Biochemical Characterization of UDP-Gal:GlcNAc ... · environment (9, 10, 15). In the complex and dynamic microbial ecosystem of the human intestine, the communication between microorganisms

JOURNAL OF BACTERIOLOGY, Jan. 2011, p. 449–459 Vol. 193, No. 20021-9193/11/$12.00 doi:10.1128/JB.00737-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of UDP-Gal:GlcNAc-Pyrophosphate-Lipid�-1,4-Galactosyltransferase WfeD, a New Enzyme from

Shigella boydii Type 14 That Catalyzes the Second Stepin O-Antigen Repeating-Unit Synthesis�†

Changchang Xu,1,2§ Bin Liu,3§ Bo Hu,3 Yanfang Han,3 Lu Feng,3,4 John S. Allingham,2Walter A. Szarek,5 Lei Wang,3,4* and Inka Brockhausen1,2*

Department of Medicine1 and Department of Biochemistry,2 Queen’s University, Kingston, Ontario, Canada; TEDA School ofBiological Sciences and Biotechnology, Nankai University, Hongda Street, TEDA, Tianjin 300457, People’s Republic of

China3; Tianjin Key Laboratory of Microbial Functional Genomics and Key Laboratory of Molecular Microbiology andTechnology, Ministry of Education, Tianjin, People’s Republic of China4; and Department of Chemistry,

Queen’s University, Kingston, Ontario, Canada5

Received 24 June 2010/Accepted 26 October 2010

The O antigen is the outer part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negativebacteria and contains many repeats of an oligosaccharide unit. It contributes to antigenic variability and isessential to the full function and virulence of bacteria. Shigella is a Gram-negative human pathogen that causesdiarrhea in humans. The O antigen of Shigella boydii type 14 consists of repeating oligosaccharide units withthe structure [36-D-Galp�134-D-GlcpA�136-D-Galp�134-D-Galp�134-D-GlcpNAc�13]n. The wfeD genein the O-antigen gene cluster of Shigella boydii type 14 was proposed to encode a galactosyltransferase (GalT)involved in O-antigen synthesis. We confirmed here that the wfeD gene product is a �4-GalT that synthesizesthe Gal�1-4GlcNAc�-R linkage. WfeD was expressed in Escherichia coli, and the activity was characterized byusing UDP-[3H]Gal as the donor substrate as well as the synthetic acceptor substrate GlcNAc�-pyrophos-phate-(CH2)11-O-phenyl. The enzyme product was analyzed by liquid chromatography-mass spectrometry(LC-MS), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and galac-tosidase digestion. The enzyme was shown to be specific for the UDP-Gal donor substrate and requiredpyrophosphate in the acceptor substrate. Divalent metal ions such as Mn2�, Ni2�, and, surprisingly, also Pb2�

enhanced the enzyme activity. Mutational analysis showed that the Glu101 residue within a DxD motif isessential for activity, possibly by forming the catalytic nucleophile. The Lys211 residue was also shown to berequired for activity and may be involved in the binding of the negatively charged acceptor substrate. Our studyrevealed that the �4-GalT WfeD is a novel enzyme that has virtually no sequence similarity to mammalian�4-GalT, although it catalyzes a similar reaction.

Lipopolysaccharides (LPSs) consist of O-polysaccharide (O-antigenic) side chains covalently linked to a core polysaccha-ride and lipid A. LPSs are found in the outer membranes ofGram-negative bacteria, where they contribute to the struc-tural integrity of the membrane and interact with the externalenvironment (9, 10, 15). In the complex and dynamic microbialecosystem of the human intestine, the communication betweenmicroorganisms and the gastrointestinal (GI) epithelium in-volves O-antigen and LPS binding molecules. Thus, the elim-ination of the O antigen may reduce virulence (2, 16, 21).Shigella is a genus of highly adapted bacterial pathogens thatcause gastrointestinal disease, such as bacillary dysentery orshigellosis. A recent survey showed that shigellosis causes ap-

proximately 165 million cases of severe dysentery and morethan 1 million deaths per year, mostly in children from devel-oping countries (10). Shigella strains are categorized into fourgroups: S. boydii, S. dysenteriae, S. flexneri, and S. sonnei, eachcontaining multiple subgroups of different serotypes, based onstructural variations in their O antigens.

O antigens consist of repeating units of oligosaccharides thatare assembled individually, followed by the polymerization ofunits to form O antigens of different lengths. The glycosyltrans-ferases involved in the biosynthesis of O antigens play a criticalrole in determining O-antigen structural diversity. The pen-tasaccharide repeating unit of S. boydii type 14 (B14) hasthe structure [36-D-Galp�134-D-GlcpA�136-D-Galp�134-D-Galp�134-D-GlcpNAc�13]n (12), suggesting the existenceof five specific glycosyltransferases: a GlcNAc-phosphotrans-ferase (WecA), three Gal-transferases, and a glucuronosyl-transferase.

Three distinct processes for the synthesis and translocationof O antigens have been described: the Wzx/Wzy-dependentpathway, the ATP binding cassette transporter-dependent pro-cess, and the synthase-dependent process (20, 25, 26). Thebiosynthesis of the S. boydii B14 O antigen that contains avariety of different sugar residues is expected to utilize the

* Corresponding author. Mailing address for Lei Wang: 23 HongdaStreet, TEDA, Tianjin 300457, People’s Republic of China. Phone:86-22-66229588. Fax: 86-22-66229596. E-mail: [email protected] address for Inka Brockhausen: Queen’s University, 94 StuartSt., Kingston, ON K7L 3N6, Canada. Phone: (613) 533-2927. Fax:(613) 549-2529. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

§ C.X. and B.L. contributed equally to the paper.� Published ahead of print on 5 November 2010.

