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Phase Variation inHelicobacterpylori Lipopolysaccharide

B J Appelmelk1,*,B Shiberu1,C Trinks1,N Tapsi1,P Y Zheng1,T Verboom1,J Maaskant1,C H Hokke2,W E C M Schiphorst2,D Blanchard3,I M Simoons-Smit1,D H vanden Eijnden3,CM J E Vandenbroucke-Grauls1
Departments of MedicalMicrobiology1 and MedicalChemistry,2 Vrije Universiteit, MedicalSchool, 1081 BT Amsterdam, The Netherlands, and RegionalBlood Transfusion Center, Nantes, France3
*

Corresponding author. Mailing address: Department ofMedical Microbiology, Vrije Universiteit, Medical School, van derBoechorststraat 7, 1081 BT Amsterdam, The Netherlands. Phone: 31 204448297. Fax: 31 20 4448318. E-mail:BJ.Appelmelk.mm@med.vu.nl.

Editor: J. R. McGhee

Received 1997 Jul 3; Revision requested 1997 Sep 5; Accepted 1997 Oct 10.

Copyright © 1998, American Society for Microbiology
PMCID: PMC107860  PMID:9423841

Abstract

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS)expresses the human blood group antigen Lewis x (Lex) in apolymeric form. Lex isβ-d-galactose-(1-4)-[α-l-fucose-(1-3)]-β-d-acetylglucosamine.Schematically the LPS structure is(Lex)n-core-lipid A. In thisreport, we show that Lex expression is not a stable traitbut that LPS displays a high frequency (0.2 to 0.5%) of phasevariation, resulting in the presence of several LPS variants in onebacterial cell population. One type of phase variation implied the lossof α1,3-linked fucose, resulting in variants that expressednonsubstituted polylactosamines (also called the i antigen), i.e.,Lex minus fucose; LPS:(lactosamine)n-core-lipid A. The switch ofLex to i antigen was reversible. A second group of variantsarose by loss of polymeric main chain which resulted in expression ofmonomeric Ley; LPS: (Ley)-core-lipid A. A thirdgroup of variants arose by acquisition of α1,2-linked fucose whichhence expressed Lex plus Ley; LPS:(Ley)(Lex)n-core-lipidA. The second and third group of variants switched back to the parentalphenotype [(Lex)n-core-lipid A]in lower frequencies. Part of the variation can be ascribed to alteredexpression levels of glycosyltransferase levels as assessed by assayingthe activities of galactosyl-, fucosyl-, andN-acetylglucosaminyltransferases. Clearly phase variationincreases the heterogeneity ofH. pylori, and this processmay be involved in generating the very closely related yet geneticallyslightly different strains that have been isolated from one patient.

Helicobacter pylori isinvolved in the pathogenesis of gastritis, gastric glandular atrophy,duodenal and gastric ulcer, gastric adenocarcinoma, andmucosa-associated lymphoid tissue lymphoma (12). Howbacteria belonging to a single species can give rise to such adiversity of disease entities is currently under investigation.H. pylori is genetically very diverse (9):strains from different sources all are different from each other at theDNA level (15). This genetic heterogeneity is in part due tothe natural competence ofH. pylori that renders thisbacterium naturally transformable (24). In addition, it hasbeen reported that recombinational events involving insertion elementsoccur (7). A mechanism used by other mucosal pathogens likeHaemophilus influenzae (19) andNeisseria spp. (30) to increase diversity isphase variation. Phase variation (also called antigenic variation) isthe reversible on-and-off switching of surface epitopes, e.g., thosepresent on adhesins or lipopolysaccharide (LPS). Switch frequencies of0.1% in these species are common. Genetically, this is paralleled byon-and-off switching of specific genes coding for surface structures,such as the glycosyltransferase genes involved in LPS biosynthesis.Often, those glycosyltransferase genes contained oligonucleotiderepeats in the 5′ end, and one mechanism for phase variation involvedreplication errors due to slipped-strand base pairing in the repeatregion. Recently, oligonucleotide repeats have been identified in thefucosyltransferase (FucT) genes ofH. pylori (8,16,23). The result of phase variation is a more versatile, moreheterogeneous microorganism that can cope better with a variety ofdifferent environments: with one set of genes switched on, themicroorganism may be able to adhere to mucosal cells of the nasopharynxbut not to survive complement attack in serum; with that particular setswitched off, the reverse may apply.

