From the Department of Biochemistry, Duke University,
Durham, North Carolina 27710 and the ¶ Department of Pharmacology
and Molecular Sciences, The Johns Hopkins University School of
Medicine, Baltimore, Maryland 21205
Received for publication, February 28, 2001, and in revised form, March 7, 2001
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ABSTRACT |
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Chlamydia trachomatis lipid A is
unusual in that it is acylated with myristoyl chains at the glucosamine
3 and 3' positions. We have cloned and expressed the gene encoding
UDP-N-acetylglucosamine 3-O-acyltransferase of
C. trachomatis (CtlpxA), the first enzyme of
lipid A biosynthesis. C. trachomatis LpxA displays
~20-fold selectivity for myristoyl-ACP over
R/S-3-hydroxymyristoyl-ACP under standard assay conditions,
consistent with the proposed structure of C. trachomatis
lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a
non-hydroxylated acyl-ACP to a hydroxyacyl-ACP. When CtlpxA
was expressed in RO138, a temperature-sensitive lpxA mutant
of Escherichia coli, five new hybrid lipid A species were
made in vivo after 2 h at 42 °C, in place of
Escherichia coli lipid A. These compounds were purified and
analyzed by matrix-assisted laser desorption ionization/time of
flight mass spectrometry. In each case, a myristoyl chain
replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coli lipid A. With prolonged growth at 42 °C, all the
ester-linked 3-hydroxymyristoyl residues were replaced with myristate
chains. Re-engineering the structure of E. coli lipid A
should facilitate the microbiological production of novel agonists or
antagonists of the innate immunity receptor TLR-4, with possible uses
as adjuvants or anti-inflammatory agents.
Lipid A is the hydrophobic anchor of lipopolysaccharide of
Escherichia coli and most other Gram-negative bacteria
(1-3). This immunostimulatory glycolipid is also known as endotoxin, an agent responsible for the severe inflammatory reaction of animals to
Gram-negative organisms via the receptor TLR-4 (4-7). The lipopolysaccharide substructure, Kdo2-lipid A (Fig. 1) is
sufficient to support the growth of E. coli under laboratory
conditions and is fully active as an endotoxin (2, 8).
The genetics and enzymology of lipid A biosynthesis have been
elucidated mainly in E. coli (1, 8). However, homologues of
key E. coli lipid A genes are present in virtually all
Gram-negative bacteria. Lipid A is made by a 9-enzyme pathway that
begins with the acylation of UDP-N-acetylglucosamine
(UDP-GlcNAc) by LpxA (Fig. 1) at the glucosamine 3-OH position (1,
8-10). UDP-GlcNAc 3-O-acyltransferases of Gram-negative
bacteria display high selectivity for the lengths and stereochemistry
of their
R-3-hydroxyacyl-ACP1
substrates, and are the primary determinants of the fatty acyl chains
that are incorporated at the 3 and 3' positions of lipid A (9, 11-14).
The crystal structure of E. coli LpxA has been solved; it is
a homotrimeric enzyme with an unusual left-handed parallel In most bacteria examined to date, only R-3-hydroxyacyl
chains are attached to the glucosamine disaccharide backbone of lipid A
(1, 3, 17). However, the 3 and 3' acyl chains of lipid A are not
hydroxylated in Chlamydia trachomatis (Fig. 2), an
intracellular Gram-negative pathogen with a biphasic life cycle and a
chronic inflammatory pathology (3, 18, 19). Ocular infections with C. trachomatis are the leading worldwide cause of
preventable blindness (20, 21). Urogenital infections with C. trachomatis are a major cause of female infertility (20, 21).
C. trachomatis also causes neonatal pneumonia, and may have
a role in the etiology of certain types of arthritis (20, 21). During
the course of an infection, C. trachomatis exchanges some of
its membrane lipids, including lipopolysaccharide, with those of its
mammalian host cell (22) and vice versa (23-25). Understanding the
biosynthesis of C. trachomatis lipid A may provide insights
into the life cycle of this pathogen.
The C. trachomatis genome contains single copy
homologues of most of the genes required for lipid A biosynthesis in
E. coli, including CtlpxA, which encodes a
protein of 280 amino acid residues with 42% identity and 50%
similarity to the 262 amino acids of E. coli LpxA (26).
Given that C. trachomatis makes a penta-acylated lipid A
molecule (Fig. 2), a homologue of the E. coli msbB gene (Fig. 1) (27, 28) is missing in C. trachomatis (26).
We now show that the presence of non-hydroxylated acyl chains at the 3 and 3' positions in C. trachomatis lipid A (Fig. 2) is
explained by the unusual substrate specificity of C. trachomatis LpxA, which is highly selective for myristoyl-ACP.
Expression of C. trachomatis lpxA in E. coli
mutant RO138, which is defective in its own lpxA gene (14,
29) at 42 °C, results in the production of structurally novel lipid
A species. Following many generations of growth at 42 °C, all of the
lipid A molecules synthesized in this construct contain myristate
residues at positions 3 and 3'. However, when analyzed 2 h after a
shift from 30 to 42 °C, residual ester-linked 3-hydroxymyristate
moieties are present only at position 3'. Genetically engineered
bacteria containing modified lipid A structures may prove useful for
the production of novel vaccines and adjuvants.
