INTRODUCTION

Haemophilus ducreyi is the Gram-negative bacterium that causes the sexually transmitted disease chancroid. Chancroid is common in developing countries and has been shown to be an independent risk factor in the transmission of the human immunodeficiency virus (1). Although there are relatively few reported cases of chancroid each year in the United States, there have been several outbreaks in urban areas, and recent studies suggest that chancroid may be greatly underreported due to inadequate methods of detection (2, 3, 4). Although chancroid is still treatable with antibiotic regimens, resistant strains of H. ducreyi have been increasing (5, 6, 7).

Lipooligosaccharides (LOS)¹ present in the outer membrane of H. ducreyi have been shown to be a virulence factor (8, 9, 10, 11). Recently, we have shown that many H. ducreyi strains synthesize LOS glycoforms that contain a terminal N-acetyllactosamine that further undergoes the addition of sialic acid (12, 13). 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 (14, 15). This mimicry may contribute to the organism's pathogenicity by allowing the bacteria to evade the host's immune system or establish infection by adhering to host cells (16). Incorporation of sialic acid in the LOS of H. influenzae, N. meningitidis, and N. gonorrhoeae has also been described (15). In addition to the ability of sialylated molecules to downregulate complement as part of a polyanionic surface, sialic acid plays important roles both in cellular recognition and the masking of epitopes, especially terminal galactose residues (17, 18). Sialylation of N. gonorrhoeae LOS has been shown to block neutrophil phagocytosis, block bactericidal antibody binding, and increase serum resistance (19, 20, 21). Also, the sialic acid containing capsules of N. meningitidis, Escherichia coli K1, and group B streptococci all have been identified as virulence factors (17, 22, 23, 24). To date, there are no conclusive results demonstrating that H. ducreyi produces a capsule (25, 26).

The following reactions (I-III) are involved in the biosynthesis of sialic acid containing molecules and are catalyzed by the enzymes shown in parenthesis to the right. In the final step, the hydroxyl group of a carbohydrate serves as the acceptor (HO acceptor) for the nucleotide-sialic acid donor, CMP-NeuAc: Sialic acid can be synthesized in bacteria by condensation of N-acetylmannosamine with either phosphoenolpyruvate (Ia) or pyruvate (Ib). The first reaction is catalyzed by NeuAc synthetase, which has been partially purified from N. meningitidis (27). This enzyme differs from the mammalian enzyme which synthesizes NeuAc-9-P (from N-acetylmannosamine-6-P and phosphoenolpyruvate), which is then dephosphorylated by a phosphorylase. Recently, it was demonstrated that NeuAc synthesis in E. coli K1 occurs only through NeuAc aldolase (Ib) (28). CMP-NeuAc synthetases catalyze the second step (II) in the pathway, forming the nucleotide-sugar donor used by sialyltransferases. These enzymes have been purified and characterized from several bacterial and animal sources (29, 30, 31, 32, 33). A LOS-specific sialyltransferase has been detected in extracts of Neisseria sp. and partially characterized (34). To date, no bacterial LOS-specific sialyltransferase has been purified to homogeneity or cloned, although a recent report characterizing a sialyltransferase deficient mutant may soon lead to cloning of the sialyltransferase gene (35). In contrast, bacterial polysialyltransferases, the enzymes that synthesize polysialic acid capsules, have been cloned (36) as have several animal sialyltransferases (37). As a group, these enzymes may be attractive targets for therapeutic intervention considering the role of sialic acid containing molecules as virulence and/or adhesion factors. In this report we detail the isolation of the CMP-NeuAc synthetase from H. ducreyi, the cloning of its gene, and expression of the enzyme in E. coli using a T7lac promoter system. Moreover, sequence analysis of CMP-NeuAc and CMP-KDO synthetases reveal a number of residues and domains conserved throughout both enzyme classes.

EXPERIMENTAL PROCEDURES

Materials

Reactive Green 19-Agarose, Cibacron Blue 3GA-Agarose, CTP, N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-manno-octulosonic acid, ampicillin, kanamycin, and SDS-PAGE molecular weight standards were from Sigma. Size exclusion chromatography standards were from Sigma and Bio-Rad. Isopropyl -D-thiogalactopyranoside was from Life Technologies, Inc. H. ducreyi strain 35000 is a typed CDC strain and was a generous gift of Dr. A. Campagnari (SUNY at Buffalo).

