From the Département de Mécanismes Moléculaires des Infections Mycobactériennes, Institut de Pharmacologie et Biologie Structurale du CNRS et de l'Université Paul Sabatier (UMR 5089), 205 route de Narbonne, Toulouse 31077 cedex 04, France
Received for publication, October 14, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Disruption of the mma4 gene
(renamed hma) of Mycobacterium tuberculosis has
yielded a mutant strain defective in the synthesis of both keto- and
methoxymycolates, with an altered cell-wall permeability to small
molecules and a decreased virulence in the mouse model of infection
(Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle,
M. A., Yu, K., Quémard, A., Smith, I., and Daffé, M. (2000) Mol. Microbiol. 36, 630-637). Assuming that the
mutant would accumulate the putative precursors of the oxygenated mycolates of M. tuberculosis, a detailed structural
analysis of mycolates from the hma-inactivated strain was
performed using a combination of matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry, proton NMR
spectroscopy, and chemical degradation techniques. These consisted most
exclusively of Mycolic acids, Numerous studies have been and are currently devoted to the structures
and biosynthesis of these acids, primarily because these substances are
specific to the Mycobacterium genus, and their metabolism is
the only clearly identified target inhibited by the major
antitubercular drug, isoniazid (5-8). With the re-emergence of
tuberculosis infections caused by multidrug-resistant strains and the
need for the development of new tuberculous drugs, deciphering the
biosynthesis pathway leading to mycolates still represents a major
objective of researchers. However, it remains that, despite the
intensive efforts of biochemists over decades (3, 9-10) and, more
recently, the help of molecular genetic (11, 12), the biosynthesis
pathway leading to mycolic acids is still poorly understood.
Nevertheless, it is currently admitted that the two known mycobacterial
fatty-acid synthases (FAS)1
participate in the formation of all types of mycolates or their precursors. FAS-I, a synthase that has been shown to be a bimodal system, is necessary to produce C16, 18 and
C22-26 saturated fatty acids, which may be either directly
incorporated in mycolates as the Mycolic acids occur usually in mycobacterial species as a mixture of
various related molecules that differ from one another by the presence
of chemical groups located on well-defined positions of their long
methylene ("meromycolic") chain. In members of the M. tuberculosis complex, three types of mycolates are commonly encountered (15, 16). The least polar mycolates, also called Bacterial Strains and Culture Conditions--
The wild-type
M. tuberculosis H37Rv (ATCC 27294) transformed with pMV261,
a replicative vector, its isogenic mutant strain referred as
hma::hyg, obtained by disruption
in the hma gene and the complemented mutant strain named
pJD, carrying the wild-type hma gene on the multicopy
plasmid pMV261 (1) were grown at 37 °C on synthetic Sauton medium as
surface pellicles for a few weeks. Kanamycin-resistant transformants of
M. smegmatis mc2155 strain were obtained after
electroporation with the replicative plasmid pMV261 containing the
wild-type hma gene cloned downstream of the hsp60
promoter (1). The resultant transformants were grown to stationary
phase in 5 ml of 7H9 medium with kanamycin (10 µg ml Purification of Mycolic Acids--
Whole cells or bacterial
residues obtained after lipid extraction with organic solvents (1) were
saponified by a mixture of 40% KOH and methoxyethanol (1:7, v/v) at
110 °C for 3 h in a screw-capped tube. After acidification,
fatty acids were extracted with diethyl ether and methylated with an
ethereal solution of diazomethane (15). The mycolate patterns of the
strains were determined by analytical thin-layer chromatography (TLC)
on Silica Gel 60 (Macherey-Nagel) using either eluent A (petroleum
ether/diethyl ether; 9:1,v/v, five runs) or eluent B (dichloromethane).
Revelation of lipid spots was performed by spraying the plates with
molybdophosphoric acid (10% in ethanol), followed by charring. The
crude mycolate fraction was obtained by precipitating an ethereal
solution of fatty acid methyl esters with methanol at 4 °C, followed
by centrifugation at 4000 × g for 20 min (24). The
different classes of mycolates were separated by chromatography on a
Florisil column irrigated with increasing concentrations of diethyl
ether (0, 10, 20, 30, and 50%, v/v) in petroleum ether, and
purification was achieved by preparative TLC using eluent A (17). To
search for the presence of ethylenic compounds, the various purified
mycolate types were analyzed and fractionated on
AgNO3-impregnated silica gel TLC plates developed with
eluent B (CH2Cl2) or eluent C (petroleum ether/diethylether; 9:1, v/v). The various purified types and sub-types
of mycolates were quantified by weighing and radiolabeling with
[14C]acetate.
Radiolabeling--
30 µl of [1-14C]acetic acid
sodium salt (2 MBq/mmol, Amersham Biosciences) were added to a 30 ml of
surface cultures of the H37Rv and
hma::hyg strains of M. tuberculosis, and the incubation was left for 24 h at
37 °C. The culture media were discarded, and the cell pellets were
saponified as described above; the resulting fatty acids were
methylated and analyzed by both silica gel-coated and argentated
(AgNO3-impregnated) TLC using dichloromethane as eluent. The radiolabeling was determined using a PhosphorImager (Amersham Biosciences).
Degradative Techniques--
Cleavage of double bonds was
performed by permanganate-periodate oxidation (25) at 30 °C in
tertiary butanol as solvent. Acetolysis of epoxymycolates was realized
by adding acetic acid to the purified mycolates at reflux for 40 h, followed by saponification to liberate the corresponding diols. The
resulting compounds were submitted to oxidative cleavage as above. The
acids obtained from the oxidative cleavages were methylated and
purified on preparative TLC with dichloromethane as eluent.
