Lepr Rev (2011) 82, 344 357

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 trisaccharide. 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 serological 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 inammatory 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.3 6 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])

344

0305-7518/11/064053+14 $1.00

q Lepra

Mycobacterium leprae Phenolic Glycolipid I

345

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;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 as M. kansasii14 (in conjunction with the M. kansasii specic 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-specic antigens.
Two contracts were awarded, one to National Jewish Hospital in Denver, CO (the Principal
Investigator, Patrick J. Brennan) based on the hypothesis that M. 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 19802 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 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 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

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J. S. Spencer and P. J. Brennan

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 after M. 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 of M. 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 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 & Shettles20 colorimetric assay. Glycolipid
I of Young19 proved to be PGL-I as shown by the subsequent isolation and full characterisation of PGL-I from human lepromatous nodules21 and formalin-xed human
lepromatous liver.22
CHEMICAL STRUCTURES OF NATIVE M. LEPRAE PGL-I, -II, -III 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 22 mg of pure PGL-I per g of
lyophilised residual tissue, which had also provided 9 1010 acid-fast M. 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 Barrow2 and Young19
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

Mycobacterium leprae Phenolic Glycolipid I

347

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 rst 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 glycosidically 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 spectrometry (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 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-aL-rhamnopyranosyl)-(1 ! 2)-3-O-methyl-a-L-rhamnopyranosyloxy]phenyl}-3-methoxy4-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 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 study25 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-methyl9,11-dihydroxyoctacosane and 3-methoxyl-4-methyl-9,11-dihydroxytriacontane
OCH3
|
CH3-(CH2) n-CH-CH2-CH-(CH2)4-CH-CH-CH2CH3
|
|
|
CH3
OH
OH

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-tetramethyltriacontanoate. These largely extracellular phthiocerol containing lipids exist in
amounts well in excess of the bacterial mass, estimated at more than 138 mg in 1 g of liver,
wet weight, containing 37 1010 M. leprae bacilli. The implications for the biology of

348

J. S. Spencer and P. J. Brennan

H3C
H3C O

CH3

O
O
O
O

(CH2)18

HO
H3CO
HO
H3CO

O
OH

O
H3CO

O
O

H3CO

OCH3

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).

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,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 puried from M. kansasii (mycoside A), the monoglycosylphenolic
diacylphthiocerol puried from M. bovis BCG (mycoside B), and a panel of glycopeptidolipids (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,26 29 and a number of incompletely O-methylated analogs were

Mycobacterium leprae Phenolic Glycolipid I

349

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
antibodies30 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 for M. 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 100 ml of serum onto a lter
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
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 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 inltration and
skin nodules, and polar lepromatous individuals with a BI . 50, concentrations which ranged
from 1 to 32 mg 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 06 10 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.

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J. S. Spencer and P. J. Brennan

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, lipoarabinomannan (LAM).42 44 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 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 1 mg/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

Mycobacterium leprae Phenolic Glycolipid I

351

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 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

352

J. S. Spencer and P. J. Brennan

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.

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,

Mycobacterium leprae Phenolic Glycolipid I

353

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 longchain 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 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
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.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.

354

J. S. Spencer and P. J. Brennan

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-I68 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.
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