(Received for publication, September 8, 1995; and in revised form, November 27, 1995)
From the
The ``core'' structure of the cell wall of Mycobacterium and related genera is unique among prokaryotes,
consisting of a covalently linked complex of mycolic acids, D-arabinan and D-galactan (mycolylarabinogalactan,
mAG), which, in turn, is linked to peptidoglycan via a special linkage
unit, --L-Rhap(1
3)-D-GlcNAc-P-.
Little is known of the biosynthesis of this complex, although it is the
site of action of several common anti-tuberculosis drugs. Isolated cell
membranes of Mycobacterium smegmatis catalyzed the
incorporation of [
C]GlcNAc from
UDP-[
C]GlcNAc into two glycolipids (1 and 2) and
of [
C]Rha from TDP-[
C]Rha
into glycolipid 2. These products were characterized as
polyprenol-P-P-GlcNAc (glycolipid 1) and polyprenol-P-P-GlcNAc-Rha
(glycolipid 2) based on sensitivity of synthesis to tunicamycin,
chromatographic characterization of the products of mild acid
hydrolysis, and mass spectral analysis of the glycosyl and polyprenyl
units. Glycolipids 1 and 2 were shown to be precursors of the linkage
unit in polymerized cell wall. The inclusion in the assays of
UDP-[
C]Galp and a preparation of cell
walls allowed the incorporation of [
C]Gal into
two further glycolipids (3 and 4). Preliminary evidence indicates a
precursor-product relationship among glycolipids 1, 2, 3, and 4. Thus,
the first steps in the biosynthesis of the mycobacterial cell wall
involve synthesis of the linkage disaccharide on a polyprenyl-P-P
carrier followed by growth of the galactan unit. Assays are thus
defined for the screening of new anti-tuberculosis drugs active against
cell wall synthesis.
The core or skeletal cell wall of members of the Mycobacterium genus consists of extensively cross-linked
peptidoglycan to which is attached the linear D-galactan
composed of alternative 5- and 6-linked -D-Galf units(1) . Attached in turn to the D-galactan are
extensively branched chains of D-Araf- containing
arabinan, the distal ends of which are almost completely esterified
with mycolic acids(2) . We have described this vast
macromolecular structure as the mycolylarabinogalactan (mAGP) (
)complex(3) . The biogenesis of the mAG portion of
this complex is under current investigation because several of the
widely used anti-tuberculosis drugs affect aspects of its synthesis and
resistance to these drugs is a serious world wide public health
problem(4) . For instance, a target for isoniazid (INH) is a
NADH-requiring 2-trans-enoyl fatty acyl reductase apparently
on the pathway to mycolic acid biosynthesis and some of the resistance
to isoniazid is due to a point mutation in the inhA gene
and/or overexpression of the target(5) . Ethambutol
specifically inhibits the synthesis of the arabinan of AG and of
lipoarabinomannan, apparently through its action on a family of
arabinosyltransferases(6) .
Some clues about the initiation
of mAG biosynthesis have arisen from earlier structural work. It has
long been known that the AG heteropolysaccharide chains are attached
through phosphodiester linkages to C-6 of a proportion of the muramic
acid residues of mycobacterial cell walls(7) . More recently,
chemical analysis of degradation fragments arising from the reducing
end of AG obtained from the cell walls of Mycobacterium
tuberculosis, Mycobacterium bovis BCG, and Mycobacterium leprae demonstrated the existence of the
terminal sequence
5)-D-Galf-(1
6)-D-Galf-(1
5)-D-Galf-(1
4)-L-Rhap-(1
3)-D-GlcNAc (1, 8) . Based on the acid lability of the 3-linked
GlcNAc unit, the presence of about equal amounts of L-Rhap-(1
3)-D-GlcNAc and muramyl-6-P
in an isolated cell wall fragment and
P NMR analysis, it
was concluded that the terminal GlcNAc residue is in phosphoryl linkage
to the 6-position of some of the muramyl residues of mycobacterial
peptidoglycan(8, 9) . Thus, this aspect of
mycobacterial cell wall structure, and, presumably, biosynthesis,
shares similarity with the teichoic acid-peptidoglycan complex of many
Gram-positive bacteria(10) . In view of the role of the
mycobacterial linkage unit as the fulcrum of cell wall integrity and as
a potential singular site for target-directed chemotherapy against
tuberculosis, we set about elucidating its biosynthesis in the belief
that such information will give rise to assays amenable to
high-throughput screening for new growth inhibitors of M.
tuberculosis.
