Haemophilus ducreyi Produces a Novel Sialyltransferase
IDENTIFICATION OF THE SIALYLTRANSFERASE GENE AND CONSTRUCTION OF MUTANTS DEFICIENT IN THE PRODUCTION OF THE SIALIC ACID-CONTAINING GLYCOFORM OF THE LIPOOLIGOSACCHARIDE*

Joel A. BozueDagger §, Michael V. Tullius, Jing WangDagger , Bradford W. Gibson, and Robert S. Munson Jr.Dagger parallel **

From the Children's Hospital Research Foundation and Departments of Dagger  Pediatrics and parallel  Medical Microbiology, Ohio State University, Columbus, Ohio 43205-2696,  Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

    ABSTRACT
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Abstract
Introduction
References

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 Omega  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

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 lambda  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

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-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.

                              
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Table I
Bacteria and plasmids

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 Omega -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 Omega -Km-2 cassette in the neuA or lst genes were saved as pRSM1682 and pRSM1896, respectively (Table I).

                              
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Table II
Primers used in this study

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 beta -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).

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% beta -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.

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 -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.

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), -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.

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 beta -chain (oxidized) (average [M - H]- = 3494.9 Da) and ACTH 1-24 (average [M - H]- = 2932.5 Da).

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 beta -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

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 lambda  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.

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 Omega -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 beta -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.

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 Omega -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 Omega -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.

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).


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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.

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.

                              
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Table III
NeuAc and glucosamine content of O-deacylated LOS

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).


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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.

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.


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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.

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.


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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.

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.


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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 (black-square), 100 µg ().


    DISCUSSION

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 lambda 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).

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.

    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 DH5alpha pcnB strain. We also acknowledge PerSeptive Biosystems (Framingham, MA) for the generous support of the MALDI-TOF instrumentation in our laboratory (to B. W. G.).

    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 beta -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.

