From the Children's Hospital Research Foundation and Departments
of Pediatrics and
Medical Microbiology, Ohio State
University, Columbus, Ohio 43205-2696, ¶ Department of
Pharmaceutical Chemistry, University of California,
San Francisco, California 94143-0446
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ABSTRACT |
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Haemophilus ducreyi, the
cause of the sexually transmitted disease chancroid produces a
lipooligosaccharide (LOS) containing a terminal sialyl
N-acetyllactosamine trisaccharide. Previously, we reported
the identification and characterization of the
N-acetylneuraminic acid cytidylsynthetase gene
(neuA). Forty-nine base pairs downstream of the synthetase
gene is an open reading frame (ORF) encoding a protein with a predicted
molecular weight of 34,646. This protein has weak homology to the
polysialyltransferase of Escherichia coli K92. Downstream
of this ORF is the gene encoding the H. ducreyi homologue
of the Salmonella typhimurium rmlB gene. Mutations were constructed in the neuA gene and the gene encoding the
second ORF by insertion of an Haemophilus ducreyi is the causative agent of
chancroid, a genital ulcer disease, which is prevalent in many
developing countries. In urban areas of the United States, chancroid
occurs sporadically as outbreaks (1-3). However, recent studies
suggest that chancroid may be greatly underreported due to inadequate
methods of detection (4, 5). Chancroid and other ulcerative sexually
transmitted diseases have been epidemiologically linked to the
heterosexual transmission of the human immunodeficiency virus in areas
where both diseases are epidemic (6, 7). Furthermore, individuals infected with human immunodeficiency virus are less responsive to
standard antibiotic treatment for chancroid (7, 8).
The mechanisms of H. ducreyi virulence are not well
understood. However, several putative virulence determinants have
recently been identified and characterized. Two different cytotoxins
have been identified that cause cytopathic effects to human foreskin fibroblasts and epithelial cells. The cytotoxins and genes that encode
them have been characterized. A hemolytic cytotoxin, encoded by the
hhdA and hhdB genes, has been identified and
demonstrated to be responsible for the cytopathic effects H. ducreyi exerts on human foreskin fibroblasts (9-11). The second
cytotoxin produced by H. ducreyi, which is toxic for
epithelial cells, is a homologue of cytolethal distending toxin, which
is produced by Campylobacter jejuni, Shigella
dysenteriae and certain Escherichia coli strains (12).
The lipooligosaccharide
(LOS)1 of H. ducreyi also is an important virulence factor. Several studies
have demonstrated that the LOS of H. ducreyi causes ulcers
in rabbits and mice (13-15). LOS also plays a role in the adherence of
H. ducreyi to human foreskin fibroblasts and keratinocytes
(16, 17). The LOS from H. ducreyi and other Gram-negative
mucosal pathogens, such as Haemophilus influenzae,
Neisseria meningitidis, and Neisseria gonorrhoeae, are structurally similar, and some of these LOS
glycoforms have been shown to mimic human antigens, such as
paragloboside and other glycosphingolipids (18-21). Molecular mimicry
may allow these organisms to evade the host's immune system. One
important aspect of molecular mimicry in these organisms is the
presence of sialic acid as a component of their LOS. Although commonly found in higher animals, sialic acids have been found in relatively few
microorganisms, and their presence is often associated with virulence
(22). For example, the sialic acid-containing capsules of N. meningitidis, E. coli K1, and group B streptococci are important virulence factors (23-26). Similarly, sialylation of the LOS of N. gonorrhoeae is now viewed as a major factor in the
organism's pathogenicity (27). Because of its terminal position on
carbohydrates, sialic acid is one of the first molecules encountered in
cellular interactions and has been found to have important roles in
cellular recognition (22).
Structural studies of the LOS glycoforms have been carried out on
several H. ducreyi strains (28-34). The LOS from these
strains are variable; however, it has been determined that the
principal glycoform expressed by most strains of H. ducreyi
terminates in N-acetyllactosamine. The terminal galactose residue of
this disaccharide acceptor is then partially substituted (approximately
one-third of the total LOS glycoforms) with a single sialic acid
residue to form the nonreducing terminal trisaccharide, sialyl
N-acetyllactosamine. Previously, we reported the purification of
N-acetylneuraminic acid cytidylsynthetase (CMP-NeuAc
synthetase) and the cloning of the gene, neuA, encoding this
enzyme (35). CMP-NeuAc synthetase is responsible for catalyzing the
condensation reaction of CTP and neuraminic acid (NeuAc) to form
CMP-NeuAc. We identified the neuA gene in a H. ducreyi Materials--
Glucosamine, NeuAc, and anhydrous hydrazine were
from Sigma. 2,5-Dihydroxybenzoic acid was from Aldrich.
Acrylamide/bisacrylamide solution (40% (w/v), 37.5:1 monomer to
cross-linker), electrophoresis quality Tris, glycine, and SDS were from
Bio-Rad. Dialysis tubing was from Spectrum (Houston, TX). Constant
boiling 6 N HCl was from Pierce.
CMP-[14C]NeuAc was from NEN Life Science Products.
Bacterial Strains, Plasmids, and Media--
The bacterial
strains and plasmids used in this study are listed in Table
I. H. ducreyi strains were
grown on chocolate agar or in brain heart infusion broth as described
previously (10). When necessary, chocolate agar was supplemented with
kanamycin at 20 µg ml Recombinant DNA Methodology--
Plasmids were isolated
utilizing Qiagen Purification kits (Qiagen, Chatswoth, CA). Restriction
enzymes and T4 DNA ligase were purchased from Life Technologies, Inc.
Electroporation of E. coli and H. ducreyi was
performed as described previously (36). Standard recombinant DNA
methods were performed as described previously (37).
DNA sequence was determined through cycle sequencing using ABI PRISM
Dye Terminator Cycle Sequencing Ready Reaction kits. Cycle sequencing
was carried out on a GeneAMP PCR System 9600 (Perkin-Elmer). The
samples were then passed through Cetri-Sep columns and run on either an
ABI 310 genetic analyzer or an ABI 377 DNA sequencer. Sequence analysis
and comparisons were performed with Lasergene (DnaStar, Madison, WI)
and GCG software (38), as well as with the NCBI Blast server.