Miscellaneous Analytical Techniques--
1H NMR
spectra of purified mycolic acid methyl esters were obtained in CDCl3
(100% D) using a Bruker AMX-500 spectrometer at 298 K. Chemical shifts
values (in ppm) were relative to the internal CHCl3
resonance (at 7.27 ppm).
MALDI-TOF mass spectra (in the positive mode) were acquired on a
Voyager-DE STR mass spectrometer (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser emitting at 337 nm. Samples were analyzed in the Reflectron mode using an extraction
delay time set at 100 ns and an accelerating voltage operating in
positive ion mode of 20 kV. To improve the signal-to-noise ratio, 150 single shots were averaged for each mass spectrum, and typically, four
individual spectra were accumulated to generate a summed spectrum. Mass
spectrum calibration was performed using the calibration mixture 1 of
the SequazymeTM peptide mass standards kit (PerSeptive
Biosystems), including known peptide standards in a mass range from 900 to 1600 Da. Internal mass calibration was performed with
sodiated synthetic corynomycolic acid
([C32H64O3 + Na]+,
Mr 519.4748) and its methyl ester derivative
([C33H66O3 + Na]+,
Mr 533.4904) or with the sodiated
C76H148O3 mycolic acid methyl ester
previously described ([M + Na]+ = 1146.1477). The
stock solutions of mycolates were prepared in chloroform, at a
concentration of 1 mM, and were directly applied on the
sample plate as 1-µl droplets, followed by the addition of 0.5 µl
of matrix solution (2,5-dihydroxybenzoic acid (10 mg/ml) in
CHCl3/CH3OH (1:1, v/v)). Samples were allowed
to crystallize at room temperature.
Infrared spectra of samples as films on NaCl discs were recorded using
a PerkinElmer Life Sciences Fourier transform IR 1600 spectrometer.
Gas chromatography (GC) of fatty acid methyl esters derived either from
the saponification of whole cells or from degradation of purified
mycolates was performed on a Hewlett-Packard 5890 series II apparatus
equipped with an OV1 capillary column (0.30 mm × 25 m) using
helium gas. The temperature separation program involved an increase
from 100 to 300 °C at the rate of 5 °C/min, followed by 10 min at
300 °C. GC-mass spectrometry (GC-MS) analyses were performed on an
HP 5889X mass spectrometer (electron energy, 70 eV) coupled to an HP
5890 series II gas chromatograph fitted with a column identical to that
used for GC. GC-MS analyses were realized in the electron impact mode.
Lipid Phenotype Associated with the Disruption of the hma
Gene--
The parent strain (H37Rv) of M. tuberculosis and
its isogenic hma-disrupted mutant exhibited similar growth
rates both on synthetic media and during the infection of the
macrophage-like THP-1 cell line (1). They were similar both in their
content in extractable lipids (20-21% of the bacterial dry weights)
and in wall-linked mycolates (10%). No obvious difference was seen between the two strains in terms of major extractable lipids, including
sulfatides, triacylglycerol, glycerol mycolate, trehalose monomycolate,
trehalose dimycolates, and phospholipids (data not shown). They also
showed identical C16-C26 fatty acid
methyl ester profiles. They differed, however, in the types of mycolic
acids esterifying trehalose and glycerol in extractable lipids, and those linked to the cell wall arabinogalactan. Although the parent strain contained the three characteristic types of mycolates, namely
Examination of the MALDI-TOF mass spectrum of the whole cell fatty acid
methyl esters of the complemented strain confirmed the above data. The
mass spectrum from the strain complemented with the wild-type
hma gene on a multicopy plasmid contained the two massifs of
peaks previously observed in the spectrum of the parent strain (17),
i.e. a massif assignable to Definition of the Types of Mycolic Acids in the hma::hyg
Strain--
The MALDI-TOF mass spectrum of the fatty acid methyl
esters from the hma-inactivated mutant contained only one
massif of peaks, in the region between m/z 1118 and 1272, corresponding to
To determine the origin of the structural difference between the
Structure of the Various
Additional data on the location of the various chemical functions in
mycolates were obtained by mass spectrometry in the electron impact
(EI) mode, notably by the occurrence of ion peaks due to rearrangements
of the aldehyde fragments derived from the pyrolysis of the molecules
(27-30). Indeed, as expected, a prominent peak at 410 m/z was observed in all the EI mass spectra of
the mycolates examined and corresponded to the hexacosanoic acid
methyl ester released from the pyrolytic cleavage of mycolates from
members of the M. tuberculosis complex (15). Furthermore, in
the EI mass spectra of the dicyclopropanated Structure of Oxygenated Mycolates of hma::hyg
Strain--
Compound E produced by the mutant in small amounts
exhibiting the lowest Rf (Fig. 2A) was
purified by chromatography on a Florisil column. It represented
5% (by weight) of the mycolic acid methyl esters of the mutant and was
analyzed by various analytical techniques. The occurrence of mycolic
structure, i.e.
Minor signals assignable to double bonds (at 5.34 ppm) were also
observed in the 1H NMR spectrum of compound E (Fig. 3).