    REFERENCES
Top
Abstract
Introduction
References

  1. Morse, S. A. (1989) Clin. Microbiol. Rev 2, 137-157[Medline] [Order article via Infotrieve]
  2. Abeck, D., and Johnson, A. P. (1992) Int. J. STD AIDS 3, 319-323[Medline] [Order article via Infotrieve]
  3. Trees, D. L., and Morse, S. A. (1995) Clin. Microbiol. Rev. 8, 357-375[Abstract]
  4. Flood, J. M., Sarafian, S. K., Bolan, G. A., Lammel, C., Engelman, J., Greenblatt, R. M., Brooks, G. F., Back, A., and Morse, S. A. (1993) J. Infect. Dis. 167, 1106-1111[Medline] [Order article via Infotrieve]
  5. Schulte, J. M., Martich, F. A., and Schmid, G. P. (1992) MMWR CDC Surveill. Summ. 41, 57-61[Medline] [Order article via Infotrieve]
  6. Jessamine, P. G., and Ronald, A. R. (1990) Med. Clin. North Am. 74, 1417-1431[Medline] [Order article via Infotrieve]
  7. Wasserheit, J. N. (1992) Sex. Transm. Dis. 19, 61-77[Medline] [Order article via Infotrieve]
  8. Telzak, E. E., Chiasson, M. A., Bevier, P. J., Stoneburner, R. L., Castro, K. G., and Jaffe, H. W. (1993) Ann. Intern. Med. 119, 1181-1186[Abstract/Free Full Text]
  9. Palmer, K. L., and Munson, R. S., Jr. (1995) Mol. Microbiol. 18, 821-830[Medline] [Order article via Infotrieve]
  10. Palmer, K. L., Goldman, W. E., and Munson, R. S., Jr. (1996) Mol. Microbiol. 21, 13-19[Medline] [Order article via Infotrieve]
  11. Alfa, M. J., DeGagne, P., and Totten, P. A. (1996) Infect. Immun. 64, 2349-2352[Abstract]
  12. Cope, L. D., Lumbley, S., Latimer, J. L., Klesney-Tait, J., Stevens, M. K., Johnson, L. S., Purven, M., Munson, R. S., Jr., Lagergard, T., Radolf, J. D., and Hansen, E. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4056-4061[Abstract/Free Full Text]
  13. Campagnari, A. A., Wild, L. M., Griffiths, G. E., Karalus, R. J., Wirth, M. A., and Spinola, S. M. (1991) Infect. Immun. 59, 2601-2608[Medline] [Order article via Infotrieve]
  14. Lagergard, T. (1992) Microb. Pathog. 13, 203-217[Medline] [Order article via Infotrieve]
  15. Tuffrey, M., Alexander, F., Ballard, R. C., and Taylor-Robinson, D. (1990) J. Exp. Pathol. 71, 233-244
  16. Gibson, B. W., Campagnari, A. A., Melaugh, W., Phillips, N. J., Apicella, M. A., Grass, S., Wang, J., Palmer, K. L., and Munson, R. S., Jr. (1997) J. Bacteriol. 179, 5062-5071[Abstract]
  17. Alfa, M. J., and DeGagne, P. (1997) Microb. Pathog. 22, 39-46[CrossRef][Medline] [Order article via Infotrieve]
  18. Mandrell, R. E., Griffiss, J. M., and Macher, B. A. (1988) J. Exp. Med. 168, 107-126[Abstract]
  19. Apicella, M. A., Mandrell, R. E., Shero, M., Wilson, M. E., Griffiss, J. M., Brooks, G. F., Lammel, C., Breen, J. F., and Rice, P. A. (1990) J. Infect. Dis. 162, 506-512[Medline] [Order article via Infotrieve]
  20. Campagnari, A. A., Spinola, S. M., Lesse, A. J., Kwaik, Y. A., Mandrell, R. E., and Apicella, M. A. (1990) Microb. Pathog. 8, 353-362[Medline] [Order article via Infotrieve]
  21. Mandrell, R. E., and Apicella, M. A. (1993) Immunobiology 187, 382-402[Medline] [Order article via Infotrieve]
  22. Schauer, R., Kelm, S., Reuter, G., Roggentin, P., and Shaw, L. (1995) in Biology of the Sialic Acids (Rosenberg, A., ed), pp. 7-67, Plenum Press, New York
  23. Timmis, K. N., Boulnois, G. J., Bitter, S. D., and Cabello, F. C. (1985) Curr. Top. Microbiol. Immunol. 118, 197-218[Medline] [Order article via Infotrieve]
  24. Jennings, H. J., Katzenellenbogen, E., Lugowski, C., Michon, F., Roy, R., and Kasper, D. L. (1984) Pure Appl. Chem. 56, 893-905
