(Received for publication, June 5, 1995)
From the
Biosynthesis of the cell envelope in mycobacteria is largely
unknown; however, several antituberculosis drugs apparently interfere
with this process. Recently, we described a lipid intermediate for the
biosynthesis of the cell wall arabinogalactan/arabinomannan of Mycobacterium smegmatis:
-D-arabinofuranosyl-1monophosphodecaprenol (Wolucka, B.
A., McNeil, M. R., de Hoffmann, E., Chojnacki, T., and Brennan, P.
J.(1994) J. Biol. Chem. 269, 23328-23335). In the
present work, by means of gas chromatography-mass spectrometry, fast
atom bombardment tandem mass spectrometry, and proton NMR, the major
pentose-containing component of the polyprenyl-P-sugar family from M. smegmatis was characterized as
-D-ribosyl-1-monophosphodecaprenol (decaprenyl-P-ribose).
Additionally, the structure of a minor arabinose-containing compound,
-D-arabinosyl-1-monophosphooctahydroheptaprenol, could be
deduced. In vivo labeling experiments with
[
C]glucose demonstrated unequivocally that
decaprenyl-P-ribose is actively synthesized in Mycobacterium
tuberculosis H37Ra and Mycobacterium avium serovar 4. It
is proposed that decaprenyl-P-ribose could be a precursor for the
biosynthesis of either some unknown ribose-containing cell envelope
polymers of mycobacteria or the arabinan part of the cell wall
arabinogalactan/arabinomannan due to the presence of a 2`-epimerase
activity at some late stages of the arabinogalactan/arabinomannan
biosynthesis.
The cell walls of all mycobacteria, including two important
human pathogens: Mycobacterium tuberculosis and Mycobacterium leprae, contain large amounts of an unusual
sugar: D-arabinofuranose (D-Ara). (
)D-Ara
is the major component of two
mycobacterial polysaccharides: arabinogalactan and arabinomannan (for
review, see (1) ). Arabinogalactan, a key element responsible
for the integrity of the lipid-rich cell envelope of
mycobacteria(2) , is covalently linked to the innermost
peptidoglycan layer and serves as a point of attachment for long chain
mycolic acids, which, in turn, interact hydrophobically with numerous
free lipids and glycolipids of the mycobacterial envelope (for review,
see (3) ). The arabinan segments of the membrane-anchored
lipoarabinomannan (4) and exocellular arabinomannan (5) are structurally similar to that of the
arabinogalactan(6) . Arabinofuranose was also found in a
glycolipid isolated from Mycobacterium avium(7) and
structurally related to the nonreducing-end motif of the
arabinogalactan-mycolate complex(8) .
The biosynthesis of D-Ara residues is largely unknown. Mycobacteria
have long been known to contain two unusual polyprenyl phosphates: a
C
-decaprenyl-P (9) and a partially saturated
C
-octahydroheptaprenyl-P(10) . Recently, a family
of endogenous glycosyl-P-polyprenols of Mycobacterium
smegmatis, comprising decaprenyl and octahydroheptaprenyl
phosphate derivatives of arabinose, ribose, and mannose, was described
and the complete structure of one component of this family,
-D-arabinofuranosyl-1-monophosphodecaprenol,
elucidated(11) . In the same work, we determined another
peculiar feature of the mycobacterial polyprenol, namely the presence
of only one internal trans-isoprene residue within the
decaprenol molecule. In vivo labeling pulse-chase experiments
demonstrated that the lipid-linked ribose, arabinose, and mannose of M. smegmatis are active biosynthetic precursors. Additionally,
it was shown that ethambutol, a key antituberculosis drug known to
inhibit arabinogalactan/arabinomannan biosynthesis(12) ,
results in the accumulation of decaprenyl-P-arabinose with a
concomitant decrease of the lipid-linked ribose content(11) .