H. pylori LPS O antigen expresses Lewis x and/or y (Fig.1), and only 15% of the strains testedlacked these blood group antigens (22). The surfacelocalization of the bacterial Lewis antigens was demonstrated byimmunoelectron microscopy (6,21). The enzymatic activity oftwo enzymes involved in LPS O-antigen biosynthesis (α1,3-FucT andβ1,4-galactosyltransferase [GalT]) has been demonstrated (1,21), but it is not clear by what mechanisms serotype diversity isgenerated.

FIG. 1.

FIG. 1

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

Thus, theH. pylori O antigen expresses structures identicalto that of the corresponding human blood group antigens present in thegastric mucosa (molecular mimicry) (2–4,6,21,22). Theidentity of these LPS epitopes with molecules of the host may play arole in the induction ofH. pylori-associated autoantibodies(1,17,18) and facilitate persistence in the gastric nicheof humans (31).

We now report that expression ofH. pylori LPS in a givenstrain is not a stable trait and thatH. pylori LPS displaysphase variation. From a single strain, we isolated various serotypes,all expressing different LPS structures and having differentglycosyltransferase levels, which demonstrates that also inH.pylori, phase variation contributes to increased heterogeneity.

MATERIALS AND METHODS

Bacterial strains and cultivation procedures.

Weinvestigated three strains, all expressing Lewis x: the laboratorystrain NCTC 11637; strain 92-1152, isolated from a patient with gastriccarcinoma; and strain 3B3, isolated from the oral cavity of a patientwith gastritis (1,22). Bacteria were stored at −80°C in20% glycerol and were subcultured on solid medium (blood agar baseplus Dent supplement) before growth in the fluid phase (brucella brothwith 3% fetal calf serum) under microaerobic conditions (1,22). Apart from B1.3 (see below), none of the strainsautoagglutinated.

MAbs.

The following murine immunoglobulin M monoclonalantibodies (MAbs) were used: MAb 54.1F6A, specific for Lewis x (G. vanDam, Leiden, The Netherlands [25]); MAb 1E52, specificfor Lewis y (R. Negrini, Brescia, Italy [1,17,18]);MAb 19-O-Le, specific for H type 2 and Lewis y (Bioprobe, Amstelveen,The Netherlands [1]); MAb 4D2, specific for H type 1(R. Negrini [1,17,18]); and MAb NAM61-1A2, specificfor i antigen (D. Blanchard, Nantes, France [5]). MAbsspecific for Lewis x did not cross-react with H type 2/Lewis y or viceversa; the anti-i MAb did not react with Lewis x, H type 2, or Lewis y.

Isolation ofH. pylori LPS mutants after UVirradiation.

Overnight cultures of strain NCTC 11637 in brucellabroth plus 3% fetal calf serum were centrifuged, suspended in 1 MMgSO4, serially diluted in phosphate-buffered saline, pH7.5 (PBS), plated, and UV irradiated. Time, distance between plates UVlight source, and dilution were chosen such that 10% of bacteriasurvived, resulting in 300 colonies per plate. Individual colonies werepicked, transferred to each of two identical plates, and grown. Thecolonies were transferred to a nitrocellulose filter (0.45-μm poresize; Millipore Corporation, Bedford, Mass.) by pressing the filter tothe surface of the plate. Mutants not expressing Lewis x were isolatedas follows: colony blots were baked (1 h at 80°C), washed three timesin PBS with 0.05% Tween 80 (PBST), and blocked with blocking buffer(Boehringer Mannheim, Almere, The Netherlands). Then blots wereincubated overnight with anti-Lewis x MAb 54.1F6a, diluted in blockingbuffer-PBST (1:1) at a concentration of 1 μg/ml. Blots were washed,incubated (1 h, 37°C) with goat anti-mouse immunoglobulinM-peroxidase (American Qualex, La Mirada, Calif.), diluted 1:1,000 inPBST plus 0.5% preimmune goat serum, and developed as describedpreviously (29). Blots were inspected under astereomicroscope at magnifications of up to ×64 and, when necessary,photographed. Nonreactive colonies were identified and subcultured atleast twice, and purity was checked by colony blotting; finally, cellswere grown in fluid medium, washed in PBS, and used in enzyme-linkedimmunosorbant assay (ELISA) and sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) for further characterization. Amodification of the procedure described above was also used: thenitrocellulose blotting paper was put on the irradiated plates, whichresulted in almost complete transfer of the bacteria to the paper.Replicas of this first colony blot to a second and third nitrocellulosepaper were made. One of the nitrocellulose replicas was put, coloniesupward, on a fresh culture plate, a procedure that kept the bacteriaalive. The remaining two blots were immunoprobed.