Construction of H. ducreyi Isogenic Mutants--
To mutate the
LOS genes of interest on plasmid pRSM1627 (Fig. 1), unique
SrfI restriction enzyme sites were introduced into the
neuA and lst genes utilizing the Chameleon
double-stranded, site-directed mutagenesis kit (Stratagene, La Jolla,
CA) according to the manufacturer's instructions. The sequences of the
mutagenic primers containing the SrfI sites as well as a
selection primer designed to change a nonessential unique restriction
enzyme site (XhoI) present in pRSM1627 into a
BglII site are shown in Table II. Plasmids containing a SrfI
site in the neuA or lst genes were identified by
restriction analysis, confirmed by determination of DNA sequence, and
saved as pRSM1681 and pRSM1712, respectively (Table I). The
Isogenic mutants of H. ducreyi were constructed as described
by Bozue et al. (36). Briefly, we observed that the
hydrolysis product of X-gal was toxic to H. ducreyi.
Further, we observed that cointegrates were readily obtained after
electroporation of H. ducreyi with a suicide plasmid
containing an insertionally inactivated gene followed by selection with
the appropriate antibiotic. Thus, the NotI fragments
containing the insertionally inactivated neuA or
lst genes were cloned into pRSM1791, a ColE1 vector that expresses
A nonpolar neuA mutant was also constructed. In this
instance, the nonpolar kanamycin cassette constructed by Menard
et al. (39) was used for insertional inactivation of the
neuA gene in SrfI-digested pRSM1681. An isogenic
H. ducreyi strain with a nonpolar mutation in the
neuA gene was constructed as described above and saved as
35000HP-RSM208.
Southern Analysis--
DNA was isolated from the H. ducreyi strains by a modified procedure (40) using the
Stratagene Chromosomal DNA isolation kit. Chromosomal DNA was digested
with BglII, subjected to electrophoresis on a 0.7% agarose
gel, and transferred to a nylon membrane using the Turbo Blotter kit
(Schleicher & Schuell). Probes were radiolabeled with 32P
using the RadPrime DNA Labeling System (Life Technologies). Hybridization and washes were performed as described (10).
Complementation of LOS Mutations--
The neuA and
lst genes were amplified by PCR using the primers shown in
Table II. The PCR products were cloned into pCR2.1, and the amplified
genes were then cloned as EcoRI fragments into EcoRI-digested pLS88. The resulting plasmids were
electroporated into the corresponding isogenic H. ducreyi
mutants, and clones were selected on chocolate agar plates containing
streptomycin. The respective plasmids were designated pNEUA and pLST.
Preparation of LOS--
LOS, used in the chemical, mass
spectrometric, and SDS-PAGE analysis, was prepared from H. ducreyi cells that were grown overnight in 0.5-1 liters of liquid
medium (yielding 140-320 mg, dry weight, of cells) and extracted using
a modified version of the hot phenol-water procedure (41-43). Typical
yields were 0.1-0.4% of the bacterial dry weight.
Alternatively, when small quantities of LOS were needed only for
SDS-PAGE analysis, LOS was extracted from H. ducreyi cells that were grown overnight on two chocolate agar plates. Cells were
suspended, washed with 10 ml of PBS containing 0.15 mM
CaCl2 and 0.5 mM MgCl2, and
extracted using the hot phenol micromethod (44).
SDS-PAGE Analysis--
Aliquots of LOS suspended in
H2O were diluted to 7.5-30 ng/µl with Bio-Rad Laemmli
sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25%
glycerol, 0.01% bromphenol blue) with 2%
LOS was visualized by silver staining according to the method of Tsai
and Frasch, with a few minor changes (46). The overnight fixing step
was shortened from overnight to 30 min, and the first three 15-min
washes were shortened to 10 min each.
SDS-PAGE analysis of the outer membrane proteins of the isogenic mutant
strains of H. ducreyi and 35000HP was performed as described
previously (10).
Preparation of O-Deacylated LOS--
To prepare a water-soluble
LOS species amenable to mass spectrometric analysis, O-acyl
groups were removed by treatment with hydrazine (47). Anhydrous
hydrazine (20-100 µl) was added to lyophilized LOS samples to give a
concentration of 1-10 µg/µl of LOS. The reaction was heated at
37 °C for 20 min with occasional vortexing. After chilling the
reactions at NeuAc and Glucosamine Analysis of O-Deacylated LOS--
NeuAc
was released from O-deacylated LOS by mild acid hydrolysis
using standard conditions (48). O-Deacylated LOS (1-3 µl, 5-20 µg) was diluted to 30 µl with water, mixed with 10 µl of 0.4 M HCl, and heated at 80 °C for 60 min. After
cooling, 12 µl of 0.4 M NaOH was added, and the samples
were frozen until ready for analysis by high pH anion exchange
chromatography with pulsed amperometric detection. The entire sample
was injected into a 500-µl loop on a Dionex HPLC system with a
CarboPac PA1 column (Dionex; 4 × 250 mm), guard column, and
in-line filter. The column was eluted with 0.4 M NaOH, and
the PAD waveform potentials were as follows: 0.05 V (0.4 s), 0.75 V
(0.2 s),
To determine the molar amount of O-deacylated LOS used for
NeuAc quantitation, the glucosamine content of the
O-deacylated LOS was determined. An aliquot of
O-deacylated LOS identical to the aliquot used for NeuAc
analysis was mixed with 100 µl of 6 N HCl and heated at
100 °C for 4 h to liberate glucosamine. After cooling, the
solution was diluted to 1 ml and lyophilized. Before analysis, the
samples were redissolved in 100 µl of H2O and injected on
the Dionex high pressure liquid chromatography system as for NeuAc
quantitation except that elution was performed isocratically with 16 mM NaOH. Quantitation of glucosamine was performed by preparing a calibration curve from injections of a glucosamine standard
(500-8000 pmol) in 100 µl of H2O. A glucosamine standard (5000 pmol) was treated in the same manner as the
O-deacylated LOS samples to estimate the amount of losses
due to sample handling.
