Identification of the gene ( lgtG ) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from Neisseria gonorrhoeae

Abstract

The lipooligosaccharide from Neisseria gonorrhoeae (GC), consists of lipid A, an oligosaccharide core and three branches, ␣, ␤, and ␥. We report the cloning of the gene (lgtG, lipooligosaccharide glycosyl transferase G) encoding the glucosyl transferase of GC that initiates the ␤ chain which consists of a lactosyl moiety. This gene contains a homopolymeric tract of cytidine [poly(C)] and we demonstrate that changes in the number of Cs in poly(C) account for the variation of ␤ chain expression in different GC strains. Biochemical analyses and mass spectrometry clearly attribute the reactivity of mAb 2C7 to the presence of the lactosyl ␤ chain. In addition, we demonstrate that in the absence of the lactosyl group, a phosphoethanolamine is added to generate a new antigenic epitope as evidenced by the gain of reactivity to mAb 2-L1-8. These results show that, like the ␣ chain, the ␤ chain of lipooligosaccharide is subject to antigenic variation.

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

“…mAb 2C7 bound none of the gonococcal LOS tested, which confirmed partial results obtained by us (18) and by Banerjee et al (21). In addition, we re-examined the binding of mAb 2C7 to 24-1 LOS.…”

“…The necessity of at least one (and perhaps both) of the Gal residues for expression of the 2C7 epitope was supported by the fact that mAb 2C7 bound to the 15253 lgtE mutant LOS that lacks the Gal moiety of each lactose on Hep [1] and Hep [2] (Table IV). These results do not support the previous suggestion that the lactose on Hep [2] is the 2C7 epitope (21).…”

“…Although we were not be able to determine which Gal residue was removed from the two lactosyl moieties present in the LOS, this lack of 2C7 binding to the ␤-galactosidase-treated 15253 LOS is clear evidence that a ␤-Gal residue at the non-reducing end is indispensable for the epitope expression. The importance of the ␤-Gal residue was also supported by the fact that mAb did not bind 15253 lgtE mutant LOS (lane 3) that lacks a Gal residue on each lactose attached to Hep [1] and Hep [2] of 15253 LOS (21). These binding studies showed that the ␤-Gal on the nonreducing end of 15253 LOS is essential for the 2C7 epitope to be expressed.…”

“…Second, inert binding of the mAb to some LOS samples has shown that LOS lacking OS on the Hep [2] may not have the 2C7 epitope (18,20). Third, immunoblot analysis using truncated gonococcal LOS has suggested the importance of a lactose on Hep [2] for expression of 2C7 epitope (21). However, those previous studies have not yet precisely confirmed whether mAb 2C7 recognizes gonococcal LOS of a specific OS elongation pattern.…”

“…Among LOS recognized by the mAb 2C7 (7,18,21), LOS produced by an isolate from disseminated gonococcal infection (DGI), 15253, is the only one whose OS structure has been determined (13). Definition of LOS structure other than 15253 LOS is essential for us to determine not only the specific OS elongation pattern that mAb 2C7 recognizes but also the common epitope expressed among various gonococcal LOS.…”