Membranes of M. smegmatis were obtained by centrifugation
of the 27,000 g supernatant (from the previous step)
at 100,000
g for 1 h at 4 °C. The supernatant was
carefully removed, and the yellow-pigmented opalescent membranes were
gently and superficially washed with buffer A and finally suspended in
0.4-0.5 ml of this buffer (protein concentration 15-20
mg/ml).
For
purposes of estimating incorporation of radioactivity into polymer, the
approach described by McArthur et al. (15) was used.
The entire reaction mixture, or a portion of it, was applied to Whatman
3MM chromatography paper which was developed in a descending fashion in
isobutyric acid, 0.5 M aqueous NHOH (5:3). The
polymer remained at the origin and was counted. The unreacted
nucleotide sugar, degraded sugar phosphate, and glycolipid
intermediates migrated down the paper(15) .
Alkali treatment of the C-labeled glycolipids served to demonstrate their alkaline
stability and to provide a first step in their purification. Excellent
recovery (about 85%) was obtained by dissolving the lipid fraction from
reaction mixtures in 0.1 ml of CHCl
/CH
OH (2:1)
followed by the addition of 0.1 ml of 0.2 M NaOH in
CH
OH and incubation at 37 °C for 20 min. Mixtures were
neutralized with 2.5 µl of glacial CH
COOH, dried,
suspended in 1.5 ml of CHCl
/CH
OH (2:1), and
0.25 ml of H
O, centrifuged, and the lower
(CHCl
) phase retained. First steps in the purification of
the glycolipids involved application of the alkali-stable lipids to a
column (7
0.5 cm) of DEAE-cellulose (acetate form) poured in
CH
OH and equilibrated in CHCl
/CH
OH
(2:1). The lipid fraction was applied in
CHCl
/CH
OH (2:1) and the column developed with 3
column volumes each of CHCl
/CH
OH (2:1),
CH
OH, and 50, 100, 200 mM, and 1 M ammonium formate in CH
OH. Salt was removed through
biphasic washings(18) . TLC of glycolipids was conducted on
plates of silica gel in
CHCl
/CH
OH/NH
OH/H
O
(65:25:0.5:3.6) which were exposed to Kodak X-Omat AR film at -70
°C and subsequently sprayed for the presence of phosphorus with a
molybdenum reagent (19) or for polyprenols with a reagent
containing p-anisaldehyde(20) . Mild acid hydrolysis
of glycolipids (21) was conducted on radiolabeled preparations
in 100 mM HCl in CHCl
/CH
OH (2:1) (1
ml) at 20 °C for 2 h or 10 mM HCl at 100 °C for 10
min. The HCl was neutralized with 0.5 N NaOH before adding
H
O (150 µl) to form a biphase, each phase of which was
counted.
Hydrolysis of glycolipids and polymer for neutral sugar
content was conducted in 2 M CFCOOH at 120 °C
for 2 h. Hydrolysis of glycolipid preparations for amino sugar content
was conducted in 4 N HCl at 100 °C for 4 h. Sugars were
analyzed in a variety of ways. Radioactive sugar preparations were
generally applied to Baker-Flex cellulose plastic sheets (J. T. Baker,
Philipsburg, NJ) or sheets from Eastman Kodak and developed three times
in formic acid/water/t-butanol/methylethyl ketone
(15:15:40:30, by volume) for neutral sugar analysis, and
1-butanol/pyridine/0.1 N HCl (5:3:2) for amino sugar analysis
followed by autoradiography. Standards of a variety of sugars (ribose,
Ara, Man, Glc, Gal, Rha, GlcNH
, GalNH
, and
mannosamine) were also run on plates and visualized by spraying with
phthalic acid, 1-butanol, aniline (22) and heating at 120
°C for 5 min. The radioactive monosaccharides released by acid
treatment were also assayed by HPLC (Dionex, Sunnyvale, CA) using a
gradient pump with pulsed amperometric detection, a Dionex CarboPac PA1
column (4
250 mm), and 15 mM NaOH eluent. Fractions
were counted, collected, and retention time compared to standards.