Materials--
Agar, tryptone, and yeast extract were from
Difco. Acetic acid, chloroform, methanol, and sodium hydroxide were
from Mallinckrodt Chemical Works. Acyl carrier protein, antibiotics,
myristic acid, (R,S)-3-hydroxymyristic acid,
(R,S)-3-hydroxypalmitic acid, and glucosamine 1-phosphate
were from Sigma. Silica Gel 60 thin layer plates (0.25 mm) were
purchased from E. Merck, Darmstadt, Germany. Restriction enzymes,
pfu polymerase, and cloning vectors pBluescript II SK(+) and
pUC18 were obtained from Stratagene. The cloning vector pET23c+ was
purchased from Novagen. T4 ligase and preparative grade
phenol/chloroform/isoamyl alcohol (25:24:1, v/v) were from Life
Technologies, Inc. Deoxynucleotide triphosphates were from Roche
Molecular Biochemicals. Custom PCR and mutagenesis primers were made by
Life Technologies, Inc. T7 sequencing primers were obtained from
Novagen. The LpxC inhibitor L-573,655 (30) was provided by Dr. A. Patchett (Merck Research Laboratories). The more potent LpxC inhibitor
LNTI-229 was synthesized by Nathan Tumey, Duke University (31). Genomic
DNA of C. trachomatis D/UW-3/CX was a gift from Dr. R. Stephens (University of California, Berkeley, CA) (26).
Bacterial Strains and Growth Conditions--
All strains used in
this work are derivatives of E. coli K12. XL1Blue was
purchased from Stratagene, and BL21(DE3)pLysS was purchased from
Novagen. The temperature-sensitive E. coli mutant RO138
(lpxA2 recA rpsL Tetr) was provided by Dr. M. Anderson, Merck Research Laboratories. RO138 is a recA
derivative of SM101 (14, 29). Constructs in which E. coli
lpxA+ was cloned either into pET23c+ (pTO1) or into
pBluescript II SK+ (pTO5) were described previously (16, 32).
Cells were generally grown at 30, 37, or 42 °C, as indicated, in LB
medium (10 g/liter Tryptone, 10 g/liter NaCl, and 5 g/liter yeast
extract adjusted to pH 7.4 with NaOH) (33), or on LB plates containing
1% (w/v) agar. Antibiotics were used when appropriate at the following
final concentrations: 100 µg/ml ampicillin, 20 µg/ml
chloramphenicol, and/or 12 µg/ml tetracycline (33).
Recombinant DNA Techniques--
Preparation of competent cells,
transformation, nucleic acid purification, and electrophoresis were
performed according to published procedures (33, 34). Plasmids were
purified using the Qiaprep miniprep spin column kit (Qiagen), and DNA
was extracted from agarose gels using the Qiaquick gel extraction spin
column kit (Qiagen). All restriction enzymes, pfu
polymerase, and T4 ligase were used according to the manufacturers'
specifications. E. coli cells were made competent for
transformation by the CaCl2 method (33). DNA sequencing was
performed on an ABIprism 377 instrument at the Duke University DNA
Analysis Facility.
Plasmid Constructs--
Plasmids used in this work are shown in
Table I. C. trachomatis lpxA
(CtlpxA) was initially cloned into the Novagen vector pET23c(+). The full-length sequences of CtlpxA at the DNA
and protein levels were found by sequence comparisons using the BLAST algorithms (35) with E. coli lpxA (36) as the probe against the C. trachomatis D/UW-3/CX genome (26). One highly
homologous gene was identified which displayed 42% identity and 50%
similarity at the protein level over the entire length of E. coli LpxA. Oligonucleotide primers to the ends of the
CtlpxA gene were synthesized with the flanking restriction
sites NdeI at the 5' end and BamHI at the 3' end
of the PCR product.
The forward primer sequence was
5'-GGAATTCCATATGACCAACATTCATCCTACAGCG-3', and the reverse
primer sequence was 5'-GCGGATCCTTAAGACTCAACGAAAGCTCCTTC-3'. The NdeI and BamHI sites, respectively, are
underlined. These primers were used at a final concentration of 0.25 µM in 100 µl of pfu polymerase PCR reaction
mixture containing 10 ng of genomic DNA. The reaction conditions were
as follows: a 94 °C melt for 5 min followed by 25 cycles of 94 °C
(melt) for 45 s, 55 °C (annealing) for 45 s, and 72 °C
(extension) for 2 min. This was followed by a 10-min run-off at
72 °C. Finally, 1 µl of this reaction mixture was subjected to a
second round of PCR, using the same conditions as the first.