Methods

H. ducreyi strain 35000 was grown on chocolate agar plates (GC Medium Base (Difco), 1% (w/v) hemoglobin (Difco), and 1% (v/v) IsoVitaleX (Becton Dickinson) or 0.1% (w/v) glucose, 0.01% (w/v) L-glutamine, 0.026% (w/v) L-cysteine hydrochloride) incubated in candle jars at 33-35 °C for 1-2 days. The bacteria were suspended in 50 mM Bis-Tris-HCl, pH 6.5 (buffer A), and used immediately for purification of the CMP-NeuAc synthetase or frozen and stored at 80 °C. E. coli strains were grown on Luria-Bertani (LB) agar plates and in LB broth along with the appropriate antibiotic (50 µg/ml ampicillin or kanamycin).

The bacterial suspension (3 g of wet weight cells in 20 ml of buffer A) was adjusted to 2 mM EDTA and 0.2 mg/ml lysozyme and was stirred for 20 min at room temperature. Unlysed cells and cellular debris were removed by centrifugation at 13,000 × g for 10 min. The supernatant was applied to two 5-ml EconoPac Q cartridges (Bio-Rad) connected in series and equilibrated in buffer A. The enzyme was eluted with 0.2 M NaCl in buffer A. The active fraction (11 ml) was applied to a 5-ml Cibacron Blue 3GA-Agarose dye column also equilibrated with buffer A. The column breakthrough (20 ml), which contained most of the enzyme activity, was concentrated to 0.5 ml with two Centricon-30 concentrators (Amicon) and applied to two Bio-Sil TSK-125 (Bio-Rad; 7.5 × 600 mm each) size exclusion columns connected in series and equilibrated with 20 mM KH₂PO₄, 0.5 mM EDTA, 20% (v/v) glycerol, pH 7.0. The active fractions (4.5 ml) were pooled, 0.5 ml of 0.5 M Tris-HCl, pH 8.5, was added to increase the pH, and the sample applied to a MonoQ HR 5/5 column (Pharmacia Biotech Inc.; 5 × 50 mm) equilibrated with 50 mM Tris-HCl, 20% (v/v) glycerol, pH 8.0. The enzyme was eluted with a KCl gradient (0.5 ml/min, 50-200 mM KCl in 40 min) in the same buffer. The active fractions (3 ml) were pooled, concentrated to 0.4 ml with a Centricon-30 concentrator, and adjusted to 1.5 M (NH₄)₂SO₄. The sample was applied to a Hydropore-5-HIC column (Rainin; 4.6 × 100 mm), equilibrated with 0.1 M KH₂PO₄, 0.5 mM EDTA, 1.2 M (NH₄)₂SO₄, pH 7.0 (buffer B), and eluted with a gradient of 0.1 M KH₂PO₄, 0.5 mM EDTA, pH 7.0 (buffer C) (0.5 ml/min, 1.2-0 M (NH₄)₂SO₄ in 40 min). The active fractions (3 ml) were readjusted to 1.2 M (NH₄)₂SO₄ and rechromatographed on the HIC column using a shallower gradient (0.4 ml/min, 1.125-0.6 M (NH₄)₂SO₄ in 52.5 min). The active fractions (2.4 ml) were pooled, concentrated, and desalted with a Centricon-30 concentrator to a volume of 0.5 ml and applied to a C4 reverse phase HPLC column (Vydac; 2.1 × 150 mm). The C4 column was equilibrated with 0.1% trifluoroacetic acid, and the proteins were eluted with a gradient of 0.08% trifluoroacetic acid, 70% CH₃CN over 70 min at a flow rate of 0.2 ml/min.

The three major peaks eluted from the HPLC C4 column were each subjected to NH₂-terminal sequence analysis using an Applied Biosystems 470A gas-phase sequencer with an on-line ABI Model 130A phenylthiohydantoin analyzer. For the fraction identified as containing the CMP-NeuAc synthetase, 43 cycles were obtained in which the amino acid residue could be assigned with confidence. The first cycle produced 4 pmol of methionine with the yield dropping to 0.1 pmol phenylalanine by the 43rd cycle. Based on typical conversion yields (30-40%), approximately 10-13 pmol (0.25-0.33 µg) of CMP-NeuAc synthetase was loaded on the sequencer.