Isolation of variants.

Spontaneously arising LPS variantswere isolated from bacteria not subjected to UV irradiation byfollowing the above-described modified protocol; i.e., bacteria werefirst grown in the fluid phase and distributed over solid media, afterwhich immunodetection took place.

ELISA.

The expression of various LPS epitopes on the parentstrain, mutants, and variants was measured in ELISA. Bacteria grown inthe fluid phase were washed in PBS, and coated at 7.5 ×106 CFU/ml on ELISA plates, and tested for reactivity withMAbs (1 μg/ml) as described elsewhere (1,22).

SDS-PAGE, silver staining, and immunoblotting.

The sizedistribution of LPS molecules of parent, mutants, and variants wasanalyzed by SDS-PAGE and silver staining. Bacterial cells were firstdigested with proteinase K and subjected to SDS-PAGE in 12% gels, andpart of the gel was silver stained for LPS (13,29). Tolocalize particular LPS epitopes to particular LPS components, part ofthe gel was electroblotted to nitrocellulose. LPS epitopes werevisualized as described above for colony blotting.

Glycosyltransferase assays.

GlcNAcβ-O-(CH2)8-COOCH3 wasgenerously donated by O. Hindsgaul (University of Alberta, Edmonton,Alberta, Canada),Galβ1→4GlcNAcβ-O-(CH2)8-COOCH3was derived therefrom as described before (11).GDP-[3H]Fuc (7.0 Ci/mmol), UDP-[3H]Gal (50Ci/mmol), and UDP-[3H]GlcNAc (30.4 Ci/mmol) werepurchased from New England Nuclear (Boston, Mass.). The radioactivenucleotide sugars were diluted to the desired specific radioactivitywith unlabeled GDP-Fuc (kind gift of H. Lönn and T. Nordberg,Biocarb, Lund, Sweden), UDP-Gal, and UDP-GlcNAc (Sigma), respectively.

Cells (1010) of each of the variants cultivated asdescribed above were sonicated in 250 μl of PBS at 0°C, using amicroprobe tip (five bursts of three s each, with intermittent coolingfor 2 min). The resulting suspension was used as an enzyme preparationin the glycosyltransferase assays. Incubation mixtures contained, in 50μl, the following: for GalT, 5 μmol of sodium cacodylate buffer (pH7.0), 1 μmol of MnCl2, 0.2 μmol of ATP, 0.25 μl ofTriton X-100, 25 nmol of UDP-[3H]Gal (specificradioactivity, 1 Ci/mol), 50 nmol ofGlcNAcβ-O-(CH2)8-COOCH3,and 1 μl of enzyme; forN-acetylglucosaminyltransferase(GlcNAcT), 5 μmol of sodium cacodylate buffer (pH 7.0), 1 μmol ofMnCl2, 0.2 μmol of ATP, 0.25 μl of Triton X-100, 25nmol of UDP-[3H]GlcNAc (specific radioactivity, 0.75Ci/mol), 50 nmol ofGalβ1→4GlcNAcβ-O-(CH2)8-COOCH3,and 10 μl of enzyme; and for FucT, 5 μmol of sodium cacodylatebuffer (pH 7.2), 1 μmol of MnCl2, 0.2 μmol of ATP, 0.25μl of Triton X-100, 50 nmol of dithiothreitol, 25 nmol ofUDP-[3H]Fuc (specific radioactivity, 6.9 Ci/mol), 50 nmolofGalβ1→4GlcNAcβ-O-(CH2)8-COOCH3,and 10 μl of enzyme. Mixtures were incubated for 1 h (GalT) or5 h (GlcNAcT and FucT) at 37°C. Radioactivity incorporated intothe acceptor substrate was determined by liquid scintillation countingafter isolation of the products by using Sep-Pak C18cartridges (Waters, Milford, Mass.) as described previously(6). Control assays lacking acceptor were carried out tocorrect for incorporation into endogenous substrates. One unit ofenzyme activity is defined as the amount of enzyme catalyzing thetransfer of 1 μmol of sugar per min.

RESULTS

Phase variation in anH. pylori LPS mutant.