  25. Jann, K., and Jann, B. (1985) in The Virulence of Escherichia coli: Reviews and Methods (Sussman, M., ed), pp. 157-176, Academic Press, Inc., Orlando, FL
  26. Bitter-Suermann, D. (1993) in Polysialic Acid: From Microbes to Man (Roth, J., Rutishauser, U., and Troy, F. A., II, eds), pp. 11-24, Birkhauser Verlag, Basel, Switzerland
  27. Smith, H., Parsons, N. J., and Cole, J. A. (1995) Microb. Pathog. 19, 365-377[CrossRef][Medline] [Order article via Infotrieve]
  28. Schweda, E. K., Sundstrom, A. C., Eriksson, L. M., Jonasson, J. A., and Lindberg, A. A. (1994) J. Biol. Chem. 269, 12040-12048[Abstract/Free Full Text]
  29. Schweda, E. K., Jonasson, J. A., and Jansson, P. E. (1995) J. Bacteriol. 177, 5316-5321[Abstract]
  30. Melaugh, W., Phillips, N. J., Campagnari, A. A., Tullius, M. V., and Gibson, B. W. (1994) Biochemistry 33, 13070-13078[Medline] [Order article via Infotrieve]
  31. Melaugh, W., Campagnari, A. A., and Gibson, B. W. (1996) J. Bacteriol. 178, 564-570[Abstract]
  32. Campagnari, A. A., Karalus, R., Apicella, M., Melaugh, W., Lesse, A. J., and Gibson, B. W. (1994) Infect. Immun. 62, 2379-2386[Abstract]
  33. Ahmed, H. J., Frisk, A., Mansson, J. E., Schweda, E. K., and Lagergard, T. (1997) Infect. Immun. 65, 3151-3158[Abstract]
  34. Gibson, B. W., Melaugh, W., Phillips, N. J., Apicella, M. A., Campagnari, A. A., and Griffiss, J. M. (1993) J. Bacteriol. 175, 2702-2712[Abstract]
  35. Tullius, M. V., Munson, R. S., Jr., Wang, J., and Gibson, B. W. (1996) J. Biol. Chem. 271, 15373-15380[Abstract/Free Full Text]
  36. Bozue, J. A., Tarantino, L., and Munson, R. S., Jr. (1998) FEMS Microbiol. Lett. 164, 269-273[CrossRef][Medline] [Order article via Infotrieve]
  37. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  38. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract]
  39. Menard, R., Sansonetti, P. J., and Parsot, C. (1993) J. Bacteriol. 175, 5899-5906[Abstract]
  40. Barcak, G. J., Chandler, M. S., Redfield, R. J., and Tomb, J. F. (1991) Methods Enzymol. 204, 321-342[Medline] [Order article via Infotrieve]
  41. Westphal, O., and Jann, K. (1965) Methods Carbohydr. Chem. 5, 83-91
  42. Johnson, K. G., and Perry, M. B. (1976) Can. J. Microbiol. 22, 29-34[Medline] [Order article via Infotrieve]
  43. Apicella, M. A., Griffiss, J. M., and Schneider, H. (1994) Methods Enzymol. 235, 242-252[Medline] [Order article via Infotrieve]
  44. Inzana, T. J. (1983) J. Infect. Dis. 148, 492-499[Medline] [Order article via Infotrieve]
  45. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  46. Tsai, C. M., and Frasch, C. E. (1982) Anal. Biochem. 119, 115-119[Medline] [Order article via Infotrieve]
  47. Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zahringer, U. (1988) Eur. J. Biochem. 177, 483-492[Abstract]
  48. Reuter, G., and Schauer, R. (1994) Methods Enzymol. 230, 168-199[CrossRef][Medline] [Order article via Infotrieve]
  49. Shaw, D. J., Guest, J. R., Meganathan, R., and Bentley, R. (1982) J. Bacteriol. 152, 1132-1137[Medline] [Order article via Infotrieve]
  50. Romana, L. K., Santiago, F. S., and Reeves, P. R. (1991) Biochem. Biophys. Res. Commun. 174, 846-852[Medline] [Order article via Infotrieve]
  51. Gibson, B. W., Engstrom, J. J., John, C. M., Hines, W., and Falick, A. M. (1997) J. Am. Soc. Mass Spectr. 8, 645-658[CrossRef]
  52. Gilbert, M., Watson, D. C., Cunningham, A. M., Jennings, M. P., Young, N. M., and Wakarchuk, W. W. (1996) J. Biol. Chem. 271, 28271-28276[Abstract/Free Full Text]