The amplified PCR product was then purified from the reaction mixture
using phenol/chloroform/isoamyl alcohol (25:24:1, v/v). The aqueous
phase of this extraction was washed two times with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1, v/v), and then mixed with
0.1 volume of 3 M sodium acetate and 2.5 volumes of 100%
ethanol at 0 °C for 30 min to precipitate the DNA. The PCR product
was then dried and re-dissolved in 50 µl of sterile water, digested
with NdeI and BamHI, purified again, and ligated
using T4 DNA ligase into similarly treated pET23c(+) vector DNA. The
ligation mixture was then transformed into CaCl2-competent XL1Blue cells. Plasmid-containing transformants were selected on LB
agar plates supplemented with 100 µg/ml ampicillin and grown overnight at 37 °C. Putative clones were re-purified on LB
ampicillin plates, inoculated into 5 ml of LB broth containing
ampicillin, and grown in shaking culture overnight at 37 °C. Plasmid
DNA was then isolated and subjected to screening by NdeI and
BamHI digestion for the presence of the insert. One positive
clone containing the CtlpxA gene was designated pCS125 and
verified by DNA sequencing.
For studies of the enzymatic activity associated with
CtlpxA, the high copy lac vector pBluescript II
SK(+) was used. CtlpxA was subcloned by restriction
endonuclease digestion of pCS125 with XbaI and
HindIII, followed by ligation into these restriction sites
of pBluescript II SK(+). One positive clone was designated pCS131. This
plasmid was transformed into RO138 and verified by restriction digestion.
Cell Extract Preparation--
The strains pCS131/RO138,
pTO5/RO138, and pBluescript II SK(+)/RO138 were grown overnight in 5 ml
of LB broth containing 100 µg/ml ampicillin, at 30 °C with 225 rpm
rotary shaking. The culture was then diluted 1:100 into 100 ml of fresh
medium, and allowed to reach A600 of ~0.6. At
this point, the cultures were shifted to 42 °C and allowed to grow
for another 2 h. At the end of this period, the cultures were
chilled on ice for 10 min and then harvested by centrifugation at
4,000 × g at 4 °C for 10 min. The cell pellets were
resuspended in 10 ml of ice-cold buffer (sterile 100 mM
potassium phosphate at pH 7.5 with 200 mM NaCl and 20%
glycerol) per 50 ml of original culture, and the cells were centrifuged
again at 4,000 × g at 4 °C for 10 min. Each washed
cell pellet was resuspended in 2 ml of the same buffer and frozen at
Substrate Preparation and Assay Conditions--
The
[
The LpxA assay follows the conversion of
[ Growth Curve Determination--
The permissive temperature for
RO138 is 30 °C. The non-permissive temperature of 42 °C inhibits
growth and reduces cell viability within one doubling time. The ability
of different constructs to grow at 42 °C was determined both on
plates and in liquid broth to assess complementation. Growth curves,
following a mid-log phase temperature shift from 30 to 42 °C, were
performed for a more detailed analysis of complementation. Growth was
followed by measuring turbidity at 600 nm on a Spectronic 21D
spectrometer (Milton Roy) or a Beckman DU-640 spectrophotometer.
Assays of Antibiotic Resistance--
Outer membrane integrity
was evaluated by examining resistance to selected antibiotics. The
assay was performed by placing 8-mm filter discs containing various
antibiotics onto a lawn of cells, freshly plated using a saturated
cotton swab from a culture at A600 ~ 0.2. After 16 h of growth, the zones of inhibition around each disc
were measured, providing an assessment of relative antibiotic sensitivity (38, 39). In our experiments, the diameter of each zone of
inhibition was measured in two dimensions, and the average diameter was
used to calculate the area of clearing (mm2). The
antibiotics used were rifampicin (30 µg/disc) and erythromycin (25 µg/disc).
Purification and Analysis of Hybrid Lipid A Species--
For
structural studies, we isolated hybrid lipid A species generated by the
E. coli lpxA2 mutant RO138 complemented with
CtlxpA, using previously established techniques (14, 40).
One liter of cells was grown in LB broth at 30 °C to
A600 = 0.7, and then shifted to 42 °C for
2 h before being harvested at A600 = 1.4.
Glycerophospholipids were removed by Bligh-Dyer extraction of the cell
pellet (14, 40). To release the lipid A, the residue was hydrolyzed at
100 °C in acetate buffer, pH 4.5, in the presence of SDS (14, 40).
This crude lipid A fraction was first purified by chromatography on a
DEAE-cellulose column (14, 40). To resolve pure EC1-EC5, several
hundred micrograms of the lipid A mixtures were spotted in a line onto
two 20 × 20-cm Silica Gel 60 plates (0.25 mm thickness), which
were developed in chloroform/pyridine/formic acid/water (50:50:16:5,
v/v), as described previously (40). A second small DEAE-cellulose
column was run after extraction of the pure lipids from the silica
plates to remove particulate and metal impurities prior to mass
spectrometry (40).
Mass Spectrometry of Hybrid Lipid A Species--
Spectra
were acquired in the negative-ion linear mode by using a Kratos
Analytical (Manchester, England) matrix-assisted laser desorption
ionization-time of flight (MALDI-TOF) mass spectrometer, equipped with
a 337-nm nitrogen laser, a 20 kV extraction voltage, and time-delayed
extraction (40). Each spectrum was the average of 50 shots. The matrix
was a mixture of saturated 5-aza-2-thiothymine in 50%
aqueous acetonitrile and 10% tribasic ammonium citrate (9:1, v/v). The
lipid A samples were allowed to dry at room temperature prior to mass
analysis. The hexa-acylated lipid A 1,4'-bis-phosphate form
from E. coli (purchased from Sigma) was used as an external standard.