Following UV irradiation, an LPS mutant (named B1.5) that lacked Lewisx expression and was strongly positive for Lewis y was isolated (forfurther characterization, see below). A colony blot of this mutant,probed with MAb 4D2, specific for H type 1, is shown in Fig.2A. There are completely nonreactivecolonies, colonies with reactive sectors only, and completely reactive(dark) colonies; this pattern indicates a spontaneous high-frequencyclonal on switching of the H type 1 epitope.

FIG. 2.

FIG. 2

(A) Colony blot of LPS mutant B1.5 probed with MAb 4D2Mab (anti-H type 1). (B) Colony blot of nonirradiated strain NCTC 11637probed with MAb NAM61-1A2 (anti-i). (C) Colony blot of nonirradiatedstrain NCTC 11637 probed with MAb 19-O-Le (anti-Lewis y). (D) Colonyblot of nonirradiated strain NCTC 11637 probed with MAb 54.1F6A(anti-Lewis x). Variant 1c (Created by potrace 1.16, written by Peter Selinger 2001-2019) expresses both Lewis x and y; variant1b (Created by potrace 1.16, written by Peter Selinger 2001-2019) expresses Lewis y but lacks Lewis x.

Isolation of LPS variants from nonirradiated bacterial cells anddemonstration of reversibility.

Nonirradiated cells of strain NCTC11637 and the two clinical isolates investigated also displayed phasevariation behavior (Fig.2B) but at a lower frequency. Figures1 and2Bshows that NCTC 11637, which expresses Lewis x, has the ability toswitch spontaneously to the expression of i antigen; strains expressingthe i antigen did not express Lewis x (Table1). Several of the colonies reactive withthe anti-i MAb but nonreactive with anti-Lewis x, including variantK4.1, were subcultured. K4.1 was tested again to detect whether itcould switch back to phenotype of the parent strain NCTC 11637. Indeed,K4.1 spontaneously switched to variants that reacted with anti-Lewis xMAbs but not with the anti-i MAb. The frequency of switching from Lewisx to i was determined in multiple experiments and equaled 49/11,080(0.44%); the switchback frequency was 17/3,095 (0.55%). One of theswitched-back variants (strain K5.1 [Table1]) was isolated.Switching from Lewis x to i-antigen expression also took place in thetwo clinical isolates tested (not shown). Forty percent of the 106clinical isolates tested proved positive for the i antigen. Parallelcolony blots of NCTC 11637 probed with 19-O-Le (anti-Lewis y) and54.1F6A (anti-Lewis x) (Fig.2C and D, respectively) showed thepresence of two other variants, i.e., variants that strongly expressedboth Lewis x and y (for example, strain 1c [see below]; frequency,0.5%) and variants that were strongly positive for Lewis y but did notexpress Lewis x (for example, strain 1b [see below]; frequency,1.5%). The majority of the colonies strongly expressed Lewis x andreacted weakly with the anti-Lewis y MAb. Three such colonies (2a, 2b,and 3c) were subcultured and characterized. Their reactivity patternwas identical to that of the parental strain (Table1).

TABLE 1.

Characterization ofH. pylori NCTC 11637 LPSmutants and variants

StrainELISAreactivity with MAbsa
Anti-Lewis yAnti-LewisxAnti-H type 1Anti-i
NCTC11637+++++++−
Mutants
 B1.5++−+−
 B2.1++−+−
 K1.4−−++
 K2.1−−++++
 K3.1−−++++
 D1.1b−−−+c
Variants
 K4.1−−+++
 K5.1−++++−
 B3.1+++−++−
 1b+++−+−
 1c++++++++−
 2a+++++++−
 2b++++++−
 3c++++++−
 3a−++++d−
a

−, optical density (OD) < 0.3; +,0.3 < OD < 1.3; ++, 1.3 < OD < 2.3; +++,OD > 2.3. 

b

D1.1 was isolated as an anti-H type1-negative variant of the K3.1 mutant. 

c

OD < 0.35. 

d

OD < 0.8. 

Immunochemical analysis of variants.