Accordingly, the suspected heterogeneity of the mycolate was resolved
by argentation chromatography. The major constituent of compound E,
called epoxy-2, exhibited a mobility higher than that of the purified
epoxymycolates of M. fortuitum, which contain a
double bond instead of a cyclopropyl group, as expected. In addition, a
minor polar lipid spot, with a mobility similar to that of the
epoxymycolates of M. fortuitum, was present in compound E. The two classes of epoxymycolates from compound E were purified by
preparative argentation chromatography and analyzed by MALDI-TOF mass
spectrometry (Table II). Epoxy-2, the least polar compound,
corresponded to cyclopropanated cis-epoxymycolates having
77-85 carbon atoms, whereas the minor and most polar constituent, epoxy-3, consisted mainly of epoxymycolates containing one double bond;
more unsaturated compounds (two double bonds or one double bond and a
cyclopropyl ring) were also present in epoxy-3, as observed for
Kinetics of Production of the Different Types of Mycolates
Synthesized by the hma::hyg Mutant Strain of M. tuberculosis--
To determine whether the Overexpression of hma in M. smegmatis and MALDI-TOF Mass
Spectrometry Analysis of Mycolates from the Transformed
Strain--
The mycolate pattern of M. smegmatis is
different from that of M. tuberculosis, especially because
the former mycobacterial species does not contain methoxy- and
ketomycolates (22). Instead, M. smegmatis elaborates
C75-C84 epoxymycolic acids, short-chain monounsaturated C60-66 mycolic acids (called We have previously shown that the introduction of the gene
cmaA from M. bovis BCG Pasteur into M. smegmatis induced the production by the recipient strain of large
amounts of methyl-branched hydroxymycolic acids and small amounts of
ketomycolic acids (22). Inactivation of the corresponding gene of
M. tuberculosis H37Rv, hma (or mma4), has resulted in the complete abolishment of the production of all the
oxygenated mycolates normally present in M. tuberculosis H37Rv, namely the methoxy-, keto-, and hydroxymycolic acids (1). It was
thus concluded that the hma gene is involved in the
synthesis of oxygenated mycolic acids and that hydroxymycolic acids
are likely to be precursors of methoxy- and ketomycolates in
M. tuberculosis. Interestingly, the
hma-disrupted mutant strain was found to produce new types
of mycolic acids absent from the parent strain, suggesting that these
molecules may represent the precursors of the oxygenated mycolates of
M. tuberculosis (17). Accordingly, the present work was
undertaken to elucidate the chemical structures of these molecules.
Application of MALDI-TOF mass spectrometry, 1H NMR
spectroscopy, and chemical degradation techniques to the analysis of
the various purified subclasses of mycolates produced by the mutant
established several facts. First, in the absence of the hma
gene, the mutant strain synthesized as much mycolates as the parent
strain, suggesting that the overall bacterial content in these
molecules is important for the normal physiology of the bacilli.
Second, both the mutant and parent strains synthesized similar amounts
of dicyclopropanated One of the questions related to the biosynthesis of mycolic acids is
the metabolic process that leads to the introduction of the specific
oxygenated functions at the distal position of the molecules. Two
classes of substrates could lead to oxygenated chemical functions after
a C-methylation (20, 35), namely a keto group, leading to an
-mycolates, composed of equal amounts of
C76-C82 dicyclopropanated (
1)
and of C77-C79 monoethylenic
monocyclopropanated (
2) mycolates, the double bond being
located at the "distal" position. In addition, small amounts of
cis-epoxymycolates, structurally related to
2-mycolates, was produced by the mutant strain.
Complementation of the hma-inactivated mutant with the
wild-type gene resulted in the disappearance of the newly identified
mycolates and the production of keto- and methoxymycolates of M. tuberculosis. Introduction of the hma gene in
Mycobacterium smegmatis led to the lowering of
diethylenic
mycolates of the recipient strain and the production of
keto- and hydroxymycolates. These data indicate that long-chain
ethylenic compounds may be the precursors of the oxygenated mycolates
of M. tuberculosis. Because the lack of production of
several methyltransferases involved in the biosynthesis of mycolates is
known to decrease the virulence of the tubercle bacillus, the
identification of the substrates of these enzymes should help in the
design of inhibitors of the growth of M. tuberculosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-branched
-hydroxylated long-chain fatty
acids (up to 90 carbon atoms), are the hallmark of the
Mycobacterium genus that comprises several human pathogens
such as Mycobacterium tuberculosis and Mycobacterium
leprae, the causative agents of tuberculosis and leprosy,
respectively. These molecules represent major cell envelope components
(40-60% of the cell dry weight) and are found covalently linked to
the cell wall arabinogalactan or esterifying trehalose and glycerol;
both types of mycolic acid-containing components are believed to play a
crucial role in the structure and function of the mycobacterial cell
envelope (1-3). Mycolic acids attached to the cell wall
arabinogalactan are organized with other lipids to form an outer
permeability barrier with an extremely low fluidity that confers an
exceptional low permeability to mycobacteria and may explain their
intrinsic resistance to many antibiotics (4). Trehalose mycolates have
been implicated in numerous biological functions related both to the
physiology and virulence of Mycobacterium tuberculosis
(3).
-branch chain or used as substrates
of the elongation system, FAS-II. The finding that isoniazid strongly and specifically inhibits InhA, an enoyl-acyl carrier protein reductase that belongs to FAS-II (13-14), is consistent with
the proposed biosynthetic pathway leading to the various types of mycolates.
-mycolates, are composed of C76-C82 fatty
acids (17) and contain two cis-cyclopropyl groups, at the
so-called "proximal" and "distal" (relative to the carboxyl
group) positions of the meromycolic chain (see Fig.