Mild Base Hydrolysis of Lipid A Species EC3 Prior to Mass
Spectrometry--
Lipid A samples were dissolved in 100 µl of 3%
aqueous triethylamine and subjected to sonic irradiation for 3 min
before being incubated at 37 °C. Portions (0.3 µl) of the reaction
mixtures were taken at 0, 2, 16, and 40 h for analysis by negative
mode MALDI-TOF mass spectrometry, as described above.
Catalytic Activity and Acyl Chain Specificity of CtLpxA--
The
structure of C. trachomatis lipid A suggests that CtLpxA
should transfer myristate specifically to
the glucosamine 3-OH of UDP-GlcNAc (Figs. 1 and
2). To examine the activity of the C. trachomatis enzyme, we cloned the CtlpxA gene
into pBluescript II SK(+) to generate plasmid pCS131. The pBluescript
II SK(+) vector (500-700 copies per cell) allows for overexpression of the CtlpxA gene behind the lac promoter. The use
of strain RO138 as the host for pCS131 facilitated direct measurement
of the acyl chain specificity of CtLpxA without interference from
E. coli LpxA (29, 41).
A crude extract of RO138/pCS131 harvested at 42 °C was assayed for
its ability to acylate UDP-GlcNAc in the presence of various acyl-ACPs
(Fig. 3 and Table II). Transfer of
myristate from myristoyl-ACP to UDP-GlcNAc was detected in this
extract, but not in a matched extract of the vector control
RO138/pBluescript II SK(+)(Fig. 3) under conditions where product
formation is linear with time. A quantitative analysis (Table
II) of the specific activities demonstrates that CtLpxA is about 20-fold more active with
myristoyl-ACP (19 ± 2.5 pmol/min/mg, n = 9) than
with (R/S)-3-hydroxymyristoyl-ACP (1.1 ± 0.27 pmol/min/mg, n = 7). EcLpxA expressed in RO138 is ~700 times more active with (R/S)-3-hydroxymyristoyl-ACP
(29,000 ± 6,500 pmol/min/mg, n = 10) than with
myristoyl-ACP (42 pmol/min/mg) (Table II). The low LpxA specific
activity of RO138/pCS131 extracts (Table II) may reflect the use of the
heterologous E. coli acyl-ACP as the donor substrate; it is
not the result of poor protein expression, since C. trachomatis LpxA can be visualized in the cytoplasmic fraction of
RO138/pCS131 by SDS-PAGE (data not shown).
Partial Complementation of the Temperature Sensitivity of RO138 by
CtlpxA--
RO138/pCS131 was able to grow on plates at 42 °C (not
shown). However, the colonies were smaller than wild type E. coli. RO138/pCS131 was also able to grow slowly in liquid shaking
culture at 42 °C, but only reached A600 of
~0.5 after 24 h.
To study the complementation of RO138 by CtlpxA in more
detail, cell density (A600) and plating
efficiencies were measured after shifting cells from 30 to 42 °C.
RO138/pCS131 and other control strains were inoculated at
A600 = 0.02 at 30 °C, and were shifted to
42 °C when A600 had reached 0.2 (Fig.
4). These growth curves confirm the
partial complementation of RO138 by CtlpxA, as indicated by
the low A600 of the RO138/pCS131 culture in
stationary phase at 42 °C (Fig. 4). However, dilution plating of
RO138/pCS131 after a shift to 42 °C showed no decrease in viable
cell count (not shown), in contrast to RO138, which loses about 4 orders of magnitude of viability after several hours at 42 °C
(29).
Antibiotic Hypersensitivity of RO138 Complemented by
CtlpxA--
To assess the impact of heterologous expression of
CtlpxA in RO138 on the integrity of the outer membrane, we
tested the sensitivity of RO138/pCS131 and several control strains to
rifampicin and erythromycin (38, 39). Significant antibiotic
hypersensitivity is observed in RO138 even at 30 °C where the cells
are able to grow, and may be the consequence of a slight reduction in
lipid A content because of low LpxA activity (29). Restoration of wild-type antibiotic resistance in RO138 at 30 °C by introduction of
lpxA on a hybrid plasmid indicates that sufficient lipid A species is again produced to restore the outer membrane barrier. Reversal of the antibiotic hypersensitivity of RO138 at 30 °C is
seen with E. coli lpxA, as well as several heterologous
lpxA genes, including lpxA from Neisseria
meningitidis and Pseudomonas aeruginosa (13, 14).
Interestingly, CtlpxA supplied to RO138 had only a small
effect on the antibiotic hypersensitivity at 30 °C (Fig.
5), suggesting that RO138/pCS131
generates insufficient or non-functional lipid A species (Fig. 5). We
favor the former scenario, since the specific activities of
RO138/pCS131 extracts are only 20 pmol/min/mg with myristoyl-ACP as the
donor substrate, compared with 150 pmol/min/mg in wild-type vector
control cells with hydroxymyristoyl-ACP as the donor (data not shown).