In the present report, we describe the isolation and structural characterization of the ribose-containing lipid of M. smegmatis (decaprenyl-P-ribose) and demonstrate that this compound is actively synthesized in M. tuberculosis H37Ra and M. avium. The presence of polyprenyl-P-ribose in mycobacteria is puzzling, since ribose-containing cell envelope components, such as polymers or glycolipids, have not been found in these organisms until now. The possible functions of decaprenyl-P-ribose in mycobacteria are discussed.
Radioactive sugars were separated by TLC on cellulose-coated glass plates (Merck, Darmstadt, Germany) in tert-butanol-methylethyl ketone-formic acid-water (8:6:3:3). Plates were autoradiographed.
Phosphate determination was performed as described earlier(11) .
H NMR and FAB-MS/MS analyses were conducted as described
previously(11) .
Our previous studies demonstrated the presence of the mild
acid-labile ribose in the lipid extracts of M. smegmatis(11) . About 75% of the lipid-ribose was recovered in the
fraction of mild alkali-stable, weakly anionic lipids, which
represented, as determined by means of H NMR and FAB-MS/MS,
a family of glycosyl monophosphopolyprenols comprising
C
-decaprenyl-P and C
-octahydroheptaprenyl-P
derivatives. The mycobacterial glycosyl-P-polyprenols could be resolved
by preparative TLC into four major bands, A-D. The fastest migrating
band A (R
= 0.46) consisted of
-D-arabinofuranosyl-1-monophosphodecaprenol (11) . Sugar analysis of the slower migrating band B (R
= 0.43) revealed the presence
of mild acid-labile ribose (340 nmol/100 g of cells) and trace amounts
of arabinose (27 nmol/100 g of cells) representing 7% of the total
sugar of band B. The mild acid-freed ribose of band B was assigned to
the D series by GC/MS analysis of its(-)-2-butylglycoside
derivative(15) . It was determined also that the band B-derived
pentoses and the organic phosphate were present in equimolar amounts.
Further structural information on band B components was obtained by
FAB-MS/MS experiments. The negative-ion FAB-MS spectrum of the purified
band B (Fig. 1A) showed the presence of an intense
signal at m/z 909 (relative intensity, 100%), corresponding to
the deprotonated molecule of decaprenyl-P-ribose (M = 910), and of a less abundant [M - 132 -
H]
fragment ion at m/z 777,
corresponding to C
-P. Signals at m/z 925 and 941
are attributable to oxidation products of the decaprenyl-P-ribose.
Moreover, a series of fragments differing by 68 mass units was observed
(at m/z 567, 499, 431, 363, and 295) derived from the loss of
5-9 isoprene units from the
-end of the isoprenoid chain of
decaprenyl-P-ribose. A very weak signal at m/z 713 (relative
intensity, 8%) indicated for the presence of trace amounts of a
C
-P-pentose (M
= 714).
Figure 1: Negative ion FAB-MS/MS analysis of the purified decaprenyl-P-ribose of M. smegmatis. A, the FAB-MS spectrum; B, the production spectrum of the m/z 909 ion.
The
product ion spectrum of the m/z 909 ion (decaprenyl-P-ribose) (Fig. 1B) was clearly different from that of
-D-arabinosyl-P-decaprenol(11) . The deprotonated
molecule of the latter compound was shown to produce, in tandem mass
spectrometry, two abundant fragments at m/z 819 and 777,
derived from the loss of a part or the entire D-arabinosyl
residue and corresponding to
[decaprenyl-PO
-(CH-CHOH]
ion
and [decaprenyl-PO
]
ion,
respectively(11) . In contrast, the tandem mass spectrum of the
deprotonated molecule of decaprenyl-P-ribose (Fig. 1B)
showed the presence of only one abundant fragment of decaprenyl-P at m/z 777 (relative intensity, 100%), whereas the
[decaprenyl-PO
-(CH-CHOH]
ion
(at m/z 819) was barely observable (relative intensity, 6%).
