Endotoxin of Neisseria meningitidis composed only of intact lipid A: inactivation of the meningococcal 3-deoxy-D-manno-octulosonic acid transferase

Abstract

Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy-D-manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar kdtA::aphA-3 mutation was created in Neisseria meningitidis via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the kdtA::aphA-3 mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IV(A) did not accumulate. The LOS structure of the kdtA::aphA-3 mutant was restored to the wild-type structure by complementation with kdtA from N. meningitidis or Escherichia coli. The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in N. meningitidis can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.

FIG. 1.

Genetic organization of kdtA locus in the meningococcal MC58 and Z2491 genomes. lpxC, UDP-3-O-3-hydroxymyristoyl N-acetylglucosamine deacetylase gene (lipid A biosynthesis); gnd, 6-phosphogluconate dehydrogenase gene (pentose phosphate pathway); murA, UDP-N-acetylglucosamine 1-carboxyvinyltransferase gene (peptidoglycan biosynthesis); HP, hypothetical protein. The locations of primers used in this study (Table 1) are also indicated.

FIG. 2.

Growth curves of N. meningitidis serogroup B wild-type strain NMB and kdtA::aphA-3 mutant NMB249. Growth in BHI broth at 37°C was monitored by measuring the optical density at 550 nm [OD (550 nm)].

FIG. 3.

Electron photomicrographs of thin sections of wild-type strain NMB (A) and kdtA::aphA-3 mutant NMB249 (B). Bacteria were grown overnight on GC agar plates at 37°C and resuspended and fixed as described in Materials and Methods. Scale bars, 100 nm.

FIG. 4.

MALDI-TOF spectra of two LOS preparations from NMB249 (A and B) and of HF-treated LOS from NMB249 (C). The spectra in panels A and B were collected in the negative mode, and the spectrum in panel C was collected in the positive mode.

FIG. 5.

Tandem MS-MS spectrum of the m/z 1577 ion of HF-treated LOS from NMB249 (A), structure and primary fragmentation of this molecule (B), and rationale accounting for the observed secondary fragments (C).

FIG. 6.

Summary of the structures of the various lipid A molecules isolated from NMB249.

FIG. 7.

(A) Southern blotting performed with the aphA-3 cassette (right panel) and wild-type kdtA (left panel) as the probes. PvuII-digested chromosomal DNA were resolved on a 0.7% agarose gel. Lanes 1, wild-type strain NMB; lanes 2, strain NMB249; lanes 3, strain NMB249/271; lanes 4, strain NMB249/274. Removal of the 754-bp BssHII fragment and insertion of the 800-bp aphA-3 cassette gave rise to the fragment of the same size recognized by both probes. An additional band in lane 3 of the left panel represents the second copy of meningococcal kdtA on the shuttle vector. (B) Tricine-SDS-PAGE analysis of LOS. Lane 1, strain NMB; lane 2, strain NMB249; lanes 3 and 4, two independent NMB249/271 transformants; lanes 5 and 6, two independent NMB249/274 transformants. (C) Western blot of the strains in panel B, probed with anti-Flag monoclonal antibody.