Article Figures & Data
Figures
- FIG. 1.
Neisserial lipooligosaccharides utilized as substrates in this study. (A) Structures of the core regions of the different LOS substrates. (B) Differences in the lipid A region after different chemical modifications. de-O-Ac, LOS after de-O-acylation by hydrazinolysis; HF-de-O-Ac, de-O-Ac LOS after dephosphorylation by incubation with HF; KOH, LOS after complete deacylation by incubation with KOH and re-N-acetylation.
- FIG. 2.
Subcellular localization of Lpt3 and Lpt6. Induced cell culture lysates (lanes 1) were clarified by centrifugation to remove inclusion bodies, followed by isolation of complete membrane fractions by ultracentrifugation (lanes 2), which were further separated into inner membrane (lanes 3) and outer membrane (lanes 4) fractions by sucrose gradient centrifugation. (A and B) Coomassie-stained 12.5% SDS-polyacrylamide gel of fractionation of lysates from induced cultures of the Lpt3-His6 expression strain CQWJR1 (A) and the Lpt6-His6 expression strain CQWJR2 (B). (C and D) Western immunoblot of the samples in panels A and B using monoclonal anti-polyhistidine peroxidase conjugate clone His-1 (Sigma Aldrich). For inner and outer membrane fractions, 10 μg total protein was loaded per lane. Note that both Lpt3-His6 and Lpt6-His6 localize to the inner membrane fraction.
- FIG. 3.
Molecular mass determination of purified Lpt3-His6 and Lpt6-His6. MALDI-TOF MS analysis of purified Lpt3-His6 (A) and Lpt6-His6 (B) and their apparent molecular masses (kDa) as determined by SDS-PAGE (insets of panels A and B, respectively). Note that the MALDI-TOF MS m/z values for Lpt3-His6 and Lpt6-His6 match closely with their predicted molecular weights (60,277.3 and 63,368.9, respectively).
- FIG. 4.
Lpt3 and Lpt6 exhibit LOS HepII 3- and 6-phosphoethanolamine transferase activities, respectively. (A) Dot blot analyses of Lpt3-His6 and Lpt6-His6 reactions (rxns) with N. meningitidis galE lpt3 de-O-Ac LOS in the absence or presence of 1 mg ml−1 phosphatidylethanolamine, using HepII 3-PEtn-specific MAb L3B5 for Lpt3-His6 reactions and HepII 6-PEtn-specific MAb L2-16 for Lpt6-His6 reactions. (B and C) Dot blot analyses of Lpt3-His6 and Lpt6-His6 reactions with N. meningitidis MC58 galE lpt3 LOS and de-O-Ac LOS, using MAb L3B5 (B) and MAb L2-16 (C), respectively. Negative controls were N. meningitidis MC58 galE lpt3 LOS and de-O-Ac LOS, 3-PEtn controls were N. meningitidis MC58 galE LOS and de-O-Ac LOS, and 6-PEtn controls were N. meningitidis 89I galE LOS and de-O-Ac LOS. Note that MAb L3B5 does not react in Lpt6-His6 reactions and MAb L2-16 does not react in Lpt3-His6 reactions.
- FIG. 5.
