From the § Department of Microbiology, Colorado State
University, Fort Collins, Colorado 80523 and the
Institute of Biological Chemistry, Academia Sinica,
Nankang, Taipei 115, Taiwan, Republic of China
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the Mycobacterium avium
complex are the most frequently encountered opportunistic bacterial
pathogens among patients in the advanced stage of AIDS. Two clinical
isolates of the same strain, numbers 397 and 417, were obtained from an
AIDS patient with disseminated M. avium complex infection
before and after treatment with a regimen of clarithromycin and
ethambutol. To identify the biochemical consequence of drug treatment,
the expression and chemical composition of their major cell wall
constituents, the arabinogalactan, lipoarabinomannan, and the surface
glycopeptidolipids (GPL), were critically examined. Through thin layer
chromatography, mass spectrometry, and chemical analysis, it was found
that the GPL expression profiles differ significantly in that several
apolar GPLs were overexpressed in the clinically resistant 417 isolate at the expense of the serotype 1 polar GPL, which was the single predominant band in the ethambutol-susceptible 397 isolate. Thus, instead of additional rhamnosylation on the 6-deoxytalose (6-dTal) appendage to give the serotype 1-specific disaccharide hapten, the
accumulation of this nonextended apolar GPL probably provided more
precursor substrate available for further nonsaccharide substitutions including a higher degree of O-methylation to give
3-O-Me-6-dTal and the unusual 4-O-sulfation on
6-dTal. Further data showed that this alteration effectively
neutralized ethambutol, which is known to inhibit arabinan synthesis.
Thus, in contrast with derived Emb-resistant mutants of
Mycobacterium smegmatis or Mycobacterium tuberculosis, which are devoid of a surface GPL layer, the
lipoarabinomannan from resistant 417 isolate grown in the presence of
this drug was not apparently truncated.
Disseminated Mycobacterium avium complex
(MAC)1 infection is the most
common bacterial infection occurring in AIDS patients, contributing
significantly to the mortality of these individuals. In the United
States, greater than 40% of HIV-positive persons whose CD4 T cell
counts are less than 50/µl of blood are afflicted with disseminated
MAC infection (1). MAC is the etiologic agent in the majority of these
cases. Treatment of disseminated MAC infection has been less than
promising, due in part to the innate resistance that MAC demonstrates
against many conventional antimycobacterial drugs, as well as the
emerging resistance of MAC to macrolide antibiotics such as
clarithromycin (2). The need clearly exists to elucidate the mechanisms
of resistance that MAC utilizes against these reagents as well as to
seek new, effective drug targets.
In the context of mycobacterium, drugs are typically developed to
target its unique cell wall. The mycobacterial cell wall skeleton, as
understood at present, consists of a peptidoglycan to which are
attached branched-chain arabinogalactan units esterified at their
distal ends with long branched-chain fatty acids, the mycolic acids
(reviewed in Refs. 3 and 4). Associated with the cell wall are other
important polymers including the lipoarabinomannan (LAM) (5) and
species-specific surface glycolipids (6). In MAC, these glycolipids are
known as glycopeptidolipids (GPLs) and are the major mycobacterial
antigens providing the basis for MAC serovariation.
Structurally, the GPLs of MAC can be divided into two general classes:
the apolar GPLs, expansively synthesized by all serovariants of MAC,
and the polar GPLs, which differ between serovars. All GPLs have in
common an N-acylated lipotetrapeptide core that bears a
rhamnosylated alaninyl C terminus (Fig.
1). The chemical difference between the
apolar and polar GPLs lies in the structure of the carbohydrate
attached to the allo-threonine residue. In apolar GPLs, this
carbohydrate is a 6-deoxytalose (6-dTal) residue whereas in polar GPLs,
this 6-dTal residue is further glycosylated with a haptenic
oligosaccharide (6, 7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 1.
Generic structure of apolar and polar
GPL.