Matrix-assisted Laser Desorption Ionization-Mass Spectrometry
(MALDI-MS) of O-Deacylated LOS--
Prior to mass spectrometric
analysis, O-deacylated LOS samples (5-50 µg) dissolved in
H2O were diluted 5-10-fold with 50% CH3CN to
give approximately 0.2-1 µg/µl of O-deacylated LOS. A small quantity (roughly 20-100 µl) of Dowex 50 × 100-200-mesh beads (NH4+ form) suspended in 50%
CH3CN was added to the O-deacylated LOS solutions, and the tubes were mildly agitated for several minutes to
desalt the O-deacylated LOS. A 1-µl aliquot of this
solution was mixed with 1 µl of 100 mM
2,5-dihydroxybenzoic acid (recrystallized from H2O) in 50%
CH3CN, and then 1 µl of the mixture was spotted on the
MALDI sample plate and allowed to air-dry. For analysis of the samples,
a Voyager-DE time-of-flight (TOF) mass spectrometer with a nitrogen
laser (337 nm) was operated in the negative ion mode using an
accelerating voltage of 20 kV, a grid voltage of 93%, a guide wire
voltage of 0.05%, and a delay time of 200 ns. The instrument was
calibrated externally using bovine insulin Sialyltransferase Assay--
H. ducreyi cells at late
log phase (A600 = 0.8) from 50 ml of liquid
culture were pelleted, washed once with 50 mM HEPES buffer (pH 7.4), and then suspended in 5 ml of the same buffer. The suspension was sonicated on ice for 10 s, six times with 20-s intervals
between pulses, and centrifuged at 4000 × g at 4 °C
for 10 min to remove unbroken cells. The crude sonicate was used in the
assay. A reaction mixture consisted of sonicate made to 70 µl with 10 mM HEPES, pH 7.4, 10 µl of 1.0% octyl Sequence of Cloned DNA--
Previously, we reported the
identification and sequence of the neuA gene, which encodes
CMP-NeuAc synthetase (35). This gene was present on an 8-kb
NotI fragment of genomic DNA obtained from a H. ducreyi
Ninety-two base pairs downstream of the lst gene is an ORF
that encodes a homologue of the rmlB gene of S. typhimurium. The rmlB gene encodes
dTDP-D-glucose 4,6-dehydratase, the second enzyme in the
rhamnose biosynthetic pathway. dTDP-D-glucose
4,6-dehydratase is responsible for the conversion of
dTDP-D-glucose to dTDP-4 keto-6-deoxy-D-glucose
(50). Twenty-two base pairs downstream of rmlB is the 5'
portion of an ORF with high homology to rmlA. The sequence
of the insert in pRSM1627 ends at the NotI site at position
5494. The rmlA gene encodes glucose-1-phosphate
thymidyltransferase, which is the first enzyme in the rhamnose
synthesis pathway. This protein is responsible for conversion of
glucose-1-phosphate to dTDP-D-glucose (50). The exact role
of these genes in H. ducreyi is undetermined, since rhamnose
has not been identified in H. ducreyi LOS.
Construction and Characterization of H. ducreyi
Mutants--
Mutants were constructed by independently constructing a
unique SrfI site in the neuA and lst
genes of pRSM1627. The
In order to verify our constructions, chromosomal DNA from the isogenic
H. ducreyi strains 35000HP, 35000HP-RSM202, and
35000HP-RSM203 was analyzed by Southern hybridization. Blots containing
BglII-digested DNA from these strains were probed with a PCR
product containing the respective gene, the
In addition to characterization by Southern analysis, the
neuA and lst mutants were examined for
differences in growth in brain heart infusion broth and outer membrane
protein profiles in comparison with the parental 35000HP strain. No
differences in growth or outer membrane protein profiles were observed
between the parental strain and the mutants (data not shown).
To determine the effects of the mutations in these genes, LOS was
isolated from strains 35000HP, 35000HP-RSM202, and 35000HP-RSM203 and
then characterized by SDS-PAGE. In Fig.
3, the LOS from H. ducreyi
strain 35000HP and the isogenic neuA and lst
mutants are shown (upper panel, lanes
1-3). The structure of 35000HP LOS and the nomenclature
used to describe the various glycoforms are presented at the
lower panel of Fig. 3. The LOS of the H. ducreyi
neuA mutant, strain 35000HP-RSM202, lacks a band previously
identified as the NeuAc-containing glycoform (Fig. 3, upper
panel, lane 2) (31). The LOS of the
lst mutant, 35000HP-RSM203, also lacks this band. In
addition to the loss of the NeuAc-containing glycoform in strain 35000HP-RSM203, the lowest band of the LOS gel is present at a much
higher concentration compared with the concentration of this glycoform
in strain 35000HP (Fig. 3, upper panel, lane
3).
Carbohydrate and mass spectrometric analyses were employed to further
verify the structure of the LOS glycoforms produced by each mutant.
Prior to analysis, the LOS was O-deacylated with anhydrous
hydrazine. This procedure results in a LOS species that contains only
two, N-linked fatty acid chains on the lipid A moiety, which
makes it far more water-soluble and directly amenable to mass
spectrometric analysis (51). The O-deacylated LOS was
subjected to mild acid hydrolysis to liberate NeuAc under conditions
commonly used for the hydrolysis of NeuAc from glycoproteins and
glycolipids (0.1 M HCl, 80 °C, 60 min) (48). Time course
studies with NeuAc-lactose and O-deacylated LOS showed that
the hydrolysis was complete after 60 min under these conditions (data
not shown). The results of NeuAc analysis of the
O-deacylated LOS from the wild-type strain 35000HP and
isogenic mutants are given in Table
III. The mol % of NeuAc was determined
by quantitation of the amount of glucosamine in an identical aliquot of
O-deacylated LOS and assumes 3 mol of glucosamine/mol of
O-deacylated LOS (one glucosamine from the oligosaccharide
and two from the conserved lipid A core). The LOS from the
neuA mutant lacked detectable NeuAc, while the
lst mutant contained less than 4% of the NeuAc observed in
the parent strain LOS. These results confirm the qualitative
SDS-PAGE analysis.