The NH₂-terminal amino acid sequence was reverse-translated employing the CompuGene program (38). Two degenerate oligonucleotides were constructed (see Table I). Oligonucleotide 1, a 23-mer containing a single inosine at position 15 and a degeneracy of 108, corresponds to the reverse translation of amino acids 1-8. Oligonucleotide 2 corresponds to the reverse translation of amino acids 14-20 and is a 20-mer with a single inosine and a degeneracy of 48. Both oligonucleotides were prepared by the Protein Chemistry Facility at Washington University School of Medicine. A DASHII library of H. ducreyi 35000 DNA (39) was plated, plaques were transferred to Hybond-N, and DNA was immobilized by UV cross-linking (Stratalinker) employing standard methodologies. Oligonucleotide 1 was end-labeled with -³²P using T4 polynucleotide kinase, and hybridization was performed overnight in 5 × SSPE (1 × SSPE = 0.18 M NaCl, 0.01 M sodium phosphate, and 1 mM EDTA, pH 7.7), 5 × Denhardt's solution, 0.5% SDS, and 20 µg/ml Salmon testes DNA at 30 °C. Following hybridization, the membrane was washed three times at 35 °C in 2 × SSPE-0.1% SDS. The membrane was then wrapped in Saran Wrap and autoradiographed. Phage plaques, which gave a positive hybridization signal, were picked and rescreened. Phage DNA was isolated from 10 ml of liquid lysates and characterized by restriction analysis.

Table I. Oligonucleotides employed in this study R = A/G, Y = C/T, H = T/C/A, and I is inosine. The sequences in bold are the NdeI site in oligonucleotide 3 and the BamHI site in oligonucleotide 4.  Oligonucleotide Sequence 1 5-ATGAARAARATHGCIATHATHCC 2 5-AARGGIATHAARGAYAARAA 3 5-AGATGCATATGAAGAAGATTGCAATCATC 4 5-CGGGATCCTGGCAATTTCTTTCATCGTTAT

The low copy number vector pWSK30 was employed for the initial subcloning (40). For subsequent studies, the DNA fragment containing CMP-NeuAc synthetase was amplified by 20 rounds of PCR using primers 3 and 4 (see Table I). The PCR product was cloned with the TA cloning kit into pCRII according to the manufacturer's instructions (Invitrogen). Plasmid vectors pT7-7 (41) and pET24b (Novagen) were employed for the preparation of the T7 expression constructs. Plasmid DNA was purified by using the Qiawell-8 plasmid kit (Qiagen, Chatsworth, CA).

DNA sequence was determined by the dideoxychain termination method employing Sequenase according to the manufacturer's directions (U. S. Biochemicals Corp.). Lasergene software (DNASTAR, Madison, WI) was employed for contig assembly and sequence analysis.

Protein sequence data bases (GenBank, Release 92.0, Dec. 1995; SWISS-PROT, Release 32.0, Dec. 1995; PIR, Release 45.0, June 1995) were searched using the search program BLAST (42), and protein sequences were aligned with the PILEUP program from the Wisconsin Sequence Analysis Package, version 8.0 (Genetics Computer Group, Inc., Madison, WI). The similarity matrix was calculated using the scoring method in Ref. 43. In pairwise comparisons, the smaller of the two sequences was taken as the denominator.

E. coli BL21(DE3) harboring the pET24 expression construct containing the CMP-NeuAc synthetase (designated pMVT1) was grown at 30 °C in LB broth containing 50 µg/ml kanamycin. After reaching an A₆₀₀ between 0.3 and 0.8, the culture was induced with 0.1 mM isopropyl -D-thiogalactopyranoside for 10 h at 30 °C. The cells were harvested and frozen at 80 °C until needed.

