions left afterM. leprae have been removed. The logic here is

that if cold acetone will solubilize the antigen, then much of it may have been lost during the

fractionation steps involved in the isolation ofM. leprae. Indeed, the prototypic procedure

18

for the isolation of both M. leprae and PGL-I from M. leprae infected armadillo livers

and spleens, still being applied, concludes: The pellet is used as a source of M. leprae.

The supernatant is lyophilized and weighed and used as a source of glycolipid.

Some of the early work of Douglas B. Young was also crucial in the discovery of PGL-I.

Upon completion of his D.Phil. at Oxford University, Dr. Young spent his initial postdoctoral

period at The Foundation for Medical Research, Worli, Bombay, and in 1981 published a key

study of mycobacterial lipids in skin biopsies from leprosy patients.

19

A relatively apolar

glycolipid (called I) was identied in skin samples from high bacillary index (BI, a measure

of the number of acid fast bacilli found in the dermis, usually the average from up to six

biopsy sites, based on a logarithmic scale from 0 at the polar tuberculoid end to 6 at the

polar lepromatous side of the clinical leprosy spectrum) lepromatous leprosy patients, absent

from normal skin samples and a collection of cultivable mycobacteria, but present in

armadillo-derived puried M. leprae. A sample of the M. leprae glycolipid I did contain

6-deoxyhexoses according to the classical Dische & Shettles

20

colorimetric assay. Glycolipid

I of Young

19

proved to be PGL-I as shown by the subsequent isolation and full charac-

terisation of PGL-I from human lepromatous nodules

21

and formalin-xed human

lepromatous liver.

22

CHEMICAL STRUCTURES OF NATIVE M. LEPRAE PGL- I , - I I , - I I I AND THE RELATED

DIM/PDIM

PGL-I occurs on the cell surface of M. leprae in copious amounts, representing up to 3%

of the total weight of the leprosy bacillus;

23

much of the PGL-I is loosely associated

with the bacillus and is sloughed off in the homogenate during processing. It is readily

extracted from the lyophilised infected armadillo liver or spleen homogenates from which

M. leprae whole cells had previously been puried. Hunter et al.

18

described in considerable

detail a protocol for the partial purication of PGL-I from this source and three alternatives

to its full purication. The present-day protocol, responsible for the pure PGL-I prepared

at Colorado State University and currently provided to the Biodefense and Emerging

Infections Research Resources Repository (BEI Resources, http://www.beiresources.org/

TBVTRMResearchMaterials/tabid/1431/Default.aspx) for distribution to leprosy researchers

worldwide, adheres closely to this protocol. Typically, yields of 22mg of pure PGL-I per g of

lyophilised residual tissue, which had also provided 9 10

10

acid-fastM. leprae per g, were

obtained. Armadillo liver and spleens with lower M. leprae titers do produce workable

quantities of PGL-I, but are heavily contaminated by host lipids, notably cholesterol. PGL-I is

very stable; 10 year old puried dried material stored at room temperature in the dark shows

no detectable degradation and no loss of serological reactivity in ELISA.

Obviously with such plentiful supplies of pure PGL-I available in the mid-1980s, full

structural elucidation was readily accomplished. Indeed, Brennan and Barrow

2

and Young

19

on the basis of elution prole and absence of amino acids (excluding C-mycosides/GPLs)

had concluded that the specic lipid of M. leprae may be mycosides of the A, B or G

J. S. Spencer and P. J. Brennan346

variety, or may be related to the glycolipid mycosides A and B from M. kansasii and

M. bovis (referring to mycobacterial lipids based on phthiocerol and known by their classical

names), which speculations proved to be correct.

Hunter and Brennan in 1981

3

rst corrected earlier false

2

impressions on the sugar

composition of PGL-I. They established that it had an inherent trisaccharide composed of

3-O-methyl-rhamnose, 2,3-di-O-methyl-rhamnose and 3,6-di-O-methyl-glucose glycosidi-

cally linked to a phenol substituent; the latter sugar, dominant epitope of the trisaccharide

entity of PGL-I, was never before found in nature. The full structure of PGL-I was reported

in 1982 by Hunter et al.

24

: partial acid hydrolysis, permethylation,

1

H NMR and

13

C NMR

established the sequence:

3,6-di-O-Me-Glcp(b1! 4)2,3-di-O-Me-Rhap(a1! 2)3-O-Me-Rhap(a1! phenol)

Acid hydrolysis of deacylated PGL-I yielded a phenolic phthiocerol and mass spectro-

metry (MS) and proton NMR of the permethylated compound demonstrated the structure:

OCH

3

|

HO-Phenyl-CH

2

-(CH

2

)

17

-CH-CH

2

-CH-(CH

2

)

4

-CH-CH-CH

2

-CH

3

|||

OH OH CH

3

Combined gas chromatography-mass spectrometry (GC-MS) showed three tetra-methyl

branched mycocerosic acids, C

30

, C

32

, and C

34

alternatively esteried to the two hydroxyl

functions of the branched phthiocerol chain. Thus, the complete elucidated structure of PGL-I

was shown to be:

29-{4-[O-(3,6,-di-O-methyl-b-D-glucopyranosyl)-(1 ! 4)-O-(2,3-di-O-methyl-a-

L-rhamnopyranosyl)-(1 ! 2)-3-O-methyl-a-L-rhamnopyranosyloxy]phenyl}-3-methoxy-

4-methyl-9,11-non-acosanediol 9,11-dimycocerosate (Figure 1).