The MALDI-TOF spectrum of the O-deacylated LOS from H. ducreyi strain 35000HP, the parent strain in this study, is shown
in Fig. 4. As was also observed by
SDS-PAGE, the LOS preparation is clearly heterogeneous. Mass
spectrometry reveals a further level of heterogeneity; in addition to
differences in the number of sugar residues in the oligosaccharide
(A5a1, A5b1,
A5, A4, A3, and A2 in
Fig. 3) each of these species may also be present with a
phosphoethanolamine (PEA) moiety, as indicated by an asterisk. The
largest peaks, A5* and A5, correspond to the
O-deacylated LOS species (with and without PEA) containing
the major oligosaccharide structure from strain 35000 (30). Likewise,
peaks A5a1* and A5a1
correspond to the addition of NeuAc to A5* and
A5, respectively (30, 31). The relative intensities of the
different LOS glycoforms agree well with the SDS-PAGE analysis. As has
been observed previously by our laboratory in MALDI-MS analysis of
O-deacylated LOS, MALDI-generated prompt fragments
corresponding to the loss of H2O and
H3PO4 are readily apparent in the spectrum
(51). Fragmentation between the O-deacylated lipid A moiety
and the oligosaccharide is also readily observed with this technique
and can be quite useful for identification of components. Adducts,
particularly of the PEA-containing species, are also commonly observed
and can be seen in Fig. 4 as well (51).
The MALDI-TOF spectra of the O-deacylated LOS from the
isogenic mutants compared with the parent strain are shown in Fig. 5. Clearly, there is no evidence of peaks
corresponding to a NeuAc-containing glycoform
(A5a1* or A5a1).
Otherwise, all four spectra appear very similar. Interestingly, the
35000HP-RSM203 mutant has a greater proportion of the A2*
and A2 components than the other mutants and the parent
strain, which was also evident by SDS-PAGE.
Complementation studies were performed. The neuA and
lst genes were independently amplified by PCR, cloned into
the shuttle vector pLS88, and saved as pNEUA and pLST. H. ducreyi strains 35000HP-RSM202(pNEUA) and 35000HP-RSM203(pLST)
were constructed. LOS was isolated from these strains and characterized
by SDS-PAGE. The NeuAc-containing glycoform was observed in the LOS
from strain 35000HP-RSM203(pLST) (Fig. 3, upper panel,
lane 5), demonstrating complementation of the
lst gene. In contrast, the NeuAc-containing glycoform was
absent from the LOS of strain 35000HP-RSM202(pNEUA), indicating that
the neuA gene on pLS88 was unable to complement the
neuA mutation in strain 35000HP-RSM202 (Fig. 3, upper
panel, lane 4). NeuAc determination and
MALDI-MS analysis of the O-deacylated LOS prepared from
these strains confirmed the qualitative analysis by SDS-PAGE (see Table
III and Fig. 6). Strain
35000HP-RSM202(pNEUA) LOS had less than 10% of the NeuAc of the LOS
isolated from the parent strain, 35000HP. In good agreement with the
SDS-PAGE results, LOS from strain 35000HP-RSM203(pLST) had the greatest
amount of NeuAc of all the clones studied.
Since only 49 base pairs separate the neuA and
lst genes, it is possible that the failure to complement the
neuA mutation is due to a polar effect on the lst
gene. Menard et al. (39) constructed a kanamycin cassette
for the construction of nonpolar mutations. The cassette contains stop
codons in all three reading frames followed by the kanamycin resistance
gene. Downstream of the kanamycin resistance gene is a Shine-Delgarno
sequence followed by a translational start. This cassette was cloned
into the SrfI site of pRSM1681 such that the portion of
neuA downstream of the kanamycin resistance gene would be
translated. The construction was verified by sequencing, and an
isogenic mutant was constructed as described above. LOS was isolated
from this mutant, designated 35000HP-RSM208. By SDS-PAGE, this LOS
lacked the NeuAc-containing glycoform (Fig. 3, upper panel,
lane 6). Strain 35000HP-RSM208(pNEUA) was
constructed, and LOS was isolated. In this strain, the neuA mutation was complemented by the neuA gene in pLS88 (Fig. 3,
upper panel, lane 7). NeuAc
determination of the O-deacylated LOS from these strains
(Table III), as well as the MALDI-MS analysis (Figs. 5 and 6), were
both in good agreement with the SDS-PAGE profiles observed in Fig. 3.
Successful complementation of both the lst and
neuA genes are consistent with the proposal that both gene products are essential for sialylation of the LOS.
The evidence we obtained from the analysis of the mutant LOS glycoforms
is consistent with the proposal that the product of the lst
gene is the sialyltransferase. However, the gene product shows no
detectable homology to the neisserial sialyltransferases (52) and with
the exception of the short region shown in Fig. 2, little homology to
the polysialyltransferase of E. coli K92 (53). We therefore
set up a direct assay to measure the ability of H. ducreyi
extracts to incorporate [14C]NeuAc into trichloroacetic
acid-precipitable material. We predicted that the acceptor would be the
LOS glycoform terminating in N-acetyllactosamine and lacking
NeuAc (A5 in Fig. 3). H. ducreyi 35000HP-RSM208
produces the lactosamine-containing glycoform of LOS (A5)
in the absence of CMP-NeuAc and produces the NeuAc-containing glycoform
of LOS (A5a1) when CMP-NeuAc is provided
in vivo by complementation of the neuA mutation.
Therefore, strain 35000HP-RSM208 must have sialyltransferase activity.
However, when sonicates were prepared from strain 35000HP-RSM208 cells
and incubated with CMP-[14C]NeuAc, incorporation of
[14C]NeuAc into trichloroacetic acid-precipitable
material was not observed. One possibility for our inability to detect
activity in this strain is that the level of activity is below our
level of detection. Both chemical and SDS-PAGE analysis of the LOS from strain 35000HP-RSM203(pLST) suggested that the sialyltransferase was
overexpressed in this strain. In order to increase the sensitivity of
our assay, we overexpressed the putative sialyltransferase in the
neuA background by constructing 35000HP-RSM208(pLST).