The cell paste (11 g) from a 3-liter culture was suspended in 40 ml of 50 mM Tris-HCl, 0.1 M NaCl, pH 7.5 (buffer D), and the cells were lysed by two passages through a French press (approximately 20,000 psi). The lysate was sonicated (four 30-s pulses at 25 watts with 2-min pauses) to shear nucleic acids and reduce its viscosity. The lysate was then centrifuged at 15,000 × g for 90 min to remove cellular debris. The supernatant (39 ml) was applied to a DEAE-cellulose column (2.5 × 13.5 cm) equilibrated with buffer D. After washing the column with 120 ml of buffer D, the enzyme was eluted with a gradient of 0.1-1.0 M NaCl of 270 ml. The active fractions were pooled (120 ml) and adjusted to 50% (NH₄)₂SO₄. The precipitate was pelleted, redissolved to 10 ml, and desalted into buffer D using a 15-ml Swift desalting column (Pierce). The sample was applied to a Reactive Green 19-Agarose dye column (2.5 × 14 cm) equilibrated with buffer D. The column was washed with 120 ml of buffer D, and most of the enzyme was then eluted with a step gradient of 50 mM Tris-HCl, 0.5 M NaCl, pH 7.5. The sample (25 ml) was adjusted to 50% (NH₄)₂SO₄, and the precipitate was pelleted. The pellet was redissolved to 4.8 ml with buffer C, and then saturated (NH₄)₂SO₄ (1.2 ml) was slowly added. The sample was centrifuged at 12,000 × g for 30 min to remove particulates before loading on the HIC column equilibrated with buffer B. The enzyme was eluted with a gradient of buffer C (1 ml/min, 1.2-0.6 M (NH₄)₂SO₄ in 40 min). Four separate runs were required to purify the entire sample. Fractions judged to be pure by SDS-PAGE were pooled (13.5 ml) and adjusted to 65% (NH₄)₂SO₄ for storage as a suspension at 4 °C.

CMP-NeuAc synthetase activity was assayed by the following procedure: 10 µl of enzyme diluted in 0.1 mg/ml bovine serum albumin (BSA) was added to 90 µl of assay mix and incubated at 37 °C for 10 min. The reaction was stopped by adding 400 µl of cold water and freezing the sample in dry ice. The final concentrations of components in the assay mixture were 1 mM CTP, 2 mM NeuAc, 20 mM MgCl₂, 0.2 M MOPS, pH 7.1, 10 µg/ml BSA, and 4-10 milliunits/ml (nmol/min/ml) CMP-NeuAc synthetase. The assay solutions were thawed directly before analysis. A 20-µl loop was completely filled by a 100-µl injection on a Rainin (Woburn, MA) HPLC system. A nucleotide analysis column (Vydac; 4.6 × 50 mm) was used to resolve CMP-NeuAc from the substrate CTP and any small amounts of CMP or CDP present. Solvent A was water and solvent B was 0.5 M ammonium formate (adjusted to pH 3.5 with formic acid). The column was operated at a flow rate of 2 ml/min, and the separation was complete in 2 min followed by a 1.5-min reequilibration cycle (Fig. 1). Quantitation of the CMP-NeuAc peak area was done using a standard curve of CMP (44). Detection was at 280 nm (_max of CMP and CMP-NeuAc at pH 3.5) using an Applied Biosystems 1000S diode array detector and was linear from 10 to 10,000 pmol. A unit is defined as the amount of enzyme that catalyzes the formation of 1 µmol of CMP-NeuAc/min under the conditions of the assay.

For calculation of the K_m for CTP and NeuAc, the enzyme was diluted with 0.2 M NaCl, 1 mM MgCl₂, 1 mg/ml BSA to 1 and 4 µg/ml, respectively. The diluted enzyme (35 µl) was added to 315 µl of assay mix preheated to 37 °C and incubated at 37 °C for up to 10.5 min. Each substrate was measured with the other held constant at a saturating concentration. NeuAc was varied from 0.25-8 mM with CTP kept at 1 mM, and CTP was varied from 0.025-0.8 mM with NeuAc at 10 mM. The final concentrations of the other components of the assay mixture were 20 mM MgCl₂, 20 mM NaCl, 0.2 M MOPS, pH 7.1, 0.1 mg/ml BSA, and 0.1 or 0.4 µg/ml CMP-NeuAc synthetase. Aliquots (100 µl) were removed from the assays at 3.5 and 7 min and frozen in dry ice along with the final time point at 10.5 min. CMP-NeuAc product for each time point was quantitated as above, except that a 50-µl loop was filled with a 100-µl injection. A straight line was fit to the three time points, and the slope was taken as the initial velocity. The K_m for CTP and NeuAc were determined by fitting the data directly to the equation v = V_max[S]/(K_m + [S]) using the computer program KinetAsyst (IntelliKinetics, State College, PA).