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Wzy/Wzx-dependent pathway, where the synthesis of the re-peating unit is initiated by WecA, catalyzing the transfer ofsugar-phosphate (GlcNAc�-phosphate) from nucleotide sugar(UDP-GlcNAc) to a lipid carrier, undecaprenol-phosphate(Und-P), at the cytoplasmic side of the inner membrane. ThewecA gene is present in the S. boydii B14 genome but outsidethe O-antigen gene cluster (1). The wecA gene is also involvedin the synthesis of bacterial polysaccharides other than the Oantigen. The extension of the chain is then mediated by specificglycosyltransferases that utilize nucleotide sugar donor sub-strates and are thought to be loosely associated with the innermembrane. In contrast, mammalian glycosyltransferases areusually membrane-bound proteins. Bacterial and mammalianglycosyltransferases, although they may have similar substratespecificities and form the same linkage, show significantly dif-ferent amino acid sequences (4). Completed repeating unitsare then flipped across the membrane to the periplasmic side(by the flippase Wzx) and are polymerized (by Wzy) to formthe O antigen under the control of a chain length regulator(Wzz). The repeating units are initially linked to the lipidcarrier through GlcNAc�-phosphate. However, the S. boydiiB14 O antigen has GlcNAc in the � linkage; thus, upon thepolymerization of the completed repeating units, the linkagemay be inverted, probably through the specific action of thepolymerase Wzy. The entire polymer is then ligated to an outercore sugar based on lipid A. Upon completion, the LPS isextruded from the inner membrane and translocated to theouter membrane (19, 26). The latter-acting enzymes have mul-tiple transmembrane regions that integrate them into the bac-terial membranes.

Genes involved in O-antigen biosynthesis are normally clus-tered between galF and gnd in Escherichia coli and Shigella andare classified into three different groups: (i) nucleotide sugarsynthesis genes involved in the synthesis of donor substrates,(ii) glycosyltransferase genes, and (iii) O-antigen-processinggenes, such as the flippase gene wzx and the polymerase genewzy. The O-antigen gene cluster of B14 has been sequencedand analyzed (10). Four putative glycosyltransferase genesfound in the B14 O-antigen synthesis gene cluster are wfeA,wfeB, wfeD, and wfeE. WfeD shares 38% identity and 57%similarity to the putative glycosyltransferase Orf9, which isinvolved in the synthesis of the E. coli O136 O antigen (ourunpublished data). Since the O antigens of B14 and E. coliO136 share only one common linkage, D-Galp�134-D-GlcpNAc (12, 23), wfeD was proposed to encode the galacto-syltransferase (GalT) that transfers Gal to GlcNAc�-PP-Undin the �1-4 linkage, which is the second step in the biosyntheticpathway of the B14 O-antigen repeating unit.

We have used biochemical approaches to assay the WfeD en-zyme activity and to characterize this enzyme. The lipid carrieranalog GlcNAc�-PO3-PO3-(CH2)11-O-phenyl [GlcNAc-PP-(CH2)11-OPh] has previously been used as a defined syntheticacceptor substrate for the characterization of glycosyltrans-ferases from E. coli serotypes O7 (�1,3-GalT WbbD), O56(�1,3-Glc-transferase WfaP), and O152 (�1,3-Glc-transferaseWfgD) (6, 17). In this work, we showed that GlcNAc-PP-(CH2)11-OPh could also serve as an exogenous substrate forWfeD from B14. We were therefore able to prove that wfeDencodes a novel �1,4-GalT.

MATERIALS AND METHODS

Materials. All reagents were purchased from Sigma unless indicated other-wise. Radioactive nucleotide sugars were purchased from Perkin-Elmer. Accep-tor substrates were synthesized as reported previously (5, 7, 13, 18). GlcNAc�-PO3-PO3-(CH2)9-CH3 was kindly provided by O. Hindsgaul, CarlsbergLaboratories, Copenhagen, Denmark.

Bacterial strains, plasmids, and expression of the wfeD gene. The putativeglycosyltransferase gene wfeD from B14 was amplified by PCR and cloned intoexpression vector pET28a (containing a cleavable His-tag-encoded sequence) toconstruct plasmid pLW1298. pLW1298 was then transformed into E. coli BL21cells. Bacteria were grown in 6 ml LB broth containing 50 �g/ml kanamycinovernight at 37°C with constant shaking (17). To induce the plasmid-derivedenzyme, the bacterial suspension was transferred into 125 ml LB broth contain-ing kanamycin and incubated for 90 min at 37°C with constant shaking. When thesuspension reached an absorbance at 600 nm of 0.8, IPTG (isopropyl-beta-thiogalactoside) was added to a final concentration of 1 mM to induce proteinexpression. Cells were grown for an additional 4 h at 30°C and were thenharvested by centrifugation for 10 min at 3,145 � g (4,000 rpm) (IEC 21000R).Pellets were washed with 5 ml phosphate-buffered saline (PBS) and resuspendedin 10 ml PBS containing 10% glycerol. Aliquots of bacteria were stored at �20°Cfor enzyme assays.

Construction of mutants. Four primers (F1, R1, FM, and RM) and threePCRs were used to construct each site-specific mutant by overlap extension. Thefirst PCR with primers F1 and RM was used to amplify DNA that contains themutation site together with upstream sequences. The second PCR with primersFM and R1 was used to amplify DNA that contains the mutation site togetherwith downstream sequences. The mutation of interest was located in the overlapregion of two amplified fragments. The overlapping fragments were mixed,denatured, and annealed to generate heteroduplexes that can be extended andthen amplified in the third PCR with primers F1 and R1. The primers used forthe construction of mutants are listed in Table 1. For each PCR, a total of 25cycles was performed under the following conditions: denaturation at 95°C for30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min. Subsequentsequencing confirmed the correct sites of mutation.

Galactosyltransferase assays. Bacterial homogenates containing WfeD wereprepared by sonication in 0.05 M sucrose buffer or other buffers as describedpreviously (13, 17). The standard assay mixtures for WfeD contained (in a totalvolume of 40 �l) acceptor substrate as indicated in Table 2, bacterial homoge-nates (0.5 to 0.6 �g protein), 0.075 M MES (2-morpholinoethanesulfonic acid)buffer (pH 7), 5 mM MnCl2, and 1 mM UDP-[3H]Gal (2,000 to 3,500 cpm/nmol).In standard GalT assays, 0.25 mM GlcNAc-PP-(CH2)11-OPh (13) was used asthe acceptor substrate, while the control assays lacked an acceptor substrate. Dueto the low solubility of GlcNAc-PP-(CH2)11-OPh in water, 10% methanol(MeOH) was present in all assay mixtures. At least two determinations werecarried out, with variation between assays of �10%. No significant inhibition ofGalT activity was noted at up to 20% MeOH or up to 7% isopropanol in the