Incorporation of [14C]NeuAc into trichloroacetic
acid-precipitable material was readily observed when sonicates of this
strain were incubated with CMP-[14C]NeuAc. The
[14C]NeuAc incorporation was time and
concentration-dependent (Fig. 7, A and B). The
activity was abolished by incubation of the sonicate at 100 °C for
10 min. An experiment was performed to demonstrate that the
lactosamine-containing LOS glycoform was the acceptor in the crude
extract. An isogenic mutant deficient in the galactose II transferase
has been constructed in our
laboratory.2 As expected, the
most complex LOS glycoform produced by this mutant contains terminal
N-acetylglucosamine (A4 in Fig. 3), and none of
the lactosamine-containing LOS glycoform is produced. This mutant is
designated 35000HP-RSM210. Strain 35000HP-RSM210(pLST) was constructed.
Sonicates prepared from this strain do not incorporate 14C-NeuAc into trichloroacetic acid-precipitable material
(Fig. 7C). We conclude that the product of the
lst gene is the H. ducreyi sialyltransferase and
that the lactosamine-containing glycoform is the substrate for the
transferase.
Previously, we reported the cloning and characterization of the
CMP-NeuAc synthetase gene, neuA, from H. ducreyi.
In order to characterize the region surrounding the neuA
gene, we determined the DNA sequence 5' and 3' of neuA.
Immediately downstream of neuA is an 888-base pair gene that
shows high homology to an H. influenzae gene (HI0871) of
unknown function and a short region of homology to the E. coli K92 polysialyltransferase. We have determined that this
888-base pair gene encodes the H. ducreyi sialyltransferase,
and we have designated this gene lst. We believe that the
H. ducreyi Lst protein is a new type of sialyltransferase. Interestingly, Lst lacks high homology to the sialyltransferases of
E. coli, N. gonorrhoeae, and N. meningitidis as well as mammalian sialyltransferases.
The LOS of H. ducreyi, like the LOS of several other
bacterial pathogens including H. influenzae, N. gonorrhoeae, and N. meningitidis, is sialylated (55,
56). The modification of bacterial LOS with NeuAc may allow these
pathogens to escape the bactericidal effect of serum. It has been
demonstrated for N. gonorrhoeae that LOS sialylation of
N-acetyllactosamine renders sensitive strains resistant to
normal human serum (57). For sialylated N. gonorrhoeae, NeuAc on the bacterial surface is able to bind factor H, a regulator of
the complement system, thereby blocking the activation of the complement pathway (58). However, neisserial strains have multiple strategies to resist the activity of complement. For example, other
gonococcal strains have been identified that do not require NeuAc-containing LOS for serum resistance (59-61). Further, an isogenic serogroup B meninogoccocal sialyltransferase mutant has been
constructed that retained its serum-resistant phenotype despite C3b
being deposited on its surface (62). Our data are consistent with the
results of Vogel and co-workers (62), who characterized the
meninogoccocal sialyltransferase mutant in that the H. ducreyi lst mutant is resistant to killing by 60% human serum (data not shown). Thus, although sialic acid-containing glycoconjugates may be
one mechanism microorganisms employ to resist complement killing,
H. ducreyi and some neisserial strains have additional mechanisms to resist the lytic effects of complement.
A second potential role of LOS sialylation may be to allow the bacteria
to either escape from or avoid host phagocytic cells in a mucosal
environment. Sialylation of the LOS produces a negative charge on the
outer surface of the bacteria (63), thus, perhaps, preventing close
contact with the negatively charged host cell surface. It has been
demonstrated for the gonococcus that sialylation leads to a reduced
invasion of some but not other cultured human epithelial and
endothelial cells (64, 65). In addition, studies have shown that LOS
sialylation decreases the adherence and the phagocytosis of gonococcal
strains by human neutrophils (66). This decrease in adherence and
phagocytosis may be responsible for the observed decreased stimulation
of an oxidative burst in neutrophils (67). Furthermore, using
sialyltransferase mutants of N. gonorrhoeae, McGee et
al. (68) have shown that sialyltransferase activity is not
necessary for gonococcal adhesion to neutrophils or required for the
human neutrophil burst. With the construction of isogenic
sialyltransferase mutants of N. gonorrhoeae, the role of LOS
sialylation will be more readily discerned.
Although the H. ducreyi lst gene does not have high homology
to other known sialyltransferases, it has significant homology to the
H. influenzae gene, orfY, (HI0871). Previously,
orfY has been studied as a potential LOS biosynthesis gene
(69). When an orfY mutant in H. influenzae strain
RM1004 was studied by Tricine-SDS-PAGE, no detectable alteration in LOS
structure was observed (69). Interestingly, when orfY was
mutated in other H. influenzae strains, RM153 or RM118,
minor alterations were observed in the LOS profiles of these mutants.
Further studies were carried out with an isogenic orfY
mutant in the H. influenzae strain RM153 in an infant-rat model of infection. No significant difference was observed when the
virulence of the mutant was compared with the virulence of the
wild-type strain (69). It remains to be determined whether the
orfY gene encodes the H. influenzae sialyltransferase.