Protein was determined using the Bio-Rad DC Protein Assay, which is a modification of the Lowry method (45), according to the manufacturer's instructions. BSA was used as a standard.

Analysis of chromatography fractions and calculation of molecular weight was performed using 15% acrylamide gels (46). Gels were stained with Coomassie Brilliant Blue R-250. Molecular weight standards used were albumin, 66000; ovalbumin, 45000; glyceraldehyde-3-phosphate dehydrogenase, 36000; carbonic anhydrase, 29000; trypsinogen, 24000; trypsin inhibitor, 20000; -lactalbumin, 14200; and aprotinin, 6500.

To estimate the native molecular weight, purified enzyme (25-200 µg) was applied to two Bio-Silect SEC 250-5 columns (Bio-Rad; 7.8 × 300 mm each) connected in series and equilibrated with 0.1 M KH₂PO₄, 0.15 M NaCl, pH 7.0. Elution was at 1 ml/min. The column was calibrated with molecular weight standards from Bio-Rad (thyroglobulin, 670000; IgG, 158000; ovalbumin, 44000; and myoglobin, 17500) and Sigma (albumin, 66000; carbonic anhydrase, 29000; cytochrome c, 12400; and aprotinin, 6500), both run separately from each other and the CMP-NeuAc synthetase. The CMP-NeuAc synthetase was exchanged into the column buffer with a 15-ml Swift desalting column, desalted with water using a Microcon-10 microconcentrator (Amicon), or resuspended from an ammonium sulfate pellet with the column buffer prior to chromatography.

The CMP-NeuAc synthetase was desalted by gradient elution from a Vydac C4 reverse phase HPLC column or by repeated concentration and dilution with water using a Microcon-10 microconcentrator. A 5 µl aliquot (5 µg) of this solution was injected into a stream of H₂O/CH₃CN (1:1) (with or without 1% acetic acid) at a flow rate of 20 µl/min, which was coupled to the electrospray ionization source of a Fison Platform quadrupole mass spectrometer (Fison, Manchester UK). The mass spectrometer was run in the positive-ion mode and scanned from m/z 600-2000 in 8.1-s intervals. Horse heart myoglobin (average M_r = 16951.5) was used as a reference compound and external calibrant, and a typical mass accuracy of ± 0.01% was obtained.

RESULTS AND DISCUSSION

The CMP-NeuAc synthetase from the H. ducreyi cell lysate was purified to homogeneity using a procedure consisting of anion exchange, dye, size exclusion, hydrophobic interaction, and reverse phase chromatography (Fig. 2). The final step using reverse phase chromatography resolved three major peaks, which were sequenced by Edman degradation. The CMP-NeuAc synthetase was identified by homology to other known CMP-NeuAc synthetases.

The relatively crude preparation of enzyme eluted from the first anion exchange column was stable to storage as a 65% ammonium sulfate suspension or 20% glycerol solution at 4 °C or as a 50% glycerol solution at 20 and 80 °C for at least several months. This material was used for initial characterization of the enzyme's properties. The enzyme has a rather broad pH optimum from 8 to 9.5, which is similar to other CMP-NeuAc synthetases (30, 31). The enzyme has a requirement for Mg²⁺, with 20 mM giving the most activity under the assay conditions used. Mn²⁺ is only 40% as effective at 20 mM, whereas Ca²⁺ gave no reaction. The divalent cation requirements of other bacterial CMP-NeuAc synthetases are similar, whereas enzymes from animal sources are capable of using Ca²⁺ and other divalent cations in addition to Mg²⁺ and Mn²⁺ (30, 32). Sulfhydryl reagents are necessary for or greatly stimulate the enzyme activity of some other CMP-NeuAc synthetases (30, 31). However, dithiothreitol was found to have little effect on enzyme activity and so was not included in purification buffers or enzyme assays. Interestingly, N-glycolylneuraminic acid was also a substrate for the H. ducreyi CMP-NeuAc synthetase. N-Glycolylneuraminic acid can be used as a substrate by CMP-NeuAc synthetases from animal tissues but not by the enzymes from E. coli or N. meningitidis (31). The monosaccharide, KDO, was not a substrate.

