Translocation and surface expression of lipidated serogroup B capsular Polysaccharide in Neisseria meningitidis

Abstract

The capsule of N. meningitidis serogroup B, (alpha2-->8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.

FIG. 1.

Genetic organization of the E. coli K1 capsule locus and the N. meningitidis capsule gene complex (cps). The genes responsible for capsule polymer biosynthesis (dotted arrows, synABCD and siaABCD), capsule transport (hatched arrows, ctrABCD), putative phospholipid substitution (black arrows, lip and lipB), and the E. coli K1 homologues of each are shown. The locations of lipA and lipB within the cps complex in the serogroup B strain NMB were similar to those of the published serogroup B MC58 meningococcal genome sequence (47) and other sequenced meningococcal genomes.

FIG. 2.

Serogroup B meningococcal capsule-specific whole-cell ELISA. The OD₄₀₅ readings of the serogroup B wild-type strain NMB were used as 100% for normalizing the OD₄₀₅ of the NMB18 (lipA::aphA-3), NMB16 (lipA::Ω), NMB19 (lipB::aphA-3), NMB17 (lipB::Ω), and the NMB1718 (lipA::aphA-3, lipB::Ω) mutants. The nonencapsulated mutant M7 (synA::Tn916) was included as a negative control (n ≥ 4).

FIG. 3.

Killing of serogroup B meningococci by normal human serum (NHS). Serum bactericidal assays were performed with the parent strain, the mutants, and the corresponding complemented mutant strains with 10% normal human serum. The solid lines represent the parent and the mutants, while the dotted lines represent the complemented mutant strains. ♦, wild-type strain NMB (solid line); □, nonpolar lipA mutant NMB18 (solid line) and complemented strain NMB1824 (dotted line); ▴, lipA::Ω mutant NMB16 (solid line) and complemented strain NMB1624 (dotted line); and *, nonpolar lipB mutant NMB19 (solid line) and complemented strain NMB19289 (dotted line).

FIG. 4.

Fluorescence-activated cell sorting analysis of capsular polysaccharide expression by serogroup B N. meningitidis. All strains were grown in GC broth, collected at early stationary phase, and processed for the fluorescence-activated cell sorting analysis as described in Materials and Methods. Panel A, SYTOX Green-stained samples of unfixed NMB (thick black line), unfixed NMB1718 (thick gray line), fixed NMB (thin black line), and fixed NMB1718 (thin gray line). Meningococci suspended in PBS without fixing (panel B) or after fixing with 70% ethanol (panel C) were incubated with either serogroup B capsule-specific monoclonal antibody (2-2-B) or serogroup C capsule-specific antibody (4-2-C) followed by R-phycoerythrin-conjugated secondary antibodies. (B and C) Solid light gray line, wild-type strain NMB with 2-2-B; solid gray line, NMB1718 mutant with 2-2-B; thick black line, capsule-deficient strain M7 with 2-2-B; thin black line, strain NMB with 4-2-C; dotted line, M7 with 4-2-C; dashed line, NMB1718 with 4-2-C.

FIG. 5.

Electron photomicrographs of immunogold-labeled intact serogroup B N. meningitidis (A to C) and meningococcal cell sections (D to F). Panels A and D show the wild-type parental strain NMB, B and E show the nonencapsulated mutant M7, and C and F are the NMB1718 (lipA lipB) mutant. Labeling was performed with the serogroup B capsule-specific monoclonal antibody as the primary antibody, and the 6-nm-gold-conjugated anti-immunoglobulin G/M antibody was used as the secondary antibody. Bars, 250 nm.

FIG. 6.

Phospholipase treatment of capsular polysaccharides purified from serogroup B strain NMB (lanes 1 to 7) and the NMB1718 (lipA lipB) mutant (lanes 8 to 14). Lanes 1 and 8, no phospholipase; lanes 2 and 9, phospholipase A2; lanes 3 and 10, phospholipase C; lanes 4 and 11, phospholipase D; lanes 5 and 12, A2 and C; lanes 6 and 13, C and D; lanes 7 and 14, A2 and C and D. The enzymatic activity of each phospholipase is indicated with phosphatidylcholine as a substrate and compared to the possible activity on the diacylphosphatidyl acid linkage of the sialic acid capsule (16). T, trypan blue; X, xylene cyanol; B, bromophenol blue.

FIG. 7.

Schematic diagram showing the method used to obtain the lipid anchor of the various capsular polysaccharide samples. The products shown in the boxes were analyzed as follows: (a) MALDI-TOF-MS analysis, (b and c) GC-MS analysis. The results of these analyses are shown in Fig. 8.

FIG. 8.

Analysis of the lipid anchor of the capsular polysaccharide from N. meningitidis. (A) Negative-ion MALDI-TOF-MS spectrum of dipalmitoyl glycerol phosphate that was liberated from the capsular polysaccharide by mild acid hydrolysis. The structure and calculated [M-H]⁻ ion at m/z 647.89 are also shown in panel A. (B) GC-MS profile (top) of the Me₃Si-1,2-di-O-palmitoyl glycerol derivatives obtained from HF treatment of the samples. The mass spectrum of this derivative is shown (bottom), and several of the major ions can be accounted for as described by Gotschlich et al. (16). (C) The GC profile of the components liberated from the samples by acid-catalyzed methanolysis and trimethylsilylation.