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Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region.

ABSTRACT: We studied capsule-defective (Cap-) serogroup B meningococcal mutants created through Tn916 or omega-fragment mutagenesis. The Cap- phenotypes were the results of insertions in three of four linked genes (synX, synC, and synD) involved in CMP-N-acetylneuraminic acid and polysialic acid capsule biosynthesis, and in ctrA the first of four linked genes involved in capsule membrane transport. Mutations in the CMP-N-acetylneuraminic acid biosynthesis genes synX and synC caused defects in lipooligosaccharide sialylation but not mutations in the putative (alpha2 -> 8)-linked polysialyltransferase (synD) or in ctrA. Reverse transcriptase PCR studies indicated that the four biosynthesis genes (synX to -D) and the capsule transport genes (ctr to -D) were separately transcribed as operons. The operons were separated by a 134-bp intergenic region. Primer extension of synX and ctrA demonstrated that transcription of the operons was divergently initiated from adjacent start sites present in the intergenic region. Both transcriptional start sites were preceded by a perfect -10 Pribnow promoter binding region. The synX to -D, but not the ctrA to -D, transcriptional start site was preceded by a sequence bearing strong homology to the consensus sigma 70 -35 promoter binding sequence. Both promoters showed transcriptional activity when cloned behind a lacZ reporter gene in Escherichia coli. Our results confirm the intrinsic relationship between polysialic acid capsule biosynthesis and lipooligosaccharide sialylation pathways in group B Neisseria meningitidis. Our study also suggests that the intergenic region separating the synX to -D and ctrA to -D operons is an important control point for the regulation of group B capsule expression through coordinated transcriptional regulation of the synX to -D and drA to -D promoters.

SUBMITTER: Swartley JS

PROVIDER: S-EPMC178160 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region.

Swartley J S JS Ahn J H JH Liu L J LJ Kahler C M CM Stephens D S DS

Journal of bacteriology 19960701 14

We studied capsule-defective (Cap-) serogroup B meningococcal mutants created through Tn916 or omega-fragment mutagenesis. The Cap- phenotypes were the results of insertions in three of four linked genes (synX, synC, and synD) involved in CMP-N-acetylneuraminic acid and polysialic acid capsule biosynthesis, and in ctrA the first of four linked genes involved in capsule membrane transport. Mutations in the CMP-N-acetylneuraminic acid biosynthesis genes synX and synC caused defects in lipooligosac ...[more]

PMID: 8763931

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Project description:Serogroup B meningococci express sialic acids on their surfaces as a modification of the lipooligosaccharide (LOS) and as capsular material consisting of alpha2,8-linked sialic acid homopolymers. The aim of this study was to elucidate the impact of each sialic acid component on the deposition of complement factor C3 and serum resistance. For this purpose, we used isogenic mutants deficient in capsule expression (a polysialyltransferase mutant) or sialylation of the LOS (a galE mutant) or both (a mutant with a deletion of the cps gene locus). Bactericidal assays using 40% normal human serum (NHS) demonstrated that both the capsule and LOS sialic acid are indispensable for serum resistance. By immunoblotting with monoclonal antibody MAb755 that is specific for the C3 alpha-chain, we were able to demonstrate that C3 from 40% NHS was covalently linked to the surface structures of meningococci as C3b and iC3b, irrespective of the surface sialic acid compounds. However, C3b linkage was more pronounced and occurred on a larger number of target molecules in galE mutants with nonsialylated LOS than in meningococci with wild-type LOS, irrespective of the capsule phenotype. C3b deposition was caused by both the classical pathway (CP) and the alternative pathway of complement activation. Use of 10% NHS revealed that at low serum concentrations, C3 deposition occurred via the CP and was detected primarily on nonsialylated-LOS galE mutants, irrespective of the capsular phenotype. Accordingly, immunoglobulin M (IgM) binding to meningococci from heat-inactivated NHS was demonstrated only in both encapsulated and unencapsulated galE mutants. In contrast, inhibition of IgA binding required both encapsulation and LOS sialylation. We conclude that serum resistance in wild-type serogroup B meningococci can only be partly explained by an alteration of the C3b linkage pattern, which seems to depend primarily on the presence of wild-type LOS, since a serum-resistant phenotype also requires capsule expression.

Dataset Information

Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region.

Publications

Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region.

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