TABLE 1. Primers used for mutagenesis

Primer Sequencea Mutationsite

F1 5�-CGCGGATCCGTGATCGATAATCTCATAAA-3�R1 5�-CCGCTCGAGTCAATTACTTCTCGTGAATATTT-3�

FM1 5�-TTAATCGGTTCTAAGGCTTATGA-3� D99ARM1 5�-TCATAAGCCTTAGAACCGATTAA-3�

FM2 5�-TCGGTTCTAAGGATTATGCTAT-3� E101ARM2 5�-ATAGCATAATCCTTAGAACCGA-3�

FM3 5�-AGGATTATGAAATAGCGCAAGC-3� E103ARM3 5�-GCTTGCGCTATTTCATAATCCT-3�

FM4 5�-ATATAAACATGTTGCGAAAAG-3� R210ARM4 5�-CTTTTCGCAACATGTTTATAT-3�

FM5 5�-TAGGAAAGCTTTTTTTATCGAT-3� R212ARM5 5�-ATCGATAAAAAAAGCTTTCCTA-3�

FM6 5�-CATGTTAGGGCTAGATTTT-3� K211ARM6 5�-AAAATCTAGCCCTAACATG-3�

a The underlined sequences indicate restriction sites. The nucleic acids beingmutated for generating the single-amino-acid mutations are indicated by bold-face type.

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assay mixtures. Mixtures were incubated for 10 min at 37°C. Reactions werequenched by the addition of 200 �l ice-cold water to the mixtures. For productisolation, 500 �l water was added, the mixture was passed through a short C18

Sep-Pak column, and the column was washed with 4 ml water. Radioactiveproduct was then eluted with 4 ml MeOH. Scintillation fluid (4.5 ml) (ReadySafe; Beckman Coulter) was added to aliquots of each 1-ml fraction, and radio-activity was determined by scintillation counting (LS6500 scintillation counter;Beckman Coulter). The radioactive product was concentrated in the first 3 ml ofMeOH eluates for high-performance liquid chromatography (HPLC) analysis.Residues were taken up in MeOH. The assays for bovine �4-GalT1 were de-scribed previously (5). Kinetic parameters were determined by using theENZFIT program.

Product identification using mass spectrometry and HPLC. The enzyme prod-uct was prepared as a radioactive product and also as a nonradioactive productfor mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis.The separation of substrates and enzyme products was achieved by HPLC usinga C18 column and acetonitrile-water mixtures at a flow rate of 1 ml/min. Forexample, the substrate GlcNAc-PP-(CH2)11-OPh and product Gal-GlcNAc-PP-(CH2)11-OPh were separated with 24% acetonitrile in water as the mobile phase.Compounds were analyzed by electrospray ionization (ESI)-MS in the negative-ion mode, as described previously (17).

Product linkage confirmation using galactosidases and NMR. The anomericconfiguration of the linkage formed in the radioactive product was determined bythe incubation of aliquots with specific galactosidases: green coffee bean �-ga-lactosidase (0.065 U/�l), jack bean (�4-specific) �-galactosidase (0.06 U/�l), E.coli (�1-4-specific) �-galactosidase, and bovine testicular (�1-3-, �1-4-, and �1-6-specific) �-galactosidase (0.095 U/�l). Aliquots of radioactive enzyme product(800 cpm) were treated in a total volume of 100 �l with 25 �l MacIlvaine buffer(0.1 M citric acid–0.2 M Na-phosphate) (pH 4.3), 10 �l of 0.1% bovine serumalbumin, and galactosidase. Mixtures were incubated for 30 min at 37°C, dilutedwith 800 �l of water, and applied onto 0.4-ml AG1x8 columns. The releasedradioactive enzyme ([3H]Gal) was eluted with 2.8 ml water, while the unreactedenzyme product stayed in the AG1x8 column.

To prepare large amounts of enzyme product for NMR, GalT assays werecarried out as follows. The incubation mixtures (a total of 8 ml) contained 10 mlbacterial homogenate in 50 mM sucrose (E. coli BL21 cells complemented withplasmid pLW1298), 2.5 �mol GlcNAc-PP-(CH2)11-OPh, 10 �mol UDP-Gal, 1mmol MES buffer (pH 7), and 50 �mol MnCl2. After incubation for 30 min at37°C, 8 ml of cold water was added, and the mixtures were applied onto C18

Sep-Pak columns. Each column was washed with 4 ml water, and the product waseluted with 4 ml MeOH. The MeOH fractions were pooled, flash-evaporated,

and redissolved in 500 �l MeOH. Aliquots of the MeOH-dissolved product werepurified by HPLC, using a C18 column and acetonitrile-water (24:76) as themobile phase. The enzyme product was dried, exchanged three times with99.96% D2O, and analyzed by 600-MHz proton and 13C NMR spectroscopy inCD3OD, as described previously (6).

Enzyme purification and analysis of enzyme protein. For the purification ofthe fusion protein His6-WfeD, bacteria (0.2 mg bacteria per ml buffer) weresonicated in sonication buffer (50 mM Tris base [pH 8.0], 500 mM NaCl, 10 mMimidazole, and 1 mM EGTA) for six cycles of 1-min sonication and 1-min waitingtime (Misonix Sonicator 3000, program 1). The homogenate was then centri-fuged at 3.9 � 104 � g for 30 min. The His-tagged fusion protein in thesupernatant was purified by affinity chromatography with a Ni2�-nitrilotriaceticacid (NTA) Sepharose Fast Flow column (Bio-Rad). Bound proteins were elutedwith a gradient of 200 to 400 mM imidazole buffer containing 50 mM Tris base(pH 8.0), 500 mM NaCl, and 1 mM EGTA. The fusion protein was analyzed bySDS-PAGE, Western blots, and GalT activity assays. Gel slices were analyzed bymatrix-assisted laser desorption ionization (MALDI) peptide mass fingerprintingat the Protein Function Discovery Facility at Queen’s University, using MALDI-time of flight (TOF) mass spectrometry.