Other chromatographic systems have been described(14) . However, for analysis of radioactive products (alditol acetates; partially permethylated oligosaccharides) a Durabond (DB)-1 fused silica column (J& Scientific, Rancho Cordova, CA) was used as described (14, 23) but as part of the Hewlett-Packard 5890 Series II Plus Gas Chromatography, coupled to the Lablogic GC-RAM radioactive counter (INUS Systems, Tampa, FL).
Samples of the
radioactive lipids were applied to silica gel TLC plates which were
developed in
CHCl/CH
OH/NH
OH/H
O
(65:25:0.5:3.6) and autoradiograms obtained (Fig. 1A).
Surprisingly clean products were obtained consisting of two closely
migrating glycolipids (GL 1 and GL 2). Conditions that resulted in
partial inhibition of [
C]GlcNAc incorporation
into the lipid fraction (i.e. prolonged storage of membranes,
some detergents) consistently resulted in a more marked inhibition of
synthesis of GL 2 (Fig. 1A), suggesting that the
formation of GL 2 involved an additional enzymatic step beyond GL 1.
Treatment of the GL 1/GL 2 mixture with 0.1 N HCl in
CHCl
/CH
OH (2:1) at 20 °C (21) resulted in over 50% loss of lipid radioactivity after 5
min; and
80% loss after 20 min. Treatment of the glycolipids with
0.1 M NaOH at 37 °C resulted in 94 and 95% recovery of
radioactivity in two experiments, supporting the evidence that these
products were polyprenyl-P based
glycolipids(24, 25, 26, 27) . To
confirm that the doublet was glycolipid in nature rather than residual
nucleotide sugar or degraded sugar-P, synthesis of GL 1/GL 2 was shown
to increase over time (Fig. 1B), and when these
products were eluted from the gel, the lipid solubility was confirmed
in the two-phase system. Attempts to better resolve the doublet were
unsuccessful; most solvents gave the false impression that the product
was homogeneous. Thus the evidence pointed to the synthesis of two
novel polyprenol-containing glycolipids, much more polar than those
described previously in mycobacteria, all of which contained neutral
sugars and decaprenol.
Figure 1:
TLC/autoradiography of the
[C]GlcNAc-labeled lipids obtained from
incubation of membranes of M. smegmatis with
UDP-[
C]GlcNAc. A, the lipid fraction
derived from the standard 1-h incubation described in Table 1(3000 cpm) was applied to thin layer plates of silica gel
(aluminum backed; 60 F
; E. Merck) and developed in
CHCl
/CH
OH/NH
OH/H
O
(65:25:0.5:3.6). Lane 1, lipid fraction from standard reaction
mixture. Lane 2, lipid fraction from standard reaction mixture
using membranes partially inactivated by prolonged storage.
Autoradiograms were obtained after 5 days of exposure. B, the
standard reaction mixture (Table 1) was applied but for variable
incubation periods. Lane 1, 5 min incubation; lane 2,
15 min; lane 3, 30 min; lane 4, 1 h; solvent,
CHCl
/CH
OH/NH
OH/H
O
(65:25:0.5:3.6).
Figure 2:
Silica gel TLC and autoradiography of the
lipids synthesized by M. smegmatis membranes from
dTDP-[C]Rha (left lane) compared to
those synthesized from UDP-[
C]GlcNAc (right
lane). The reaction mixture described in Table 1, experiment
3, was scaled up 8-fold to obtain sufficient quantity of the
[
C-Rha-labeled GL 2 for analysis. Other conditions
are described in the legend to Fig. 1.
The
[C]GlcNAc-labeled GL 1/GL 2 mixture was
subjected to strong acid hydrolysis (4 N HCl, 100 °C, 4
h), partitioned between CHCl
and H
O, and the
aqueous phase dried repeatedly and applied to cellulose TLC plates and
developed in 1-butanol/pyridine/0.1 N HCl (5:3:2).