1A). The more polar "M" and "K" mycolates consist of
C82-C89 substances (17) and contain a
cis- or a trans (with an adjacent methyl
group)-cyclopropyl group at the proximal position, and a methoxy- or a
keto- group (with an adjacent methyl group) at the distal position
(Fig. 1A). These discrete
structural variations in mycolates may be of crucial biological
importance, because it has been shown that mutations resulting in the
loss of these chemical functions profoundly modify the permeability of
the cell envelope to solutes and severely affect the virulence of the
mutant strains in experimental infections (1, 18, 19). Accordingly, the
enzymatic systems that introduce the chemical modifications in the
mycolic acid chain merit special attention. Based on C-alkylation mechanisms (20), a biosynthetic pathway that may explain the action of
specific S-adenosylmethionine-dependent
methyltransferases on ethylenic precursors leading to methyl branches
and cyclopropanes in mycolates has been postulated (11). Similar
C-alkylation mechanisms have been proposed to explain the synthesis of
keto- and methoxymycolic acids: the transformation of the distal double bond of a precursor into a secondary hydroxyl group with an adjacent methyl-branch using an
S-adenosylmethionine-dependent methyltransferase coded by the gene mma4 (21). A gene with the same function
in Mycobacterium bovis BCG, first called cmaA
(22), when introduced in Mycobacterium smegmatis has been
shown to confer to the latter organism the ability to produce
ketomycolic acids. In addition, the transformant produced large amounts
of hydroxymycolic acids with an adjacent methyl-branch, structurally
related to the ketomycolic acids of M. bovis BCG. Trace
amounts of these hydroxymycolic acids have been also detected in
mycobacterial species producing keto- and/or methoxymycolates, further
supporting the hypothesis that hydroxymycolic acids can be the
precursors of both keto- and methoxymycolic acids (23). Finally, a
mutant strain of M. tuberculosis in which the
mma4 gene (thereafter called hma for
hydroxymycolic acid) has been
inactivated and shown to be devoid of both keto- and methoxymycolic
acids (1, 17). Although this last experiment clearly established the
biosynthetic relationships between the three oxygenated mycolates of
M. tuberculosis, the chemical structures of precursor
molecules used by Hma to produce these oxygenated mycolates remain
unknown. Interestingly, analysis of the mass spectra of the fatty acids
from the mutant strain indicated the accumulation of new substances
structurally related to
-mycolates and absent from the parent strain
(17). In an attempt to identify these putative precursors of oxygenated
mycolic acids of M. tuberculosis, a detailed analysis of the
mycolic acid content of the hma knock-out mutant was
performed using the combination of matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, proton nuclear magnetic resonance (1H NMR) spectroscopy,
and various chemical degradation techniques.
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Fig. 1.
Structures of the major mycolic acids of
M. tuberculosis H37Rv (A) and
hma::hyg mutant
(B). The total carbon number refers to
data obtained from the MALDI-TOF mass spectra. The mass values of the
major homologues are represented in boldface. The values of
m1 (the number of methylene groups in the distal
part) are indicated; m2 and
m3 depend on the presence or the absence of
methyl branch adjacent to the double bond and cyclopropane on the
meromycolic acid chains. m2 and
m3 = 26, 28 ( ,
-mycolates); 33-35
(M, methoxymycolates); 33, 35 (K, ketomycolates);
30, 32 (
1, dicyclopropanated mycolates); 29, 31, 33 (
2, monoethylenic mycolates); 29-33 (E,
epoxymycolates). t indicates the trans
conformation of the proximal cyclopropane that is always accompanied by
a methyl branch, explaining the odd-numbered values of the total
carbon number.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1)
as described previously (22) because of the observed frequent loss of
the plasmid in 100-ml cultures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-, methoxy-, and ketomycolates, the mutant contained most exclusively
-mycolates (Fig.
2A). Because the two strains
contained similar amounts of mycolic acids, the mutant strain produced
more
-mycolates than the parent strain to compensate for the absence of oxygenated mycolates. Nevertheless, the lack of production of
oxygenated mycolates resulted in a profound alteration of the cell
envelope permeability (1). That this phenotype was due to the
inactivation of the hma gene was shown by the
complementation of the mutant strain with the wild-type hma
gene. Analysis of the mycolate profile of the complemented strain
showed that the production of both keto- and methoxymycolates was
restored (Fig. 2A), but the ratio between the two oxygenated
mycolates differed from that of the parent strain. This difference,
attributed to the overexpression of the hma gene in the
mutant, was found to affect the permeability of cell wall barrier of
the complemented strain to chenodeoxycholate. The complemented strain
exhibited a lower initial rate of uptake and less accumulation of the
probe compared with the parent strain but higher than that of the
hma-inactivated mutant (data not shown).
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Fig. 2.
Thin-layer chromatography of the methyl
esters of mycolic acids from M. tuberculosis.
A: silica gel as adsorbent: 1, H37Rv
(pMV261); 2, hma::hyg
mutant; 3, hma::hyg mutant
complemented with the wild-type hma gene; 4,
hma::hyg mutant (methanol-insoluble
esters); ,
-mycolates; M, methoxymycolates;
K, ketomycolates; E, epoxymycolates. Solvent:
petroleum ether/diethylether 9:1 (v/v, five runs). B:
argentation chromatography of
-mycolates from: 1,
H37Rv (plasmid pMV261); 2,
hma::hyg mutant; 3,
hma::hyg mutant complemented with the
wild-type hma gene (pJD).
1,
2, and
3 refer to dicyclopropanated, monoethylenic, and
monocyclopropanated, and diethylenic-
mycolates, respectively.
Solvent: dichloromethane. Visualization for A and
B: molybdophosphoric acid followed by charring.
Arrows indicate the solvent front.
-mycolates (at
m/z 1146 to 1202) and another attributable to
oxygenated mycolates (at m/z 1218 to 1330). This
latter massif was absent from the mass spectrum of fatty acid methyl
esters from the hma-inactivated mutant (17). In agreement
with the TLC data (Fig. 2A), the content in peaks
corresponding to methoxymycolic acids (pseudomolecular ion peaks at
m/z 1262 for C83 and at
m/z 1290 for C85) was very low in the
mass spectrum of the complemented strain, compared with that of
ketomycolic acids (pseudomolecular ions at m/z
1246 for C82, 1274 for C84, and 1302 for
C86). Pseudomolecular ion peaks, at
m/z 1288 (C85) and
m/z 1316 (C87) were attributed to the
presence of small amounts of ketomycolic acids with odd-carbon number,
commonly occurring in ketomycolates with one
-branch trans cyclopropane (17). It follows then that the production of oxygenated mycolates by the complemented strain was due to the
presence of the hma gene.
