Most meningococcal LOS is heterogeneous and variable. In order to understand the antigenic variation of LOS at the gene level, we examined nine lgt genes on three chromosomal loci (lgt-1,2,3) encoding glycosyltransferases responsible for the biosynthesis of LOS in over 30 strains of N. meningitidis. DNA hybridization, PCR and nucleotide sequence data revealed that the 7 genes (lgtABCDEHZ) in lgt-1 locus and the lgtG in lgt-3 locus were hypervariable while the lgtF gene in lgt-2 locus was conserved. In contrast, in 51 strains of N. gonorrhoeae examined, no variability in the composition or organization of the lgt loci was observed. In addition to the above two pathogenic Neisseria, 18 strains of commensal Neisseria representing 12 species were also analyzed. Based on the genetic organization of the lgt genes in Neisseria species, the lgt-1 locus was classified into 8 types and the lgt-3 locus was classified into 4 types. Four types of lgt-1 (II, III, IV and VIII) and one type of lgt-3 (IV) were novel genetic organizations not reported previously. Based on the three lgt loci, 10 LOS genotypes of N. meningitidis could be distinguished. Phylogenetic analysis revealed a new gene cluster, lgtH, which separated it from other LOS homologous genes. The genetic diversity of the lgt loci provides fundamental information toward understanding the heterogeneity and antigenic variation of Neisseria LOS. To modify LOS for vaccine purpose, the lgtH and lpxL2 (htrB2) genes were cloned into the plasmids of E. coli. The recombinant plasmids containing kanamycin selective marker were transformed to N. meningitidis 6275 ( L3 prototype strain) and the lgtH knocking-out mutants were constructed. On SDS-PAGE, the size of LOS in lgtH mutants was reduced. Comparison of LOS structures between wildtype and lgtH mutant by MALDI-TOF mass spectrometry and sugar analysis is ongoing. The construction of lpxL2 mutant that alters and detoxify the lipid A is planned.