Autoradiography showed the presence of just one radioactive sugar,
corresponding to GlcNH
. Hydrolysis with 2 M CF
COOH (2 h, 120 °C) of the acidic
[
C]Rha-labeled GL 2, followed by cellulose TLC
in t-butanol/ethylmethylketone/formic acid/water (40:30:15:15)
and autoradiography, showed only [
C]Rha (results
not shown).
It was obvious throughout that the quantities of GL 1
and 2 generated in scale-ups of the standard reaction mixtures were too
small for adequate chemical characterization. Accordingly, the standard
reaction mixture was increased 158 times (that containing
UDP-[C]GlcNAc was increased 46 times; the
corresponding UDP-GlcNAc-cold reaction was increased 112 times). A
total of 1.142
10
cpm were incorporated into
lipids. These were applied to three preparative (20
10 cm)
plates, developed in
CHCl
/CH
OH/NH
OH/H
O and
subjected to autoradiography. The other known mycobacterial
sugar-containing polyprenyls, C
-P-Man and
C
-P-Man (24, 25, 26) and
C
-P-Araf(27) , ran much faster in this
solvent (R
0.5-0.7), and hence we knew that
the GL 1/GL 2 mixture was not contaminated with other known
polyprenyl-containing glycolipids. In excising GL 1 and 2 from the
preparative plate, every effort was made to separate them. However,
subsequent analytical TLC showed that they were heavily contaminated
with each other. The purified products were subjected to mild acid
hydrolysis (10 mM HCl, 100 °C, 10 min) partitioned within
a mixture of CHCl
/CH
OH/H
O (4:2:1),
and the lipid in the organic phase subjected to MS analysis as
described(33) . EI-MS of the product from the GL 1-rich
preparation showed an apparent molecular ion peak at m/z = 484
(M-H
PO
-sugars)
, similar to
that of the octahydroheptaprenol fragment arising from the
6-O-mycolyl-
-D-mannopyranosyl-1-monophosphoryl-octahydroheptaprenol
(Myc-PL) recently described(33) . A dominant ES-MS fragment (m/z 582) in the GL 2-rich preparation was also indicative of
octahydroheptaprenol-P [(M-sugar(s) +
(H)]
(27) . Hence both products seemed to
contain the C
octahydroheptaprenol rather than the
decaprenol usually found in mycobacterial
products(24, 25, 26, 27, 34) ,
or undecaprenol. The proposed structures, C
-P-P-GlcNAc and
C
-P-P-GlcNAc-Rha, would explain the much greater polarity
of GL 1/GL 2 compared to C
-P-Manp,
C
-P-Manp, or C
-P-Araf.
However, much greater quantities of the two glycolipids are required
for more thorough characterization of the type recently applied to the
polyprenyl of the C
-P-Araf(27) and the
Myc-PL (33) of M. smegmatis.
The C-labeled aqueous-soluble fractions from mild acid
hydrolysis of the GL 1 and GL 2 preparation were combined and processed
as described (3) (Fig. 3B, inset), seeking
chemical evidence for the presence of the
-L-Rhap-(1
3)-D-GlcNAc unit. The
neutral, dried, combined hydrolysates were reduced with
NaB[
H]
and subjected to the NaOH
methylation procedure of Ciucanu and Kerek(35) . The
methylation reaction was quenched with H
O and the
per-O-methylated products extracted with CHCl
, O-acetylated, and subjected to GC on the Durabond DB-1 fused
silica column and compared to the authentic, derivatized L-Rhap-(1
3)-GlcNAc linkage unit (3) (Fig. 3). Singly and by co-chromatography, both
showed identical retention times of 12.95 min. The
per-O-methylated glucosaminitol derivative arising from GL 1
was not obvious in any of these work-ups; presumably, it was lost in
the initial purification of the per-O-methylated products.
