| Abstract: | Neisseria meningitidis, but notHaemophilus influenzae, damage cultured human endothelial cells. We have undertaken a study to generate genetically and structurally defined lipopolysaccharide (LPS) mutant strains of meningococci for functional studies to assess the role of surface exposed oligosaccharides in imparting specificity of toxic damage to human endothelial cells. TheIsi1gene, which had been shown to be involved in LPS biosynthesis ofNeisseria gonorrhoeae, was amplified by PCR and cloned. Nucleotide sequence analysis confirmed the identity of the clone and revealed homology withIsi1ofN. gonorrhoeaeand therfaFgene ofSalmonella typhimuriumwhich encodes a heptosyl-2-transferase involved in LPS biosynthesis. The identity of the clonedIsi1gene, as a functionalrfaFhomologue, was confirmed by the complementation of aS. typhimurium rfaFmutant using a P22 phage sensitivity test. AnIsi1mutant meningococcal strain was constructed, and structural analysis of the mutant LPS molecule revealed a single heptose in the core structure, consistent with a heptosyl-2-transferase deficient mutant. In order to investigate the relative cytotoxicities of meningococci expressing native and altered LPS, wild type,Isi1, andgalEstrains were compared in cytotoxicity assays using human umbilical vein endothelial cells (Huvecs) in culture. Analysis using Huvecs derived from several individuals (cords) showed that the three phenotypes were almost equally cytotoxic. Removal of the terminal portion (galEmutant) or the majority (Isimutant) of the oligosaccharide did not effect LPS-mediated cytopathic damage to Huvecs in a culture suggesting that the oligosaccharide portion did not play a major role in cytotoxicity. |