Subsequently, Hunter and Brennan

25

discovered two other minor phenolic glycolipids,

apparent autolytic products of PGL-I, one of which, PGL-III, was chemically dened; it

simply lacked the 3-O-methyl substituent of the terminal 3,6-di-O-methyl-glucose of PGL-I.

An important outcome of this study

25

was the denition of the phthiocerol dimycerosate

(known as both DIM and PDIM, dimycocerosyl phthiocerol/phthioceryl dimycocerosate) of

M. leprae as consisting of a mixture of two phthiocerol homologues, 3-methoxyl-4-methyl-

9,11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9,11-dihydroxytriacontane

OCH

3

|

CH

3

-(CH

2

)

n

-CH-CH

2

-CH-(CH

2

)

4

-CH-CH-CH

2

CH

3

| | |

OH OH

CH

3

n 16 or 18 and the hydroxyl functions are acylated by a mixture of three mycocerosic

acids, 2,4,6,8-tetramethylhexacosanoate, 2,4,6,8-tetramethyloctacosanoate and 2,4,6,8-tetra-

methyltriacontanoate. These largely extracellular phthiocerol containing lipids exist in

amounts well in excess of the bacterial mass, estimated at more than 138mg in 1 g of liver,

wet weight, containing 37 10

10

M. leprae bacilli. The implications for the biology of

Mycobacterium leprae Phenolic Glycolipid I 347

leprosy of a bacillus within a copious environment of exotic lipids of its own making has

never been thoroughly explored.

IDENTIFYING THE IMMUNOLOGIC EPITOPE OF PGL- I

Polyclonal rabbit antisera raised against M. leprae whole cells and pooled sera from

lepromatous leprosy patients reacted strongly with both intact puried PGL-I and the

deacylated form derived from alkaline hydrolysis,

46

whereas healthy control sera or serum

from individuals infected with M. tuberculosis or other atypical mycobacterial infections

were uniformly negative. Reactivity to a structurally closely related triglycosylphenolic

diacylphthiocerol puried from M. kansasii (mycoside A), the monoglycosylphenolic

diacylphthiocerol puried from M. bovis BCG (mycoside B), and a panel of glyco-

peptidolipids (GPLs) isolated from different members of the M. avium-M. intracellulare-M.

scrofulaceum (MAIS) complex that contain short type-specic tetra- or trisaccharide

antigenic determinants were not cross-reactive with the rabbit or leprosy patient sera.

5

The dissected puried components of PGL-I, including the phenolic phthiocerol core, the

mycocerosic acids, and deglycosylated PGL-I also showed no reactivity, indicating that the

reactive component resided within the trisaccharide moiety. Syntheses of the trisaccharide,

26,

27

the terminal disaccharide,

2629

and a number of incompletely O-methylated analogs were

H

3

C

O

H

3

C

H

3

CO

H

3

CO

H

3

CO

OCH

3

H

3

CO

HO

HO

OH

CH

3

(CH

2

)

18

O

O

O

O

O

O

O

O

O

O

Figure 1. Structure of PGL-I showing C32 mycocerosic acid; C30 and C34 are also found (gure courtesy of

Dr. Michael McNeil at Colorado State University).

J. S. Spencer and P. J. Brennan348

used in inhibition assays to eventually show that all of the exquisite specicity and

recognition by leprosy patient anti-PGL-I polyclonal IgM antibodies and mouse monoclonal

antibodies

30

were directed against the terminal disaccharide, mainly towards the nonreducing

3,6-di-O-methyl-b-D-glucopyranosyl moiety, with a specic requirement for both the 3- and

6-O-methyl substituents. These studies demonstrated that the trisaccharide structure is unique

and specic forM. leprae PGL-I, the reason for its utility as a reagent to assist in the diagnosis

of leprosy or categorising patients based on anti-PGL-I titer to make better decisions on

treatment regimens.

DETECTION OF PGL- I ANTIGEN IN SERUM FROM LEPROSY PATIENTS

Soon after the method of purifying PGL-I from infected armadillo tissues was described,

similar methods showed that PGL-I was extractable from a number of biological specimens

from leprosy patients, including skin lesions,

31

serum samples

32,33

and urine.

34

Detection of

PGL-I in serum samples was quite simple; drying as little as 100ml of serum onto a lter

paper disc and extracting lipid material with CHCl

3

/CH

3

OH, 2:1 followed by fractionation on

small silicic acid columns. PGL-I antigen extracted from serum samples was readily

identied by dot-blot ELISA using rabbit polyclonal anti-PGL-I antiserum or monoclonal

antibody,

35

methods that had greater sensitivity than using TLC or high-pressure liquid

chromatography. Untreated lepromatous leprosy patients classied as BL or LL according

to the Ridley-Jopling classication system

36

were positive for serum PGL-I detection at

between 88%

37

and 96%.

38

The levels of PGL-I in the serum correlated with the BI, with the

highest levels detected in multibacillary (MB) individuals with diffuse skin inltration and

skin nodules, and polar lepromatous individuals with a BI.50, concentrations which ranged

from 1 to 32mg of PGL-I per ml. As the BI decreased, the ability to detect PGL-I in individual

samples was lower, with less than half of those MB patients with a BI of,31 being positive,

and no detection in TT/BT individuals with a BI of 0. PGL-I levels did not vary signicantly

with differences in the duration of pre-existing disease, with the disability index, or in

those patients who experienced Type 2 ENL (erythema nodosum leprosum) reactions after

beginning MDT.