Ethambutol (Emb) is an antimycobacterial agent typically given in combination with a macrolide antibiotic to treat disseminated MAC infection (8). Previous studies have demonstrated that Emb acts to inhibit the biosynthesis of the arabinan-containing components of the mycobacterial cell wall, the arabinogalactan and LAM (9). Furthermore, Emb-resistant M. smegmatis strains were found to synthesize truncated LAM when grown in the presence of Emb (10). However, the biochemical consequence of Emb treatment in species such as MAC complex that are additionally endowed with surface dominant GPLs have not been addressed.
We report here the chemical alterations of the major cell wall
components of MAC as a consequence of drug treatment. Two clinical isolates of the same strains from an AIDS patient with disseminated MAC
infection before and after treatment with a regimen of clarithromycin and Emb were investigated. By definition, these are virulent strains, and the post-treatment isolate is naturally and clinically
drug-resistant. It was found that the LAM structures were not
apparently altered, but profound differences on the GPL expression
profiles were observed between Emb/clarithromycin-resistant and
-susceptible isolates of MAC. We hypothesize that the altered
physicochemical properties of GPLs probably contribute to drug
resistance by inhibiting the entrance of some antimicrobial agents,
including ethambutol, into the cell. The dose-dependent
action of Emb on arabinan synthesis (9, 10) was therefore neutralized
with no apparent effect on LAM.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemical Reagents-- All chemical reagents were of highest grade from Aldrich unless otherwise specified. Milli-Q® water was used for all chemical reactions.
Mycobacteria--
Clinical isolates 397 and 417 of MAC from an
AIDS patient with disseminated MAC infection were obtained from Denver
Health and Hospitals. Isolate 397 was isolated at the time of diagnosis of disseminated MAC infection and showed clinical and in
vitro susceptibility to clarithromycin and Emb as determined by
disk diffusion and macrobroth dilution (11). Isolate 417 was isolated after the patient had suffered breakthrough bacteremia while being treated with a regimen containing clarithromycin and Emb and also showed in vitro resistance to these drugs. These isolates
were determined by pulsed-field electrophoresis following RFLP
(restriction fragment length polymorphism) to be the same strain. MAC
strain 2151 is a laboratory strain that was originally isolated from a
patient with pulmonary MAC infection. This strain was selected based on
its ability to yield distinct, stable colony morphotypes on solid media
(12); the smooth transparent (SmT) and smooth opaque (SmO) morphotypes
were used in this study. All isolates of MAC were maintained in
Middlebrook 7H11 broth with 20% glycerol at 70 °C.
Growth Conditions of Isolates-- All isolates were grown in Middlebrook 7H11 broth with oleric acid/albumin/dextrose/catalase enrichment supplement and various concentrations of Emb at 37 °C for 10-12 days. Colony morphologies of MAC 2151 broth cultures were monitored periodically by streaking onto Middlebrook 7H11 agar plates containing oleric acid/albumin/dextrose/catalase enrichment.
Extraction and Purification of GPLs--
MAC cells were
separated from the culture medium by centrifuging at 8500 rpm. Cells
were then freeze-dried and extracted with chloroform/methanol (2:1 by
volume) at room temperature for 24 h. The solvent extract was
dried under a steady stream of nitrogen and then deacylated by the
method of Brennan and Goren (13). GPLs were visualized by TLC using
Silica Gel 60 plates (Merck) with chloroform/methanol/water (30:8:1 by
volume) as the developing solvent and -naphthol/sulfuric acid as the
spray reagent. GPLs were separated by preparative TLC on 20 × 20 silica gel 60 plates under the same conditions, except that
-naphthol/sulfuric acid was used only to identify GPLs in side bands
cut from the plates. Corresponding bands on the main part of the plates
were extracted from the scraped off silica gel with chloroform/methanol
(2:1 by volume). The purity of the individual bands was confirmed by re-TLC.