Western blot analysis was performed in duplicates with rabbit antibody againstthe His tag as the primary antibody (kindly provided by P. Davies, Queen’sUniversity) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG as thesecondary antibody (from Promega). Ni2�-NTA-purified and diluted cell lysatesof the wild type and mutants (3 to 5 �g protein in 20 �l) were diluted 5:1 in 4 �lSDS loading buffer consisting of 1 mM Tris-HCl (pH 6.8), 1% SDS, 10%glycerol, and 1% �-mercaptoethanol and run on SDS-PAGE gels (8% gel).Proteins were electrophoretically transferred onto nitrocellulose and thenprobed sequentially with anti-His antibody (1:5,000) and anti-rabbit IgG (1:10,000). Labeling was visualized with 10 ml of a 1:1 mixture of luminol reagentand peroxide solution (Millipore). Prestained protein standards (Fermentas)were used to calibrate the gels. The densitometric analysis of the relative bandintensity of the Western blot film was performed by using ImageJ1.43 software(9), which measured the optical density of the protein bands and indicated theapproximate relative amounts of His-tagged protein in wild-type and mutantWfeD. The enzyme activities were normalized according to relative amounts ofprotein detected on Western blots.

Effect of amino-acid-specific reagents. To evaluate the role of amino acids inactivity, the crude and purified enzyme preparations were incubated with amino-acid-specific reagents prior to the assays for 10 min at room temperature inreaction mixtures lacking UDP-Gal. The reaction was initiated by the addition ofUDP-Gal to the mixtures. Diethyl pyrocarbonate (DEPC) (reacts with His),iodoacetic acid (IAA) (reacts with Cys), p-hydroxyphenylglyoxal (HPG) (reactswith Arg), and 5,5�-dithio-bis-2-nitrobenzoic acid (DTNB) (reacts with Cys) wereused at a 0.2 mM concentration in the assay. The disulfide-bond-reducing agentdithiothreitol (DTT) was used at 0.2 to 3 mM in the assay mixture, and �-mer-captoethanol was used at 1 to 30 �M.

RESULTS

WfeD enzyme activity and kinetics. Under standard assayconditions, the reaction rate of WfeD in bacterial homoge-nates using 1 mM UDP-Gal and 0.25 mM GlcNAc-PP-(CH2)11-OPh acceptor in the presence of 5 mM Mn2� ions waslinear up to at least 20 min. The reaction rate was proportionalto the enzyme protein concentration up to 0.016 mg/ml (notshown). Untransformed control BL21 cells without pLW1298showed a low background (�5%) compared to transformedBL21 cells, suggesting a high specific activity in the crudeenzyme preparation (about 8 �mol/h/mg). The crude enzymepreparation retained at least 60% of the initial activity afterstorage at 4°C for up to 9 weeks. The decay of the activity afterstorage at �20°C was similar to that at 4°C. However, after 5months of storage at �20°C, the activities were decreased to�5% of the original. The kinetic parameters for the crudeenzyme were an apparent Km for UDP-Gal of 0.1 mM and anapparent Vmax of 11 �mol/h/mg. For the acceptor substrateGlcNAc-PP-PhU, the apparent Km was 0.4 mM and the ap-parent Vmax was 12 �mol/h/mg (data not shown).

TABLE 2. Activities of purified WfeD with GlcNAc derivativestested as substrates and inhibitorsa

Compound Activity (%)

GlcNAc �-PO3-PO3-(CH2)11-OPh ................................................100GlcNAc �-PO3-PO3-(CH2)6-OPh.................................................. 39GlcNAc �-PO3-PO3-(CH2)16-OPh ................................................ 92GlcNAc �-PO3-(CH2)11-OPh......................................................... 11GlcNAc �-PO3-PO3-(CH2)9-CH3 .................................................. 74GlcNAc �-(CH2)11-OPh .................................................................�5GlcNAc �-(CH2)11-OPh .................................................................�5GlcNAc �-CO-CH2-P-(CH2)11-OPh .............................................�5GlcNAc �-(3-isoquinolinyl) ............................................................�1GlcNBu �-(2-naphthyl)...................................................................�1GlcNAc �-(2-naphthyl) ...................................................................�1GlcNBu �-S-(2-naphthyl) ...............................................................�1GlcNAc �-Bn ...................................................................................�1GlcNAc �-Bn ...................................................................................�1GlcNAc .............................................................................................�1GlcNAc �-S-Ph ................................................................................�14-Deoxy-GlcNAc �-Bn....................................................................�14-F-4-Deoxy-GlcNAc �-Bn.............................................................�14-Deoxy-GlcNAc..............................................................................�16-Deoxy-GlcNAc..............................................................................�16-S-GlcNBu �-(2-naphthyl) ............................................................�1GlcNBu �-NH-(2-naphthyl) ...........................................................�1

a GalT assays were carried out as described in Materials and Methods, usingthe compounds as acceptor substrates at a 0.2 mM concentration in the assaymixture.

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Enzyme purification. WfeD was purified using Ni2�-NTAaffinity chromatography to a specific activity 16-fold higherthan that of the crude bacterial homogenate, considering thetotal amount of protein in both preparations. Based on theamino acid composition, the molecular mass of WfeD wascalculated to be 30 kDa (protein molecular mass calculator;Science Gateway). SDS-PAGE showed a band at 30 kDa thatwas induced by IPTG (Fig. 1A). Western blotting, using anti-body to the His tag, also showed a major protein band at 30kDa for both the solubilized crude enzyme and the purifiedenzyme (Fig. 1B). MALDI peptide mass fingerprinting of gelslices identified the 30-kDa band as WfeD (data not shown).For the purified enzyme, the apparent Km for UDP-Gal was0.25 mM, with a Vmax of 40 �mol/h/mg, measured with 1 mM

GlcNAc-PP-PhU (data not shown). The Km for the acceptorsubstrate GlcNAc-PP-(CH2)11-OPh was 0.10 mM, with a Vmax

of 42 �mol/h/mg, measured with 1 mM UDP-Gal (data notshown). The kinetic studies were performed by two indepen-dent repeats, with differences of �10%.