Immunochemical data forall mutants and variants isolated by us from strain NCTC 11637 areshown in Table1 and Fig.3 and4. Variant K4.1 (Fig.3a) expressed anLPS similar to that of the parent strain; both in ELISA and in blotanalysis, K4.1 (Fig.3b) reacts with the anti-i MAb but not withanti-Lewis x. Variant K5.1, a switchback variant isolated from K4.1,behaves serologically like the parent strain: the O antigen expressedLewis x, not i antigen. We hypothesized that the switch from parent toK4.1 implied the loss of α1,3-fucose, which was regained uponswitching from K4.1 to K5.1. Serologically, the mutants K1.4, K2.1, andK3.1 were identical to the K4.1 variant. Figure4 shows that variant 1bhad lost its polymeric main chain and now synthesized alow-molecular-mass LPS expressing Lewis y but not Lewis x.Serologically, mutants B1.5 and B2.1 are identical to variants B3.1 and1b. Strain 1c expressed a polymeric Lewis x ladder, runs of the ladderbeing capped with Lewis y and with slightly more runs in thelower-molecular-weight zone compared to the parent. Variant 3a reactedless than the parent or not at all with 4D2 in ELISA or blot analysis,respectively. This variant expressed LPS that upon SDS-PAGE yielded asmear instead of a ladder (see silver stain and blot in Fig.4).

FIG. 3.

FIG. 3

(a) SDS-PAGE and silver stain. Lane 1, NCTC 11637; lane2, K4.1; lane 3, K5.1. (b) Blot (after SDS-PAGE) probed with MAb54.1F6A (anti-Lewis x). (c) Blot probed with MAb NAM61-1A2 (anti-i).

FIG. 4.

FIG. 4

(a) SDS-PAGE and silver stain. Lane 1, NCTC 11637; lane2, 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).

Additional data on switch frequencies ofH. pylori NCTC11637, LPS mutants, and variants are shown in Table2. The switching behavior of mutants wassimilar to that of the variants: mutant K1.4 (Lewis x−,i+) switched back to Lewis x+, i−at a frequency of 0.2%. One K1.4 derived switchback variant (calledK6.1 [data not shown]) was isolated and found to be able to switch toLewis x−, i+, which is further proof of thereversibility of the Lewis x-to-i-antigen switch. In contrast, mutantsand variants that were Lewis x−, Lewis y+ didnot switch back (mutant B1.5, variant B3.1) or did so in extremely lowfrequencies (variant 1b, switchback frequency, 1/1,400). We also didnot find switchbacks of variant 1c (Lewis x+, Lewisy+). The serotype that most often is expressed by coloniesof NCTC 11637 (strong Lewis x+, weak Lewis y+;variants 2b and 3c) switched to strong expression of Lewisy+ at frequencies of 0.28 to 1%.

TABLE 2.

Frequencies of LPS phase variation inH.pylori NCTC 11637, its mutants, and its variants

StrainMAbEstimated no. of colonies on blotsNo. (%) ofpositive colonies
K1.4mutantAnti-Lewisx3,0005 (0.2)
K6.1variantaAnti-i5,00039 (0.78)
B1.5mutantAnti-Lewis x9000 (0)
B3.1 variantAnti-Lewisx6,3860 (0)
1b variantAnti-Lewisx1,4001 (0.07)
2b variantAnti-Lewisy1,85019 (1)
3c variantAnti-Lewisy6,40018 (0.28)
1c variantAnti-Lewisx3,2000b
Anti-Lewis y
a

K6.1 is a phase variant (Lewisx+,i−) isolated from K1.4 mutant. 

b

Number of negative colonies. 

Glycosyltransferase assays.

To study the enzymatic basis forthe phenotypic differences observed, the activities of three relevantglycosyltransferases were assayed in some of the variants. Variousactivities were found (Table3), withhighest GalT activity in the parental strain (NCTC 11637) and highestFucT activity in variant 1c, while the activity of GlcNAcT was belowthe detection limit in variant 1b. Except for the latter variant, theGlcNAcT, GalT, and FucT activities appeared to vary over ranges of 2-,8-, and 60-fold, respectively, suggesting that the gene coding for thelatter enzyme in particular might be a target of mechanisms that causesphase variations.

TABLE 3.