Derivatization and FAB-MS Analysis-- Intact GPLs were analyzed by FAB-MS both in the native deacylated forms and after perdeuteromethylation as described previously (14). Mild methanolysis was performed with 0.5 N methanolic-HCl (diluted from 3 N methanolic HCl, Supelco) at room temperature for 3 h, and the reagent was then removed by evaporation under a stream of nitrogen. Perdeuteromethylated samples were also analyzed directly by FAB-MS after standing in 0.5 N methanolic HCl at room temperature for several minutes in a time course experiment to monitor directly the extent of degradation. FAB mass spectra were obtained on an Autospec mass spectrometer (Micromass, Manchester, UK) fitted with a cesium ion gun operated at 26 kV. All samples were redissolved in methanol for loading onto the probe tip coated with m-nitrobenzyl alcohol as matrix. Triethanolamine was used as matrix for negative ion mode.
GC-MS Analyses--
Partially methylated/trideuteromethylated
alditol acetates were prepared from underivatized and
perdeuteromethylated GPLs by hydrolysis (2 M
trifluoroacetic acid, 121 °C, 2 h), reduction (10 mg/ml sodium
borodeuteride, 25 °C, 2 h), and acetylation (acetic anhydride,
100 °C, 1 h). For glycosyl composition analysis, GC-MS was
carried out on a Hewlett-Packard model 5890 gas chromatograph connected
to a Hewlett-Packard 5790 mass-selective detector. Alditol acetates
were chromatographed on a SP 2380 capillary column (30 m, 0.25-mm inner
diameter, 0.20-µm film thickness; Supelco) using a temperature
gradient of 30 °C/min to 190 °C and then 4 °C/min to
250 °C. For linkage analysis, GC-MS was carried out using a Hewlett-Packard gas chromatograph model 6890 connected to a
Hewlett-Packard 5973 mass-selective detector. The sample was dissolved
in hexane prior to splitless injection into an HP-5MS fused silica
capillary column (30 m, 0.25-mm inner diameter; Hewlett-Packard). The
column head pressure was automatically adjusted under electronic
pneumatic control to give a constant flow rate of 1 ml/min using helium as carrier gas. The initial oven temperature was held at 60 °C for 1 min, increased to 90 °C in 1 min, and then to 290 °C in 25 min.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinical isolates 397 and 417 of MAC from an AIDS patient with disseminated MAC infection were determined to be the same strain by comparing large restriction fragments of genomic DNA separated by pulsed field gel electrophoresis (15). The pretreatment isolate 397 showed (via disk diffusion on 7H11 agar) in vitro susceptibility to clarithromycin, whereas the post-treatment isolate showed in vitro resistance. The Emb MIC for the pretreatment isolate was determined to be 16 µg/ml, whereas the post-treatment isolate was shown to have an Emb minimal inhibitory concentration of >256 µg/ml. These isolates were grown with (20 µg/ml) and without the addition of Emb, and total GPLs, LAM, and arabinogalactan were extracted for structural analysis. In addition, different morphotypes (SmO and SmT) of another laboratory strain (strain 2151, serotype 2) that was originally isolated from a sputum sample of a patient with pulmonary MAC infection were also analyzed in parallel.
Comparative Profiles of GPLs from Isolates 397 and 417--
It was
found that the expression profiles of GPLs from strain 2151 were
similar irrespective of morphotypes or the Emb dose used in culture.
Likewise, the addition of up to 20 µg/ml Emb did not change
significantly the GPL profile of the drug-resistant isolate 417. In
contrast, the expression profiles of GPLs from the two clinical
isolates differed significantly in that a number of GPLs were
overexpressed in 417, the clinically resistant strain (Fig.