37

Monitoring the serum levels of PGL-I after initiating multidrug therapy (MDT) was

proposed as a means to ascertain the efcacy of drug treatment, since the active synthesis and

release of PGL-I was shown to be a marker of M. leprae viability when metabolically

maintained in vitro.

39,40

Following the rst administration of MDT, levels of serum PGL-I

in patients showed a dramatic decline in concentration, likely reecting the rapid killing of

bacilli and cessation of new PGL-I synthesis. Successful treatment generally gave rise to

low circulating PGL-I antigen (less than 100 ng/ml) within 12 months of MDT, even in

individuals with the highest BI. Although the BI detected in skin lesions of patients decreases

relatively slowly at a rate of 0610 per year with effective chemotherapy, none of the serum

samples obtained from any patient treated for at least 18 months had measurable levels of

PGL-I antigen.

Despite the initial promise of using PGL-I antigen detection to monitor successful MDT

treatment of MB leprosy patients, this method was not applicable for most individuals with

a BI under 30 or for PB patients, and the purication of PGL-I from serum samples

is somewhat labour intensive. Eventually, this technique fell by the wayside in favour of

detecting serum antibodies to either PGL-I or other M. leprae antigens.

Mycobacterium leprae Phenolic Glycolipid I 349

DETECTION OF PGL- I ANTIBODY IN SERUM FROM LEPROSY PATIENTS

After the initial purication and characterisation of M. leprae PGL-I, it was determined that

the most immunogenic portion of the glycolipid resided with the novel trisaccharide attached

to the phenolic residue. As had been shown earlier with the blood group antigens and the

type-specic GPLs of the MAIS complex, antisera are readily raised that can differentiate

subtle structural differences in their oligosaccharide haptens. It was shown early on that

individuals with a high BI reective of a high bacillary load almost universally showed a high

titer of IgM antibodies to PGL-I,

41

which were almost exclusively directed against the

terminal disaccharide. The fact that the antibody response to PGL-I was mainly of the IgM

class indicates the T cell independent nature of the response to this glycolipid antigen, unlike

the predominant IgG response to the major M. leprae carbohydrate antigen, lipoarabino-

mannan (LAM).

4244

Attempts to develop serological assays using native PGL-I were at rst

problematic due to the apolar nature of the puried glycolipid and its lack of solubility in

aqueous buffers commonly used in immunodiffusion or ELISA assays. This issue of

solubility was overcome initially by incorporating native PGL-I into liposomes, which could

then be shown to form a reactive precipitate by Ouchterlony gel immunodiffusion.

4

Young and Buchanan

6

partially solved the problem by deacylation of PGL-I, i.e. removal of

the mycocerosic acids with alkali. Sonication of native PGL-I in phosphate buffered saline

containing 1mg/ml of the detergent sodium deoxycholate enabled the antigen to be

efciently coated onto microtiter plate wells in a PGL-I ELISA assay that reacted with

leprosy patient sera.

45

It was later determined that the use of the detergent Tween, commonly

used in buffers in ELISA assays, was problematic, as the detergent interacted with the lipid

portion of the molecule and caused its detachment from the plastic of the ELISA plate wells.

This problem was alleviated by avoiding the use of detergents in all blocking and wash

buffers, and by increasing the concentration of bovine serum albumin to 3% in all buffers

used throughout the procedure. In addition, it was determined that PGL-I solubilised in 100%

ethanol and dried overnight onto ELISA plate wells was rmly immobilised onto the plastic;

this is now an aspect of the routine procedure. Using this detergent-free protocol, ELISA

assays were developed using polyclonal or monoclonal reagents that were shown to be highly

specic to the sugar entities of PGL-I, and amenable to the detection of the anti-PGL-I titer in

leprosy patients or household contacts. This development provided the ability for the rst

time to perform routine screens of populations in high prevalence areas to gain knowledge of

the clinical and epidemiological signicance of detectable antibody titer to this antigen,

which allowed an assessment of the potential risk that this posed in eventual progression from

infection to disease.

46

DEVELOPMENT OF SYNTHETIC PGL-I NEOGLYCOCONJUGATES

Upon denition of the trisaccharide structure of PGL-I, a number of laboratories developed

synthetic strategies for the production of the terminal monosaccharide and disaccharide

haptens or the entire trisaccharide to allow for chemical coupling to water soluble carrier

molecules such as bovine or human serum albumin (BSA, HSA); such polyvalent structures

had the advantage of multiple hapten substitutions (up to 40) on each polypeptide backbone,

and, being water soluble, were amenable to the development of assays more facile than

conventional ELISA. The rst of these neoantigens e-N-1-[1-deoxy-2,3-di-O-methyl-4-O-

(3,6-di-O-methyl-b-D-glucopyranosyl)rhamnitol]-lysyl-BSA - essentially a coupling of

J. S. Spencer and P. J. Brennan350

the synthetic terminal disaccharide to the lysine residues in the BSA backbone by reductive

amination, proved highly sensitive in ELISA and showed good concordance with the

native glycolipid in analysis of serum samples from leprosy patients.

47

A second generation

of products, one of which is still being generated by the Colorado group and named

ND-O-BSA/HSA (natural disaccharide-octyl-BSA or -HSA), involved synthesis of

the terminal monosaccharide, disaccharide and indeed the entire trisaccharide but as the

8-methoxy-carbonyloctyl sugar(s)

4850

in order to provide a linker arm, which, by using

the strategy of Lemieux et al.

