The Role of Mycobacterium leprae Phenolic Glycolipid I (PGL-I) in ...

The Role of Mycobacterium leprae PhenolicGlycolipid 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 define 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, confirmation and management of lepromatous leprosy. PGL-I has

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

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

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

thereby facilitating the persistence of M. leprae/leprosy.

Introduction

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

the discovery in the early 1970’s 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 first 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) specific for M. leprae was first 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.3–6 The history of how native PGL-I

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

Lepr Rev (2011) 82, 344–357

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

was first discovered, purified, chemically characterised, the sugar epitopes identified 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-specific antigen, a glycolipid, came from two separate

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

success in defining the surface/cell wall species and serovar/serotype-specific 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;7–9 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. kansasii14 (in conjunction with theM. kansasii specific LOS (for a review, see [7]).

On the heels of the discovery that M. leprae could readily replicate in the armadillo1 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-specific 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

specific serodiagnosis of leprosy. A second contract was awarded to Pacific Medical Center

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

of M. leprae specific 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 19802 first 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 CHCl3-CH3OH to

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

of the classical Ouchterlony agar gel immunodiffusion technique17 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 identified 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 procedure18

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 identified 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 þ 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 purified M. leprae. A sample of the M. leprae glycolipid I did contain

6-deoxyhexoses according to the classical Dische & Shettles20 colorimetric assay. Glycolipid

I of Young19 proved to be PGL-I as shown by the subsequent isolation and full charac-

terisation of PGL-I from human lepromatous nodules21 and formalin-fixed 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 purified. Hunter et al.18 described in considerable

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

to its full purification. 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 2·2mg of pure PGL-I per g of

lyophilised residual tissue, which had also provided 9 £ 1010 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 purified 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 Barrow2 and Young19

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

had concluded that the specific 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 19813 first corrected earlier false2 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, 1H NMR and 13C 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:

OCH3|

HO-Phenyl-CH2-(CH2)17-CH-CH2-CH-(CH2)4-CH-CH-CH2-CH3|||

OH OH CH3

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

branched mycocerosic acids, C30, C32, and C34 alternatively esterified 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 Brennan25 discovered two other minor phenolic glycolipids,

apparent autolytic products of PGL-I, one of which, PGL-III, was chemically defined; 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 study25 was the definition 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

OCH3

|CH3-(CH2) n-CH-CH2-CH-(CH2)4-CH-CH-CH2–CH3

| | |OH OH CH3

n ¼ 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 1·38mg in 1 g of liver,

wet weight, containing 3·7 £ 1010 M. leprae bacilli. The implications for the biology of


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 purified PGL-I and the

deacylated form derived from alkaline hydrolysis,4–6 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 purified from M. kansasii (mycoside A), the monoglycosylphenolic

diacylphthiocerol purified 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-specific tetra- or trisaccharide

antigenic determinants were not cross-reactive with the rabbit or leprosy patient sera.5

The dissected purified 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,26–29 and a number of incompletely O-methylated analogs were

H3C

OH3C

H3CO

H3CO

H3COOCH3

H3CO

HO

HO

OH

CH3

(CH2)18

O

O

O

O

O

O

O

OO

O

Figure 1. Structure of PGL-I showing C32 mycocerosic acid; C30 and C34 are also found (figure courtesy ofDr. Michael McNeil at Colorado State University).


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

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

antibodies30 were directed against the terminal disaccharide, mainly towards the nonreducing

3,6-di-O-methyl-b-D-glucopyranosyl moiety, with a specific requirement for both the 3- and6-O-methyl substituents. These studies demonstrated that the trisaccharide structure is unique

and specific 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 samples32,33 and urine.34 Detection of

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

paper disc and extracting lipid material with CHCl3/CH3OH, 2:1 followed by fractionation on

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

identified 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 classified as BL or LL according

to the Ridley-Jopling classification system36 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 infiltration and

skin nodules, and polar lepromatous individuals with a BI.5·0, concentrations which ranged

from 1 to 32mg of PGL-I per ml. As the BI decreased, the ability to detect PGL-I in individualsamples was lower, with less than half of those MB patients with a BI of,3·1 being positive,

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

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 efficacy 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 first administration of MDT, levels of serum PGL-I

in patients showed a dramatic decline in concentration, likely reflecting 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 0·6–1·0 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 3·0 or for PB patients, and the purification 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.


DETECTION OF PGL- I ANTIBODY IN SERUM FROM LEPROSY PATIENTS

After the initial purification 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-specific 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 reflective 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).42–44 Attempts to develop serological assays using native PGL-I were at first

problematic due to the apolar nature of the purified 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 Buchanan6 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

efficiently 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 firmly 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

specific 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 first

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

the clinical and epidemiological significance 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 definition 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 first 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


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)48–50 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 onthe 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 significantly to sensitivity or specificity.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 flow 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 flanked 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 flanking 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 specifically 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 field use in resource limited

settings. In an evaluation of one type of anti-PGL-I lateral flow 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 97·4% of MB leprosy

patients, 40% of PB patients, and 28·6% of household contacts.54 The specificity of this test


was 90·2%, 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

classification 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 flow test include a simple

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

that could assist in the classification of confirmed leprosy patients under field 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 field conditions of heat and humidity, positives could be easily

distinguished by minimally trained staff, and the concordance, sensitivity and specificity 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 flow device to detect anti-PGL-I antibody reactivity in leprosy patient serum samples. C, controlline; T, test line. Positive reactive serum shown on the left with a strong band at the T line; negative pattern on thedevice on the right. Courtesy of Dr. Sang-Nae Cho, Yonsei University College of Medicine, Seoul, Korea.


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

those already diagnosed with leprosy for the purposes of defining 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, disfigurement 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

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

basement membrane in the basal lamina. The specific 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, fibronectin, 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, first 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 E2 (PGE2, 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

inflammatory 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.65–69

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

proliferate in the hostile intracellular environment.


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 identification 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, specific 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 efficacy 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-I68 such that questions on the specific

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.

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