2). Based on their relative mobility on
the TLC plate, these bands were numbered I through VII, with I being
the least polar (highest mobility on the TLC plate). Although bands
similar to I-V were apparently present in 397, it is clear that
isolate 397 is predominantly characterized by a single GPL band, with a
TLC mobility similar to band V of 417. These results indicated that fundamental changes in the range of GPL synthesized have been induced
in the drug-resistant strain resulting from in vivo drug treatment. On the other hand, in vitro culture in the
presence of Emb has minimal effect on the growth or expression of the
GPLs.
|
Characterization of Individual GPL Species-- To ascertain if GPLs of similar structures were synthesized in both isolates 397 and 417 and to delineate the chemical differences among the individual bands observed on TLC, the more prominent bands I-VII from 417 and V from 397 were purified by preparative TLC and characterized in detail. In addition, since band IV appeared to be the most overexpressed band in 417, the corresponding band in 397, which was of relatively minute amount, was also purified for further analysis.
Direct FAB-MS analysis of the perdeuteromethyl derivatives has proved
to be highly effective in the past in the analysis of apolar GPLs,
which are typically heterogeneous in fatty acyl chain length and the
presence/absence of natural O-Me groups on the saccharide
residues (for the most recent example, see Ref. 14). Abundant
characteristic fragmentation across the peptide bond allows unambiguous
assignment of the respective saccharide appendages on the
allo-Thr and alaninol residues (see Fig.
3). In particular, the complementary
fragment ions 1 and 5 define the molecular mass
heterogeneity on the fatty acyl residues and the total number of
O-Me substituents on the dHex. Mass intervals between ions
1, 2', and 3' allow confirmation of the fatty acyl-Phe-allo-Thr-Ala sequence, whereas ions
2 and 4 define the respective glycosyl appendages
on the allo-Thr and alaninol. All ions carry the same fatty
acyl heterogeneity profiles except ion 5, which is usually a
single ion for each isolated GPL band, since the separation of GPLs on
TLC is mainly dependent on the degree of O-methylation on
the dHex rather than the fatty acyl heterogeneity. The mass data
obtained from analysis of the apolar GPL fractions are summarized in
Table I; representative mass spectra for
purified band IV from 417 and band V from 397 are shown in Figs.
4 and 5,
respectively. The presence of negative charge on the most polar GPLs in
bands VI and VII of 417 drastically changed the fragmentation pattern, and these were considered separately.
|
|
|
|
From the results obtained, apolar GPLs found in bands I-VI from 417 can be classified into six major species (designated GPLs 1-6; column 2 of Table I) based on the different substituents on the two dHex, 6-dTal and Rha. For each GPL species, further heterogeneity was contributed by fatty acyl substituents that were best defined by the fragment ion fatty acyl-Phe+ (ion 1) produced. Under this consideration, there are essentially four distinct groups of fatty acyl chains, designated a-d, with c and d being detected by FAB-MS analysis as minor components in all GPLs. Fatty acyl groups a and b could both incorporate three Me-d3 upon derivatization, whereas groups c and d only incorporated one. Since fragment ions 1, 2', and 3' do not include any of the saccharide moiety (see Fig. 3), their m/z values are the same for all GPLs classified as having the same fatty acyl heterogeneity (mass e.g. GPL 1a to 6a. The fatty acyl chains designated as a is 34 mass units higher than b, and so is the mass relationship between c and d. Further microheterogeneity was observed within each group, which comprised signals of 14 mass units apart, i.e. a difference that could be attributed to the number of CH2 units. The exact structural details of these fatty acyl chains were not pursued further.
The degree and position of O-methylation on the two dHex attached to allo-Thr and alaninol, respectively, were further corroborated by GC-MS analysis of the glycosyl compositions (data not shown). In summary, the least polar GPL 1 carries a 3-O-Me-6-dTal and a 3,4-di-O-Me-Rha. GPLs 2 and 3 both have two O-Me on their dHex but differ in their distribution (the former with a 6-dTal and a 3,4-di-O-Me-Rha, the latter with a 3-O-Me-6-dTal and a 3-O-Me-Rha). These two GPLs were fairly similar in polarity and not well resolved from each other. Hence, a substantial amount of GPL 2 (GPL 2b and GPL 2c) were also found in band III. GPL 4 have only one O-Me, namely a 6-dTal and a 3-O-Me-Rha, which were also found in band V. Finally, both GPL 5 and GPL 6 were found to have an additional Rha extending from 6-dTal and either a 3,4-di-O-Me-Rha (GPL 5) or a 3-O-Me-Rha (GPL 6) on the alaninol.