51

allowed attachment to the e-amino groups of the lysines on

the polypeptide backbone, and provided distance between the reactive hapten and the

polypeptide. Another generation of products pioneered by Fujiwara,

52,53

chose methyl

3( p-hydroxyphenyl) propionate as the linker arm, since, in the native PGL-I, the

p-hydroxylphenyl group may contribute to the requirements for evocation of and binding to

anti-glycolipid antibodies. Indeed, Fujiwara still produces for the research community the

trisaccharide segment of PGL-I in the form of the p-(2-methoxycarbonylethyl)phenyl

glycoside coupled to BSA by the acyl azide method, which he terms NT-P-BSA (natural

trisaccharide-propyl-BSA). Gigg et al.

28

had previously produced the terminal disaccharide

carrying the allyl linker arm and Brett et al.

45

described the coupling of such a disaccharide to

BSA generating the glycoconjugate with the propyl group. Comparative serological testing in

ELISA of NT-O-BSA, ND-O-BSA and NT-P-BSA against sera from leprosy patients and

control populations showed concordance; the presence of the innermost sugar or the phenyl

group apparently did not contribute signicantly to sensitivity or specicity.

50

USE OF SYNTHETIC GLYCOCONJUGATES IN TESTS TO ASSESS PGL- I ANTIBODY

IN LEPROSY ENDEMIC AREAS

With the development of these di- and trisaccharide synthetic neoglycoconjugates, there

followed a number of assay formats and devices amenable to the testing of individuals at risk

in leprosy endemic areas. The simplest of these is a lateral ow device that contains a

nitrocellulose detection strip that has two 1mm wide lines deposited in parallel, one with the

neoglycoconjugate to detect human IgM antibodies to PGL-I (the test line, T), and the other

containing human IgM as a positive control line (C). The nitrocellulose strip is anked by a

reagent pad that can receive a serum or whole blood sample with diluent, which is wicked

towards the nitrocellulose by an absorbent pad at the opposite end. The nitrocellulose

detection strip and anking pads are encased in a plastic support with a sample application

port and an open rectangular viewing area over the test and control lines. As the sample

travels towards the nitrocellulose, it picks up a colloidal gold-labeled anti-human IgM reagent

that specically binds to human IgM antibody and gives a positive reaction to the control

and/or test lines. Samples that contain anti-PGL-I antibodies will display two visible lines,

one being the positive test line against the neoglycoconjugate which is semiquantitative,

varying in intensity depending on the anti-PGL-I titer (Figure 2); those without any detectable

antibodies develop a single positive control line.

The results are rapid, being easily read in about 10 minutes, and can be interpreted by

individuals with minimal training, all of which are well-suited for eld use in resource limited

settings. In an evaluation of one type of anti-PGL-I lateral ow device, the ML Flow test,

there was agreement in detecting a positive test between a standard ELISA assay and the ML

Flow 91% of the time, with the ability to detect a positive reaction in 974% of MB leprosy

patients, 40% of PB patients, and 286% of household contacts.

54

The specicity of this test

Mycobacterium leprae Phenolic Glycolipid I 351

was 902%, which was very good considering that the controls included a sizable number of

healthy individuals and those with other skin diseases, including Buruli ulcer, from three

different endemic countries. It was found that these tests were useful in the correct

classication of MB versus PB individuals after diagnosis,

55

as in general, those with high

levels of anti-PGL-I antibody had a correspondingly high bacillary load, while those lacking

antibodies were likely to have a negative BI.

56

Other simple tests that preceded the development of the lateral ow test include a simple

dipstick and a particle agglutination test. The ML Dipstick was developed as a simple format

that could assist in the classication of conrmed leprosy patients under eld conditions.

57,58

The dipstick was coated with two bands, one containing ND-O-BSA and the other a control

anti-human IgM. It was incubated with whole blood or serum mixed with diluent containing

the detection reagent, anti-human IgM coupled to colloidal gold, with reactivity to the

ND-O-BSA band indicating a positive reaction. The dipsticks and reagents were shown to be

stable under tropical eld conditions of heat and humidity, positives could be easily

distinguished by minimally trained staff, and the concordance, sensitivity and specicity of

the dipstick with the ELISA assay showed consistently high agreement at various cutoff

values. Another simple test, the gelatin particle agglutination test, was developed by

activating gelatin particles with tannic acid, followed by mixing with NT-P-BSA.

59

Sensitised particles mixed with serial two-fold dilutions of serum in U-shaped wells were

observed for end point agglutination, which could easily be discerned by visual examination,

with cut-offs for positivity generally being between 1:64 and 1:128 serum dilutions.

The sensitised particles could be lyophilized for stable long-term storage and reconstituted

for use. The concordance rates between particle agglutination and the indirect ELISA assay

was generally .90% in all groups tested, including leprosy patients and their contacts,

Figure 2. Lateral ow device to detect anti-PGL-I antibody reactivity in leprosy patient serum samples. C, control

line; T, test line. Positive reactive serum shown on the left with a strong band at the T line; negative pattern on the

device on the right. Courtesy of Dr. Sang-Nae Cho, Yonsei University College of Medicine, Seoul, Korea.

J. S. Spencer and P. J. Brennan352

TB patients and healthy controls. Thus, these tests have been reliably used to categorise

those already diagnosed with leprosy for the purposes of dening treatment regimens and

identifying those contacts of index cases most at risk of developing this disease based on

a positive anti-PGL-I test.

ROLE OF PGL- I IN THE INFECTIVITY OF SCHWANN CELLS AND THE IMMUNE

RESPONSE

M. leprae displays a characteristic tropism for peripheral nerves, and as a result of Schwann

cell (SC) invasion, initiates a process that eventually destroys the functional integrity of

the nerve, which is the leading cause of neuropathy, disgurement and disability in

individuals with leprosy. Myelinated and non-myelinated nerves have associated SC-axon

units that are surrounded by a basal lamina, and a number of mechanisms have been

proposed for how M. leprae binds to and enters the SC.