Recently, we demonstrated that the FAB-MS/MS technique can be applied
for the determination of the anomeric configuration of glycosyl esters
of nucleoside pyrophosphates (16) (
)and
polyisoprenyl phosphates.
In the latter work, it was shown
that the fragmentation pattern of polyisoprenyl-P-sugars depends on
cis/trans configuration of the phosphodiester and 2`-hydroxyl groups of
the glycosyl residue. Compounds with cis configuration, as in
-D-mannopyranosyl-P-undecaprenol and
-D-Ara
-P-decaprenol, give two abundant
fragments corresponding to
[polyisoprenyl-PO
-(CH-CHOH)]
(B) and [polyisoprenyl-HPO
]
(A), with the relative intensity ratio (B/A) of about 1. In
contrast, compounds with trans configuration of the above-mentioned
groups, as in
-D-mannopyranosyl-P-undecaprenol, give an
abundant [polyisoprenyl-HPO
]
fragment and practically no
[polyisoprenyl-PO
-(CH-CHOH)]
ion in tandem mass spectrometry; consequently, the relative
intensity ratio of these two ions (B/A) is very low (close to 0.01) for
compounds with trans configuration. Since the mycobacterial
decaprenyl-P-ribose produces, in tandem mass spectrometry, only one
abundant ion corresponding to
[decaprenyl-HPO
]
, and the
relative intensity ratio of
[decaprenyl-PO
-(CH-CHOH)]
ion
to [decaprenyl-HPO
]
ion is very
low (0.06), it can be deduced that this compound has trans
configuration of the phosphodiester and 2`-hydroxyl groups of its D-ribosyl residue. Therefore, the D-ribosyl residue
of the mycobacterial decaprenyl-P-ribose must be
-linked to
decaprenyl-P.
The 500-MHz H NMR spectrum of the
decaprenyl-P-ribose preparation from M. smegmatis (Fig. 2) shows all signals characteristic for a
glycosylated decaprenyl-P derivative (11) . Proton resonances
of the phospholipid part (decaprenyl phosphate) are, as expected,
almost identical with those observed for the purified
decaprenyl-P-arabinose(11) . However, signals at
0.83 ppm
and 1.2 ppm are not derived from the C
-P compound and can
be assigned to the protons of methyl and methylene groups of the
octahydroheptaprenyl-P contaminant. To estimate the molar ratio of the
decaprenyl-P-ribose versus the C
-P compound in
band B, we compared the integration values measured for the proton
signals of the trans-methyl groups of the
- and the
isoprene unit(s) adjacent to it (at
1.55 ppm) with that of the
methyl protons of the
-isoprene residue (at
1.69 ppm). The
mycobacterial C
-octahydroheptaprenol has four saturated
isoprene units at the
-end (10) and, thus, is expected to
contain no isoprenyl trans-methyl groups. Indeed, the mycobacterial
C
-P-mannose recently purified in our laboratory does not
give any signal at
1.55 ppm. (
)Therefore, the signal
at
1.55 ppm of the band B spectrum (Fig. 2) (integral 6.0)
corresponds to 6 protons of the two isoprenyl trans-methyl groups of
the mycobacterial decaprenyl-P derivative (11) . To the signal
at
1.69 ppm (integral, 3.5) contribute three methyl protons of
the
-residue of both, C
- and C
-P
derivatives. Therefore, the molar ratio of the octahydroheptaprenyl-P
compound versus decaprenyl-P-ribose in band B was
approximately 1 to 6. The position of the individual protons of the
ribosyl residue of decaprenyl-P-ribose (Fig. 2) was determined
through sequential selective decoupling experiments. The anomeric
proton signal of the decaprenyl-P-ribose (a doublet at
5.41 ppm, J
= 4.11 Hz, J
< 1.5 Hz) is upperfield-shifted in comparison with that
measured for the commercially available
-D-ribofuranosyl
1-phosphate standard (from Sigma) (a doublet of doublets at
5.77
ppm, J
= 6.29 Hz, J
= 3.31
Hz). It is known from the literature that the upperfield shift of the
anomeric proton signal of D-ribofuranosyl derivatives is
characteristic for
-linked residues(18) . Therefore, it
can be concluded that the D-ribosyl residue of the
mycobacterial decaprenyl-P-ribose is
-linked to decaprenyl
phosphate. This is in agreement with the above-described results
obtained in FAB-MS/MS experiments. Since, in nature, ribose is always
found in the furanose ring-form, we propose the following structure for
the mycobacterial
-D-ribosyl-1-monophosphodecaprenol (Fig. 3).