Confirmation of Gal transfer and newly synthesized linkageas Gal�1-4GlcNAc. To confirm the synthesis of enzyme prod-uct, assay mixtures were applied onto C18 Sep-Pak columns,and the enzyme product and substrate were eluted withMeOH. These fractions were then subjected to HPLC analysis.Using a C18 column and an acetonitrile/water ratio of 24:76 asthe mobile phase, the product eluted at 25 min and the sub-strate eluted at 33 min, allowing a good separation of the twocompounds. The fractions eluted with MeOH from the Sep-

FIG. 1. Expression and purification of WfeD and six mutants. (A) SDS-PAGE analysis of proteins. Wild-type WfeD and six mutants, elutedfrom the Ni2�-NTA Sepharose Fast Flow column, as well as wild-type bacterial lysates (before IPTG induction) were run on reducing SDS-PAGEgels. Molecular mass (M) markers are indicated on the right. The band of the wild-type enzyme at 30 kDa was analyzed by peptide massfingerprinting and found to be due to WfeD. (B) Western blot analysis of purified wild-type WfeD and six mutants (D99A, E101A, E103A, R210A,R212A, and K211A). An anti-His tag antibody was used to detect the WfeD protein. All of the mutants, as well as the wild-type enzyme purifiedon a Ni2�-NTA Sepharose Fast Flow column, showed a protein band of 30 kDa. Bovine �4-GalT (33 kDa) without a His tag was used as thenegative control.



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Pak column were also analyzed by electrospray-MS analysis(Fig. 2) and showed peaks at m/z 626 for the substrate GlcNAc-PP-(CH2)11-OPh and m/z 788 for the product Gal-GlcNAc-PP-(CH2)11-OPh. This showed that only one Gal residue (m/z 162)had been added to the substrate. Since the next sugar in therepeating unit is also a �1-4-linked Gal, it was consideredpossible that WfeD is a bifunctional enzyme. However, therewas no peak at m/z 950 that could have been due to Gal-Gal-GlcNAc-PP-(CH2)11-OPh. Thus, under our reaction condi-tions, WfeD did not have a dual activity.

Galactosidases were used to determine the anomeric linkagebetween Gal and GlcNAc in the [3H]Gal-GlcNAc-PP-(CH2)11-OPh enzyme product synthesized by purified WfeD. The re-leased free [3H]Gal was separated by an AG1x8 column thatbinds the uncleaved product. We showed that the enzymeproduct was resistant to green coffee bean �-galactosidase,indicating that Gal was not � linked. In contrast, treatmentwith jack bean (�1,4-specific) �-galactosidase and bovine tes-ticular (�1-3-, �1-4-, and �1-6-specific) �-galactosidase re-leased most of the radioactivity (52% and 60%, respectively) ofthe radioactivity from the disaccharide product. Therefore, itcan be concluded that Gal was � linked in the enzyme productand likely in the 1-4 linkage.

The 1H-NMR spectrum of the product showed a new doubletat 4.345 ppm with a coupling constant of 7.6 Hz, which is indic-ative of H-1 of Gal in the � linkage (Table 3). The spectrum of theenzyme product of �3-GalT WbbD [Gal�1-3GlcNAc-PP-(CH2)11-OPh] also showed a doublet for Gal H-1 at 4.35 ppm(17). To determine whether Gal was �1-3, �1-4, or �1-6 linked toGlcNAc, we conducted two-dimensional NMR (2D-NMR) ex-periments, including correlation spectroscopy (COSY) (Fig. 3A)and heteronuclear single quantum coherence (HSQC) (Fig. 3B),

which identified most of the carbon and proton shifts of theGlcNAc residue. The chemical shifts of GlcNAc H-1, H-2, andH-3 were slightly different in the product compared to the sub-strate. However, there was a large downfield shift of GlcNAc H-4from 3.33 ppm in the substrate to 3.626 ppm in the product anda large shift from the C-4 signal of 71.9 in the substrate to 79.1 inthe product. This indicates that Gal was linked to the 4 position ofGlcNAc and the Gal�1-4GlcNAc linkage in the enzyme product.Therefore, these results show that WfeD is a UDP-Gal:GlcNAc-R�1,4-Gal-transferase.

Donor substrate specificity. The donor substrate specificitystudy of the enzyme in the crude cell lysate was carried out byusing the standard assay but replacing 0.5 mM UDP-Gal withseveral other nucleotide sugars, i.e., 0.25 mM UDP-Glc, 0.4mM UDP-GalNAc, 0.4 mM UDP-GlcNAc, 0.55 mM UDP-xylose, 0.4 mM GDP-Man, and 0.2 mM CMP-sialic acid. Thesenucleotide sugars yielded enzyme activities of less than 1% ofthe activity observed with UDP-Gal. This indicated a strictspecificity of WfeD for UDP-Gal as the donor substrate andthe absence in bacterial homogenates of other types of glyco-syltransferase activities acting on the substrate GlcNAc-PP-(CH2)11-OPh. It also showed that the 4-epimerase present inE. coli did not convert significant amounts of UDP-Glc toUDP-Gal within the 10-min incubation time.

Role of pyrophosphate in acceptor substrate specificity. Inorder to define further the structural requirements of WfeDfor the acceptor substrate, a number of neutral and negativelycharged phosphate-containing analogs of GlcNAc (5, 7, 8, 13,18) were tested as acceptor substrates (Table 2). The neutralcompounds included both �-and �-anomers of GlcNAc-benzyl;�-anomers of GlcNBu-benzyl, GlcNAc-naphthyl, GlcNAc-iso-quinolinyl (5), as well as GlcNAc�-OCOCH2-COO(CH2)11-OPh;

FIG. 2. Electrospray mass spectrometry of the WfeD product Gal-GlcNAc-PP-(CH2)11-OPh. The disaccharide lipid product was synthesizedfrom GlcNAc-PP-(CH2)11-OPh with a bacterial homogenate containing WfeD Gal-transferase, purified by Sep-Pak C18 columns, and analyzed byelectrospray mass spectrometry (negative-ion mode). Both m/z 626 (substrate) and m/z 788 (product) represent [M � H]. The absence of m/z 950indicates that only one Gal residue was transferred. amu, atomic mass unit (equal to daltons).