  53. Steenbergen, S. M., Wrona, T. J., and Vimr, E. R. (1992) J. Bacteriol. 174, 1099-1108[Abstract]
  54. Perez-Casal, J., Caparon, M. G., and Scott, J. R. (1991) J. Bacteriol. 173, 2617-2624[Medline] [Order article via Infotrieve]
  55. Parsons, N. J., Patel, P. V., Tan, E. L., Andrade, J. R., Nairn, C. A., Goldner, M., Cole, J. A., and Smith, H. (1988) Microb. Pathog. 5, 303-309[Medline] [Order article via Infotrieve]
  56. Fox, A. J., Jones, D. M., Scotland, S. M., Rowe, B., Smith, A., Brown, M. R., Fitzgeorge, R. G., Baskerville, A., Parsons, N. J., Cole, J. A., and Smith, H. (1989) Microb. Pathog. 7, 317-318[Medline] [Order article via Infotrieve]
  57. Smith, H., Cole, J. A., and Parsons, N. J. (1992) FEMS Microbiol. Lett. 79, 287-292[Medline] [Order article via Infotrieve]
  58. Ram, S., Sharma, A. K., Simpson, S. D., Gulati, S., McQuillen, D. P., Pangburn, M. K., and Rice, P. A. (1998) J. Exp. Med. 187, 743-752[Abstract/Free Full Text]
  59. Rice, P. A., Blake, M. S., and Joiner, K. A. (1987) Antonie Leeuwenhoek 53, 565-574[Medline] [Order article via Infotrieve]
  60. Rice, P. A., and Kasper, D. L. (1977) J. Clin. Invest. 60, 1149-1158[Medline] [Order article via Infotrieve]
  61. Schoolnik, G. K., Buchanan, T. M., and Holmes, K. K. (1976) J. Clin. Invest. 58, 1163-1173[Medline] [Order article via Infotrieve]
  62. Vogel, U., Claus, H., Heinze, G., and Frosch, M. (1997) Med. Microbiol. Immunol. (Berl.) 186, 159-166[CrossRef][Medline] [Order article via Infotrieve]
  63. Swanson, J. (1991) in Neisseriae 1990 (Achtman, M., Kohl, P., Marchal, C., Morelli, G., Seiler, A., and Thiesen, B., eds), pp. 391-396, Walter de Gruyter, Berlin
  64. van Putten, J. P., and Robertson, B. D. (1995) Mol. Microbiol. 16, 847-853[Medline] [Order article via Infotrieve]
  65. van Putten, J. P. (1993) EMBO J. 12, 4043-4051[Abstract]
  66. Kim, J. J., Zhou, D., Mandrell, R. E., and Griffiss, J. M. (1992) Infect. Immun. 60, 4439-4442[Abstract]
  67. Rest, R. F., and Frangipane, J. V. (1992) Infect. Immun. 60, 989-997[Abstract]
  68. McGee, D. J., Chen, G. C., and Rest, R. F. (1996) Infect. Immun. 64, 4129-4136[Abstract]
  69. Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol. 22, 951-965[Medline] [Order article via Infotrieve]
  70. Hammerschmidt, S., Birkholz, C., Zahringer, U., Robertson, B. D., van Putten, J., Ebeling, O., and Frosch, M. (1994) Mol. Microbiol. 11, 885-896[Medline] [Order article via Infotrieve]
  71. Robertson, B. D., Frosch, M., and van Putten, J. P. (1994) J. Bacteriol. 176, 6915-6920[Abstract]
  72. Munoz, P., Llancaqueo, A., Rodriguez-Creixems, M., Pelaez, T., Martin, L., and Bouza, E. (1997) Arch. Intern. Med. 157, 213-216[Abstract]
  73. Preston, A., Mandrell, R. E., Gibson, B. W., and Apicella, M. A. (1996) Crit. Rev. Microbiol. 22, 139-180[Medline] [Order article via Infotrieve]
  74. Hammond, G. W., Lian, C. J., Wilt, J. C., and Ronald, A. R. (1978) Antimicrob. Agents Chemother. 13, 608-612[Medline] [Order article via Infotrieve]
  75. Spinola, S. M., Orazi, A., Arno, J. N., Fortney, K., Kotylo, P., Chen, C. Y., Campagnari, A. A., and Hood, A. F. (1996) J. Infect. Dis. 173, 394-402[Medline] [Order article via Infotrieve]
  76. Wang, R. F., and Kushner, S. R. (1991) Gene (Amst.) 100, 195-199[CrossRef][Medline] [Order article via Infotrieve]
  77. Dixon, L. G., Albritton, W. L., and Willson, P. J. (1994) Plasmid 32, 228-232[CrossRef][Medline] [Order article via Infotrieve]


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