Analysis of bands IV and V from isolate 397 indicated that the GPLs were similar in structure to the corresponding ones from isolate 417. In particular, GPL 5a were the dominant species in band V, but a small proportion were also present in band IV. The molecular ions observed included m/z 1335, 1349, 1357, 1371, 1385, 1399, 1413, with m/z 1371 being the most abundant species that shifted to m/z 1592 after perdeuteromethylation (Fig. 5). The characteristic fragment ions afforded by the perdeuteromethyl derivatives firmly established that this major GPL species of 397 was indeed similar to GPL 5a of 417. Another minor group of related GPLs that probably contained three O-Me on their dHex (m/z 1385/1413 shifted to 1589/1617 after perdeuteromethylation) were also found in band IV. The low abundance of these species did not allow further characterization. Apart from these minor amounts of disaccharide-containing GPLs, the dominant components in band IV of 397 were identified as similar to GPL 4a and 4b of 417 (Fig. 4). Major [M + Na]+ molecular ions of m/z 1367 and 1401 were afforded by the perdeuteromethyl derivatives, complete with the characteristic fragment ions expected including m/z 971/1005 and 1130/1164.
FAB-MS Analysis of the Total GPL Profiles--
Although other
bands from 397 were not analyzed individually, FAB-MS analysis on the
total GPLs did reveal that most of other apolar GPLs can be found in
isolate 397 based on the molecular ions detected for both
nonderivatized and perdeuteromethyl derivatives (Fig.
6, A and C). These
apolar GPLs were, however, of minor amounts relative to GPL 5 as was
obvious from the intensity of the bands observed on TLC analysis.
Notably, when total GPL were analyzed by FAB-MS, the less abundant
species could not be detected. Thus, GPL 4 appeared to be absent or
very minor in 397 (Fig. 6, A and C), whereas GPL
5 were not detected in isolate 417 (Fig. 6, B and
D).
|
In negative ion mode, most of the molecular ion signals afforded
corresponded to [M H]
of the species observed
in the positive ion mode with the exception of a cluster of signals at
m/z 1205, 1219, 1233, 1253, and 1267, which were
only present in the spectrum of 417 (data not shown), indicating that
417 additionally synthesized a group of negatively charged polar GPLs.
These unusually modified GPLs, designated GPL 7, were subsequently
localized to bands VI and VII, consistent with their being the most
polar GPLs and their apparent absence in 397.
Identification of Negatively Charged Polar GPLs in 417--
Band
VII from 417 did not afford strong molecular ion signals when first
analyzed directly in positive ion mode. However, in negative ion mode,
two major molecular ions were observed at m/z
1219 and 1253 (Fig. 7B).
Similar analysis showed that a pair of related molecular ions were also
present in band VI at m/z 1233 and 1267 (Fig.
7A) in addition to the molecular ions at
m/z 1319/1347, which corresponded to [M H]
of the most abundant species, GPL 6a.
After perdeuteromethylation, band VII afforded two major pairs of
positive ions at m/z 1350/1384 and 1452/1486,
with a mass difference of 102 units between the two (Fig.