Figure 3:
Comparison by GC of (A) the
NaB[H
]-reduced,
per-O-methylated, per-O-acetylated linkage
disaccharide from the mAGP complex of M. bovis BCG and (B) the similarly derived product obtained from the in
vitro generated [
C]GlcNAc-labeled GL 1/GL 2
mixture of M. smegmatis. The derivatization of the authentic
linkage unit has been described(8) . The derivatization of the
linkage disaccharide from GL 1/GL 2 is summarized in the inset. GC was conducted on a fused silica DB-1 column coupled
to the Lablogic GC-RAM radioactive counter.
In order to examine the nature of the polymer synthesized under
these conditions, membranes were incubated with
UDP-[C]GlcNAc in the presence of 0.1 mM UDP-Gal and 0.05 mM dTDP-Rha and the Percoll-60 cell wall
fraction for various times. The entire reaction mixtures were applied
to strips of Whatman 3MM and chromatographed overnight in isobutyric
acid/0.5 M NH
OH (5:3). The areas around the
solvent front, corresponding to GL 2/GL 3 were excised and counted.
Likewise, the material at the origin, the cell wall polymer, was
counted. Incorporation into both populations was linear over the course
of the experiment (Fig. 4). Thus, the kinetics were more
reminiscent of the relationship between the dolichol-bound
oligosaccharide precursors and the core region of yeast mannoproteins (36) , which also involves a GlcNAc-containing (chitobiose)
linkage (37) , than that of the simpler mycobacterial
polyprenol-P-Man precursors and mycobacterial
mannan(24, 34) , indicative of a greater similarity to
yeast mannoprotein synthesis(37) . The labeled polymer was
hydrolyzed, subjected to cellulose TLC and autoradiography as described
for GL 1/GL 2. Only GlcNH
was present; there was no
evidence for synthesis of muramic acid and hence of peptidoglycan,
under these conditions. Application of the approach ( Fig. 3and (3) ) used to identify the
Rha(1
3)-[
C]GlcNAc linkage region in the GL
1/GL 2 mixture produced the radiolabeled disaccharide from the polymer.
Figure 4:
Time course of incorporation of
[C]GlcNAc from
UDP-[
C]GlcNAc into polymerizer linkage region
and the GL-containing lipid fraction. The incubation conditions were a
variation of those described under ``Experimental
Procedures,'' i.e. 50 µl (0.9 mg of protein) of
membrane, 74 µl (0.7 mg of protein) of Percoll-60, ATP (0.06
mM), UDP-Gal (0.02 mM), TDP-Rha (0.01 mM),
UDP-[
C]GlcNAc (0.5 µCi). Total volume, 160
µl. Ten such tubes were installed, incubated at 37 °C for the
indicated times, stopped by the addition of 1 ml of
C
H
OH, and the entire reaction mixture applied
to sheets of Whatman 3MM paper which were developed overnight in
isobutyric acid/NH
OH (5:3), cut into strips and counted.
The origin represented the polymer, while the solvent front contained
the glycolipids(8) .
Figure 5:
TLC autoradiographic comparison of the
[C]GlcNAc-containing lipids obtained without (lane 1) and with (lane 2) the Percoll-60 cell wall
fraction. TLC autoradiography conditions are described in the legend to Fig. 1. Standard incubation conditions were applied in the case
of lane 1 and these conditions with the added presence of 1.4
mg of Percoll-60 applied.
Figure 6:
TLC autoradiographic comparison of the
glycolipids synthesized by M. smegmatis membranes/cell wall
(Percoll-60) from UDP-[C]GlcNAc (lane/experiment 1), dTDP-[
C]Rha (lane/experiment 2), and UDP-[
C]Gal (lane/experiment 3). The assay mixture contained the
following: membranes (1.6 mg), Percoll-60 (1.4 mg), ATP (0.06
mM), UDP-Gal (0.02 mM, experiments 1 and 2),
UDP-GlcNAc (0.02 mM, experiments 2 and 3), dTDP-Rha (0.1
mM, experiments 1 and 3), UDP-[
C]Gal (1
µCi, experiment 3), UDP-[
C]GlcNAc (1
µCi, experiment 1), TDP-[
C]Rha (250,000 cpm,
experiment 2), and buffer A to a total volume of 320 µl. Total cpm
incorporated into lipids were: 21,340 (experiment 1), 7,500 (experiment
2), and 22,380 (experiment 3). Samples were applied to TLC plates as
described in the legend to Fig. 1.