Analysis of Lpt3-His6 and Lpt6 reactions by CE-MS. Shown are CE-MS spectra for N. meningitidis MC58 galE lpt3 de-O-Ac LOS (substrate) (A), the Lpt3-His6 reaction using N. meningitidis MC58 galE lpt3 de-O-Ac LOS as the substrate (B), and the Lpt6-His6 reaction using N. meningitidis MC58 galE lpt3 de-O-Ac LOS as the substrate (C). Spectra were obtained in negative-ion mode, using precursor ion scanning for de-O-Ac lipid A (m/z 951.0). See the text for details regarding the minor peaks observed.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain/plasmid Descriptiona Source/reference Strains Neisseria meningitidis MC58 L3 immunotyping reference strain 20 MC58 galE L3 galE 12 MC58 galE lpt3 L3 galE lpt3::Emr 19 89I L4 immunotyping reference strain 89I galE L4 galE::Emr 39 Escherichia coli DH5α F− φ80 LacZ M15 (lacZY-argF) supE44 thi-1 gyrA96 recA1 endA1 relA1 hsdR17 BL21(DE3) F− ompT hsdSB(rB− mB −) gal dcm (DE3) Novagen CQW50 Lpt3-His6 expression strain; BL21(DE3) containing pCQJR3; Kmr This study CQW51 Lpt6-His6 expression strain; BL21(DE3) containing pCQJR7; Kmr This study Plasmids pET28a T7 expression vector, N- and C-terminal His6 tags; 5.4 kb; Kmr Novagen pET30a T7 expression vector, N- and C-terminal His6 tags; 5.4 kb; Kmr Novagen pCQJR3 Lpt3-His6 expression vector; PCR product of CQ-160 and CQ-162, containing lpt3, cloned into the NdeI and XhoI sites of pET30a; 6.8 kb; Kmr This study pCQJR7 Lpt6-His6 expression vector; PCR product of CQ-222 and CQ-223, containing lpt6, cloned into the NcoI and XhoI sites of pET28a; 7.0 kb; Kmr This study ↵ a Em, erythromycin.
- TABLE 2.
CE-MS analyses of Lpt3-His6 and Lpt6-His6 reactions with various LOS substrates
Sample Observed ion (m/z)a Relative intensityd Molecular mass (Da) Proposed compositione [M + 2H]2+ [M − 2H]2− Observed Calculated N. meningitidis MC58 galE lpt3 de-O-Ac LOS 1,070.5 1 2,143.0 2,143.04 Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) Lpt3-His6 + de-O-Ac LOS 1,132.0 0.39 2,266.0 2,266.09 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) 1,070.5 1 2,143.0 2,143.04 Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) Lpt6-His6 + de-O-Ac LOS 1,132.0 0.18 2,266.0 2,266.09 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) 1,070.5 1 2,143.0 2,143.04 Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) de-O-Ac HF LOS 992.5b , c 1 1,983.0 1,983.08 Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac-HF) Lpt3-His6 + de-O-Ac HF LOS 1,054.0b , c 0.31 2,106.0 2,106.03 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac-HF) 992.5b , c 1 1,983.0 1,983.08 Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac-HF) Lpt6-His6+ de-O-Ac HF LOS 992.5b , c 1 1,983.0 1,983.08 Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac-HF) KOH LOS 886.0 1 1,774.0 1,774.38 Hex·HexNAc·Hep2·Kdo2·HexNAc2·(PO4)2 Lpt3-His6 + KOH LOS 947.5 0.11 1,897.0 1,897.43 PEtn·Hex·HexNAc·Hep2·Kdo2·HexNAc2·(PO4)2 886.0 1 1,774.0 1,774.38 Hex·HexNAc·Hep2·Kdo2·HexNAc2·(PO4)2 Lpt6-His6 + KOH+NAc LOS 886.0 1 1,774.0 1,774.38 Hex·HexNAc·Hep2·Kdo2·HexNAc2·(PO4)2 N. meningitidis 89I galE de-O-Ac LOS 1,132.0c 1 2,266.0 2,266.09 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) Lpt3-His6 + de-O-Ac LOS 1,193.5c 0.57 2,389.0 2,389.14 PEtn2 ·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) 1,132.0c 1 2,266.0 2,266.09 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) N. meningitidis MC58 galE de-O-Ac LOS 1,132.5 1 2,267.0 2,266.09 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) Lpt6-His6 + de-O-Ac LOS 1,194.0 0.12 2,390.0 2,389.14 PEtn2 ·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) 1,132.5 1 2,267.0 2,266.09 PEtn·Hex·HexNAc·Hep2·Kdo2·lipid A (de-O-Ac) ↵ a Obtained using precursor scanning for de-O-Ac lipid A in negative-ion mode or HexNAc in positive-ion mode.
↵ b Contained minor loss-of-water ions ([M − H2O + 2H]2+).
↵ c Contained minor sodium adducts ([M + H + Na]2+ or [M − 3H + Na]2−).
↵ d Values are based on assigning a value of 1 to the largest peak in each spectrum.
↵ e Boldface indicates PEtn groups added by Lpt3 or Lpt6, as appropriate.
