The Neisseria meningitidis Serogroup A Capsular Polysaccharide O-3 and O-4 Acetyltransferase
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m313552200
ISSN1083-351X
AutoresSeshu K. Gudlavalleti, Anup Datta, Yih‐Ling Tzeng, Corie Noble, Russell W. Carlson, David S. Stephens,
Tópico(s)Bacterial Infections and Vaccines
ResumoNeisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, α1→6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA–D (sacA–D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60–70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted ∼26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the d-mannosamine capsule of N. meningitidis serogroup A. Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, α1→6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA–D (sacA–D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60–70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted ∼26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the d-mannosamine capsule of N. meningitidis serogroup A. Neisseria meningitidis serogroup A is responsible for the massive epidemics of meningococcal meningitis and septicemia that periodically affect sub-Saharan Africa, China, South America, and other parts of the world. The serogroup A capsular polysaccharide (CPS) 1The abbreviations used are: CPS, capsular polysaccharide; mAb, monoclonal antibody; Ni-NTA, nickel-nitrilotriacetic acid; IPTG, isopropyl-β-d-thiogalactopyranoside; ELISA, enzyme-linked immunosorbent assay. that confers serogroup specificity is composed of repeating units of α1→6-linked ManNAc 1-phosphate that is O-acetylated (1Liu T.Y. Gotschlich E.C. Jonssen E.K. Wysocki J.R. J. Biol. Chem. 1971; 246: 2849-2858Abstract Full Text PDF PubMed Google Scholar). Although there is evidence of other glycosidic linkages (2Bundle D.R. Smith I.C.P. Jennings H.J. J. Biol. Chem. 1974; 249: 2275-2281Abstract Full Text PDF PubMed Google Scholar), the principal linkage between monomer ManNAc residues in this polysaccharide is the α1→6 phosphodiester bond involving the hemiacetal group of carbon 1 and the hydroxyl group of carbon 6 of the mannosamine residues. Serogroup A CPS is structurally distinct from the other disease-causing meningococcal serogroups, B, C, Y, and W-135, which are composed of or contain sialic acid (1Liu T.Y. Gotschlich E.C. Jonssen E.K. Wysocki J.R. J. Biol. Chem. 1971; 246: 2849-2858Abstract Full Text PDF PubMed Google Scholar, 3Bhattacharjee A.K. Jennings H.J. Kenny C.P. Martin A. Smith I.C. Can. J. Biochem. 1976; 54: 1-8Crossref PubMed Scopus (133) Google Scholar, 4Bhattacharjee A.K. Jennings H.J. Kenny C.P. Martin A. Smith I.C.P. J. Biol. Chem. 1975; 250: 1926-1932Abstract Full Text PDF PubMed Google Scholar). Meningococcal serogroups C, Y, W-135, and H also express O-acetylated capsules. Interestingly, the serogroup B CPS is not O-acetylated. The genes encoding the putative CPS O-acetyltransferases OatC and OatWY, responsible for the O-acetylation of meningococcal serogroup C and serogroups W-135 and Y, respectively, have been recently identified (5Claus H. Borrow R. Achtman M. Morelli G. Kantelberg C. Longworth E. Frosch M. Vogel U. Mol. Microbiol. 2004; 51: 227-239Crossref PubMed Scopus (84) Google Scholar). Other pathogens such as pneumococcal serotype 9V, Salmonella enterica serovar typhi Vi, Staphylococcus aureus serotypes 5 and 8, and Escherichia coli K1 (6Orskov F. Orskov I. Sutton A. Schneerson R. Lin W. Egan W. Hoff G.E. Robbins J.B. J. Exp. Med. 1979; 149: 669-685Crossref PubMed Scopus (146) Google Scholar) also express O-acetylated capsules. The biological importance of O-acetylation of CPS appears to be species- or subspecies-dependent. In some pathogens, O-acetylation of capsules is involved in immune recognition (6Orskov F. Orskov I. Sutton A. Schneerson R. Lin W. Egan W. Hoff G.E. Robbins J.B. J. Exp. Med. 1979; 149: 669-685Crossref PubMed Scopus (146) Google Scholar, 7Szu S.C. Li X.R. Stone A.L. Robbins J.B. Infect. Immun. 