7D). In negative ion mode, [M
H]
molecular ions were observed at m/z 1406/1440,
therefore allowing the assignment of the molecular ions signals in the
positive mode (m/z 1452/1486) as disodiated, and
loss of a moiety such as sodium sulfite (102 mass units) yielded the
fragment ions at m/z 1350/1384. Similarly, in
addition to those ions corresponding to GPL 6, signals were present at
m/z 1449/1483 and 1347/1381 in the mass spectrum of perdeuteromethylated band VI (Fig. 7C), consistent with
the presence of related GPLs having one additional O-Me
group on their dHex, as compared with those of band VII.
|
From the data above, it was clear that the negatively charged GPLs detected in bands VI and VII were GPL 7 detected in the analysis of total GPLs from 417. The loss of the charged group (80 mass units plus a sodium) as the primary fragment ions induced in positive ion mode strongly suggested that GPL 7 have a basic core structure similar to the apolar GPL but further substituted with either a phosphate or sulfate group. Indeed, mild methanolysis treatment of band VII resulted in the removal of an 80 mass unit moiety, giving [M + Na]+ molecular ions at m/z 1163/1197, with fragment ions at m/z 835/869 corresponding to the expected protonated lipopeptide core (data not shown). The mild acid lability of the negative charged group indicated that it was most likely a sulfate substituent, since phosphate is usually stable under these conditions. Further perdeuteromethylation of the desulfated sample yielded molecular and fragment ions identical to those of GPL 4a/4b (data not shown). Together with the glycosyl composition data, which also identified a 6-dTal and a 3-O-Me-Rha in band VII, it could be concluded that GPL 7 were sulfated analogs of GPL 4a/4b.
To further define the location of the sulfate group,
perdeuteromethylated band VII was treated with mild methanolic HCl and analyzed directly by FAB-MS in positive ion mode. Apart from the molecular ions (m/z 1452/1486) and fragment ions
(m/z 1350/1384) corresponding to loss of sodium
sulfite as described above, additional fragment ions allowed for
further assignment of the structure. It was reasoned that if the
sulfate was localized on the 3-O-Me-Rha, a further loss of a
perdeuteromethylated O-Me-Rha(OH)/6-dTal (177/197 mass
units) would be observed. On the other hand, if the sulfate was located
at the 6-dTal, loss of a perdeuteromethylated
O-Me-Rha/6-dTal(OH) (194/180 mass units) might be induced.
Fig. 8 showed that a further loss of 194 and 180 mass units from m/z 1350/1384 were
observed, giving the pairs of ions at m/z
1156/1190 and 1170/1204, respectively. Loss of both dHex residues gave
the ions at m/z 976/1010. In addition, ions at
m/z 1130/1164 corresponded to elimination of the
6-dTal residue (ion 4 in Fig. 3), retaining a fully
perdeuteromethylated 3-O-Me-Rha. Further linkage analysis
performed on the perdeuteromethyl derivatives revealed the presence of
a 4-linked 6-dTal and a terminal 3-O-Me-Rha (Fig.
9D, peaks
6 and 3, respectively), whereas if the sample was
desulfated before perdeuteromethylation, a terminal 3-O-Me-Rha and a terminal 6-dTal were identified (Fig.
9C, peaks 3 and 1). These
data firmly established that the sulfate group was located on the C-4
position of the 6-dTal and represented a further modification on GPL
4a/4b. The detailed structure of the related GPLs
in band VI with one additional O-Me substituent was not
further investigated, since the presence of the more abundant GPL 6 made interpretation of data less definitive.
|
|
Structural Alterations in the GPLs Expressed in Drug-resistant
Isolate 417--
In summary, GPL 5 with a Rha-6-dTal appendage on
allo-Thr and a 3,4-di-O-Me-Rha on the alaninol is
the dominant GPL species in 397. Three major peaks were detected in the
GC-MS linkage analysis of band V performed on the perdeuteromethyl
derivatives of both 417 and 397 (Fig. 9B), corresponding to
a terminal Rha (peak 2), a terminal 3,4-di-O-Me
Rha (peak 4), and a 2-linked 6-dTal (peak 5). The
data therefore confirmed that the disaccharide determinant on GPL 5 is
the previously defined hapten of MAC serovar 1 (7). Indeed, among the
collection we have, only monoclonal antibodies raised against MAC
serovar 1 reacted strongly with the GPLs from both the clinical
isolates 397 and 417, further indicating that the same sero-specific
GPL 5 is present in both. The anomeric configurations were not
determined for Rha and 6-dTal of the clinical isolates. However, as
known from our previous work and the stringent recognition by the
monoclonal antibody against MAC serovar 1, all deoxyhexoses are
expected to be in -configuration, as shown in Fig. 1 for the generic
GPL structure of MAC.