Immediately downstream of the H. ducreyi lst gene,
homologues to the rmlB and rmlA genes of other
Gram-negative bacteria were identified. In Salmonella
typhimurium, the rml gene cluster, rmlBCAD, is responsible for the conversion of glucose to rhamnose and subsequent incorporation of rhamnose into the O antigen (50). In the cloned fragment of H. ducreyi DNA in pRSM1627, the rmlA
gene was truncated at a genomic H. ducreyi NotI restriction
site. To sequence the remaining portion of the rmlA gene and
to determine if any of the other genes from the rml gene
cluster were present in H. ducreyi, a portion of the
remaining 5.5-kb fragment of H. ducreyi DNA from the
original For Neisseria, the rmlBAD genes are present, but
the rmlC gene has not been identified. Mutation of any of
the rml genes in N. gonorrhoeae did not affect
LOS biosynthesis or lead to any observed phenotype (71). In H. influenzae, only the rmlB homologue has been identified
(69). No observed difference was detected in the LOS structure of the
rmlB mutant for H. influenzae strain RM7004 when
the purified LOS was run on a Tricine-SDS-PAGE gel. However, when the
rmlB gene was disrupted in H. influenzae strain RM153, it was reported that a minor change was observed in the LOS
structure. More interesting, the virulence of the rmlB
mutant strain of RM153 was tested in the infant-rat model of infection, and the mutant strain was greatly attenuated for infection. The H. ducreyi rmlB mutant will be tested for its virulence in
the various models of H. ducreyi infection. The mutants that
lack the sialic acid-containing glycoform of LOS will also be studied in detail to gain an appreciation for the role of sialic acid in the
pathogenesis of H. ducreyi disease.
kanamycin cassette, and isogenic
strains were constructed. LOS was isolated from each strain and
characterized by SDS-polyacrylamide gel electrophoresis, carbohydrate,
and mass spectrometric analysis. LOS isolated from strains containing a mutation in neuA or in the second ORF, designated
lst, lacked the sialic acid-containing glycoform.
Complementation studies were performed. The neuA gene and
the lst gene were each cloned into the shuttle vector pLS88
after polymerase chain reaction amplification. Complementation of the
mutation in the lst gene was observed, but we were unable
to complement the neuA mutation. Since it is possible that
transcription of the neuA gene and the lst gene
were coupled, we constructed a nonpolar mutation in the neuA gene. In this construct, the neuA
mutation was complemented, suggesting transcriptional coupling of the
neuA gene and the lst gene. Sialyltransferase
activity was detected by incorporation of 14C-labeled NeuAc
from CMP-NeuAc into trichloroacetic acid-precipitable material when the
lst gene was overexpressed in the nonpolar neuA mutant. We conclude that the lst gene encodes the H. ducreyi sialyltransferase. Since the lst gene product
has little, if any, structural relationship to other
sialyltransferases, this protein represents a new class of sialyltransferase.
INTRODUCTION
Top
Abstract
Introduction
References
library. In order to determine whether additional
genes relevant to LOS biosynthesis were closely linked to the
neuA gene, we determined the DNA sequence 5' and 3' of the
neuA gene. The gene encoding the H. ducreyi
sialyltransferase was identified as well as homologues of the
rmlB (rfbB) and rmlA (rfbA)
genes of many Gram-negative bacteria. Isogenic mutants of H. ducreyi were constructed that were deficient in both the CMP-NeuAc
synthetase and the sialyltransferase.
EXPERIMENTAL PROCEDURES
1, streptomycin at 20 µg
ml
1, and/or X-gal at 40 µg ml
1. All
E. coli strains were grown on Luria-Bertani (LB) plates or
in LB broth. When necessary, this medium included X-gal at 40 µg
ml
1 and/or the appropriate antibiotics. Ampicillin was
used at 50 µg ml
1, kanamycin was at 20 µg
ml
1, and streptomycin was at 20 µg
ml
1.
Bacteria and plasmids
-Km-2
cassette from pJRS102.0 was then cloned into the unique SrfI
site constructed in the neuA and lst genes in
pRSM1681 and pRSM1712, respectively. Constructions were verified by
restriction analysis, and plasmids containing the
-Km-2
cassette in the neuA or lst genes were saved as
pRSM1682 and pRSM1896, respectively (Table I).
Primers used in this study
-galactosidase. Cointegrates were constructed in H. ducreyi 35000HP by electroporation with plasmid DNA followed by selection on chocolate plates containing kanamycin. Kanamycin-resistant clones were then streaked for isolation on chocolate agar containing kanamycin and X-gal. Isogenic H. ducreyi mutants were
identified as large white colonies, whereas cointegrates appear as
small blue colonies on this medium. After verification of the
constructions by Southern hybridization (see below), the
neuA and lst mutants were saved as strains
35000HP-RSM202 and 35000HP-RSM203, respectively (Table I).
-mercaptoethanol. Typically, samples were diluted 7-700-fold with this buffer to obtain
these concentrations. The samples were heated in a boiling water bath
for 5 min and allowed to cool before loading 100-200 ng of LOS into
the sample wells. A 16% acrylamide resolving gel with a 4% stacking
gel (14 cm × 16 cm × 0.75 mm) was used to separate the LOS
(45). A current of 15 mA was applied for 5 h. Bromphenol blue
migrates off the end of the gel after approximately 4.5 h under
these conditions.
20 °C for at least 10 min, chilled acetone (8-10
volumes) was slowly added to destroy the hydrazine and precipitate the
O-deacylated LOS. The precipitate was pelleted by
centrifugation (14,000 × g for 120 min, 0-4 °C), and the supernatant was carefully removed. The pellet was suspended in
a second, equivalent amount of chilled acetone, and the centrifugation was repeated. After removing the supernatant, the pellet was dissolved in a small volume of water and lyophilized.
0.15 V (0.4 s). Quantitation was performed by treating known
quantities of NeuAc (100-6400 pmol) under exactly the same hydrolysis
conditions to prepare a calibration curve.
-chain (oxidized)
(average [M
H]
= 3494.9 Da) and ACTH 1-24
(average [M
H]
= 2932.5 Da).
-glucoside, 10 µl of 50 mM MgCl2, and 10 µl of
CMP-[14C]NeuAc. Boiled crude extract was used for
negative control. The samples were incubated at 30 °C for 30 min,
and then approximately 2 ml of cold 5% trichloroacetic acid was added
to each tube. trichloroacetic acid-precipitated material was collected
by vacuum filtration through a 0.45 µ filter (Millipore Corp.;
Bedford, MA). The trichloroacetic acid-insoluble precipitate was washed
three times with 2 ml of 5% trichloroacetic acid and air-dried, and
incorporation of [14C]NeuAc was determined by
scintillation counting.