1991; 59: 4555-4561Crossref PubMed Google Scholar). For meningococcal serogroup A CPS, there is a dramatic reduction in the immunogenicity of the polysaccharide observed upon removal of the O-acetyl groups by chemical treatment (8Berry D.S. Lynn F. Lee C.H. Frasch C.E. Bash M.C. Infect. Immun. 2002; 70: 3707-3713Crossref PubMed Scopus (113) Google Scholar). The general genetic organization of CPS genes of N. meningitidis is similar to that of other bacterial systems such as Haemophilus influenzae, E. coli K1, etc., that are classified (9Roberts I.S. Annu. Rev. Microbiol. 1996; 50: 285-315Crossref PubMed Scopus (525) Google Scholar, 10Whitfield C. Roberts I.S. Mol. Microbiol. 1999; 31: 1307-1319Crossref PubMed Scopus (413) Google Scholar) as group II capsules. It is usually composed of a unique biosynthetic genetic cassette and conserved genes involved in translocation of the CPS. The genetic cassette responsible for the biosynthesis of the serogroup A capsule is composed of an ∼5-kb nucleotide sequence located (Fig. 1) between ctrA, the outer membrane capsule transporter, and galE, the UDP-glucose 4-epimerase (11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google Scholar). Four open reading frames (designated mynA–D or sacA–D) are cotranscribed as an operon (11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google Scholar) and are not found in the genomes of other meningococcal serogroups or in Neisseria gonorrhoeae. Separated from ctrA by a 218-bp intergenic region, mynA is predicted to encode a 372-amino acid protein that has homology to the E. coli UDP-N-acetyl-d-glucosamine 2-epimerase. MynB has been hypothesized to be the capsule polymerase, linking individual UDP-ManNAc monomers together, whereas MynD has been predicted to be involved either in CPS transport assembly or in cross-linking of the capsule to the meningococcal cell surface (11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google Scholar). In this study, we demonstrate that mynC (744 bp) encodes an O-acetyltransferase (247 amino acids) that transfers acetyl groups to the ManNAc residues of the serogroup A CPS. Materials and Bacterial Strains—The bacterial strains, plasmids, and primers used in this study are described in Table I. The meningococcal serogroup A strains were originally isolated during an outbreak in Nairobi, Kenya, in 1989 (12Pinner R.W. Onyango F. Perkins B.A. Mirza N.B. Ngacha D.M. Reeves M. DeWitt W. Njeru E. Agata N.N. Broome C.V. J. Infect. Dis. 1992; 166: 359-364Crossref PubMed Scopus (68) Google Scholar) and were provided by the Centers for Disease Control and Prevention (Atlanta, GA). Strain F8229 (CDC1750) is encapsulated and was isolated from the cerebrospinal fluid of a patient with meningitis. Strain F8239 (CDC16N3) is an unencapsulated variant originally isolated as a serogroup A strain from the pharynx of an asymptomatic carrier. These strains belong to clonal group III-I and are closely related to strains that have caused epidemics in Saudi Arabia, Chad, Ethiopia, and other parts of the world. Monoclonal antibody (mAb) 14-1-A (13Zollinger W.D. Boslego J. Froholm L.O. Ray J.S. Moran E.E. Brandt B.L. Antonie Leeuwenhoek. 