However, the inclusion of
UDP-[C]Galp in the assay had the most
dramatic effect. Only GL 3 and Gl 4 and material at the origin, perhaps
higher homologs, became labeled, indicating growth of the galactan
chain on the polyprenol-P-P-GlcNAc-Rha unit. The lipid products from
these three reactions were subjected to mild acid hydrolysis and the
water soluble products chromatographed on paper against the
Rha(1
3)GlcNH
standard (Fig. 7). The profile
bears out the conclusion that [
C]Gal from
UDP-[
C]Gal was selectively incorporated into
higher glycolipid intermediate(s). Radioactive GC of the
NaB[
H]
reduced, methylated and
acetylated [
C]Gal oligosaccharide preparation,
as described for the derivatized linkage disaccharide (Fig. 3),
confirmed the dominance of products with retention times indicative of
[
C]Gal-containing tri- and tetrasaccharide.
Figure 7:
Paper chromatography of the lipid-linked
oligosaccharides released from the [C]GlcNAc,
[
C]Rha, and
[
C]Gal-labeled GL 1-4 by mild acid
hydrolysis. Samples of the
C-labeled lipid fractions from
the three assays described in Fig. 6were subjected to mild acid
hydrolysis (0.1 N HCl in CHCl
/CH
OH
(2:1), 25 °C, 4 h) and neutralized (recovery of water-soluble
radioactivity was 84%, 27% (due to the presence of the alkali-stable
GPLs) and 83%, respectively). Radioactivity was applied to sheets of
Whatman 3MM, developed in isobutyric acid/0.5 M NH
OH (5:3) overnight, and the strips cut into 1-cm
sections and counted. GlcNH
and linkage disaccharide
(Rha-GlcNH
) were run on parallel strips and located with
aniline-phthalate.
In order to demonstrate that the [C]Gal
transferred from the UDP-[
C]Galp precursor appeared as [
C]Galf in
GL 3 and GL 4, the NaB[
H]
reduced and
per-O-methylated oligosaccharide mixture from the
[
C]Gal-labeled lipid fraction was hydrolyzed and
alditol acetates prepared(1) . Radioactive GC showed two
products coincident with t-Galf (i.e. 2,3,5,6-tetra-O-Me-1, 4-di-O-Ac-galactitol) and
5-linked Galf (i.e. 2,3,6-tri-O-Me-1,4,5-tri-O-Ac-galactitol) among
the range of Galf derivatives derived from cell wall galactan.
In addition, hydrolysis (2 M CF
COOH) of the
[
C]Gal-labeled lipids and analysis by Dionex
HPLC on the Dionex CarboPac PA1 column (a categorical means of
distinguishing Gal and Glc) established that all of the lipid
radioactivity was in Gal and not in Glc.
In these experiments, no
direct evidence was provided that GL 3 and GL 4 were derived from GL
1/GL 2, i.e. a precursor-product relationship was not
demonstrated. In order to generate preliminary evidence to this effect,
tubes containing UDP-[C]GlcNAc and the standard
reaction mixture were incubated at 37 °C for 30 min, followed by
the addition of ``cold'' UDP-Gal as a substrate for further
synthesis and more UDP-GlcNAc as a chase. Tubes were then incubated
further for variable times (Fig. 8). The emergence of the
[
C]GlcNAc-containing GL 3 and GL 4 and also,
apparently, a GL-5 was evident, particularly after the longer
incubation periods. The other most distinctive quantitative effect of
this form of chase was a steady loss of radioactivity from the total
lipid fraction (175,000 cpm/reaction mixture/0.8 mg of protein at O
chase time (tube 1), compared to 70,000 cpm after the 90 min chase).
Over this period, the GL 1/2 combination lost over half of its
radioactivity, and incorporation into GL 3 and GL 4 increased 4-fold
and 15-fold, respectively.