As noted above, the low UDP-GlcNAc acyltransferase specific activity in
extracts of RO138/pCS131 (Table II) may reflect the fact that the
C. trachomatis enzyme does not efficiently utilize the
heterologous E. coli myristoyl acyl-carrier protein as the
donor substrate.
Biosynthesis of Hybrid Lipid A Species in RO138/pCS131 at
42 °C--
Since RO138/pCS131 grows slowly and retains viability at
42 °C (Fig. 4), it may be synthesizing novel lipid A species with myristoyl chains at position 3 and/or 3' of lipid A. We initially examined the structures of the lipid A species made by a mid-log phase
culture of RO138/pCS131, after being shifted to 42 °C for 2 h.
Separation of the five major lipid A species present in this strain
under these conditions required a combination of ion-exchange and thin
layer chromatography. Purification of the lipid A obtained from
RO138/pCS131 on a DEAE-cellulose column revealed that the five lipid
A-like components, designated EC1 to EC5 in Fig.
6, eluted with 240 mM
ammonium acetate in the aqueous component. Prior to mass spectrometry,
EC1-EC5 were resolved from each other by preparative TLC (40).
Negative ion MALDI-TOF Mass Spectrometry of Lipid A Species
in RO138/pCS131--
The negative-ion
MALDI-TOF spectra (Fig. 7 and Table III)
are consistent with the idea that EC1 and EC2 are hepta- and
hexa-acylated lipid A 1,4'-bis-phosphate species,
respectively (Fig. 2). The molecular ions [M-H]
EC3 is of particular interest as it is a penta-acylated lipid A
molecule (Fig. 6) with [M-H]
About one-third of E. coli lipid A molecules contain a
diphosphate substituent at position 1 in place of the more predominant monophosphate group (not shown in Fig. 1) (40, 43). Based on their mass
spectra (Fig. 7 and Table III), EC4 and EC5 are interpreted as
diphosphate variants of EC2 and EC3, respectively (Fig. 2). No
wild-type E. coli lipid A, with [M-H] Positive Ion MALDI-TOF Mass Spectrometry of Lipid A Species in
RO138/pCS131--
The positive ion mode spectra of EC1, EC2, and EC4
(Fig. 8 and Table III) show that the
B1+ ions of these species are identical to those expected
for the B1+ ion of E. coli wild-type lipid A
(14, 40), whereas the B2+ ions for these three compounds,
like their [M-H]
In EC3 and EC5 (Fig. 8), the size of B1+ ions are each 16 atomic mass units smaller than what would be expected for a lipid A
species isolated from an E. coli msbB mutant (Fig. 1) that
is otherwise wild type with respect to lipid A biosynthesis (42). The
B2+ ions of EC3 and EC5 are each 32 atomic mass units
smaller than would be the case if all four primary lipid A acyl chains
were (R)-3-hydroxymyristate (Fig. 8). Therefore in these
species there is a loss of 16 atomic mass units in both the proximal
and distal units, consistent with the view that myristoyl groups are
present both at positions 3 and 3' (Fig. 2).
To prove that the myristate chains in our hybrid lipid A molecules are
indeed ester linked, EC3 (Fig. 2) was treated with 3% aqueous
triethylamine for 40 h. At the times indicated in Fig. 9, the EC3 samples were analyzed by
MALDI-TOF mass spectrometry in the negative mode. The gradual loss of
two 210 atomic mass units fragments from EC3 clearly demonstrates that
both of the myristate chains of EC3 are ester linked (Figs. 2 and
9).
UDP-N-acetylglucosamine 3-O-acyltransferase,
the product of the lpxA gene, attaches the primary
ester-linked hydroxyacyl chains found at positions 3 and 3' of E. coli lipid A (Fig. 1) (9-11). LpxA is required for bacterial
growth and is an important determinant of the structure of lipid A (13,
14, 29). The 3' (R)-3-hydroxyacyl chain is further acylated
in wild-type E. coli and Salmonella typhimurium
with a secondary myristoyl chain to generate the 3'-acyloxyacyl unit
(Fig. 1) (28, 42). The absence of the secondary myristoyl chain, as
seen in msbB mutants of E. coli and S. typhimurium (Fig. 1), greatly reduces virulence (44, 45) and
diminishes the ability of the mutant bacteria to stimulate cytokine
production in human macrophages (42), a process mediated by TLR-4 (6). The lipid A of C. trachomatis is unusual in that it contains
myristoyl instead of (R)-3-hydroxymyristoyl chains at
positions 3 and 3' (Fig. 2) (18, 19). C. trachomatis
therefore cannot generate a 3'-acyloxyacyl unit (Fig. 2), and its lipid
A lacks potency when tested as an inducer of cytokines in human cells
(46).