1987; 53: 403-411Crossref PubMed Scopus (24) Google Scholar) against the meningococcal serogroup A CPS was generously provided by Dr. Wendell Zollinger (Walter Reed Army Institute of Research). Restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA). Nickel-nitrilotriacetic acid (Ni-NTA)-agarose gravity flow matrix and anti-pentahistidine monoclonal antibodies were purchased from QIAGEN Inc. (Valencia, CA). The B-PER 6xHis fusion protein purification kit was purchased from Pierce. Nucleotide primers were synthesized at MWG Biotech (High point, NC). [14C]Acetyl-CoA and 4-nitrophenyl acetate were purchased from Sigma. Automated DNA sequence analysis was performed with the Prism dye deoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA), and completed reactions were run on an ABI Model 377 automated DNA sequencer at the Microchemical Facility of Emory University.Table IStrains, plasmids, and primers used in this studyStrains/plasmids/primersDescription/sequenceRef./SourceStrainsN. meningitidisF8229N. meningitidis serogroup A strain (CDC1750)11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google ScholarNmA001NmA with chromosomal mynC::aphA-3 mutationThis studyNmAwtc1F8229 carrying pGS205 (mynC)This studyNmAnpc1NmA001 carrying pGS205 (mynC)This studyE. coliDH5αCloning strain58Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8186) Google ScholarBL21(DE3) LysSExpression strainNovagenPlasmidspCR2.1TA cloningStratagenepUC18Cloning vector, Ampr59Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11461) Google ScholarpUC18KSource of aphA-3 (Kmr) cassette15Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (616) Google ScholarpFLAG-CTCCloning vector for FLAG fusionSigmapYT250Meningococcal shuttle vector (Emr)60Tzeng Y.-L. Datta A. Kolli V.K. Carlson R.W. Stephens D.S. J. Bacteriol. 2002; 184: 2379-2388Crossref PubMed Scopus (67) Google ScholarpGS201SE57-SE61 PCR product cloned into pCR2.1This studypGS202aphA-3 cloned into blunted SspI site of pGS201This studypGS203Full-length mynC obtained from SG005 (NdeI) and SG006 (XhoI) PCR product cloned into NdeI-XhoI-digested pET20bThis studypGS204Full-length mynC with His tag obtained from SG007 (HindIII) and SG008 (EcoRI) PCR product cloned into pCR2.1This studypGS205HindIII-EcoRV-digested fragment of pGS204 ligated with HindIII-SmaI-digested fragment of pFLAG-CTCThis studypGS206BglI-digested fragment of pGS205 subcloned into EcoRV site of pYT250This studyPrimersSE56AATCATTTCAATATCTTCACAGCC11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google ScholarSE57TTACCTGAATTTGAGTTGAATGGC11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google ScholarSE61CAAAGGAAGTTACTGTTGTCTGC11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google ScholarYT79CATCATAACGGTTCTGGCAAATATTC60Tzeng Y.-L. Datta A. Kolli V.K. Carlson R.W. Stephens D.S. J. Bacteriol. 2002; 184: 2379-2388Crossref PubMed Scopus (67) Google ScholarYT80CTGTATCAGGCTGAAAATCTTCTCTC60Tzeng Y.-L. Datta A. Kolli V.K. Carlson R.W. Stephens D.S. J. Bacteriol. 2002; 184: 2379-2388Crossref PubMed Scopus (67) Google ScholarSG005GAACATATGTTATCTAATTTAAAAAACThis studySG006TTACTCGAGATATATATTTTGGATTATGGTThis studySG007GGAGATATACATAAGCTTTCTAATTTAAAAThis studySG008AGCGAATTCTCAGTGGTGGTGGTGGTGGTGThis study Open table in a new tab Growth Conditions—Meningococcal strains were grown with 3.5% CO2 at 37 °C on gonococcal base agar (Difco) supplemented with 0.4% glucose and 0.68 mm Fe(NO3)3 or in gonococcal broth containing the same supplements and 0.043% NaHCO3. Brain/heart infusion medium (37 g/liter) with 1.25% fetal bovine serum was used when kanamycin selection was required. The antibiotic concentrations used for E. coli strains were 100 μg/ml ampicillin, 50 μg/ml kanamycin, and 300 μg/ml erythromycin, and those for N. meningitidis were 80 μg/ml kanamycin, 60 μg/ml spectinomycin, and 3 μg/ml erythromycin. E. coli strain DH5α cultured on LB medium was used for cloning and propagation of plasmids. Meningococci were transformed by the procedure of Janik et al. (14Janik A. Juni E. Heym G.A. J. Clin. Microbiol. 