60

PGL-I has been shown to bind

specically to laminins, which are glycoproteins that are involved in the assembly of the

basement membrane in the basal lamina. The specic interaction of PGL-I with laminin was

shown by binding assays to be directed towards the laminin-2 domain, while there was no

binding to other human matrix proteins, such as collagen, bronectin, or heparan sulfate

proteoglycan.

61

Removal of the trisaccharide portion of PGL-I, but not removal of the long-

chain mycocerosic acid residues, abrogated the ability of PGL-I to bind to laminin-2,

suggesting that the unique sugar residues are the reason for nerve tropism. Once the SCs have

been invaded, they seem to lack the ability to kill intracellular bacilli, and large numbers

of bacteria proliferate within these cells and macrophages within the peripheral nerves.

M. leprae appears to be able to perturb the lipid homeostasis of infected cells resulting in the

formation of cytoplasmic organelles known as lipid bodies (LB),

62

which are primarily

responsible for the appearance of foamy macrophages in lesion sites found in lepromatous

leprosy but not tuberculoid lesions, rst described by Virchow in 1863.

63

The lipids in these

vesicles are mainly host-derived, but their formation is an active process that requires viable

bacilli. Recent studies showed that M. leprae-induced LB biogenesis correlated with

increased prostaglandin E

2

(PGE

2

, a potent immune modulator shown to downregulate Th1

responses and bactericidal activity towards intracellular pathogens) and IL10 and decreased

IL-12 and nitric oxide production in infected SCs,

64

conditions that would favour survival

of the bacteria. Inhibition of biogenesis by a fatty acid synthase inhibitor abolished this effect

and enhanced the ability of SCs to kill intracellular bacilli. It appears that LB formation

creates intracellular conditions favourable to the survival and replication of M. leprae.

The bacilli likely use these LBs as a nutritional source, and accumulation of LBs in infected

SCs generates an innate immune response that allows for permissive growth of the bacilli

within the nerve. PGL-I has been shown to play an important role in downregulating the

inammatory immune response, inhibits dendritic cell maturation and activation, facilitates

entry of bacilli into macrophages and SCs, and scavenges potentially cytocidal oxygen

metabolites in vitro, all of which would promote the survival of intracellular bacilli.

6569

The role of PGL-I is likely crucial to the ability of M. leprae to invade, survive and

proliferate in the hostile intracellular environment.

Mycobacterium leprae Phenolic Glycolipid I 353

THE FUTURE OF PGL-I IN THE SERODIAGNOSIS AND IMMUNOPATHOGENESIS OF

LEPROSY

Although individuals who test positive for anti-PGL-I antibodies have about an 8-fold higher

risk to develop leprosy,

70

screening for PGL-I antibodies in the general population is not

useful, because not every person who develops a positive anti-PGL-I titer will progress to a

diseased state,

71

and the vast majority of active or potential PB cases are negative for PGL-I

antibody. Nevertheless, an assessment of anti-PGL-I antibody titer among contacts would aid

in the identication of those positive individuals who may be most at risk of developing

the disease, which would allow for better follow-up and reduce the level of transmission.

In addition, the test is valid to classify newly diagnosed leprosy patients for the purpose of

providing the correct treatment regimen. In combination with PGL-I, specic reactivity

against M. leprae recombinant protein antigens ML0405 and ML2331, which have been

engineered into a fusion protein called LID-1, has shown promise in the development of a

tool for the assessment of treatment efcacy and disease relapse,

72

and may be more

effective at the PB end of the disease spectrum. Despite the limited availability of rapid tests

due to the lack of interest from industry, a number of governmental health organisations

within countries where leprosy prevalence is high have expressed an interest in providing

resources for the development and use of these tests for screening those found to be at risk

for coming down with leprosy. Regardless, serology as such will always have limited

application in the diagnosis of early leprosy on account of the requirement of measurable

quantities of antibodies, themselves synonymous with lepromatous leprosy, otherwise

readily amenable to diagnosis. Attention nowadays has turned towards diagnostics based on

T-cell responses to novel M. leprae antigens,

73

with the potential to detect the earliest

evidence of M. leprae infection. An equally pressing but more intractable question, the role

of the copious phthiocerol-based lipids in the particular immunopathogenesis of leprosy may

have received a boost from recent developments. Arising from knowledge of the genome

sequence of several isolates/strains of M. leprae, the underlying genetics and enzymology

of PDIM and PGL-I biosynthesis is now understood.

74,75

Consequently Mycobacterium

bovis BCG has now been engineered to express PGL-I

68

such that questions on the specic

role of PGL-I, anchored on a living mycobacterium, in disease onset and progression, can

be now addressed.

Acknowledgements

Support from the National Institute of Allergy and Infectious Diseases/NIH through contract

N01-AI-25469 and grant R37-AI-18357 over a 30 year period. More recently, the IDEAL

(Initiative for Diagnostic and Epidemiological Assays for Leprosy) Consortium has supported

our research on leprosy diagnostics.

References

1

Kirchheimer WF, Storrs EE. Attempts to establish the armadillo (Dasypus novemcinctus Linn.) as a model for the

study of leprosy. I. Report of lepromatoid leprosy in an experimentally infected armadillo. Int J Lepr, 1971; 39:

693702.