We have now shown that the presence of the myristate chain at the 3 and
3' positions of C. trachomatis lipid A is due to the unusual
substrate specificity of CtLpxA. Selectivity for myristoyl-ACP over
(R)-3-hydroxymyristoyl-ACP is apparent both in
vitro and in vivo, when CtlpxA is expressed
in E. coli RO138 (Figs. 2, 3, and 6-9). All previously
characterized UDP-GlcNAc 3-O-acyltransferases display a high
degree of selectivity for 3-hydroxyacyl-ACPs over nonhydroxylated
acyl-ACPs (11-14). Although the x-ray structure of E. coli
LpxA has been solved at 2.6-Å resolution (15), the substrate-binding
pocket is not well characterized, since enzyme-acyl-ACP complexes have
not yet been crystallized.
The incomplete complementation of the temperature
sensitivity of RO138 by CtlpxA (Fig. 4) may be due to the
low specific activity of CtLpxA (Table II) when E. coli
myristoyl-ACP is the donor substrate. To confirm this idea, the
specific activity of CtLpxA with C. trachomatis
myristoyl-ACP as the substrate should be investigated. It may be
possible to improve CtlpxA complementation of RO138 by
simultaneous expression of C. trachomatis acpP, the
structural gene for acyl carrier protein (26).
The presence of 3-hydroxymyristate at the 3' position of several of the
hybrid lipid A species (EC1, -2, and -4) (Fig. 2) seen in RO138
complemented by CtlpxA is explained by the procedure used to
grow this construct. In the experiment shown in Fig. 6, cultures were
grown at 30 °C to A600 of ~0.7, and then
shifted to 42 °C for 2 h, during which time the
A600 increased to ~1.4. Therefore, about half
the lipid A species obtained for mass spectrometry were derived from
RO138/pCS131 growing at 30 °C, where the E. coli LpxA2
mutant protein would be partially functional (29). We do not believe
that CtlpxA itself incorporated the 3-hydroxymyristate found in EC1,
-2, and -4 (Figs. 2 and 6-9), since prolonged exponential growth of
RO138/pCS131 at 42 °C results in the complete disappearance of
species EC1, -2, and -4, and their replacement by EC3 and EC5 (Fig. 2)
together with small amounts of novel palmitate-containing variants of
EC3 and EC5 (not shown).
The fact that 3-hydroxymyristate is present only in the distal
glucosamine units of EC1, -2, and 4 (Figs. 2 and 8) is very interesting. This finding suggests that LpxB, the lipid A disaccharide synthase (47-49) and/or LpxH, the UDP-2,3-diacylglucosamine hydrolase (50) are highly selective for the structures of their
UDP-2,3-diacylglucosmaine substrates (Fig. 1). The incorporation of
(R)-3-hdyroxymyristate exclusively into the 3' lipid A
position of distal units of EC1, -2, and -4 (Fig. 2) might occur in two
ways. 1) E. coli LpxH (Fig. 1) might have a higher binding
affinity for UDP-2,3-diacylglucosamine species containing myristate at
the 3-position, thereby inhibiting cleavage of
UDP-2,3-diacylglucosamine bearing hydroxymyristate at position 3, and
resulting in selective formation of lipid X species that are acylated
with myristate at position 3. 2) LpxB might select against
UDP-2,3-diacylglucosamine species containing myristate at position 3, forcing this material to be cleaved by LpxH, and expanding the lipid X
pool with molecular species containing myristate (Fig. 1). The
advantage of both mechanisms over random incorporation is that the
chances for generating a 3'-acyloxyacyl group are maximized, ensuring
virulence during animal infections (44, 45).
The relevance of the unusual specificity of its UDP-GlcNAc
3-O-acyltransferase to the biology of C. trachomatis is unknown. C. trachomatis lipid A is known
to lack potent endotoxin activity (46). Being an obligate intracellular
pathogen, C. trachomatis may require the presence of a lipid
A species with low intrinsic endotoxin activity, so as not to activate
the innate immune system excessively. To test this hypothesis, the
feasibility of deleting C. trachomatis lpxA and replacing it
with E. coli lpxA could be explored. Providing E. coli
msbB in addition to E. coli lpxA might also be of
interest. These changes in the lipid A pathway should convert C. trachomatis lipid A to a more potent endotoxin. However, we cannot
exclude the possibility that the unusual C. trachomatis lipid A has some other biological function, such as the inhibition of
phagolysosome fusion, which is a critical event for the intracellular survival of C. trachomatis (20, 21).
Our ability to produce lipid A species in E. coli that bear
some resemblance to those found in C. trachomatis now
provides facile access to a new series of interesting endotoxin
analogs. The immunostimulatory activities of these compounds need to be examined. More generally, the use of Gram-negative bacteria with modified lipid A structures may prove to be an effective approach to
the development of new vaccines, justifying the exploration of
additional lpx gene chimeras.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix
secondary structure (15). The location of the active site has been
surmised based on site-directed mutagenesis (16), but crystal
structures of enzyme-substrate complexes are not yet available.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains and constructs
20 °C. The cells were then thawed on ice and broken by one passage
through a French pressure cell at 14,000 psi. The cell lysates were
centrifuged at 10,000 × g for 20 min at 4 °C to
remove large debris, and the supernatants (extracts) were collected and
stored at
80 °C. Before use in the activity assays, the protein
concentrations of these extracts were quantified using the Pierce
bicinchoninic acid assay kit (37). The data were collected using a
Molecular Devices SpectraMax 250 plate reader, and analyzed relative to
a bovine serum albumin standard with the software package Softmax Pro. The extracts were also characterized by 15% SDS-PAGE (Bio-Rad mini-protean system) to assess the extent of LpxA overproduction.