1976; 4: 71-81Crossref PubMed Google Scholar). E. coli strains were transformed with a Gene-Pulser (Bio-Rad) according to the manufacturer's protocol. Construction of Meningococcal Non-polar mynC Mutant NmA001— An internal 745-bp fragment of mynC, produced by PCR amplification using primers SE57 and SE61 (11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google Scholar) and the chromosomal DNA of strain F8229 as a template, was cloned into pCR2.1 to yield pGS201. The aphA-3 fragment obtained from pUC18K (15Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (616) Google Scholar) with EcoRI and HincII digestion and filled in with Klenow polymerase was inserted into the unique SspI site of mynC in pGS201 to generate pGS202. The correct orientation of aphA-3 was confirmed by colony PCR and direct sequence analysis of pGS202. An ScaI-linearized pGS202 plasmid was used to transform meningococcal serogroup A strain F8229 to generate NmA001. The correct homologous recombination of the aphA-3 cassette into the mynC coding sequence was confirmed by PCR with cassette-specific primers and chromosome-specific primers. Overexpression and Purification of Meningococcal MynC—The complete coding sequence of mynC was obtained by PCR amplification using primers SG005 (NdeI) and SG006 (XhoI) (Table I). The PCR product, digested with NdeI and XhoI, was subsequently cloned into pET20b(+) cut with the same enzymes to yield pGS203, resulting in a C-terminal His6 fusion. Plasmid pGS203 was purified and subjected to DNA sequence analysis to confirm the intact mynC sequence and the C-terminal His tag fusion. pGS203 was then transformed into E. coli expression strain BL21(DE3) pLysS. One liter of LB culture of the MynC-overexpressing strain was induced with 1 mm isopropyl-β-d-thiogalactopyranoside (IPTG) for 5 h. The harvested cells were resuspended in 15 ml of lysis buffer (50 mm sodium phosphate (pH 8.0), 300 mm NaCl, 10 mm imidazole, 1% (v/v) Tween 20, 1 mm phenylmethylsulfonyl fluoride, and 1 mg/ml lysozyme), left on ice for 30 min, and sonicated 10 times for 30 s with 30-s cooling intervals. The cell debris was removed by centrifugation at 14,000 × g for 15 min at 4 °C. The overexpressed protein was purified under native conditions on Ni-NTA matrices following the supplier's protocol with a modification in column washing. Briefly, the crude extract was incubated with 2 ml of 50% suspension of Ni-NTA-agarose for 1 h before packing into a column. The column was washed with 5 ml each of 10, 20, and 40 mm imidazole in lysis buffer without lysozyme and then eluted with 5 ml of buffer containing 250 mm imidazole. The MynC protein was also extracted and purified using a B-PER protein extraction kit (Pierce), containing a lysis reagent with a proprietary mild nonionic detergent in 20 mm Tris-HCl (pH 7.5), following the manufacturer's instructions. The purified MynC fractions obtained by either method were concentrated separately using Centricon YM-3 centrifugal filters (Millipore Corp., Bedford, MA) after SDS-PAGE analysis and dialyzed in storage buffer (50 mm HEPES (pH 7.05), 5 mm MgCl2, 100 mm NaCl, and 1 mm EDTA). The protein concentration was determined with a BCA protein assay kit (Pierce) using bovine serum albumin as the standard. Complementation of the NmA001 Mutant—An intact copy of mynC under the control of the tac promoter was constructed on a meningococcal shuttle vector. Full-length mynC with a C-terminal His tag was amplified from pGS203 using primers SG007 (HindIII) and SG008 (EcoRI) (Table I). The amplified PCR product was cloned into pCR2.1 to yield pGS204. The mynC insert was subsequently released from pGS204 with HindIII and EcoRV digestion and ligated into the HindIII and SmaI sites of pFLAG-CTC to generate pGS205 with mynC under the control of the lac promoter. The construct was confirmed by PCR using vector-specific primers YT79 and YT80. The pGS205 plasmid was then cut with BglI, filled in with Klenow, and ligated into the EcoRV site of the meningococcal shuttle vector pYT250(ErmR), yielding pGS206. The pGS206 construct was methylated with HaeIII methylase, and the reaction mixture was used directly to transform wild-type strain F8229 and non-polar mynC mutant NmA001, yielding NmAwtc1 and NmAnpc1, respectively. Meningococcal Membrane and Cytosolic Preparations—Meningococcal membranes and cytosol were separated by the method of Clark et al. (16Clark V.L. Campbell L.A. Palermo D.A. Evans T.M. Klimpel K.W. Infect. Immun. 