Despite this observation, the expression of sero-specific GPL 5 was
clearly down-regulated in drug-resistant 417, relative to the
expression of other GPLs. Consistent with this observation is the
enhanced level of GPL 4, since a larger proportion of its 6-dTal
residue was not further glycosylated with another Rha. Our data further
suggested that the accumulated GPL 4 probably provided more precursor
substrate available for further nonsaccharide substitutions such as
O-methylation (to give the less polar GPL 1-3) and the
unusual addition of a sulfate group to the 6-dTal (to give GPL 7).
Thus, the most pronounced overall effect as a consequence of drug
resistance is the enhanced expression of apolar GPLs at the expense of
polar GPL determined to be serotype 1.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GPLs of MAC have been associated with the capsular-like matrix that surrounds the bacillus and forms an electron transparent zone in phagocytic vesicles (16). It has been clearly demonstrated that the GPLs survive within the intraphagosomal environment, where they are resistant to degradation by lysosomal enzymes and possibly also inhibit phagosome-lysome fusion (17, 18). However, their actual contribution to the pathogenesis of disseminated MAC infection and the intracellular survival of bacilli has been equivocal (19). Both the virulent SmT and avirulent SmO colony forms express GPLs, whereas the rough variants, which have variously been described as virulent (20) or avirulent (21), are completely devoid of this surface antigen (22). Furthermore, the SmT variants have been consistently shown to have an increased resistance to antibiotics (23, 24), therefore implicating colony morphology and hence GPL expression in drug resistance.
It is as yet unclear how the altered expression profile of GPLs as defined in this paper contributed to the apparent drug resistance. Nonetheless, it is significant that the prime consequence of drug resistance in the clinical isolates is the apparent inhibition of rhamnosylation on the 6-dTal of apolar GPL. Since the polar GPLs of all MAC serovars characterized to date are oligoglycosylated by extension from this Rha-6-dTal disaccharide unit (6), this phenomenon may be relevant to all other serovars whereby their polar GPLs may be similarly targeted and down-regulated.
Although little is known about the biosynthesis of GPLs, Belisle et al. (25) have demonstrated that apolar GPL of M. smegmatis can serve as a precursor for the synthesis of MAC serovar 2 polar GPL, for which a 22-27 kilobase pair gene cluster termed ser2 was identified. More recently, a gene termed rtfA, which encodes for the rhamnosyltransferase essential for the synthesis of serovar-specific oligosaccharides, was identified (26). Yet, the possible sequence of order for O-methylation versus the addition of Rha on 6-dTal and further glycosylation to polar GPL is largely unknown. The accumulation of GPL 4 in isolate 417 argues that further O-methylation or sulfation was more likely to be an incidental modification in the presence of more precursor intermediates rather than an obligatory competitive "stop" signal upon further glycosylation of GPL 4.
The location of sulfate on the 6-dTal makes it a novel GPL structure not previously reported. The only other sulfated GPLs are those from Mycobacterium fortuitum biovar peregrinum, characterized by López Marín et al. (27) with sulfated Rha on the alaninol. We cannot rule out the possibility that sulfated apolar GPL may be normally present in serovar 1 and other serotypes in low amounts and that this unusual modification has hithertho escaped detection. Likewise, it is obvious that most, if not all, of the other apolar GPLs detected are also normally present in isolate 397. The extreme heterogeneity associated with these apolar GPLs, as first demonstrated by Brennan and Goren (13) chemically, was a consequence of both O-methylation and the fatty acylation. The former resulted in 3-O-Me or 3,4-di-O-Me Rha on the alaninol, and 6-dTal or 3-O-Me 6-dTal on allo-Thr. In the absence of more information on the molecular basis of acquired resistance in MAC, the significance of down-regulation of the serotype 1 GPL 5, concomitant with more extensive O-methylation and sulfation, remains unanswered.