RESULTS
DashII clone. The DNA sequence upstream and downstream
of this gene was determined. Five additional ORFs were identified (Fig.
1). At the 5'-end of the sequenced region
is a homologue of the menA gene of E. coli and
H. influenzae. The menA gene encodes for the
enzyme 1,4-dihydroxy-2-naphthoate octaprenyltransferase, which is
involved in the biosynthesis of menaquinone (49). Downstream of the
menA gene, a small ORF of 345 base pairs was identified that
is transcribed in the opposite direction of menA. The ORF encodes for a homologue of YadR, a conserved hypothetical protein of
unknown function that is found in H. influenzae, E. coli, and other bacteria. The next gene on this fragment of DNA
was the neuA gene, which we previously described (35).
Forty-nine base pairs downstream of neuA, a gene
encoding a protein with an Mr of 34,646 was
identified. We have designated the gene lst (for lipooligosaccharide
sialyltransferase). The putative product of the
lst gene has significant homology to protein HI0871 of
H. influenzae, a hypothetical protein of unknown function.
Extremely weak homology to the polysialyltransferase of E. coli K92 (score 31, E value 6.9) was also observed
using the BlastP algorithm. Stronger local homology to the E. coli K92 polysialyltransferase was identified using the
Lipman-Pearson algorithm. Between residues 246 and 291, the
lipooligosaccharide sialyltransferase is 40% identical to residues
167-211 of the polysialyltransferase (Fig. 2).
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Fig. 1.
Open reading frame map of the sequenced
portion of pRSM1627. The genes and direction of transcription are
shown. The rmlA gene is truncated by a genomic
NotI site at the plasmid/insert junction.
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Fig. 2.
Lipman-Pearson alignment showing homology
between residues 246 and 291 of the lst gene product
and the polysialyltransferase of E. coli K92. The
numbers correspond to the amino acid residues of the
respective LST and NeuS proteins. Identical amino acids are printed
between the two sequences, conservative substitutions are
identified by a colon, less conserved substitutions are
identified by a period, and gaps are identified by a
dash.
-Km-2 fragment was cloned into the
SrfI site in each of these genes to form pRSM1682 and
pRSM1896, respectively. Previously, isogenic mutants in H. ducreyi were constructed by electroporation of a linearized
plasmid construct containing an insertionally inactivated gene,
followed by selection with the appropriate antibiotic. After numerous
unsuccessful attempts to construct isogenic neuA and lst mutants by electroporation with
NotI-linearized pRSM1682 or pRSM1896, we devised a new
strategy to construct isogenic mutants. This new strategy exploited the
observation that we could use
-galactosidase as a counter-selectable
marker to select for H. ducreyi mutants. We constructed a
suicide vector containing the lacZ gene driven off of the
trc promoter with a unique NotI site. In separate
constructions, the NotI fragments containing the
insertionally inactivated neuA and lst genes were
cloned into the vector pRSM1791. The newly generated plasmids pRSM1895
and pRSM1903, which contained the insertionally inactivated genes, were
individually transformed into 35000HP. Cointegrates were selected on
chocolate agar containing kanamycin. Individual clones were then
streaked on chocolate agar containing both kanamycin and X-gal. On
these plates, isogenic mutants were easily recognized, since they
appeared as large white colonies, whereas H. ducreyi
cointegrates were small and blue. Isogenic mutants were constructed in
the neuA and lst genes. These strains are
designated 35000HP-RSM202 and 35000HP-RSM203, respectively.
-Km-2 fragment, and
vector pRSM1791 (data not shown). When chromosomal DNA from strain
35000HP was probed with the LOS biosynthetic genes, a single band of
approximately 6.1 kb was observed. For the neuA and
lst mutants, single bands were also observed, but the size
of the fragments was approximately 8.3 kb. When probed with the
-Km-2 fragment, no bands were apparent with 35000HP DNA, but
single bands were observed for the mutants of approximately 8.0 kb in size. When probed with vector pRSM1791, no bands were observed
with 35000HP or the LOS mutants.
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Fig. 3.
Structure, nomenclature, and
SDS-polyacrylamide gel of LOS isolated from strain 35000HP
and the neuA and lst mutants of
H. ducreyi. Upper panel,
silver-stained SDS-PAGE of LOS from 35000HP (lanes
1 and 8), LOS from strain 35000HP-RSM202
(neuA mutant) (lane 2), LOS from
strain 35000 HP-203 (lst mutant) (lane
3), LOS from strain 35000HP-RSM202(pNEUA) (lane
4), LOS from strain 35000HP-RSM203(pLST) (lane
5), LOS from strain 35000HP-RSM208 (nonpolar neuA
mutant) (lane 6), and LOS from strain
35000HP-RSM208(pNEUA) (lane 7). The
arrow indicates the position of the NeuAc-containing LOS
glycoform that is missing from the neuA and lst
mutants. The letters to the right refer to the
proposed glycoforms to which the bands are believed to correspond in
the structure shown in the lower panel. When
NeuAc is not present in the LOS, the A5b1 and
A5b2 glycoforms from the second biosynthetic
pathway are more readily observed. Lower panel,
as H. ducreyi strains produce a complex mixture of LOS
glycoforms, we have used the generalized nomenclature previously
proposed to describe Neisseria and Hemophilus
lipooligosaccharides, where glycoform heterogeneity was referred to by
which core heptose the oligosaccharide chain (or branch) is attached
(73). In the nomenclature shown, we refer to the different heptose
positions for chain attachment but add a letter to further distinguish
between different biosynthetic branches and a number for the position
of the terminal monosaccharide in the branch. For example, the major
glycoform in strain 35000HP terminates in Gal and is designated
A5 because it is the fifth monosaccharide in the A-branch
extending from Hep-I. There are two possibilities for chain extension
at this point. Either NeuAc can be added and is designated
A5a1 because it is a branching point and an
extension of A5, or a second biosynthetic pathway can add
an additional lactosamine, which would be designated
A5b2. The branch heptose in italics
(Hep) is
D-glycero-D-manno-heptose,
while the three core heptoses (HepI-III) are of the
L-glycero-D-manno
configuration.