Figure 2:
The 500-MHz H NMR spectrum of
the decaprenyl-P-ribose. The sample was obtained from 44 g of M.
smegmatis cells. The purified decaprenyl-P-ribose (
150 nmol
based on sugar estimation) was dissolved in a mixture of deacidified
CDCl
(330 µl) and CD
OD (120 µl).
Extension of the 3.5-5.5 ppm region (uppercurve).
Figure 3:
The proposed structure of the
decaprenyl-P-ribose,
-D-ribofuranosyl-1-monophosphodecaprenol, of M.
smegmatis.
The minor C-P-pentose component
represented about 15% of the total polyprenyl-P-pentoses of band B, as
determined by
H NMR. This value matches quite well with the
amount of arabinose detected in band B (7% of total sugar). The FAB-MS
analysis further confirmed the presence of only small amounts of a
C
-P-pentose (Fig. 1A). Moreover, the
product ion spectrum of the deprotonated C
-P-pentose
molecule (M
= 714) showed the presence of
two abundant fragments at m/z 581 and 623(19) ,
corresponding to an
[octahydroheptaprenyl-HPO
]
ion
(relative intensity, A = 96%) and an
[octahydroheptaprenyl-PO
-(CH-CHOH)]
fragment derived from the cleavage across the pentose ring
(relative intensity, B = 85%), respectively. The relative
intensity ratio B/A was 0.88, thus indicating that the compound has cis
configuration of the phosphate and 2`-hydroxyl groups of the pentosyl
moiety,
as in the case of
-D-arabinofuranosyl-P-decaprenol. Taken together, the
presented data suggest that the minor component of band B is
-D-arabinosyl-1-monophospho-octahydroheptaprenol.
Quantification of the arabinose found in band A (as
C
-P-Ara; 105 nmol/100 g of cells) and in band B (as
C
-P-Ara; 27 nmol/100 g of cells) demonstrated that the
C
-P-arabinose represented about 20% of the total
polyprenyl-P-arabinose (C
- and C
-P-arabinose)
isolated from M. smegmatis. Accordingly, the
decaprenyl-P-ribose (340 nmol/100 g of cells) was recovered from M.
smegmatis cells in amounts almost 3-fold higher than for the
polyprenyl-P-arabinose, thus indicating that the former compound is the
major pentose-containing lipid intermediate in this
organism(11) .
To examine if the lipid-ribose is
characteristic only for M. smegmatis or if it is a common
lipid intermediate of mycobacteria, in vivo labeling
experiments were performed with M. tuberculosis and M.
avium cells. In the previous studies(11) , we demonstrated
that the family of polyprenyl-P-sugars of M. smegmatis can be
rapidly labeled by in vivo incubation of cells with D-[U-C]glucose. Preliminary experiments
with M. tuberculosis H37Ra and M. avium serovar 4
indicated that the efficiency of incorporation of
[
C]glucose label into the lipid-linked sugars in
these organisms is lower than in M. smegmatis, whereby longer
incubation time and higher label are needed to obtain similar yields of
labeling. This fact is probably related to the existence of an
efficient permeability barrier in M. tuberculosis and M.
avium(20) or to differences in the biochemical systems
responsible for glucose uptake and assimilation in mycobacteria. M.
tuberculosis and M. avium cells were labeled in vivo with [
C]glucose for 2 h and 45 min,
respectively, and the fraction of
C-polyprenyl-P-sugars
was isolated, as described under ``Experimental Procedures.''