-mycolic acids, but was much more complex
than that of the wild-type. Although the massif of
-mycolic acids
from the parent strain consisted of four major pseudomolecular [M + Na]+ ions corresponding to
-mycolic acids composed of
C76, C78, C80, and C82,
that of the mutant strain contained at least 10 major ion peaks that
differed from one another by 14 atomic mass units, indicating the
existence of mycolic acids with odd and even carbon numbers
(C78 to C82, Table
I). To analyze further the mycolic acids
of the hma mutant, the mycolate-enriched methanol
precipitate was first examined by TLC (Fig. 2A). This
analysis confirmed the predominance of
-mycolic acid methyl esters
and showed the presence of an additional polar compound with a mobility
lower than that of ketomycolic acid methyl esters. The methanol
precipitate was then fractionated by chromatography on a Florisil
column. The least polar fraction, consisting exclusively of
-mycolic
acid methyl esters, was analyzed by 1H NMR spectroscopy
comparatively to
-mycolates from the parent and complemented strains
(Fig. 3). The three spectra of
-mycolates contained the expected signals attributed to
cyclopropanated mycolates. The isolated methylene proton
resonances (at 1.29 ppm, broad signal), signals due to terminal
methyl groups (at 0.85 ppm, triplet), methyl ester (at 3.71 ppm,
singlet), methine located at postion C-2 (at 2.50 ppm, multiplet), and
signals assignable to cis-cyclopropyl proton resonances (at
0.35, 0.45, and 0.70 ppm) were clearly identified. In addition, the
1H NMR spectrum of the
-mycolates from the
hma mutant contained signals attributable to ethylenic
proton resonances (at 5.34 ppm) and those of methylene adjacent to
double bonds (at 2.00 ppm). These latter signals were absent from the
spectra of
-mycolates isolated from the parent and complemented
strains. Thus, the analysis of the 1H NMR spectra confirmed
the structural difference observed by MALDI-TOF mass spectrometry
between the
-mycolates from the hma mutant and those of
the parent strain and showed the occurrence of double bonds in the
mycolates produced by the mutant strain. Interestingly, the
1H NMR spectra of the
-mycolates from the parent (Fig.
3) and complemented strains (data not shown) were superimposable,
indicating that the production of ethylenic compounds by the mutant
strain was due to the inactivation of the hma gene.
Comparative data from MALDI-TOF mass spectrometry of mycolic acids
methyl esters from M. tuberculosis H37Rv (parent strain),
hma (hma::hyg mutant), and pJD (hma::hyg
complemented with the wild-type hma gene)
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Fig. 3.
1H NMR spectra of mycolic acids
methyl esters from M. tuberculosis. A,
-mycolates purified from the parent H37Rv strain;
B,
-mycolates purified from the
hma::hyg mutant strain; C,
epoxymycolates purified from the from
hma::hyg mutant strain. Spectra were
recorded in CDCl3 at 500 MHz. Shift values are expressed in
ppm relative to the internal CHCl3 (7.2 ppm).
-mycolates from the mutant strain and those of the parent and
complemented strains, the
-mycolates from the mutant were further
analyzed by AgNO3-impregnated TLC (Fig. 2B), a
layer that is known to have an affinity for cis-ethylenic
bonds. Two major lipid spots, called
1 and
a2, were detected and isolated by preparative AgNO3-impregnated TLC and represented 90-95% (by weight)
of the
-mycolic acid methyl esters from the mutant strain. A lipid
spot with a lower mobility, called a3 (Fig. 2B),
was also detected. The spots corresponding to
2- and
3-mycolates were not detected in mycolates either of the
parental strain or the hma-complemented strain (Fig.
2B). The
1-mycolates from the mutant strain
exhibited the same mobility on AgNO3-impregnated TLC as the
dicyclopropanated mycolic methyl esters from the parent strain, whereas
the
2- and
3-mycolates had a migration
consistent with the presence of one and two cis-ethylenic
double bonds, respectively. Quantification experiments indicated that
1-mycolates represented 45% (by weight) of the
mycolates isolated from the mutant, a percentage comparable to that of
dicyclopropanated
-mycolates in the parent strain. It was thus
concluded that the disruption of the hma gene of M. tuberculosis resulted in the normal production of
dicyclopropanated
-mycolates, the accumulation of
2-mycolates (45-50% of the
-mycolic acid methyl
esters) and small amounts of
3-mycolates.
-Mycolates of hma::hyg Mutant
Strain--
The various
-mycolic acid methyl esters purified by
argentation chromatography from the hma-inactivated strain
were studied by 1H NMR spectroscopy (Fig.
4) and MALDI-TOF mass spectrometry (Fig. 5). From the 1H NMR spectra
it was possible to conclude that
1-mycolates from the
mutant contained two cis-cyclopropyl and no double bond, as found in the
-mycolates from the parent and complemented strains.
2-Mycolic acids contained one cis-ethylenic
bond and one cis-cyclopropyl, whereas
3-mycolic acids contained only ethylenic double bonds (Fig. 4). The MALDI-TOF mass spectrum of a1-mycolates from
the strain hma was surperimposable on that of
dicyclopropanated
-mycolates from the parent strain H37Rv and
consisted of peaks corresponding to C74-C84
-mycolates (Fig. 5 and Table II).
Major peaks in the spectrum of
2-mycolates from the
hma-inactivated strain corresponded to the odd-numbered
carbon atoms (C75 to C83), consistent
with the 1H NMR data that indicated the presence of one
double bond and one cyclopropyl in these molecules. The mass spectrum
of
3-mycolates was more complex. The major series of
peaks corresponded to compounds possessing C76 to
C80 and two cis-double bonds (Fig. 5 and Table II). Two minor series of peaks were also observed and could tentatively be attributed to mycolates having either (i) three
cis-double bonds, (ii) two cis-double bonds and a
cyclopropyl, or (iii) two cis-double bonds and a
trans-double bond with an adjacent methyl branch. The
postulated cyclopropyl and trans-double bond were not
observed in the 1H NMR spectrum of
3-mycolates (Fig. 4), probably due to their relative
small abundance in the mixture. Interestingly, minor amounts of
-mycolates possessing three unsaturation centers have been reported
to occur in mycobacterial strains (26).