J. S. Spencer and P. J. Brennan354

2Brennan PJ, BarrowWW. Evidence for species-specic lipid antigens inMycobacterium leprae. Int J Lepr, 1980;

48: 382387.

3

Hunter SW, Brennan PJ. A novel phenolic glycolipid from Mycobacterium leprae possibly involved in

immunogenicity and pathogenicity. J Bacteriol, 1981; 147: 728735.

4

Payne SN, Draper P, Rees RJW. Serological activity of puried glycolipid fromMycobacterium leprae. Int J Lepr,

1982; 50: 220221.

5

Cho S-N, Yanagihara DL, Hunter SW et al. Serological specicity of phenolic glycolipid I from Mycobacterium

leprae and use in serodiagnosis of leprosy. Infect Immun, 1983; 41: 10771083.

6

Young DB, Buchanan TM. A serological test for leprosy with a glycolipid specic for Mycobacterium leprae.

Science, 1983; 221: 10571059.

7

Aspinall GO, Chatterjee D, Brennan PJ. The variable surface glycolipids of mycobacteria: Structures, synthesis of

epitopes, and biological properties. In: Horton D (ed). Advances in Carbohydrate Chemistry and Biochemistry.

Academic Press, San Diego, CA, 1995; pp. 169242.

8

Brennan PJ, Goren MB. Structural studies on the type-specic antigens and lipids of the Mycobacterium avium-

Mycobacterium intracellulare-Mycobacterium scrofulaceum serocomplex. J Biol Chem, 1979; 254: 42054211.

9

Brennan PJ, Aspinall GO, Nam Shin JE. Structure of the specic oligosaccharides from the glycopeptidolipid

antigens of serovars in the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum

serocomplex. J Biol Chem, 1981; 256: 68176822.

10

Hunter SW, Murphy RC, Clay K et al. Trehalose-containing lipooligosaccharides. J Biol Chem, 1983; 258:

1048110487.

11

Chatterjee D, Bozic CM, Knisley C et al. Phenolic glycolipids of Mycobacterium bovis: new structures and

synthesis of a corresponding seroreactive neoglycoprotein. Infect Immun, 1989; 57: 322330.

12

Daffe

M, Lacave C, Laneelle M-A, Laneelle G. Structure of the major triglycosyl phenol-phthiocerol of

Mycobacterium tuberculosis (strain Canetti). Eur J Biochem, 1987; 167: 144160.

13

Daffe

M, Cho S-N, Chatterjee D, Brennan PJ. Chemical synthesis and seroreactivity of a neoantigen containing

the oligosaccharide hapten of the Mycobacterium tuberculosis-specic phenolic glycolipid. J Inf Dis, 1991; 163:

161168.

14

Fournie J-J, Riviere M, Puzo G. Structural elucidation of the major phenolic glycolipid from Mycobacterium

kansasii. I. Evidence for tetrasaccharide structure of the oligosaccharide moiety. J Biol Chem, 1987; 262:

31743179.

15

Caldwell HD, Kirchheimer WF, Buchanan TM. Identication of a Mycobacterium leprae specic protein

antigen(s) and its possible application for the serodiagnosis of leprosy. Int J Lepr, 1979; 47: 477483.

16

Draper P. Protocol 1/79: Purication ofM. leprae. Annex 1 to the Report of the Enlarged Steering Committee for

Research on the Immunology of Leprosy (IMMLEP) Meeting of 78 February. World Health Organization,

Geneva, 1979; p. 4.

17

Crowle AJ. Immunodiffusion. 2nd edn., Academic Press, New York, 1973; pp. 247303.

18

Hunter SW, Stewart C, Brennan PJ. Purication of phenolic glycolipid from armadillo and human sources.

Int J Lepr, 1985; 53: 484486.

19

Young DB. Detection of mycobacterial lipids in skin biopsies from leprosy patients. Int J Lepr, 1981; 49:

198204.

20

Dische Z, Shettles LB. A specic color reaction of methylpentoses and a spectrophotometric micromethod for

their determination. J Biol Chem, 1948; 175: 595603.

21

Vemuri N, Khandke L, Mahadevan PR et al. Isolation of phenolic glycolipid I from human lepromatous nodules.

Int J Lepr, 1985; 53: 487489.

22

Izumi S, Sugiyama K, Fujiwara T, et al. Isolation of the Mycobacterium leprae-specic glycolipid antigen,

phenolic glycolipid-I, from formalin-xed human lepromatous liver. J Clin Microbiol, 1985; 22: 680682.

23

Gaylord H, Brennan PJ. Leprosy and the leprosy bacillus: recent developments in characterization of antigens and

the immunology of the disease. Ann Rev Microbiol, 1987; 41: 645675.

24

Hunter SW, Fujiwara T, Brennan PJ. Structure and antigenicity of the major specic glycolipid antigen of

Mycobacterium leprae. J Biol Chem, 1982; 257: 1507215078.

25

Hunter SW, Brennan PJ. Further specic extracellular phenolic glycolipid antigens and a related diacylphthiocerol

from Mycobacterium leprae. J Biol Chem, 1983; 258: 75567562.

26

Fujiwara T, Hunter SW, Cho S-N et al. Chemical synthesis and serology of disaccharides and trisaccharides

of phenolic glycolipid antigens from the leprosy bacillus and preparation of a disaccharide protein conjugate

for serodiagnosis of leprosy. Infect Immun, 1984; 43: 245252.

27

Fujiwara T, Aspinall GO, Hunter SW, Brennan PJ. Chemical synthesis of the trisaccharide unit of the species-

specic phenolic glycolipid from Mycobacterium leprae. Carbohydrate Res, 1987; 163: 4152.