In order to clone the gene for the H. ducreyi CMP-NeuAc synthetase, the amino-terminal sequence was reverse translated, and two degenerate oligonucleotides were constructed (Table I). Approximately 1200 clones from a DASHII library of strain 35000 DNA were screened with oligonucleotide 1, and seven hybridization-positive clones were identified. After plaque purification and rescreening, DNA was prepared from each clone and characterized by restriction analysis and by Southern hybridization. Two of the clones were identical, and the additional five clones had overlapping restriction maps. One clone was chosen for further characterization. H. ducreyi DNA fragments of 5.5 and 8 kb were identified in a NotI digest and cloned into NotI-digested pWSK30, and the mixture was transformed into E. coli DH5. Clones containing either the 5.5- or the 8-kb fragment were identified. Southern analysis using oligonucleotides 1 and 2 indicated that the H. ducreyi CMP-NeuAc synthetase gene was localized to the 8-kb fragment. A clone containing the 8-kb NotI fragment, designated pRSM1627, was saved. Oligonucleotide 1 was used as a sequencing primer and sequence was determined with Sequenase. Additional primers were constructed, and the complete DNA sequence of the H. ducreyi CMP-NeuAc synthetase gene (neuA) was determined in both directions. The position and direction of transcription of the H. ducreyi CMP-NeuAc synthetase gene in pRSM1627 was determined by PCR analysis (Fig. 3).

An open reading frame of 684 base pairs encoding a 228-residue protein was identified (Fig. 4). An inverted repeat characteristic of a rho-independent transcriptional terminator was identified 63 nucleotides downstream of the termination codon. Putative promoter sequences were not identified.

A T7 expression system was employed to generate large quantities of the H. ducreyi CMP-NeuAc synthetase. The gene was first amplified by PCR and recloned by TA cloning. The 5 PCR primer contained an NdeI site (Table I, oligonucleotide 3), and the 3 primer contained a BamHI site (Table I, oligonucleotide 4). The sequence of the clone in the TA vector was again determined to verify that there were no PCR errors. The CMP-NeuAc synthetase gene was initially cloned as a NdeI-BamHI fragment into pT7-7, which had been digested with the same enzymes. The construct was readily transformed into E. coli strains lacking a source of T7 polymerase; however, no transformants were obtained with competent BL21(DE3) cells, suggesting that the synthetase gene product was being expressed and was toxic to the cells. Transformants were obtained after cloning the gene as a NdeI-BamHI fragment into the more tightly regulated T7lac pET24 vector. Initial expression experiments at 37 °C and inducing with 1 mM isopropyl -D-thiogalactopyranoside showed the synthetase to be the most abundant protein in cell lysates and enzyme activity levels in a crude lysate to be several hundred times higher than from H. ducreyi lysates. However, a large portion of the enzyme (approximately 75%) was found in the insoluble cell pellet. Lower temperatures (20-30 °C) and isopropyl -D-thiogalactopyranoside concentration (0.1 mM) favored more soluble enzyme, although insoluble CMP-NeuAc synthetase was still found in the lysate pellet under the best conditions identified.

