Phase variation in Helicobacter pylori lipopolysaccharide

Abstract

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le(x)) in a polymeric form. Le(x) is beta-D-galactose-(1-4)-[alpha-L-fucose-(1-3)]-beta-D-acetylglucosamine. Schematically the LPS structure is (Le(x))n-core-lipid A. In this report, we show that Le(x) expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of alpha1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le(x) minus fucose; LPS: (lactosamine)n-core-lipid A. The switch of Le(x) to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le(y); LPS: (Le(y))-core-lipid A. A third group of variants arose by acquisition of alpha1,2-linked fucose which hence expressed Le(x) plus Le(y); LPS: (Le(y))(Le(x))n-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le(x))-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and N-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

FIG. 1

Structures of Lewis-related antigens and H. pylori LPS. Abbreviations: Gal, d-galactose; Fuc, l-fucose; GlcNAc, N-acetyl-d-glucosamine.

FIG. 2

(A) Colony blot of LPS mutant B1.5 probed with MAb 4D2 Mab (anti-H type 1). (B) Colony blot of nonirradiated strain NCTC 11637 probed with MAb NAM61-1A2 (anti-i). (C) Colony blot of nonirradiated strain NCTC 11637 probed with MAb 19-O-Le (anti-Lewis y). (D) Colony blot of nonirradiated strain NCTC 11637 probed with MAb 54.1F6A (anti-Lewis x). Variant 1c () expresses both Lewis x and y; variant 1b () expresses Lewis y but lacks Lewis x.

FIG. 3

(a) SDS-PAGE and silver stain. Lane 1, NCTC 11637; lane 2, K4.1; lane 3, K5.1. (b) Blot (after SDS-PAGE) probed with MAb 54.1F6A (anti-Lewis x). (c) Blot probed with MAb NAM61-1A2 (anti-i).

FIG. 4

(a) SDS-PAGE and silver stain. Lane 1, NCTC 11637; lane 2, strain 1b; lane 3, strain 1c; lane 4, strain 2b; lane 5, strain 3a; lane 6, strain 3c. (b) Blot probed with MAb 54.1F6A (anti-Lewis x). (c) Blot probed with MAb 1E52 (anti-Lewis y).

FIG. 5

Proposed schematic LPS structures of variants and their interrelationships. The actual number of Lewis x repeats in NCTC 11637 is higher than indicated. The question mark in the NCTC 11637 LPS structure indicates that this LPS contains nonfucosylated lactosamines, adjacent to the core. dd-Hep, d-glycero-d-manno-heptose. For other abbreviations, see the legend to Fig. 1.