32P]-UDP- GlcNAc and
(R,S)-3-hydroxymyristoyl-ACP substrates were prepared as
previously described (13, 14). Myristoyl-ACP (kindly provided by Dr.
S. Carty of Duke University Medical Center) was prepared by the same
enzymatic method as the hydroxymyristoyl-ACP.
-32P]UDP-GlcNAc to
[
-32P]UDP-(3-O-acyl)-GlcNAc based on a
mobility shift during TLC on a silica plate as previously described (9,
13, 14), with minor modifications. Each 10-µl reaction contained 40 mM HEPES, pH 8.0, 1 mg/ml bovine serum albumin, 0.2 mg/ml
L-573,655 (or 0.02 mg/ml LNTI-229 as indicated), 10 µM
(R,S)-3-hydroxymyristoyl-ACP or myristoyl-ACP (as
indicated), 10 µM UDP-GlcNAc,
[
-32P]UDP-GlcNAc (2 × 105 dpm/tube),
and an appropriate amount of extract. L-573,655 and LNTI-229 prevent
the further metabolism of the lpxA reaction product. The
assays were carried out at 30 °C, and time points were analyzed by
spotting 2.5-µl portions of each reaction mixture onto a silica TLC
plate. The plate was developed in chloroform/methanol/water/acetic acid
(25:15:4:2, v/v) and analyzed with a PhosphorImager (Molecular Dynamics
Storm 840 system).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
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Fig. 1.
Role of LpxA in E. coli
lipid A biosynthesis. E. coli LpxA
selectively acylates the glucosamine 3 position of UDP-GlcNAc with
(R)-3-hydroxymyristate, as indicated in red,
eventually resulting in the incorporation of
(R)-3-hydroxymyristate residues at positions 3 and 3' of
E. coli lipid A (1, 51). The lengths of the acyl chains are
indicated by the numbers at the bottoms of
selected structures. About one-third of the E. coli K12
lipid A species are modified with a diphosphate unit (not shown) at
position 1 in place of the more abundant monophosphate substituent
(40). The Kdo disaccharide of the inner core of E. coli
lipopolysaccharide is required for the completion of lipid A acylation
by HtrB and MsbB.
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Fig. 2.
Proposed structures of C. trachomatis lipid A and of hybrid lipid A molecules EC1 to
EC5. C. trachomatis lipid A is unusual in that it is
acylated with myristate at positions 3 and 3' (19), as shown in
blue. The lipid A structure indicated for C. trachomatis is the predominant molecular species (18, 19). Based
on their genomic DNA sequences (26, 36), we propose that the pathways
for lipid A biosynthesis are generally the same in C. trachomatis and E. coli, indicating that C. trachomatis LpxA should be selective for myristoyl-ACP. C. trachomatis lacks the msbB gene (26, 36), which encodes
the enzyme that attaches the secondary myristate chain present at
position 3' in E. coli lipid A (Fig. 1), consistent with the
structure of C. trachomatis lipid A (19). Numbers
at the bottom of the structure indicate the lengths of the
acyl chains. For the hybrid lipid A species generated by RO138/pCS131
at 42 °C, myristate chains are blue, whereas
(R)-3-hydroxymyristate chains are red. Positions
3 and 3' are indicated to emphasize that any remaining
(R)-3-hydroxymyristate chains present in lipid A species of
RO138/pCS131 are selectively incorporated into the distal glucosamine
unit. No wild type E. coli lipid A molecules are present in
RO138/pCS131. The B1+ and B2+ fragments (53)
are indicated for species EC1 only. Palmitate is present on the
proximal unit of EC1, which is a minor component, and is incorporated
by the regulated enzyme PagP (54) (not shown in Fig. 1).
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Fig. 3.
Selectivity in vitro of
C. trachomatis LpxA for myristoyl-ACP. Extracts
of RO138/pBluescript, RO138/pCS131, and RO138/pTO5 were assayed at
30 °C for 8 min in a 10-µl reaction mixture containing 40 mM HEPES, pH 8.0, 1 mg/ml bovine serum albumin, 0.02 mg/ml
of the LpxC inhibitor LNTI-229, 10 µM acyl-ACP, and 10 µM [ -32P]UDP-GlcNAc (2 × 106 cpm/nmol). The reactions were stopped by spotting
2-µl portions onto a Silica Gel 60 TLC plate. Conversion of
[
-32P]UDP-GlcNAc to
[
-32P]UDP-3-O-(acyl)-GlcNAc is shown by
migration during thin layer chromatography in the solvent
chloroform/methanol/water/acetic acid (25:15:4:2, v/v), as indicated.
Product formation was linear with time, and was quantified using a
Molecular Dynamics PhosphorImager.
Substrate specificity of C. trachomatis versus E. coli LpxA
View larger version (17K):
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Fig. 4.