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and �- and �-anomers of GlcNAc-(CH2)11-OPh (18). Using theAG1x8 anion-exchange method to isolate the product, theseneutral substrates showed activities of less than 8% of theactivity with the standard substrate. The charged compoundsincluded GlcNAc�-PP-(CH2)6-OPh, GlcNAc�-PP-(CH2)16-OPh, and GlcNAc�-PP-(CH2)9-CH3, having a pyrophosphategroup but modifications in the lipid moiety. All of these com-pounds containing GlcNAc-PP showed high levels of activity(39 to 92% of the activity with the standard substrate). Thisindicates that the structure of the lipid moiety in the acceptoris of minor importance, although a hydrophobic group may berequired. In order to examine the role of the pyrophosphate inthe substrate, we tested GlcNAc�-P-(CH2)11-OPh, which hasonly one phosphate group, and found 11% activity comparedto the identical substrate having two phosphates (Table 2).WfeD thus has an absolute requirement for at least one phos-phate group and requires the pyrophosphate group in theacceptor for full activity.

Effect of detergents on enzyme activity. In order to deter-mine whether the enzyme activity is affected by the presence ofmicelle-forming detergents, Triton X-100, Tween 80, or NP-40(nonyl phenoxylpolyethoxylethanol) was added to the crudecell homogenate in the assay mixture at a 0.1% (vol/vol) con-centration. The presence of detergent yielded WfeD enzymeactivities between 80 and 90% of the activities measured in the

absence of the detergent (Table 4). As the concentration ofTriton X-100 increased from 0 to 0.5% in the reaction mixture,WfeD activity decreased but was maintained at about 60% ofthe original activity, up to 6% Triton X-100.

Divalent cation requirement for WfeD enzyme activity. Theinvolvement of divalent cations as cofactors in GalT catalysis issuggested by the presence of the DxD motif (3). Previousreports stated that the presence of a cofactor such as Mn2� isessential for the activity of bovine �4-GalT (5) and �3-GalTWbbD from E. coli (17), both of which have a GT-A fold anda DxD motif in the UDP-Gal binding site. WfeD has a DYEIEsequence resembling a DxD motif and is a member of theGT26 family of glycosyltransferases with a predicted WecG-TagA fold (www.CAZy.org).

To examine the cofactor requirement of WfeD, assays werecarried out in the presence of 5 mM Mn2� or, instead of Mn2�,with Mg2�, Cu2�, Ca2�, Pb2�, Ni2�, or EDTA or no additionin the negative control. The highest stimulation of activity wasobtained in the presence of Mn2� ions, but Ca2� and Mg2�

were also effective. Surprisingly, Pb2� ions, often inhibitory toenzymes, and Ni2� ions, which are not usually studied as gly-cosyltransferase cofactors (22), were both shown to inducehigh levels of enzyme activity in the bacterial homogenate aswell as in the purified enzyme (Fig. 4A and B). A further testshowed that the effect of Pb2�, Ni2�, and Mn2� on enzyme

TABLE 3. 1H- and 13C-NMR parameters established from COSY, NOESY, and HSQCa

Product H Shift (ppm) J coupling (Hz) C Shift (ppm)

GlcNAc�-PP-(CH2)11-OPhGlcNAc H-1 5.53 (5.64) C-1 96

H-2 3.97 C-2 55H-3 3.75 C-3 72.9H-4 3.33 (3.45) C-4 71.9

GlcNAc�-PP-decanol (7)GlcNAc H-1 5.45 3.2, 7.2 C-1 94.5

Gal�1-4GlcNAc-PP-(CH2)11-OPh (WfeD)GlcNAc H-1 5.561 C-1 94.7

H-2 4.027 C-2 53.1H-3 3.846 C-3 69.4H-4 3.626 C-4 79.1

Gal H-1 4.345 7.6 C-1 103.4H-2 3.538 6.9, 10.1 C-2 70.8H-3 3.465 2.9, 9.5 C-3 73.1H-4 3.792 2.5 C-4 68.8

Gal�1-3GlcNAc-PP-(CH2)11-OPh (17)GlcNAc H-1 5.58 C-1 94.8

H-2 4.14 C-2 52.4H-3 3.84 C-3 80.3

Gal H-1 4.35 C-1 104.0

Glc�1-3GlcNAc-PP-(CH2)11-OPh (6)GlcNAc H-1 5.54 C-1 95.0

H-2 4.14 C-2 54.0H-3 3.87 C-3 82.8H-4 3.42 C-4 70.3

Glc H-1 4.41 C-1 105.0H-2 3.18 C-2 74.5H-3 3.37 C-3 77.6H-4 3.33

a Shown is a comparison of NMR parameters between substrates and �3-GalT, �4-GalT, and �3-Glc-transferase enzyme products. NOESY, nuclear Overhauserenhancement spectroscopy.



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FIG. 3. NMR spectroscopy (600 MHz) of the WfeD enzyme product. (A) 1H-1H correlation COSY spectrum. The WfeD enzyme product wasexchanged in D2O and dissolved in CD3OD, and 1H and 13C spectra were acquired by using a 600-MHz Bruker spectrometer. The 1H-1H COSYspectrum is shown. (B) HSQC spectrum. Spectra were acquired by using a 600-MHz Bruker spectrometer. The chemical shifts of H-4 and C-4 ofGlcNAc exhibit a major change compared to the substrate, due to the attachment of Gal to GlcNAc in the �1-4 linkage. The 1H-13C correlationspectrum (HSQC) is shown.



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activity was concentration dependent (Fig. 4C). HPLC analysisconfirmed the identity of the enzyme product formed in thepresence of these metal ions. This finding may uncover a novelrole that Pb2� plays in the metabolism of bacteria. Mammalian�4-GalT was also tested in the presence of Pb2� salts at a 5mM concentration in the assay and showed a slight stimulationof activity, which was 11% of the activity found in the presenceof 5 mM Mn2�.

Effect of amino-acid-specific reagents on WfeD activity.There are many His, Arg, and Lys residues in WfeD that maypossibly be involved in the binding of the negatively chargedsubstrates. In order to examine the involvement of specificamino acids in catalysis, the standard assay was carried out inthe presence of amino-acid-specific reagents. The inclusion of0.2 mM DEPC in the assay mixture showed a 31% inhibition ofactivity, while 0.2 mM HPG showed a 50% inhibition (Fig. 5).This suggests a potential role of His and Arg in protein struc-ture or catalysis. The alkylating agent IAA (0.2 mM) showed a20% inhibition, indicating that one or both of the Cys residuesmay be involved in protein structure or catalysis. DTNB (0.2mM), which binds to reduced SH groups of Cys, inhibited theactivity by 85%. DTT (0.2 mM), which reduces disulfide bonds,stimulated the enzyme almost 5-fold at concentrations of 0.1 to3 mM. �-Mercaptoethanol also increased the activity 4-fold at1 to 30 mM concentrations in the assay. This suggested that atleast one reduced Cys residue may be required for full activity.