28

Gigg R, Payne S, Conant R. The allyl group for protection in carbohydrate-chemistry. 14. Synthesis of 2,3-di-O-

methyl-4-O-(3,6,-di-O-methyl-b-D-glycopyranosyl)-L-rhamnopyranose (and its a-propyl glycoside) a haptenic

portion of the major glycolipid from Mycobacterium leprae. J Carbohydr Chem, 1983; 2: 207223.

29

Fujiwara T, Hunter SW, Brennan PJ. Chemical synthesis of disaccharides of the specic phenolic glycolipid

antigens from Mycobacterium leprae and of related sugars. Carbohydr Res, 1986; 148: 287298.

Mycobacterium leprae Phenolic Glycolipid I 355

30

Young DB, Khanolkar SR, Barg LL, Buchanan TM. Generation and characterization of monoclonal antibodies

to the phenolic glycolipid of Mycobacterium leprae. Infect Immun, 1984; 43: 183188.

31

Venkatesan K, Singh HS, Bhardwaj VP, Ramu G. Isolation, purication and quantication of phenolic glycolipid-

1 from human leprosy skin tissues. Trans R Soc Trop Med Hyg, 1985; 82: 321323.

32

Young DB, Harnisch JP, Knight J, Buchanan TM. Detection of phenolic glycolipid I in sera from patients with

lepromatous leprosy. J Infect Dis, 1985; 152: 10781081.

33

Cho S-N, Hunter SW, Gelber RH et al. Quantitation of the phenolic glycolipid of Mycobacterium leprae and

relevance to glycolipid antigenemia in leprosy. J Infect Dis, 1986; 153: 560569.

34

Kaldany RR, Maasho K, Ohman R et al. Methods of detection of a specic Mycobacterium leprae antigen in

the urine of leprosy patients. Scand J Immunol, 1987; 25: 3743.

35

Cho S-N, Shin J-S, Daffe

M et al. Production of monoclonal antibody to phenolic glycolipid of Mycobacterium

tuberculosis and its use in detection of the antigen in clinical isolates. J Clin Microbiol, 1992; 30: 30653069.

36

Ridley DS, Jopling WH. Classication of leprosy according to immunity. A ve group system. Int J Lepr, 1966;

34: 255273.

37

Cho S-N, Cellona RV, Fajardo TT et al. Detection of phenolic glycolipid-I antigen and antibody in sera from

new and relapsed lepromatous patients treated with various drug regimens. Int J Lepr, 1991; 59: 2531.

38

Roche PW, Britton WJ, Neupane KD et al. The response to chemotherapy of serum Mycobacterium leprae-

specic antigen in multibacillary leprosy patients. Am J Trop Med Hyg, 1991; 44: 702708.

39

Franzblau SG, Harris EB, Hastings RC. Axenic incorporation of [

14

C] palmitic acid into the phenolic glycolipid-I

of Mycobacterium leprae. FEMS Microbiol Lett, 1989; 48: 407411.

40

Mistry Y, Antia NH, Mukherjee R. Correlation of bacterial viability with uptake of [

14

C] acetate into phenolic

glycolipid-1 of Mycobacterium leprae within Schwannoma cells. J Biosci, 1989; 14: 3745.

41

Young DB, Dissanayake S, Miller RA et al. Humans respond predominantly with IgM immunoglobulin to the

species-specic glycolipid of Mycobacterium leprae. J Inf Dis, 1984; 149: 870873.

42

Hunter SW, Gaylord H, Brennan PJ. Structure and antigenicity of the phosphorylated antigens from the leprosy

and tubercle bacilli. J Biol Chem, 1986; 261: 1234512351.

43

Gaylord H, Brennan PJ, Young DB, Buchanan TM. MostMycobacterium leprae carbohydrate-reactive antibodies

are directed to lipoarabinomannan. Infect Immun, 1987; 55: 28602863.

44

Sousa AO, Henry S, Maro

ja FM et al. IgG subclass distribution of antibody responses to protein and

polysaccharide mycobacterial antigens in leprosy and tuberculosis patients. Clin Exp Immunol, 1998; 111: 4855.

45

Brett SJ, Draper P, Payne SN, Rees RJW. Serological activity of a characteristic phenolic glycolipid from

Mycobacterium leprae in sera from patients with leprosy and tuberculosis. Clin Exp Immunol, 1983; 52: 271279.

46

Douglas JT, Cellona RV, Fajardo TT et al. Prospective study of serological conversion as a risk factor for

development of leprosy among household contacts. Clin Diagn Lab Immunol, 2004; 11: 897900.

47

Cho S-N, Fujiwara T, Hunter SW et al. Use of an articial antigen containing the 3,6-di-O-methyl-

b-D-glycopyranosyl epitope for the serodiagnosis of leprosy. J Infect Dis, 1984; 150: 311322.

48

Chatterjee D, Douglas JT, Cho S-N et al. Synthesis of neoglycoproteins containing the 3,6-di-O-methyl-

b-D-glucopyranosyl epitope and their use in serodiagnosis of leprosy. Glycoconjugate J, 1985; 2: 187208.

49

Chatterjee D, Cho S-N, Brennan PJ. Chemical synthesis and seroreactivity of O-(3,6-di-O-methyl-

b-D-glucopyranosyl)-(1! 4)-O(2,3-di-O-methyl-a-L-rhamnopyranosyl)-(1! 9)-oxynonanoyl-bovine serum

albumin The leprosy-specic, natural disaccharide-octyl-neoglycoprotein. Carbohydr Res, 1986; 156: 3956.