The enzyme was the major component of the soluble cell lysate (Fig. 5). This greatly simplified the purification and allowed a homogeneous preparation of enzyme to be obtained in only three steps. For this, successive anion exchange, Green 19 dye, and hydrophobic interaction chromatography steps were used, and 20 mg of pure protein per liter of culture was obtained. In the final purification step a single, large peak was eluted from an HIC column using a shallow gradient (23.5 column volumes over 40 min) (Fig. 6). A summary of the purification of the recombinant enzyme is given in Table II. The enzyme ran as a single band by SDS-PAGE with a molecular mass of 25.6 kDa. The enzyme also appeared homogeneous by reverse phase chromatography. The calculated average mass of 25440.6 Da was confirmed by electrospray mass spectrometry with an accuracy to within 1.4 Da (Fig. 7). By size exclusion chromatography the enzyme's molecular mass was calculated as 47-74 kDa in 0.1 M KH₂PO₄, 0.15 M NaCl, pH 7.0 buffer and thus appears to exist as a dimer or possibly larger species. The concentration of enzyme affected the retention time, with higher concentrations shifting the peak to earlier retention times (higher molecular mass). Whether the enzyme was resuspended from an ammonium sulfate suspension or desalted with the column buffer or water, most of the enzyme eluted as a higher molecular weight species with little evidence for the presence of a monomeric form. However, if the enzyme was left at 4 °C overnight in water or column buffer, both dimer and monomer (26-28 kDa) were detected by size exclusion chromatography. Incubation overnight at 4 °C in 0.2 M NaCl (no phosphate buffer) resulted in very little of the monomeric species. The retention time of the monomer showed little dependence on enzyme concentration. The rat liver enzyme was identified as a dimer by size exclusion chromatography (32). The E. coli enzyme is thought to be active as a monomer but did form dimers and larger aggregates in some buffers (47). The N. meningitidis enzyme also possibly exists as a dimer based on size exclusion chromatography results (29). The apparent K_m for CTP and NeuAc were found to be 0.035 mM and 0.26 mM (mean of three experiments), respectively, at pH 7.1. The K_m for NeuAc is similar to that measured for the N. meningitidis enzyme, but the K_m for CTP is much lower than values obtained for previously described activities (see Ref. 30 for review). A possible reason for such a large difference is that most other CMP-NeuAc synthetases have been assayed at a higher pH where activity is greater, whereas the H. ducreyi enzyme was assayed at a more physiological pH.

Table II. Purification of the recombinant H. ducreyi CMP-NeuAc synthetase expressed in E. coli Chromatography step Protein Units Specific activity Purification Recovery mg µmol/min µmol/min/mg fold % activity Lysate 718 3400 4.74 1 100 DEAE-cellulose 372 2222 5.97 1.26 65 Green 19 110 1382 12.56 2.65 41 HIC 61 772 12.66 2.67 23

There are now five sequences in the sequence data bases identified as CMP-NeuAc synthetases. A sixth sequence from Campylobacter coli is not identified as such but is highly similar (ptmB, U25992; identified as being involved in the posttranslational modification of flagellin). All six sequences are from bacteria. Twenty residues from the NH₂ terminus of the rat liver enzyme are known, but the sequence is not similar to any of the bacterial sequences. The H. ducreyi CMP-NeuAc synthetase is most similar to the H. influenzae and N. meningitidis sequences being 65 and 45% identical (79 and 62% similar), respectively (Table III). These three enzymes all have nearly the same molecular weight, whereas the E. coli and Streptococcus agalactiae enzymes are nearly twice as large. The H. ducreyi CMP-NeuAc synthetase is 32% identical with the E. coli and S. agalactiae enzymes. Taken as a whole, the CMP-NeuAc synthetases have 32-65% identity and 50-79% similarity with each other. The CMP-NeuAc synthetases also have homology to the four known CMP-KDO synthetase sequences (11-18% identity, 32-39% similarity). Although the specificity of these enzymes for their sugar substrate is high, they catalyze a very similar reaction in which CMP is transferred from CTP to either NeuAc or KDO; both of which are -keto acid sugars (48, 49, 50). These proteins do not contain the HiGH motif that is involved in nucleotide binding in the recently elucidated cytidylyltransferase superfamily (51). The highest homology is found in the NH₂-terminal region. The two previously noted regions of highest similarity between the E. coli NeuA and KdsB proteins largely holds true for the larger set of sequences as well (residues 6-10 and 46-51 in H. ducreyi NeuA) (47). There are only a few residues conserved throughout in these two enzyme classes, and these are likely to be involved in catalysis and binding of the common substrate MgCTP (Fig. 8). A conserved arginine and lysine are found nine residues apart in the NH₂ terminus along with a third arginine or lysine in between. Chemical modification and site-directed mutagenesis of the E. coli CMP-NeuAc synthetase suggests that arginine and lysine are important for enzyme activity (52, 53). In addition, there is almost a complete lack of negatively charged amino acids in this NH₂-terminal region. There is also a third region of high similarity with a conserved glutamine, proceeded by several hydrophobic residues, and a conserved proline four residues apart (residues 98-107 in H. ducreyi NeuA). The residues in between are conserved within each class but differ between the two classes ((P/V)TS and GDE for CMP-NeuAc and CMP-KDO synthetases, respectively).

