Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide.

Abstract

StlmmaryA locus involved in the biosynthesis of gonococcal lipooligosaccharide (LOS) laas been cloned from gonococcal strain F62. The locus contains five open reading frames. The first and second reading frames are homologous, but not identical, to the fourth and fifth reading frames, respectively.Interposed is an additional reading frame which has distant homology to the Escherichia coli rfaI and r~J genes, both glucosyl transferases involved in lipopolysaccharide core biosynthesis. The second and fifth reading frames show strong homology to the lex-1 or lic2A gene of Haernophilus influenzae, but do not contain the CAAT repeats found in this gene. Deletions of each of these five genes, of combinations of genes, and of the entire locus were constructed and introduced into parental gonococcal strain F62 by transformation. The LOS phenotypes were then analyzed by SDS-PAGE and reactivity with monoclonal antibodies. Analysis of the gonococcal mutants indicates that four of these genes are the glycosyl transferases that add GalNAc/31 ~ 3Gal~l 4GlcNAcB1 ~ 3Gal/31 ~ 4 to the substrate GlcB1 ~ 4Hep ~ R of the inner core region.The gene with homology to E. coli ~I/rfaJ is involved with the addition of the or-linked galactose residue in the biosynthesis of the alternative LOS structure Galotl -~ 4Gal/31 ~ 4G1c~1 ~ 4Hep R. Since these genes encode LOS glycosyl transferases they have been named IgtA, lgtB, IgtC, IgtD, and IgtE. The DNA sequence analysis revealed that lgtA, IgtC, and IgtD contained poly-G tracts, which, in strain F62 were, respectively, 17, 10, and 11 bp. Thus, three of the LOS biosynthetic enzymes are potentially susceptible to premature termination by reading frame changes. It is likely that these structural features are responsible for the high-frequency genetic variation of gonococcal LOS.W 'hile Neisseria species commonly colonize many mammalian hosts, human beings are the only species subject to invasive disease by members of this species. Neisseria rneningitidis is the etiologic agent for septicemia and meningitis that may occur in epidemic form. Neisseria gonorrhoeae is the causative agent of gonorrhea and its manifold complications. These organisms, particularly the gonococcus, have proved remarkably adept at varying the antigenic array of their surface-exposed molecules, notably their adhesive pili and opacity-related proteins. The genetic mechanisms for the variation of pilus (1-4) and opacity-related (5-7) protein expression are in the main well understood. Like other gramnegative bacteria the Neisseria species carry LPS in the external leaflet of their outer membranes (8). In contrast to the high molecular weight LPS molecules with repeating O-chains seen in many enteric bacteria, the LPS of Neisseria species is of modest size and therefore is often referred to as lipooligosaccharide (LOS) 1. Although the molecular size of 1 Abbreviations used in this paper: CMP-NANA, N-acetyl neuraminic acid cytidine monophosphate; GC, gonococcus; LOS, lipooligosaccharide; wt, wild-type.

Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyl transferases.

“…The desired clones were identified by colony hybridization (15) using 32p-labeled oligonucleotide matching the reverse complement of 2573-2589. wild type (wt) gives rise to two principal LOS bands, the lacto-N-neotetraose structure with and without the terminal GalNAc substitution. F62 Al expresses mainly Gakxl-4Gal~l-4Glc as cl chain, whereas F62 AS is limited to a lactosyl group (9). Note that F62 wt and the two MS11 variants give patterns identical to those published by Kerwood et al (2).…”

“…GC on GC agar (12) were grown overnight, suspended and diluted in proteose peptone liquid medium (9), spread on GC agar, and allowed to grow for "'18 h to yield ,'ol,000 small colonies. The agar cultures were overlaid with nitrocellulose filters (Schleicher & Schuell, Inc., Keene, NH), lifted off, and air-dried for 60 rain.…”

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

“…Genotyping N. meningitidis LOS-We determined the LOS genotypes of 7946, 8024, and 7993 by sequencing lgtA, lgtB, lgtE (28,32), lgtG (33), lpt3 (34), and lpt6 (35) with the use of primers in Table 3. The 6GF primers amplify both lpt6 and lgtG, which are on opposite strands, along with short flanking regions.…”

“…We used PCR to amplify lgtABCDE from 1291wt and the 1291a-c mutants with the use of primers in Table 3 (28,29) and sequenced the resulting amplicons. We used the DNA sequence numbering for the lgtABCDE region of gonococcal strain, F62, as described in the National Center for Biotechnology Information database under accession number U14554 to orient the sequences to the literature (29).…”

Structural and Immunochemical Characterization of aNeisseria gonorrhoeae Epitope Defined by a Monoclonal Antibody 2C7; the Antibody Recognizes a Conserved Epitope on Specific Lipo-oligosaccharides in Spite of the Presence of Human Carbohydrate Epitopes

“…12), which confirmed a previous suggestion (13). Recent studies have identified genes encoding a set of glycosyltransferases for the biosynthesis of the specific OS structures (21,32,33). Gonococci add a glycose moiety sequentially on the lactose of Hep [1] and synthesize a lactoneotetraose and its N-acetylgalactosaminylated form.…”