50

Chatterjee D, Cho S-N, Stewart C et al. Synthesis and immunoreactivity of neoglycoproteins containing the

trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae. Carbohydr Res, 1988; 183: 241260.

51

Lemieux RU, Baker DA, Bundle DR. A methodology for the production of carbohydrate-specic antibody.

Can J Biochem, 1977; 55: 507512.

52

Fujiwara T, Izumi S, Brennan PJ. Synthesis of 3,6-di-O-methylglucosyl disaccharides with methyl

3-(p-hydroxyphenyl)propionate as a linker and their use in the serodiagnosis of leprosy. Agric Biol Chem,

1985; 49: 23012308.

53

Fujiwara T, Izumi S. Synthesis of the neoglycoconjugates of phenolic glycolipid-related trisaccharides for the

serodiagnosis of leprosy. Agric Biol Chem, 1987; 51: 25392547.

54

Bu

hrer-Se

kula S, Smits HL, Gussenhoven GC et al. Simple and fast lateral ow test for classication of leprosy

patients and identication of contacts with high risk of developing leprosy. J Clin Microbiol, 2003; 41:

19911995.

55

Buhrer-Se

kula S, Sarno EN, Oskam L et al. Use of ML dipstick as a tool to classify leprosy patients. Int J Lepr,

2000; 68: 456463.

56

Klatser PR, Cho SN, Brennan PJ. The contribution of serological tests to leprosy control. Int J Lepr, 1996; 64:

S63S66.

57

Buhrer-Se

kula S, Cunha MGS, Ferreira WA, Klatser PR. The use of whole blood in a dipstick assay for detection

of antibodies to Mycobacterium leprae: a eld evaluation. FEMS Immunol Med Microbiol, 1998; 21: 197201.

58

Bu

hrer-Se

kula S, Cunha MG, Foss NT et al.Dipstick assay to identify leprosy patients who have an increased risk

of relapse. Trop Med Int Health, 2001; 6: 317323.

J. S. Spencer and P. J. Brennan356

59

Izumi S, Fujiwara T, Ikeda M et al. Novel gelatin particle agglutination test for serodiagnosis of leprosy in the

eld. J Clin Microbiol, 1990; 28: 525529.

60

Scollard DM, Adams LB, Gillis TP et al. The continuing challenges of leprosy. Clin Microbiol Rev, 2006; 19:

338381.

61

Ng V, Zanazzi G, Timpl R et al. Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of

Mycobacterium leprae. Cell, 2000; 103: 511524.

62

Mattos KA, DAvila H, Rodrigues LS et al. Lipid droplet formation in leprosy: Toll-like receptor-regulated

organelles involved in eicosanoid formation andMycobacterium leprae pathogenesis. J Leukocyte Biol, 2010; 87:

371384.

63

Virchow R. Die krankhaften Geschwu

lste. August Hirschwald, Berlin, 1863.

64

Mattos KA, Oliveira VG, DAvila H et al. TLR6-driven lipid droplets inMycobacterium leprae-infected Schwann

cells: Immunoinammatory platforms associated with bacterial persistence. J Immunol, 2011; In press.

65

Schlesinger LS, Horwitz MA. Phenolic glycolipid-I of Mycobacterium leprae binds complement component C3

in serum and mediates phagocytosis by human monocytes. J Exp Med, 1991; 174: 10311038.

66

Murray RA, Siddiqui MR, Mendillo M et al. Mycobacterium leprae inhibits dendritic cell activation and

maturation. J Immunol, 2007; 178: 338344.

67

Sinsimer D, Fallows D, Peixoto B et al. Mycobacterium leprae actively modulates the cytokine response in na

ve

human monocytes. Infect Immun, 2010; 78: 293300.

68

Tabouret G, Astarie-Dequeker C, Demangel C et al. Mycobacterium leprae phenolglycolipid-1 expressed by

engineeredM. bovis BCG modulates early interaction with human phagocytes. PLoS Pathog, 2010; 6: e1001159.

69

Chan J, Fujiwara T, Brennan PJ et al. Microbial glycolipids: possible virulence factors that scavenge oxygen

radicals. Proc Natl Acad Sci, 1989; 86: 24532457.

70

Van Beers SM, Hatta M, Klatser PR. Patient contact is the major determinant in incident leprosy: implications

for future control. Int J Lepr, 1999; 67: 119128.

71

Cunaman A, Jr, Chan GP, Douglas JT. Risk of development of leprosy among Culion contacts. Int J Lepr, 1998;

66: S78A.

72

Duthie MS, Hay MN, Rada EM et al. Specic IgG antibody responses may be used to monitor leprosy treatment

efcacy and as recurrence prognostic markers. Eur J Clin Microbiol Inf Dis, 2011; In press.

73

Geluk A, Spencer JS, Bobosha K et al., and on behalf of the IDEAL Consortium. From genome-based in silico

predictions to ex vivo verication of leprosy diagnosis. Clin Vaccine Immunol, 2009; 16: 352359.

74

Constant P, Pe

rez E, Malaga W et al. Role of the pks15/1 gene in the biosyntheisis of phenolglycolipids in the

Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl

esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene. J Biol Chem,

2002; 277: 3814838158.

75

Pe

rez E, Constant P, Lemassu E et al. Characterization of three glycosyltransferases involved in the biosynthesis

of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex. J Biol Chem, 2004; 279:

4257442583.

Mycobacterium leprae Phenolic Glycolipid I 357