Table III. Similarity matrix for CMP-NeuAc and CMP-KDO synthetase proteins Protein Accession Number CMP-NeuAc synthetases CMP-KDO synthetases 1 2 3 4 5 6 7 8 9 1 HDa NeuA U54496 65b 45 32 32 13 14 14 16 (79) (62) (51) (50) (37) (35) (34) (35) 2 HI SiaB L45913 46 33 34 13 11 13 15 (63) (53) (50) (35) (33) (34) (33) 3 NM SynB X78068 34 35 17 18 14 17 (54) (53) (37) (36) (33) (36) 4 EC NeuA P13266 35c 16 15 15 15 (51) (39) (39) (37) (37) 5 SA CpsF U19899 18 17 15 14 (38) (36) (38) (32) 6 EC KdsB A26322 65 46 36 (79) (62) (55) 7 HI KdsB L44702 45 37 (62) (56) 8 EC KpsU P42216 38 (58) 9 CT KdsB U15192 a The abbreviations used are the same as in Fig. 8. b The percentage of identical residues is given for each pair of proteins from the sequence alignment shown in Fig. 8. The percentage of identical plus similar residues is given in parentheses. As a class, the CMP-NeuAc synthetases are 32-65% identical and share 50-79% similarity. Likewise, the CMP-KDO synthetases are 37-65% identical and share 55-79% similarity. The two classes are 11-18% identical and 32-39% similar. c The values for the E. coli K1 NeuA and the S. agalactiae CpsF sequence comparison are for residues 1-261. When the entire proteins were compared, the values were lowered to 27% identity and 44% similarity due to less homology in their carboxyl-terminal region.

The NH₂-terminal regions of CMP-NeuAc and CMP-KDO synthetases also share some homology with other nucleotidyltransferases that catalyze the general reaction: where NTP is a nucleotide triphosphate. The sequence alignment of several representative sugar nucleotidyltransferases is shown in Fig. 9. The conserved domains are aligned with a consensus sequence of ADP-glucose synthetases that is believed to be involved in phosphate binding (54). This domain, along with a second, were shown to be highly conserved in glucose-1-phosphate nucleotidyltransferases (55). The second domain is not found in CMP-NeuAc, CMP-KDO, or UDP-N-acetylglucosamine synthetases. Based on the conserved positive charges and high sequence homology in the NH₂ terminus, chemical modifications and site-directed mutagenesis experiments with the E. coli enzyme, and homology between CMP-NeuAc synthetases and other nucleotidyltransferases, it is quite possible that the NH₂-terminal region is involved in binding CTP. There are several other conserved residues and domains within the CMP-NeuAc and CMP-KDO synthetases that are not shared between the classes and may be involved in sugar substrate specificity.

Regarding the role of CMP-NeuAc synthetase in H. ducreyi, it has been recently reported that many strains contain terminal lactosamine and sialylated lactosamine as part of their LOS (12, 13). These epitopes are also a component of human blood group antigens and have important roles in molecular and cellular recognition (16, 18). Understanding the function of these sugars in the interaction of H. ducreyi with host cells should lead to a better understanding of the pathogenesis of this organism. Recent studies have shown that H. ducreyi can adhere to and invade various human cells (56, 57, 58, 59), and LOS appears to play an important role in this process. Sialic acid may offer the bacteria more protection from the host immune defenses, but exposed terminal galactose may be necessary for the initial infection as has been suggested for N. gonorrhoeae LOS (60). An isogenic mutant of H. ducreyi incapable of producing sialylated LOS will enable a better understanding of the role of sialic acid and the terminal galactose in adhesion to and invasion of host cells and susceptibility of the organism to host defenses. Such a mutant is currently under construction.

The importance of sialylated molecules in general and as virulence factors in pathogenic bacteria in particular make understanding the biosynthesis of sialic acid containing molecules an area of great interest. Further study will hopefully better define the role of sialic acid and LOS in the pathogenesis of H. ducreyi. With large quantities of recombinant CMP-NeuAc synthetase now available, more detailed characterization of this enzyme can be accomplished, including screening conditions for crystallographic analysis. Along with the information gained from conserved residues and domains in the nine sequences of CMP-NeuAc and CMP-KDO synthetases now available, site-directed mutagenesis and active site labeling with reactive substrate analogs will hopefully lead to a better understanding of the residues necessary for substrate binding and catalysis in CMP-NeuAc synthetases.