The purified
C-polyprenyl-P-sugar fractions were submitted
to mild acid hydrolysis and the
C-sugars released to the
water phase were separated by TLC and visualized by autoradiography.
Comparison of the TLC patterns revealed that the sugar composition of
the glycosyl-P-polyprenol families isolated from M. tuberculosis and M. avium (Fig. 4) is very similar to that of M. smegmatis: ribose, mannose, arabinose, and some still
unidentified oligosaccharides are the major
C-labeled
glycosyl residues. Additionally, in the case of M. avium, an
unknown
C-sugar component, which migrated just behind
ribose and co-migrated with cold xylose/lyxose standard, could be
observed (Fig. 4, lane1). These results
demonstrate clearly that decaprenyl-P-ribose was actively synthesized
in the all mycobacteria tested, including M. tuberculosis H37Ra, M. avium serovar 4, and different strains of M. smegmatis, and, moreover, that it represents one of the
predominant lipid-linked intermediates in these organisms.
Figure 4:
TLC of C-sugars released by
mild acid from the purified polyprenyl-P-sugar fraction of M. avium (lane 1) and M. tuberculosis (lane 2)
after in vivo labeling with
[U-
C]glucose. Cells of M. avium serovar
4 and M. tuberculosis H37Ra were incubated with
[
C]glucose for 45 min and 2 h, respectively, and
extracted with 95% ethanol at 70 °C for 15 min. The dried ethanolic
extracts were partitioned into a mixture of chloroform-methanol-water
(8:4:3), and the organic layers were washed with theoretical upper
phase. The lipid extracts were submitted to mild alkaline hydrolysis,
and the alkali-stable lipids were chromatographed on a DEAE-cellulose
column. The polyprenyl-P-sugar fraction of M. avium and M.
tuberculosis were hydrolyzed in 10 mM HCl at 100 °C
for 5 min; free sugars were recovered in the water phase after
partitioning and, subsequently, resolved by TLC. The radioactive spots
were visualized by autoradiography.
D-Ribose is a common component of cell envelope
polysaccharides, such as lipopolysaccharides and capsular
polysaccharides, in many Gram-negative bacteria, and it always occurs
as the -furanosyl residue(21) . For example,
-D-ribofuranose was found in the lipopolysaccharides of Pseudomonas aeruginosa(22, 23) , Salmonella sp.(24, 25) , Shigella sp.(26) , Proteus sp., and spotted fever group
rickettsia strains (27) , and of some
myxobacteria(28) ; in capsular K antigens of Escherichia
coli strains(29, 30) ; and also in a
type-specific, teichoic acid-like capsular polysaccharide
(polyribosylribitol phosphate) of Haemophilus influenzae(31) . Surprisingly, the nature of
-D-ribofuranosyl donor for the biosynthesis of these
bacterial polysaccharides is still unknown(32) . Recently,
ribosyl units were also found in N-linked oligosaccharides of
two eukaryotic organisms, namely in trypanosomatids Blastocrithidia
culicis(33) and Endotrypanum
schaudinni(34) . However, there is no report in the
literature on ribose-containing cell envelope polymers from
mycobacteria.