Figure 8:
Demonstration of a precursor-product
relationship between GL 1/GL 2 and GL 3/GL 4. All eight reaction tubes
contained membranes, ATP, dTDP-Rha, and
UDP-[C]GlcNAc to allow synthesis of
[
C]GlcNAc-containing GL 1 and GL 2. After 30 min
of incubation at 37 °C, 0.22 mM UDP-Gal was added to each
tube as additional substrate, 0.22 mM UDP-GlcNAc as chase and
Percoll-60 fractions. Tubes were then incubated for 0 min (lane
1), 10 min (lane 2), 20 min (lane 3), 45 min (lane 4), 60 min (lane 5), and 90 min (lane
6). The lipids were extracted from reaction mixtures,
chromatographed, exposed to x-ray film for 2 weeks as described in the
legend to Fig. 1, and individual lanes also
counted.
Present success in defining the early stages of mycobacterial
cell wall synthesis arose from the realization of chemical, and hence
biosynthetic, similarities with the cell walls of Gram-positive
bacteria(38) . Early chemical studies of Gram-positive cell
walls had revealed that teichoic acids released from cell walls by
treatment with dilute acid contained a phosphate group esterified at C6
of some of the muramic acid residues in the wall
peptidoglycan(7) . Subsequent investigation of the ribitol
teichoic acid of Staphylococcus aureus demonstrated an
attachment to peptidoglycan by a discrete ``linkage unit''
containing GlcNAc-1-P and 2 or 3 glycerol-P residues (39) .
From detailed analysis of teichoic acid attachments in a wide range of
species, it is now clear that linkage units consist of a
disaccharide-1-phosphate (N-acetylmannosaminyl-N-acetylglucosamine-1-phosphate)
unit with a small number (1-3 depending on the species) of
glycerol-P residues attached to the N-acetylmannosamine
(ManNAc)(10) . This unit is attached, in turn, to muramic acid
in peptidoglycan through the GlcNAc-1-P, while the teichoic acid chain
proper, composed of ribitol-P units, is linked through a phosphodiester
to the terminal glycerol-P residue of the linkage unit. The structure
is thus:
(ribitol-P)-(glycerol-P)
-ManNAc-GlcNAc-1-P-MurNAc
. . . (40) . Of more direct relevance to this study, linkage
units are also involved in cell wall attachment of a polysaccharide in Micrococcus luteus, and of teichoic acids in actinomycete and
related bacteria(10) . The GlcNAc-1-P link is highly
susceptible to acid hydrolysis, accounting for the ease of extraction
of teichoic acid from the cell wall under acidic conditions and the
retention of a phosphate group on muramic acid. The similar
susceptibility of the mycobacterial arabinogalactan-peptidoglycan
linkage, and the isolation of (Galf)
Rha-GlcNAc units (8) demonstrated the existence of
distinct but analogous linkage units in mycobacteria. Thus, current
evidence indicates that the whole of the mycolylarabinogalactan (mAG)
complex of mycobacterial cell wall is covalently linked to
peptidoglycan through a crucial disaccharide linker unit attached to
the nonreducing terminus of the galactan of mAG in the following
arrangement:
-D-Galf-(
1
4)-
-L-Rhap-(1
3)-D-GlcNAc-(1
P
6)Mur-N-glycolyl (8) . Presumably only the occasional muramic acid residue is so
occupied(8) . Yet to be resolved is the old evidence that
attachments not involving phosphorus also exist(41) . The
special version of linkage unit formed in mycobacteria also extends to
a broad range of Mycobacterium, Rhodococcus, and Nocardia spp.(42, 43) .