Mutagenesis to identify crucial amino acids. The WfeDenzyme has a DYEIE sequence resembling a DxD motif,found in virtually all putative glycosyltransferases (3). To eval-uate the role of the acidic amino acids in this motif in enzymeactivity, mutations of single acidic amino acids were made. Wetherefore mutated individually Asp99, Glu101, and Glu103 tothe neutral Ala. The D99A and E103A mutants were highlyactive, but the E101A mutant exhibited minimal enzyme activ-ity. This suggested that Glu101 in the DYE sequence has thecatalytic function of an acidic residue in the DxD sequenceobserved for other glycosyltransferases. Since the donor andacceptor substrates both have a negatively charged pyrophos-phate, we proposed that positively charged clusters of aminoacids may be important for substrate binding. We thereforemutated Arg210, Arg212, and Lys211 of the RKR sequence atthe N terminus of the enzyme to Ala. R210A and R212Amutants were active, while the K211A mutant lacked activity(Fig. 6). This indicated an important role of Lys211 in eithersubstrate binding, catalysis, or protein structure. The GalTactivity of the mutants was also tested with Pb2�, and productformation was confirmed by HPLC. The replacement of

Glu101 by Ala greatly reduced enzyme activity in the presenceof either Mn2� or Pb2�, although a 60-fold-higher activity wasseen for the Lys211Ala mutant in the presence of Pb2� than inthe presence of Mn2�. Western blots of Ni2�-NTA-purifiedbacterial extracts harboring the mutant enzymes showed thatall of the mutants were expressed at 30 kDa (Fig. 1B). Thesingle bands seen on Western blots indicated that none of thewild-type or mutant enzymes were degraded.

FIG. 4. Divalent cation requirement of WfeD. (A) Cofactor re-quirement of the crude WfeD enzyme. Freshly homogenized 1:10-diluted E. coli cells were used as the crude enzyme. The activities areshown as �mol/h/mg. EDTA (5 mM) and H2O were used as negativecontrols. Assays were carried out in duplicates, and bars indicate thedifferences between duplicates. Cu2� ions did not stimulate the activ-ity. (B) Cofactor requirement of the purified WfeD enzyme. The pu-rified WfeD solution (stored for 3 weeks) was used for the assay. Theactivities are shown as �mol/h/mg. EDTA (5 mM) and H2O were usedas negative controls. Assays were carried out in duplicates, and barsindicate the differences between duplicates. The products obtained byusing Mn2�, Pb2�, and Ni2� ions were confirmed by HPLC. (C) Con-centration effects of divalent metal ions. The activity of WfeD (using1:10-diluted crude enzyme homogenate) was measured after the addi-tion of MnCl2, Pb(OAc)2, or NiSO4 at different concentrations in theassay mixture. Activity is indicated as �mol/h/mg protein.

TABLE 4. Effects of detergents on crude WfeD enzyme activitya

Treatment Mean activity (%)

No detergent ...........................................................................100 6Triton X-100 ........................................................................... 86 4Tween 80 ................................................................................. 92 5NP-40 ....................................................................................... 86 10

a WfeD was assayed in the presence of 0.1 % (vol/vol) NP-40 (nonyl phenoxyl-polyethoxylethanol 40), Tween 80, or Triton X-100. The standard assay mixturewithout any detergent added (“no detergent”) was used as a positive control.Treatments with detergents were carried out in duplicates, with the variation ofthe duplicates indicated.



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DISCUSSION

We have shown that the second step of the repeating-unitassembly in B14 is catalyzed by WfeD, the gene product ofwfeD. Analysis of the WfeD enzyme product structure byHPLC, mass spectrometry, NMR, and galactosidase digestionclearly showed that WfeD adds one Gal residue in the �1-4linkage to GlcNAc�-pyrophosphate-lipid. There are severalother putative glycosyltransferase genes in the B14 O-antigengene cluster (wfeA, wfeB, and wfeE) that may encode the gly-cosyltransferases necessary to add the remaining sugars. Basedon the structure of the B14 O antigen, these enzymes areexpected to be a second �1,4-Gal-transferase, a �1,6-glucu-ronosyltransferase, and an �1,4-Gal-transferase. The third res-idue of the repeating unit, which is also a �1-4-linked Gal, islikely to be added by another enzyme that remains to be iden-tified and is expected to have the same specificity for UDP-Galbut a different acceptor substrate specificity. As there is verylittle sequence similarity between the enzymes encoded by thegenes in the B14 O-antigen gene cluster (wfeA, wfeB, andwfeE) that could help to predict their specific functions, anal-yses similar to those described here are required to determinetheir roles in O-antigen assembly.

The synthetic acceptor substrate GlcNAc�-PP-(CH2)11-OPhis an amphipathic molecule with similarity to the endogenoussubstrate GlcNAc�-PP-Und that acts as a lipid carrier in O-antigen biosynthesis and is exposed at the cytoplasmic side ofthe inner membrane (26). We have shown that GlcNAc�-PP-(CH2)11-OPh is an excellent in vitro acceptor substrate forWfeD. The advantage of GlcNAc�-PP-(CH2)11-OPh is thatthe substrate and the enzymatic reaction product can bereadily detected and separated by HPLC. We have previouslyshown that this compound was also an excellent substratefor the �1,3-Gal-transferase WbbD from E. coli strainVW187 (8, 17) and the �1,3-Glc-transferases WfgD andWfaP from E. coli serogroups O152 and O56, respectively(6). A similar compound was also shown to be an acceptorfor a GalNAc-transferase from E. coli O86:H2 (28). All ofthese enzymes add the second sugar residue to GlcNAc inthe repeating-unit assembly.

Detergent treatment of crude WfeD cell homogenates sug-

gested that the enzyme can be solubilized but is not activatedby detergents. Although the purified protein appeared to be95% pure, it was purified only 16-fold compared to the spe-cific activity in bacterial sonicates. This suggests that the en-zyme was partially insoluble under our conditions and re-mained associated with the membrane; thus, a substantialportion of the total enzyme was not purified. However, theintracellular localization of the WfeD protein remains to beconfirmed, for example, by cell fractionation.