Partial complementation of the temperature
sensitive growth of RO138 by C. trachomatis lpxA on
pCS131. The indicated strains were grown overnight at 30 °C on
LB broth containing 100 µg/ml ampicillin, and then diluted to
A600 = 0.02. The diluted cultures were grown for
several more hours at 30 °C until A600 = 0.2, and were shifted to 42 °C at time 0. At 42 °C, RO138 rapidly lost
viability and gradually lysed (29). RO138/pCS131 did not undergo lysis
or lose viability at 42 °C, but it failed to reach the higher cell
density characteristic of wild type cells, such as SM105, or RO138
complemented with wild type E. coli lpxA (not shown).
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Fig. 5.
Antibiotic sensitivity of RO138 and various
strains. Antibiotic susceptibility was determined by measuring the
zones of clearing (mm2) around antibiotic discs placed on
lawns of the indicated strains, grown overnight on LB plates, as
described previously (31). The strains are further described in Table
I.
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Fig. 6.
Ion exchange chromatography of lipid A
species from RO138/pCS131 grown at 42 °C. Crude lipid A from a
1-liter culture of RO138/pCS131, grown at 42 °C for 2 h, was
prepared as previously described by hydrolysis at pH 4.5 at 100 °C
in the presence of SDS (40). A 2-ml DE52-cellulose column (acetate
form) was equilibrated with chloroform/methanol/water (2:3:1, v/v) (40,
52), and following sample application in the same solvent mixture, was
eluted by increasing the ammonium acetate concentration in the aqueous
component, as indicated by the first number (mM) below each
fraction (3 ml each). Lipid species were detected by spotting 20-µl
portions of each fraction onto a Silica Gel 60 TLC plate, which was
developed in the solvent chloroform/pyridine/formic acid/water
(50:50:16:5, v/v), followed by charring with 10% sulfuric acid in
ethanol. The five lipid A species, labeled EC1 to EC5, were further
purified by preparative thin layer chromatography on the 100-500-µg
scale (40).
for
these two compounds at m/z 2019.5 and m/z 1781.9, respectively, are 16 atomic mass units smaller than those expected for
hepta- or hexa-acylated lipid A 1,4'-bis-phosphates from
wild-type E. coli, which would be observed at m/z
2035.8 and 1797.4, respectively. These results suggest that one
hydroxymyristoyl chain has been replaced by a myristoyl moiety in both
EC1 and EC2 (Figs. 2 and 7 and Table III).
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Fig. 7.
Negative ion MALDI-TOF mass spectrometry of
hybrid lipid A species EC1 to EC5. The hybrid lipids were purified
as indicated in the legend to Fig. 6, and analyzed as described under
"Experimental Procedures." Proposed structures and predicted masses
are given in Fig. 2 and Table III.
Summary of MALDI-TOF mass spectrometry of RO138/pCS131 lipid A hybrids
at m/z 1554.3 (Figs. 2 and 7 and Table III). The latter value is 32 atomic mass units
lower than that expected for a penta-acylated E. coli lipid
A species in which the secondary myristate chain at position 3' is
missing, as would be the case for lipid A isolated in mutant defective
in msbB (42) (Fig. 1). The molecular ion of EC3 (Fig. 7)
therefore suggests that both the 3 and 3'-hydroxymyristoyl chains have
been replaced with myristate (Fig. 2). A secondary myristate chain
cannot be incorporated at position 3' into a lipid A species of this
kind (Fig. 2).
expected at m/z 1797.4 (40), was seen in any of the lipid A fractions isolated from RO138/pCS131 (Fig. 7).
value, are each 16 atomic mass units
smaller. This finding demonstrates conclusively that myristate replaces
hydroxymyristate only at position 3 on the proximal sugar of these
species, and that the 3' position occupied by
(R)-3-hydroxymyristate as in wild-type E. coli
lipid A (Fig. 2).
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Fig. 8.
Positive ion MALDI-TOF mass spectrometry of
hybrid lipid A species EC1 to EC5. The masses of the major ions,
including B1+, B2+, [M + Na]+ are
indicated (53). Proposed structures and predicted masses are given in
Fig. 2 and Table III.
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Fig. 9.
Negative ion MALDI-TOF mass spectrometry of
triethylamine-treated EC3. Purified EC3 was treated with 3%
aqueous triethylamine at 37 °C for the indicated times to cleave the
ester-linked acyl chains. The pattern is consistent with loss of two
myristoyl moieties, as expected for a compound with the structure of
EC3 (Fig. 2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
---|
We thank Suzanne M. Ramirez of the Johns Hopkins University School of Medicine for selected MALDI-TOF analyses of partially purified lipid A preparations isolated from RO138/pCS131 grown exclusively at 42 °C.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM-51310 (to C. R. H. R.) and GM54882 (to R. J. C.).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.
§ Supported by National Institutes of Health Training Grant GM08558 in Biological Chemistry to Duke University.
¶ To whom correspondence should be addressed. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz@biochem.duke.edu.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M101868200
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ABBREVIATIONS |
---|
The abbreviations used are: ACP, acyl carrier protein; PCR, polymerase chain reaction; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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