Characterization of the Gene Cassette Required for Biosynthesis of the (α1→6)-Linked N -Acetyl- d -Mannosamine-1-Phosphate Capsule of Serogroup A Neisseria meningitidis

Abstract

ABSTRACT The (α1→6)-linked N -acetyl- d -mannosamine-1-phosphate meningococcal capsule of serogroup A Neisseria meningitidis is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene, ctrA , and galE , encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from ctrA by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to ctrA and that transcription of the ORFs was initiated from the intergenic region by a ς-70-type promoter that overlapped the ctrA promoter. The first ORF exhibited 58% amino acid identity with the UDP- N -acetyl- d -glucosamine (UDP-GlcNAc) 2-epimerase of Escherichia coli , which is responsible for the conversion of UDP-GlcNAc into UDP- N -acetyl- d -mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A N. meningitidis -specific operon located between ctrA and galE .