The WfeD enzyme was found to be surprisingly stable aftercold storage. The kinetic properties of the purified WfeD �1,4-Gal-transferase resemble those of previously studied mamma-lian and bacterial glycosyltransferases that have similar activi-ties but unrelated amino acid sequences (3, 5, 14, 17, 26),suggesting a common catalytic mechanism.

A number of GlcNAc-pyrophosphate-lipid analogs provedto be good substrates, independent of the lipid structure. Thus,the main groups that are expected to bind to the enzyme arethe sugar and the pyrophosphate phosphate group. Modifica-tions of the pyrophosphate group eliminated the activity andbinding to the enzyme, although the compound having a singlephosphate did have 11% activity. None of the GlcNAc analogstested, which were inactive as substrates, bound to and inhib-ited WfeD, and we previously reported similar findings forWbbD (8) and for WfgD and WfaP (6). Thus, the pyrophos-phate group may be an essential group responsible for thebinding of the acceptor to the enzymes that recognize GlcNAc-PP-lipid. In the development of an enzyme inhibitor, therefore,the pyrophosphate group must be intact or present as a closelyrelated structural analog.

The DxD motif found in many glycosyltransferases wasshown previously to be involved in divalent-metal-ion-stimu-lated enzyme catalysis (3, 24). WfeD has a DxExE sequence ina central position of the protein, and we have shown by mu-tagenesis that the second acidic residue in this sequence

FIG. 5. Effect of amino acid reagents on activity of WfeD. Amino-acid-specific reagents were preincubated with purified enzyme for 10min, prior to the assays. The percentages of activities are shown rela-tive to the positive control (no addition of amino acid reagents). Thereagent treatments were applied in duplicates, and bars indicate vari-ations between duplicates. DEPC acts on His, HPG acts on Arg, andDTNB and IAA react with the SH group of Cys.

FIG. 6. GalT activities of mutant enzymes. GalT activities of crudecell homogenates containing site-specific WfeD K211A, R212A,R210A, E103A, E101A, and D99A mutants as well as the wild-typeenzyme were assayed using 1 mM UDP-Gal and 0.25 mM GlcNAc-PP-(CH2)11-OPh acceptor substrate in the presence of 5 mM MnCl2.The activities are shown as �mol/h/mg. The wild-type and mutantenzymes were tested in duplicates, and bars indicate the differencesbetween the duplicates. Activities were normalized according to therelative amount of His-tagged protein. The E101A and K211A mu-tants showed greatly reduced GalT activities.



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(Glu101) is essential for activity. Thus, the divalent-metal-ion-supported nucleophilic attack by Glu (or Asp) appears to be acommon mechanism in the catalysis of GalT. This motif iswithin a hydrophilic region of the protein and may be part ofthe UDP-Gal binding site.

The WfeD protein has been classified as a member of theGT26 family of glycosyltransferases with a WecG-TagA fold(www.CAZy.org), many of which act on sugar-pyrophosphatelipids. However, there is no structure available for other trans-ferases with this predicted fold or a possible arrangement ofthe catalytic region. Mammalian �1,4-Gal-transferase is almostidentical in its specificity but has a GT-A fold within the GT7family. The divalent metal ion may complex the pyrophosphategroup of UDP-Gal, thus facilitating the nucleophilic action ofthe Glu residue on the 4-hydroxyl of GlcNAc to be galacto-sylated (24). It is surprising that several metal ions, includingMn2�, Ni2�, and Pb2�, can stimulate WfeD activity, especiallygiven that Pb2� is usually toxic to enzymes. However, in apreviously reported protein kinase C study, Pb2� was alsofound to activate the enzyme at very low concentrations (11).The slight stimulation of mammalian �4GalT by Pb2� saltssuggests that this metal ion can also bind to the mammalianenzyme and replace the function of Mn2� ions, although thishad not been considered previously. Further structural analysisof the WfeD protein may confirm that Pb2� is forming acomplex with UDP-Gal or may reveal other functions for Pb2�

suggesting the mechanism of its stimulation and to determinewhy its stimulation is much more pronounced in the K211Amutant.

Several putative glycosyltransferases with sequence similar-ity to WfeD (see Fig. S1 in the supplemental material) have theRKR sequence or a similar cluster of positively charged aminoacids and are also assumed to bind two substrates containingpyrophosphate bonds. The RKR motif in WfeD may interactwith one or more phosphates of the acceptor pyrophosphategroup. The mutation of Lys211, but not of the adjacent Argresidues, resulted in a loss of activity, suggesting that eitherthere might be a specific interaction between the positivelycharged amino acid and the negatively charged acceptor or itmay be directly involved in catalysis or in an essential structuralfunction.

WfeD has two Cys residues, one of which (Cys148) is highlyconserved. Cys reagents that restore free SH bonds stimulatedthe activity, while reagents that covalently modify Cys inhibitedthe activity. This suggests that at least one of the two Cys-SHgroups participates in catalysis or is essential for the structureof the protein. A similar finding was made with core 2 GlcNAc-transferase, which forms intramolecular disulfide bonds butrequires one Cys-SH for activity (27). Cys148 in WfeD may bethis critical Cys residue. This could be verified by mutagenesisas well as peptide analysis by mass spectrometry. X-ray crys-tallography will help to further unravel the enzyme-substrateinteractions and enzyme protein structure and mechanisms ofWfeD.

ACKNOWLEDGMENTS

This work was supported by a grant from the Natural Sciences andEngineering Research Council of Canada; the Canadian Cystic Fibro-sis Foundation; the Chinese National Science Fund for DistinguishedYoung Scholars (grant 30788001); NSFC General Program grants

30670038, 30870070, 30870078, 30771175, and 30900041; the TianjinResearch Program of Application Foundation and Advanced Technol-ogy (grant 10JCYBJC10000); National 863 Program of China grants2006AA020703 and 2006AA06Z409; National 973 Program of Chinagrant 2009CB522603; and National Key Programs for InfectiousDiseases of China grants 2008ZX10004-002, 2008ZX10004-009,2009ZX10004-108, and 2008ZX10003-005.

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