1987; 55: 1359-1364Crossref PubMed Google Scholar) from the complemented meningococcal mynC strain NmAnpc1. Briefly, the pellet from a 500-ml culture of NmAnpc1 carrying pGS206, induced overnight with 1 mm IPTG, was used to prepare the inner and outer membranes and cytosolic fractions. The pellet was suspended in 2 ml of buffer containing 1 mm EDTA, 50 mm Tris, and 20% sucrose (pH 8.0) with 1 mg/ml lysozyme for 30 min at 4 °C. The cell suspensions were diluted with 20 ml of Tris buffer to generate spheroplasts and were sonicated three times each for 30 s in an ice bath with 30-s resting intervals. The cell debris was removed by centrifugation at 10,000 × g for 15 min at 4 °C. The supernatant was freeze-thawed once at –70 °C before ultracentrifugation at 100,000 × g for 90 min at 4 °C. The pellet, containing the meningococcal membrane fraction, was washed with Tris buffer. The level of contamination of the membrane fraction with cytoplasmic components was assessed by determining the activity of a cytoplasmic enzyme, malate dehydrogenase (17de Maagd R.A. Lugtenberg B.J. J. Bacteriol. 1986; 167: 1083-1085Crossref PubMed Google Scholar), for both fractions. The membrane fractions were 97–98% pure. The cytosolic proteins were precipitated by 5% trichloroacetic acid and suspended in 2 ml of 1 m Tris (pH 6.8). Total membrane was solubilized with 2 ml of 2% N-lauroylsarcosine (Sarkosyl) in 10 mm HEPES (pH 7.4) for 1 h at room temperature using an orbital shaker. Soluble inner membrane components and insoluble outer membrane components were separated by ultracentrifugation at 100,000 × g for 2 h at 4 °C. The outer membrane pellet was suspended in 500 μl of 1 m Tris (pH 6.8). The diluted inner membrane proteins were precipitated by 5% trichloroacetic acid, and the pellet thus obtained was suspended in 500 μlof1 m Tris (pH 6.8). Subcellular fractions were resolved on 10% SDS-polyacrylamide gels. The loadings were standardized based on the same amount of starting meningococci (500 ml of meningococcal culture pellet at A550 = 1 contains ∼2.5 × 1011 cells) and analyzed by Western blotting. To examine the nature of the membrane association of MynC, membrane solubilization experiments were performed as described (18Finberg K.E. Muth T.R. Young S.P. Maken J.B. Heitritter S.M. Binns A.N. Banta L.M. J. Bacteriol. 1995; 177: 4881-4889Crossref PubMed Google Scholar). Briefly, the membrane pellets were extracted with 5 ml of buffer (100 mm sodium phosphate (pH 7.6), 0.2 mm dithiothreitol, 20% sucrose, and 0.2 m KCl) containing 1% Triton X-100, 1 m NaCl, or 6 m urea for 30 min at room temperature (urea), at 30 °C (Triton X-100), or on ice (buffer alone and buffer with NaCl). Samples were centrifuged at 130,000 × g for 1 h at 4 °C after the extraction. Proteins in the soluble fractions were precipitated with 5% trichloroacetic acid, and the precipitates obtained were washed two times with acetone, dried, and resuspended in 1 m Tris (pH 6.8) before an equal volume of 2× SDS-PAGE sample buffer was added. CPS Extraction and Structural Characterization—CPS was extracted from 2 liters of meningococcal cultures using the standard method of Gotschlich et al. (19Gotschlich E.C. Liu T.Y. Artenstein M.S. J. Exp. Med. 1969; 129: 1349-1365Crossref PubMed Scopus (267) Google Scholar). Briefly, the overnight cultures were