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Fig. 4.
1H NMR spectra of the subtypes
of -mycolic acids methyl esters from the
hma::hyg mutant strain of
M. tuberculosis.
1,
2, and
3 refer to the subtypes of
-mycolates as revealed by argentation chromatography shown in Fig.
2B. The conditions are the same as in Fig. 3.
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Fig. 5.
MALDI-TOF mass spectra of the various
subtypes of -mycolates of the
hma-inactivated mutant strain isolated by argentation
chromatography. The
1 subtype contains two
cis-cyclopropane rings, as found in the
-mycolates of the
wild-type H37Rv strain; the
2 series has an odd number
of carbon atoms and contains one cis-cyclopropane ring and
one cis double bond; the major homologues of the
3
subtype contain two cis double bonds. Values indicate the
masses of the corresponding sodium adducts (M+23). For experimental
conditions, see "Experimental Procedures."
Data from MALDI-TOF mass spectrometry of the methyl mycolic acids
methyl esters from the hma mutant strain
-mycolates from the
parent and complemented strains and
1-mycolates from the
hma-inactivated mutant strain, peaks were observed and
corresponded to the "meroaldehydes" with 52, 54, and 56 carbon
atoms, respectively, at 740, 768, and 796 m/z.
However, differences were seen between the EI mass spectra of
- and
1-mycolates in the relative abundances of peaks that allowed the determination of m1 values, i.e. the
number of methylene groups in the distal part of the molecule (Fig. 1).
These resulted from the interactions between the meroaldehydes and the
distal cyclopropane (27, 28). Although the major fragmentation peaks of
the meroaldehydes were observed at 459 and 487 m/z in the spectrum of
-mycolates from the
parent strain, the corresponding peaks in the spectrum of the
1-mycolates from the hma-inactivated mutant strain were seen at m/z 459 and 487 but also at
higher mass values (m/z 515 and 543). These data
indicated that, although the mutant strain elaborated
1-mycolates with chain lengths identical to those of the
-mycolates of the parent strain, the two strains differed one
another by the m1 values of dicyclopropanated mycolates (Fig. 1). A more complex pattern of peaks was seen in the region of
meroaldehydes of the spectrum of
2-mycolates isolated
from the hma-inactivated mutant strain. This was probably
due to the occurrence of a double bond that stabilizes the secondary
fragmentation ions. To circumvent this difficulty and localize the
double bond in
2-mycolates, the molecules were first
submitted to an oxidative cleavage (25), a method that cleaves the
compounds at the level of the ethylenic bond to yield mono- and
dicarboxylic acids. When the resulting fatty acids were esterified,
purified, and analyzed by GC and GC-MS, heptadecanoic and nonadecanoic
acid methyl esters were identified as the monoesters, establishing the
values of m1 (Fig. 1). Similarly, the purified long chain
diester cleavage products were analyzed by MALDI-TOF mass spectrometry,
comparatively to the
-carboxylic acid methyl ester of similar
structure isolated from Mycobacterium phlei (17). The
MALDI-TOF mass spectrum of the long chain diacid methyl esters gave
peaks at m/z values 968, 996, and 1024, consistent with the occurrence of molecules with chain lengths of
C60, C62, and C64 for the free
diacids. Thus, the data on the cleavage products showed that a
cyclopropyl group, insensitive to oxidative cleavage, was located at
the proximal position of the
2-mycolates and confirmed
that intact
2-mycolic acids contained the odd-numbered
carbon atoms as deduced from the analysis of their intact
MALDI-TOF mass spectra.
-branched
-hydroxylated fatty acid, in
the compound was confirmed by the detection of hexacosanoic acid methyl
ester released upon a pyrolytic cleavage on GC. Analysis of the IR
spectrum of compound E revealed the presence of an infrared absorption
band at 850 cm
1 corresponding to the absorption band of a
cis-epoxy group, that of a trans-epoxy ring in
mycolates being observed at 900 cm
1 (31). These data were
confirmed by NMR spectroscopy in that the 1H NMR spectrum
of compound E from the hma-inactivated strain of M. tuberculosis showed a signal at 2.90 ppm (Fig. 3), whereas the
characteristic resonance of the trans-epoxymycolic acid
methyl esters (bearing a methyl branch adjacent to the epoxy group)
from Mycobacterium fortuitum was seen at 2.71 ppm (31-33).
The observed chemical shift values were consistent with those reported
for synthetic epoxy acids (34) in which the resonances of
cis- and trans-epoxide were seen at 2.90 and 2.65 ppm, respectively. The cis configuration of the epoxy ring
in compound E from the hma-disrupted mutant strain was also
supported by the absence of the characteristic doublet at 1.10 ppm
observed in the epoxymycolates from M. fortuitum and
M. smegmatis (31-33) and assigned to the methyl branch
adjacent to the epoxy ring. The 1H NMR spectrum of compound
E also showed the presence of the three characteristic signals of a
cis-cyclopropyl ring (Fig. 3). To characterize compound E
further, the purified mycolate was analyzed by MALDI-TOF mass
spectrometry (Table II). Pseudomolecular ion peaks were seen at 1176, 1204, 1232, 1260, and 1288 m/z, corresponding to
free acids with 77 to 85 carbon atoms. Analysis of the EI mass spectrum
confirmed the molecular mass determination of compound E in that the
constitutive mycolic acid methyl esters gave the expected ion peaks
corresponding to pyrolytic cleavage products: an ion peak at 410 m/z attributed to the released hexacosanoic acid
methyl ester and a series of peaks of "anhydromeroaldehydes" having
51, 53, and 55 carbon atoms at 724, 752, and 780 m/z. The values of m1 in
compound E were determined by oxidative cleavage of the epoxide, which
was first transformed into an
-diol and then cleaved by periodate to
yield long chain fatty acids and dicarboxylic acids. The resulting
reaction products were methylated, purified, and analyzed by GC-MS and
MALDI-TOF mass spectrometry (data not shown). The data allowed the
location of the epoxy group in compound E at the same position as the
distal ethylenic bond in the
2-mycolates (Fig.