Recent studies of the mechanism of action of Emb have been in accordance with the suggestion that the antimycobacterial activity of this drug is achieved through the inhibition of arabinan biosynthesis (10, 28). Specifically, this seems to be mediated through the inhibition of an array of arabinosyltransferases responsible for the polymerization of arabinan in arabinogalactin and LAM. Khoo et al. (10) have reported in their study a unique phenomenon observable in LAM of Emb-resistant strains of M. smegmatis. When the wild type strain was rendered antibiotic-resistant and then grown in the presence of Emb, normal arabinogalactan was produced by the cells, but the synthesized LAM was "truncated." Although LAM from MAC have not been previously characterized and more work is needed to elucidate the fine structure, preliminary data (SDS-polyacrylamide gel electrophoresis Western blot and neutral sugar analysis) on LAMs extracted from all isolates revealed no apparent truncation, regardless of the strain, morphotype, susceptibility to Emb, or concentration of Emb in the culture media. These results were in contrast to the observed effect of Emb on M. smegmatis (10) or M. tuberculosis,2 which do not display the additional surface layer of GPL. Therefore, it is conceivable that MAC may adopt different resistant mechanism to counteract Emb, probably by utilizing and changing the exact physicochemical nature of their dominant GPLs.
However, this strategy did compromise certain physiological functions for in vivo growth in immunocompetent individuals. Our initial experiment3 has shown that although isolate 417 is a drug-resistant isolate from one AIDS patient, it nevertheless cannot grow in immunocompetent mice. We do not know if the induced biochemical/physiological changes in altered expression of GPL and other undetermined effects reflect an irreversible adaptation to a drug-treated immunocompromised environment. It may also be argued that the presence of a sufficient amount of the serospecific GPL instead of more copious amounts of the apolar ones is essential to counter host immune response for mycobacterial survival. Since in AIDS patients or in in vitro culture this need for immunomodulation has largely been removed, drug resistance can be achieved by making more of the apolar GPLs at the expense of polar ones without adverse effect.
Finally, it should be noted that the type of glycosylation present on
the GPLs was known to dramatically affect the immunomodulatory potential of these unique lipids. For example, Barrow and colleagues (29) have demonstrated that mice injected with GPLs have a reduced lymphocyte response to mitogen-induced blastogenesis, a phenomenon observed in MAC infections. However, a similar in vitro
response was obtained only when a chemically deglycosylated form of the GPL was used (30, 31). GPLs from serovars 4 and 8 were found to induce
secretion of TNF from human peripheral blood monocytes, but only the
serovar 8 GPL and deglycosylated serovar 4 GPL induced secretion of
prostaglandin E2 (30, 31). The altered expression profiles
of GPLs therefore have further ramifications beyond conferring a drug
resistance phenotype, which should allow us to dissect further the
functional roles of GPLs as well as their biosynthesis.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants NIAID AI-37139, TW 00943, and AI 41925 (to D.C.) and by Academia Sinica and National Science Council (Taipei) Grant NSC 88-2113-M-001-003 (to K. H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 970-491-7495; Fax: 970-491-1815; E-mail: delphi{at}lamar.colostate.edu.
2 D. Chatterjee, unpublished results.
3 J. Torrelles, T. Osborne, K.-H. Khoo, D. Chatterjee, and A. Cooper, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MAC, M. avium complex; SmO, smooth opaque morphotype; SmT, smooth transparent morphotype; LAM, lipoarabinomannan; GPL, glycopeptidolipid; 6-dTal, 6-deoxytalose; Emb, ethambutol; GC, gas chromatography; MS, mass spectrometry; FAB, fast atom bombardment.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|