treated with a final concentration of 1% Cetavlon, a polycationic detergent that precipitates the polyanionic polysaccharides. The precipitate was collected by centrifugation and resuspended in water, and CaCl2 was then added to a final concentration of 1 mm to separate the polysaccharide from the detergent. Nucleic acids were precipitated from the solution by adding 25% (v/v) ethanol, followed by centrifugation. CPS in the supernatant was subsequently precipitated by ethanol at a final concentration of 80% (v/v). Contaminating protein, traces of Cetavlon, and other low molecular mass contaminants were removed with proteinase K digestion and extensive dialysis against a buffer composed of 10% ethanol, 50 mm NaCl, and 5 mm Tris. CPS was further purified by Sephacryl 200 gel filtration column using 50 mm ammonium formate elutions. Column fractions were tested for neutral sugar estimation by phenol sulfuric acid assay (20Dubois M. Anal. Chem. 1956; 28: 350-356Crossref Scopus (40800) Google Scholar). Void volume fractions were pooled and concentrated by speed vacuuming and analyzed by deoxycholate-PAGE and Alcian blue staining (21Reuhs B.L. Carlson R.W. Kim J.S. J. Bacteriol. 1993; 175: 3570-3580Crossref PubMed Google Scholar). Compositional and NMR Analyses of CPSs—Compositional analysis of purified CPS was performed on the alditol acetate derivatives of the sugars after removing the phosphate groups by hydrogen fluoride treatment of the purified serogroup A CPS. The alditol acetate derivatives were analyzed by combined gas chromatography/mass spectrometry using a 30-m SP2330 capillary column (Supelco) (22Stevenson T.T. Furneaux R.H. Carbohydr. Res. 1991; 11: 195-211Google Scholar). Lyophilized wild-type or mutant CPS powder (5 mg) was dissolved in D2O (99.999 atom % D; Sigma) to a uniform concentration of 5 mg/ml. Solutions were agitated with vortexing for 10 min at room temperature, followed by low speed centrifugation at 7200 × g for 10 min to eliminate undissolved material. Aliquots (600 μl) of the supernatant were transferred to 5-mm NMR tubes and placed in a sonication bath for 10 min to eliminate air bubbles trapped on the inner wall of the NMR tube. NMR spectra were acquired on a Varian Unity 500 NMR spectrometer equipped with a 5-mm pulsed field gradient triple resonance probe and a high precision temperature controller (+0.1 °C) and under the control of VNMR Version 6.1B or on a Varian Inova 500 spectrometer equipped with a 5-mm pulsed field gradient inverse detection heteronuclear probe running under VNMR Version 6.1C and Solaris Version 2.8. One-dimensional proton NMR spectra were collected at 25 °C using a standard one-pulse experiment. The transmitter was set at the HDO frequency (4.78 ppm). Standard spectral acquisition conditions are as follows: collection of 64K data points over a spectral window of 8000 Hz, acquisition time of 4.096 s, and a relaxation delay of 26 s, giving a recycle time of 30 s. Typically, 64 scans were averaged. Spectra were Fourier-transformed after applying a 0.2-Hz line broadening function. Integrations were performed using subroutines built into the VNMR software. Hydrophobic Interaction Chromatography—The cell-surface hydrophobicity of meningococcal strains was tested using a modified method of Karlyshev et al. (23Karlyshev A.V. Linton D. Gregson N.A. Lastovica A.J. Wren B.W. Mol. Microbiol. 