1B).
-mycolates from the mutant strain (see above and Table II). It was
thus concluded that compound E consists mainly of cyclopropanated
cis-epoxymycolates and contained minor ethylenic constituents with two degrees of unsaturations showing a structural relationship with
-mycolates of the mutant strain.
- and epoxymycolates
that are accumulated in the hma-inactivated mutant arise as a
consequence of secondary reactions catalyzed by other enzymes, cells
from the parent and mutant strains of M. tuberculosis were
labeled at different time points of their growth phases by
[1-14C]acetate, and the resulting mycolates were purified
and analyzed by radio-TLC. Cells from both log phase (6 days old) and
stationary phase (23 days old) bacteria, produced roughly equal amounts
of the dicyclopropanated (
1) and monoethylenic
(
2) mycolates (Fig. 6).
Although the very minor epoxymycolates were poorly labeled (roughly 4%
of mycolate labeling), as expected, they were seen in all the growth
stages of the mutant strain. It was thus unlikely that the new
-mycolates, which are produced in the hma-inactivated mutant, arise from secondary reactions. This conclusion was further supported by the results of pulse-chase experiments (data not shown) in
which young cells were labeled with [14C]acetate for
24 h and then transferred in fresh sterile media for 2-33 days.
No significant change was observed in the kinetics of incorporation and
amounts of incorporated radioactivity into the various classes of
lipids.
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Fig. 6.
[1-14C]Acetate labeling of
mycolates in log (6-day-old)- and stationary (23-day-old)-phase grown
cells from the parent (H37Rv) and isogenic
hma::hyg mutant strains of
M. tuberculosis. The values represent the
relative percentages of radioactivity determined in each class of
mycolates after separation on specific TLC (for H37Rv,
silica gel G; for hma::hyg mutant,
NO3Ag-impregnated silica gel). See legend of Fig. 2 for
abbreviations. In both cases, the eluent was dichloromethane. Detection
was realized first with a PhosphorImager then with molybdophosphoric
acid reagent to characterize types of mycolates.
'), and
diunsaturated C75-C82
-mycolic acids.
Interestingly, the
-mycolates of M. smegmatis are
structurally similar to the
2-mycolates of the hma::hyg mutant strain of M. tuberculosis (17, 22). Therefore, M. smegmatis was used
as a worthwhile host to address further the question of the possible
biosynthetic relationship between the ethylenic mycolates that
accumulated in the hma::hyg mutant strain and the oxygenated mycolates whose production was abolished in
the mutant. Accordingly, the hma gene from M. tuberculosis was cloned and overexpressed in M. smegmatis, and the resulting transformant clones were screened by
TLC analysis for the presence of keto- and hydroxymycolates (22).
Interestingly, both TLC (data not shown) and MALDI-TOF mass
spectrometry analyses (Fig. 7) showed
that the amounts of
-mycolates was severely reduced, compared with
the parent strain carrying the vector alone, in all the clones
producing keto- and hydroxymycolates, in addition to the usual mycolic
acids of M. smegmatis. Analysis of the MALDI-TOF mass
spectra showed that the parent strain of M. smegmatis
produced mainly C77 and C79
-mycolic acids
(Fig. 7A) and the overexpression of the M. tuberculosis hma gene in M. smegmatis strain led to the
synthesis of mainly C78 and C80 keto- and
hydroxymycolates (Fig. 7B). These data are consistent with
the hypothesis that
-mycolic acids or precursor molecules are used
to produce the keto- and related hydroxymycolates in mycobacterial
strains expressing the hma gene.
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Fig. 7.
Partial MALDI-TOF mass spectra of the fatty
acid methyl esters from the mc2155 strain
(A) and the hma-overexpressing strain
(B) of M. smegmatis. The various
types and pseudomolecular masses of mycolates are indicated: ,
-mycolates; E, epoxymycolates; K,
ketomycolates; H, hydroxymycolates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mycolates exhibiting identical structures,
indicating that the mutation did not affect the production of this type
of mycolates. Third, the mutant produced large amounts of a new type of
mycolates, namely a mixture of monoethylenic monocyclopropanated
-mycolates, and a tiny amount of cis-epoxy-containing monocyclopropanated mycolates; the ethylenic
-mycolates were synthesized throughout the various growth phases of the mutant, indicating that they did not arise from secondary reactions. Fourth, complementation of the mutant strain with the wild-type hma
gene resulted in the disappearance of the new types of mycolates and the production by the complemented strain of all the types of mycolates
of the parent strain. Finally, introduction of the hma gene
in M. smegmatis led to the lowering of diethylenic
-mycolates of the recipient strain and the production of keto- and
hydroxymycolates. Our results demonstrate that the observed changes in
the structure of mycolates of the mutant are specifically due to the
inactivation of the hma gene and raise some important
questions regarding the biosynthesis of mycolic acids.
-methylated ketone (Fig.
8A), as known for
menaquinones, or an ethylenic group (Fig. 8B); in this latter case a water molecule should participate in the
sulfonium-mediated addition mechanism, as proposed for Mma4 function
(21). Because only cis-ethylenic mycolate accumulates
significantly in the hma-inactivated mutant, it is more
attractive to postulate that the ethylenic distal double bond occurring
in the new
-mycolates of the mutant strain is the substrate of
Mma4/Hma. In the "ethylenic hypothesis" (Fig.