In the present work, we described a second member of
the glycosyl-P-polyprenol family of M. smegmatis,
-D-ribosyl-1-monophosphodecaprenol, and showed that
decaprenyl-P-ribose is actively synthesized in M. tuberculosis and M. avium. Paradoxically, the function of
decaprenyl-P-ribose in mycobacteria remains obscure. One possibility is
that
-D-ribosyl-P-decaprenol could serve as a ribosyl
donor for the synthesis of still unknown cell envelope or extracellular
polysaccharides of mycobacteria. Such ribose-containing polysaccharides
might be, in fact, overlooked in mycobacteria, since the purification
procedures for mycobacterial cell wall polysaccharides, commonly used
by previous researchers, involved steps, such as strong alkaline
hydrolysis, RNase treatment and extensive extractions, which would lead
to the destruction or removal of certain kinds of ribose
polymers(31) . It is worthy of notice that, in this case, the
mycobacterial
-D-ribosyl-P-decaprenol would then serve as
a donor of unusual
-linked ribosyl units of the putative polymer,
since glycosyl transfer reactions from a glycosyl-P-polyprenol to a
glycosyl acceptor occur with inversion of the configuration at the C-1`
position of glycosyl residue(32) . Unexpectedly, indirect
evidence for the existence of a D-ribofuranosyl-transferase
activity in M. smegmatis was obtained during the studies on
the mechanisms of drug resistance in mycobacteria(35) . Dabbs et al. (35) demonstrated that M. smegmatis and some other fast-growing mycobacteria inactivate rifampin, a
key drug against tuberculosis and leprosy, through an unusual
glycosylation mechanism, namely the addition of an
-D-ribofuranosyl unit to the rifampin molecule. We can
speculate that the
-D-ribofuranosyl-P-decaprenol
described here could act as a donor of
-D-ribofuranosyl
units for the modification of rifampin in mycobacteria.
Another
possible explanation for the existence of decaprenyl-P-ribose and the
absence of ribose-containing cell envelope polymers in mycobacteria is
that decaprenyl-P-ribose could be a precursor for the synthesis of some
of the D-arabinofuranosyl residues of arabinogalactan and
lipoarabinomannan in mycobacteria due to the presence of a hypothetical
2`-epimerase activity. Such 2`-epimerase could act either at the level
of the lipid-linked oligosaccharides, as it was proposed in
archaebacteria (36) or at the level of the final polymer, as it
occurs in the biosynthesis of heparin (37) and bacterial
alginic acid(38) , but apparently not at the level of
decaprenyl-P-pentose, since our in vitro enzymatic trials for
the conversion of C-labeled decaprenyl-P-ribose into
decaprenyl-P-arabinose and vice versa were unsuccessful.
This hypothesis would help to explain the observation that
polyprenyl-P-ribose is present in severalfold higher amounts than
polyprenyl-P-arabinose in mycobacteria and the fact that
antimycobacterial drugs (ethambutol and isoniazid), known to inhibit
the formation of arabinogalactan-mycolate complex of the mycobacterial
cell walls, result in the inhibition of decaprenyl-P-ribose synthesis
and the accumulation of decaprenyl-P-arabinose(11) .
There
are several reports in the literature suggesting that the proposed
2`-epimerization of D-ribofuranose to the corresponding D-arabinofuranose derivative probably occurs in other
bacteria. For example, in Streptomyces antibioticus, which
belongs to the same order of Actinomycetales as mycobacteria,
a 2`-epimerase is apparently responsible for the conversion of the
ribosyl residue of adenosine (or its derivative) into D-arabinofuranosyl residue and the production of D-Ara-A nucleoside analogue(39) . Additionally, both
pentofuranoses, D-Ara and D-Rib
, were found in the lipopolysaccharides of Pseudomonas maltophilia(40) . And finally, it was
suggested that an NAD/NADP-dependent sugar epimerase-like nolK gene of Azorhizobium caulidans(41) might be
involved in the biosynthesis of D-arabinosyl residues present
in the Nod factors of this symbiotic bacterium(17) .
We
propose that, for the synthesis of decaprenyl-P-ribose, mycobacteria
could employ the same -D-ribofuranosyl donor as other
bacteria known to produce
-D-ribofuranosyl-containing
cell envelope polysaccharides. The nature of this compound is still
unknown(32) . Biosynthetic studies aiming at the identification
of the
-D-ribofuranosyl donor in mycobacteria are now in
progress.