2000; 35: 529-541Crossref PubMed Scopus (195) Google Scholar). Disposable plastic columns packed with octyl-Sepharose CL-4B (Sigma) to a height of 2 cm were washed with 10 ml of buffer A (0.2 m ammonium sulfate in 10 mm sodium phosphate buffer (pH 6.8)). Meningococci collected from overnight plate cultures were suspended in phosphate-buffered saline to an absorbance of 10, and a 100-μl aliquot was gently pipetted onto the surface of the column and eluted with 5 ml of buffer A. A 100-μl cell suspension diluted directly into 5 ml of buffer A was also prepared as a control. The A600 of both the column flow-through and control samples was determined. Results were calculated as the A600 of the flow-through divided by that of the control and are expressed as a percentage of cells adsorbed to the column. Serum Bactericidal Assay—A serum bactericidal assay was performed as described previously (24Kahler C.M. Martin L.E. Shih G.C. Rahman M.M. Carlson R.W. Stephens D.S. Infect. Immun. 1998; 66: 5939-5947Crossref PubMed Google Scholar) using pooled normal human serum at a final concentration of 10% (v/v) with a 30-min incubation at 37 °C. Heat-inactivated normal human serum was used as a control. Immunoblots—Serogroup A wild-type and mynC mutant NmA001 CPSs were resolved on 15% deoxycholate-polyacrylamide gels and transferred onto polyvinylidene difluoride membrane using a transfer buffer of 25 mm Tris, 192 mm glycine (pH 8.3), and 20% methanol. An identical gel was stained with Alcian blue to visualize capsules. Membranes were blocked with 3% bovine serum albumin in Tris/Tween buffer (0.5 m Tris (pH 7.5), 0.9% NaCl, and 0.05% Tween 20). Serogroup A capsule-specific mAb 14-1-A (13Zollinger W.D. Boslego J. Froholm L.O. Ray J.S. Moran E.E. Brandt B.L. Antonie Leeuwenhoek. 1987; 53: 403-411Crossref PubMed Scopus (24) Google Scholar) was used as the primary antibody at 1:1000 dilution, whereas alkaline phosphatase-conjugated goat antimouse IgG + IgM (Organon Teknika Corp., West Chester, PA) was used at 1:5000 dilution. All incubations were done at room temperature for 1 h. Blots were developed in 20 ml of alkaline phosphatase buffer (0.1 m Tris (pH 9.5), 0.1 m NaCl, and 0.5 mm MgCl2) containing 40 μl of 10% nitro blue tetrazolium in 70% N,N-dimethylformamide and 30 μl of 5-bromo-4-chloro-3-indolyl phosphate (50 mg/ml in N,N-dimethylformamide). Colony immunoblots were processed similarly using nitrocellulose membranes. After the meningococci were lifted, the membranes were allowed to air-dry for 30 min at room temperature and then blocked for 1 h with 5% bovine serum albumin in Tris/Tween buffer. Protein samples for Western blots were resolved by a 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes as described above. Anti-pentahistidine monoclonal antibodies were used as primary antibodies at 1:1000 dilutions. Whole Cell ELISA—ELISAs were performed using the methods reported previously (11Swartley J.S. Liu L.J. Miller Y.K. Martin L.E. Edupuganti S. Stephens D.S. J. Bacteriol. 1998; 180: 1533-1539Crossref PubMed Google Scholar) with the following modifications. Aliquots (50 μl) at 1:9 dilution of meningococcal suspensions (A550 = 0.1) were applied to microtiter plates and dried overnight at 37 °C. mAb 14-1-A was used at 1:30,000 dilution, and alkaline phosphatase-conjugated goat antimouse secondary antibody (Organon Teknika Corp.) was used at 1:10,000 dilution. All incubations were performed at 37 °C. Colorimetric Estimation of Capsule O-Acetylation—O-Acetylation of purified CPSs was also measured colorimetrically according to the method described by Hestrin (25Hestrin S. J. Biol. Chem. 1949; 180: 249-261Abstract Full Text PDF PubMed Google Scholar). Aliquots of CPS samples (500 μl) were incubated with an equal volume of 0.035 m hydroxylamine in 0.75 m NaOH for 10 min at 25 °C, followed by the addition of 1 m of perchloric acid (500 μl) and 70 mm ferric perchlorate in 0.5 m perchloric acid (500 μl). The pink color resulting from the presence of O-acetyl groups was quantified at 500 nm with a known amount of ethyl acetate as the standard. p-Nitrophenyl Acetate Assay—Acetylesterase activity was tested with the synthetic substrate p-ni
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