8B), the hydroxyl group formed after the methylation step of
the ethylenic bond could be either transformed into a methoxyl group or
oxidized into a ketone. In both cases, an oxidoreduction step would be
necessary to obtain either the keto- or the methoxymycolic acids.
Importantly, the values of m1 in the new
-mycolates are identical to those found in oxygenated mycolates of the parent strain
and shorter than those observed in the
-mycolates of the parent
strain (Fig. 1). Interestingly, the difference in chain lengths between
-mycolates and oxygenated mycolates have been previously observed
(17, 36) and shown to be due to the specificity of the
methyltransferases mainly with respect to the
-end of the growing
mero acyl chain (37).
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Fig. 8.
Possible reaction mechanisms for the
synthesis of oxygenated mycolic acids mediated by the Hma
methyltransferase (adapted from Ref. 20). A,
the "ketone hypothesis"; B, the "ethylenic
hypothesis."
Based on the kinetics of production of -mycolates and related
oxygenated compounds in Mycobacterium microti (38) and other mycobacterial species (39) on the one hand, and the chain lengths of
these two classes of substances, on the other hand, it has been
suggested that these molecules are synthesized by different enzymatic
systems. Accordingly, the common precursor, if any, would not be a full
"meromycolic" diethylenic compound that would then be modified by
different methyltransferases, before or after the Claisen-type
condensation step, to yield
- or/and oxygenated-mycolic acids (for a
review see Ref. 3). By showing that the hma-disrupted mutant
produced dicyclopropanated
-mycolic acids as the parent strain and
in comparable amounts, our data are consistent with the existence of
one biosynthetic system devoted to the synthesis of
-mycolic acids
and another machinery involved in the synthesis of oxygenated mycolic
acids; only the latter one would be altered by the disruption of the
hma gene. Both systems may contain a mixture of "core"
enzymes whose activities are necessary for the synthesis of the
meromycolic chain but may differ one from another by the association of
additional specific enzymes involved in the introduction of various
chemical groups in the meromycolic chain. The accumulation of large
amounts of ethylenic
-mycolates in the hma-inactivated
mutant, comparable to those of oxygenated mycolates of the parent
strain, reinforces the hypothesis that ethylenic long chain fatty acid
derivatives may be used for the biosynthesis of oxygenated mycolates in
the parent and the hma-complemented strain. In the absence
of the hma gene, these putative precursors would be used by
the machinery originally devoted to the synthesis of oxygenated
mycolates and yield distal-ethylenic compounds. The remaining unsolved
question is the discrepancy between the chain lengths of the postulated
precursors and those of the final products. Although the mutant
produced C75-C83 monoethylenic
monocyclopropanated
-mycolates (
2) and cis-epoxy
monocyclopropanated mycolates, the parent and complemented strains
synthesized C81-C89 methoxy- and
ketomycolates, 4-6 carbon atoms longer than the
-mycolates (17,
26). Examination of the detailed structures of mycolic acids, however,
pointed to the observation that the additional carbons in oxygenated
mycolates are not located between the methyl end and the oxygenated
group but distributed in the other parts of the meromycolic chain.
Accordingly, one can postulate that the introduction of an oxygenated
function at this position induces enough hydrophilicity in the long
carbon chain to slightly disturb the specificity toward chain lengths
of the system in charge of the synthesis of the methylenic chain. As a
consequence, the oxygenated groups should be introduced before
completion of the meromycolic chain.
Several genes coding for methyltransferases have been identified in the
genome of M. tuberculosis (40). These enzymes are assumed to
be responsible for the introduction of subtle variations in the mycolic
acid structure, variations that may have profound effects on the
physiology and virulence of the tubercle bacillus; for instance, the
replacement of a cyclopropane ring by a double bond in -mycolates
esterifying trehalose totally abolishes the formation of cords
typifying virulent tubercle bacilli and profoundly affects the
virulence of the mutant strain (19). Similarly, the lack of production
of keto- and methoxymycolates in M. tuberculosis results in
both a change of the permeability and an attenuation of virulence of
the mutant strain (1). These observations, in addition to the fact that
the biosynthesis pathway leading to mycolic acids is the target of the
most effective antituberculous drug, isoniazid, suggest that
methyltranferases may represent good targets for the development of new
antituberculous drugs. In this respect, the identification of putative
precursors of oxygenated mycolates should help in the design of
substrate analogs that would be tested as inhibitors of the enzymatic
activity of the Hma protein. The individual methyltransferases are not
essential for the bacterial growth, but, because their crystal
structures are highly similar (41), the inhibitors of one enzyme may
also abolish the activities of other methyltransferases, resulting in
an additive effect that may be either lethal or bacteriostatic for the
micro-organism.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Eugenie Dubnau (Public Health Research Institute, Newark, NJ) for critically reading the paper and Dr. Annaik Quémard (Institut de Pharmacologie et Biologie Structurale (IPBS), Toulouse, France) for friendly discussion. We also thank Drs. Fabienne Bardou and Stéphanie Ducasse (IPBS, Toulouse, France) for the generous gift of some lipid samples and Jean-Dominique Bounery (IPBS, Toulouse, France) for expert assistance in the GC-MS experiments.
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FOOTNOTES |
---|
* This work was supported in part by the Ministère de l'Education Nationale, de la Recherche et de la Technologie (Program Action Concertée Incitative: Molécules et Cibles Thérapeutiques), and CNRS (France).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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 33-561-175-569; Fax: 33-561-175-994; E-mail: mamadou.daffe@ipbs.fr.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210501200
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ABBREVIATIONS |
---|
The abbreviations used are: FAS, fatty-acid synthase; BCG, bacillus Calmette-Guérin; GC, gas chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; EI, electron impact.
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