The type of linkage unit that attaches teichoic acid to peptidoglycan in the Gram-positive cell wall is highly conserved among a wide range of only distantly related species indicating that it confers a significant advantage in either the synthesis or the properties of the cell wall(10) . Likewise, we have attributed comparable significance to the mycobacterial linkage unit(38) . The initial studies of teichoic acid synthesis in S. aureus demonstrated the key role played by the linkage unit in initiation of new teichoic acid polymer chains(10) . A membrane fraction from mechanically disrupted bacteria catalyzed the synthesis of a trace of ribitol teichoic acid from the precursor CDP-ribitol. However, addition of the precursors of the linkage unit, UDP-GlcNAc, and CDP-glycerol, dramatically stimulated teichoic acid synthesis. More detailed investigations, including pulse-radiolabeling experiments, showed that the biosynthetic system catalyzed the incorporation of GlcNAc-1-P from UDP-GlcNAc into a lipid molecule which in turn gave rise to other lipids in the presence of CDP-glycerol. When CDP-ribitol was added, radioactivity from these lipids appeared in the newly synthesized teichoic acid(44) . The lipophilic part of the lipids was shown to be an undecaprenolphosphate of the type involved in peptidoglycan synthesis, and the initial transfer to it of GlcNAc-1-P was very sensitive to inhibition by the antibiotic tunicamycin(44) . In the case of the M. luteus polysaccharide, the initial biosynthetic reaction is also the tunicamycin-sensitive transfer of GlcNAc-1-P to a polyprenyl phosphate carrier lipid(45) . Thus, these experiments demonstrated that the first stage in teichoic acid and M. luteus polysaccharide synthesis was the assembly of linkage unit on the polyisoprenol-phosphate carrier lipid, and that this linkage unit-lipid then acted as the primer on which the polymer was assembled from CDP-ribitol.
This pioneering work on the
biogenesis of the cell wall of Gram-positive bacteria provided the
framework for the present experiments. Based solely on the precedents
of teichoic acid and M. luteus polysaccharide synthesis, it
was possible to propose the following initial steps for the first
stages of mAG synthesis in Mycobacterium: polyprenyl-P +
UDP-GlcNAc polyprenyl-P-P-GlcNAc + UMP;
polyprenyl-P-P-GlcNAc + dTDP-Rha
polyprenyl-P-P-GlcNAc-Rha
+ dTDP; polyprenyl-P-P-GlcNAc-Rha + UDP-Gal
polyprenyl-P-P-GlcNAc-Rha-Gal + UDP; and
polyprenyl-P-P-GlcNAc-Rha-Gal + UDP-Gal
polyprenyl-P-P-GlcNAc-Rha-Gal-Gal + UDP.
The evidence presented in this report that these steps represent the initial events in mycobacterial cell wall biogenesis is firm, although based mostly on comparative radiolabeling experiments, and sensitivity of products to acid, base, and tunicamycin. The paucity of tangible quantities of polyprenyl-PP-GlcNAc (the proposed structure for glycolipid 1) and polyprenyl-P-P-GlcNAc-Rha (glycolipid 2) precluded characterization of the polyprenyl component. The answer to this question may lie in the isolation of glycolipid 2, which appears present in relatively large, steady state levels. The question of the nature of the polyprenyl carrier is an important one, since mycobacteria to date have yielded only decaprenols (24, 25, 27) and heptaprenols (25, 33) as their version of the bactoprenols, but never the common undecaprenol.
Subsequent steps in
cell wall biogenesis are less well established. UDP-Galp is a
very effective substrate for the Galf-containing galactan
component of mAG ()and, according to present results, of the
apparent glycolipid intermediates. In Salmonella enterica,
UDP-Galp is a precursor of the Galf in T1
polysaccharides(46) . In Penicillium charlosii, the
Galf of UDP-Galf can be transferred to the
polysaccharide galactocarylose(47) . Stevenson et al. (48) based on genetic analysis concluded that a single enzyme
can convert UDP-Galp to UDP-Galf through a 2-keto
intermediate and that orf6 is the gene involved. Present
results would indicate that this enzyme is membrane-bound. Recently, we
described the presence of the rfb (rhamnose biosynthetic)
genes close to a new insertion-like element (ISL445) in the genome of M. tuberculosis, which included the 3`-region of the rfbB gene, the whole rfbC gene, and the 5` region of
the rfbA gene; however, the rest of the rfbA gene and
the following rfbD gene were not obvious in this region. (
)Accordingly, the genetics and enzymology of mAG-linkage
unit synthesis show the promise of unique molecular principles to match
the novelty of the biochemical pathway described herein. Thus, this
work represents a return to the topic of mycobacterial cell wall
biogenesis which proved an intractable problem in previous times, and
the assays and intermediates described pave the way for screens for new
anti-tuberculosis drugs to counteract the serious problem of drug
resistance.