NeuAc and glucosamine content of O-deacylated LOS
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Fig. 4.
MALDI-TOF spectrum of
O-deacylated LOS from H. ducreyi
35000HP. The spectrum is quite similar to that obtained for
strain 35000 previously by our laboratory (51). All of the major peaks
and most of the minor peaks have been assigned and are based on
previous characterization of the LOS from strain 35000 and other
wild-type strains by our laboratory (30-32, 51). Prompt fragments,
which correspond to the loss of H2O and
H3PO4, are shown by the large,
downward pointing arrows. A number of
salt adducts were observed, primarily of PEA-containing species, and
are indicated by the small, horizontal
arrows. It is thought that these PEA-containing glycoforms
bind metal ions very tightly because the PEA is bound to phosphate,
forming a pyrophosphate linkage (51). Two fragments corresponding to
cleavage of the A5a1* O-deacylated
species into oligosaccharide (A5a1* OS) and
lipid A moieties are observed at the lower end of the mass scale. The
species lower in mass is due to the loss of CO2 (44 Da)
from the 2-keto-3-deoxyoctanoic acid of the oligosaccharide. Other
oligosaccharide and lipid A fragments are not shown here because they
are off scale at lower mass, but some are shown in Figs. 5 and 6.
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Fig. 5.
MALDI-TOF spectra of
O-deacylated LOS from H. ducreyi
isogenic mutant strains compared with 35000HP. The spectrum
of 35000HP is the same as in Fig. 4 and is shown here for comparison.
All four spectra are quite similar, except for the absence of NeuAc
containing glycoforms (A5a1 and
A5a1*) from all three of the mutants.
OS, oligosaccharide is abbreviated.
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Fig. 6.
MALDI-TOF spectra of
O-deacylated LOS from H. ducreyi
complemented mutant strains compared with 35000HP. The
spectrum of 35000HP is the same as in Fig. 4 and is shown here for
comparison. The NeuAc-containing glycoforms
(A5a1 and A5a1*) are
seen in both 35000HP-RSM208(pNEUA) and 35000HP-RSM203(pLST) but not
35000HP-RSM202(pNEUA). The increase in the amount of
A5a1 and A5a1*
glycoforms in 35000HP-RSM203(pLST) compared with 35000HP is quite
dramatic.
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Fig. 7.
Characteristics of the
sialyltransferase activity. A, crude sonicate was
incubated for 30 min with CMP-[14C]NeuAc. The reaction
was stopped with cold 5% trichloroacetic acid, and the incorporated
14C was determined as trichloroacetic acid-precipitable
material. The results are the mean of duplicate determinations.
B, 80 µg of protein was incubated with
CMP-[14C]NeuAc as described under "Experimental
Procedures." At the indicated time points, the reaction was stopped
with cold 5% trichloroacetic acid, and the incorporated
14C was determined as trichloroacetic acid-precipitable
material. The results are the mean of duplicate determinations.
C, 50- and 100-µg aliquots of sonicate from strains
35000HP-RSM208 (nonpolar neuA mutant), 35000HP-RSM208(pLST),
or 35000HP-RSM210(pLST) (galactosyltransferase II mutant
overexpressing sialyltransferase) were incubated for 30 min, and
incorporated NeuAc was determined as described above. The results are
the mean of duplicate determinations. Sonicates were prepared from
strain 35000HP-RSM208, 50 µg ( ), 100 µg (
), strain 35000HP-RSM208(pLST), 50 µg (
), 100 µg (
); and from
strain 35000HP-RSM210(pLST), 50 µg (
), 100 µg (
).
DISCUSSION
DASHII clone was sequenced (data not shown). However, no
other rml genes were identified downstream from the
rmlA homologue in H. ducreyi. A mutant was
constructed in the rmlB gene, and although there were
changes in the relative concentrations of the LOS glycoforms (data not
shown), the rmlB mutant produces all of the known LOS
glycoforms. Thus, the role of the rmlB and rmlA
genes in H. ducreyi remains to be determined, since all of the glycoforms are produced and rhamnose has not been detected in
H. ducreyi. rml (rfb) genes have also
been identified in H. influenzae, N. gonorrhoeae,
and N. meningitidis (69-71). In addition to the presence of
rml genes in Gram-negative bacteria, homologues have also
been identified in Gram-positive bacteria, such as Streptococcus pneumoniae (72).
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ACKNOWLEDGEMENTS |
---|
We thank Laurie Tarantino and Huachun Zhong
for excellent technical assistance, James Kaper for providing the
kanamycin cassette constructed by Menard et al. (39), and
Gerard Barcak for the E. coli DH5pcnB strain.
We also acknowledge PerSeptive Biosystems (Framingham, MA) for the
generous support of the MALDI-TOF instrumentation in our laboratory (to
B. W. G.).
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FOOTNOTES |
---|
* This study was supported by Public Health Service Grants AI34967 and AI38444 (to R. S. M.) and AI31254 (to B. W. G.). The Core DNA Sequencing Facility at the Children's Hospital Research Foundation was supported by National Institutes of Health Grant HD34615, and the UCSF Mass Spectrometry Facility was funded by National Center for Research Resources Grant RR01614. This work was presented in part at the Annual Meeting of the American Society for Microbiology, May 21, 1998.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF101047.
§ Recipient of a National Research Service Award (P32-AI09813).
** To whom correspondence should be addressed: Children's Hospital Research Foundation, Room W402, 700 Children's Dr., Columbus, OH 43205. Tel.: 614-722-2680; Fax: 614-722-3273; E-mail: munsonr{at}pediatrics.ohio-state.edu.
The abbreviations used are:
LOS, lipooligosaccharide(s); CMP-NeuAc synthetase, N-acetylneuraminic acid cytidylsynthetase; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; MS, mass
spectrometry; TOF, time of flight; kb, kilobase pair(s); ORF, open
reading frame; PEA, phosphoethanolamine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
2 S. Sun, B. W. Gibson, N. J. Philips, and R. S. Munson, Jr., manuscript in preparation.
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REFERENCES |
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