Glycosyltransferase activities inH. pylori11637 LPS variants

Strain orvariantGlycosyltransferase activity
GalT (mU/1010cells)GlcNAcT (μU/1010cells)FucT (μU/1010 cells)
NCTC1163720.033.846.0
K4.19.359.415.3
K5.16.728.23.6
1b2.8<0.119.6
1c7.329.9216

DISCUSSION

In this report, we show thatH. pylori LPS displaysphase variation. Reversible Lewis x-to-i-antigen switching wasobserved, at frequencies of about 0.4%. We also observed switchesimplying loss of polymeric main chain (Lewis x−, Lewisy+; for example, variant 1b) as well as switches implyingstrong expression of Lewis y (Lewis x+, Lewisy+; for example, variant 1c). By chemical means, strainNCTC 11637 was shown to express Lewis x (2), but in the pastwe have described the presence of low amounts of Lewis y in this strainas measured by serology (1); this finding was confirmed inthis study. We assume that serology is more sensitive thanstructural-chemical methods. The presence of nonfucosylatedpolylactosamine stretches (i.e., the i antigen) has already beendemonstrated by chemical methods (2). Our report is thefirst one on phase variation inH. pylori LPS; however,Bukholm et al. (5a) have reported the occurrence ofreversible variants in colony morphology.

In previous reports (1,18), we have suggested a pathogenicrole forH. pylori LPS-induced anti-Lewis antibodies thatcross-react with gastric mucosal antigens of the host. To further studythe role of Lewis x, we isolated UV-induced Lewis x−mutants (Table1). By serendipity, we discovered that a mutant (B1.5[Fig.2A]) displayed clonal expression of the epitope recognized byMAb 4D2, specific for H type 1 antigen. However, no H type 1 antigencan be detected in LPS of strain NCTC 11637 by structural-chemicalmeans, and possibly the reactivity with strain 11637 represents across-reaction. Due to these uncertainties, we did not study phasevariation of this epitope any further.

In contrast, the events taking place during phase variation of Lewis xto i antigen (and vice versa) can be understood at the molecular level,based on serological data (Table1; Fig.3) and on the primarystructure of the LPS ofH. pylori NCTC 11637 (Fig.1). Wepostulate that the Lewis x antigen of strain 11637 switches to the iantigen by loss of the α1,3-linked fucose. A summary of thepostulated LPS structures of the various variants and their possibleinterrelationships is shown in Fig.5.The chemical structures of the LPS of the variants is underinvestigation. The presence of FucT enzymatic activity inH.pylori has been reported (1,6), and hence it wasconceivable that this Lewis x-to-i-antigen switch actually involved theon and off switching of the gene coding forH. pyloriα3-FucT. Variant K4.1 shows FucT activity yet is Lewis x negative.Whether the measurement of FucT activities of a value in this respectis not clear, because in the past it has been shown that strainsstrongly positive for Lewis x may have lower FucT activities thanstrains expressing less Lewis x (6). It is also conceivablethat genes coding for enzymes of the pathway leading to the donorsubstrate for FucT, GDP-Fuc, are involved in the Lewis x-to-i phasevariation.

FIG. 5.

FIG. 5

Proposed schematic LPS structures of variants and theirinterrelationships. The actual number of Lewis x repeats in NCTC 11637is higher than indicated. The question mark in the NCTC 11637 LPSstructure 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.

We observed the occurrence of i antigen to Lewis x switches first instrains that had been obtained from NCTC 11637 after UV irradiation(strains K1.4, K2.1, and K3.1). Later, switches were shown to occuralso in nonirradiated cells. The reversibility of the phenomenon wasinvestigated extensively, and we demonstrated that both spontaneousLewis x-to-i-antigen switches and i antigen-to-Lewis x switchesoccurred at the same frequency, 0.44 and 0.55%, respectively. By usingour colony blot procedures, we succeeded in isolating variants that hadspontaneously switched from Lewis x to i antigen (e.g., K4.1) and avariant (K5.1) that had switched back again from i antigen to Lewis x.Mutants K1.4, K2.1, and K3.1 (all i+) also switched backspontaneously to Lewis x-positive cells, and one Lewis x-positivevariant (K6.1 [data not shown]) that again switched to i+at a frequency of 0.78% was isolated. In short, phase variation in theLewis x to i antigen is reversible.

The presence in human sera of antibodies to the i antigen has beenreported; these antibodies belong to the group of so-called coldagglutinins (CAs) (20). CAs recognize epitopes onerythrocytes and cause agglutination. As manyH. pyloristrains express the i antigen, it is possible that they induce CAs.

Second, we also studied mutants and variants that strongly expressedLewis y and were negative for Lewis x (B1.5, B2.1, B3.1, and 1b [Table1; Fig.4]). Analysis of the lengths of these LPSs by SDS-PAGE showedthat the polymeric main chain was lost. Apart from the core, only asingle band could be seen. Strains that express Lewis y only, asdetermined both by chemical-structural analysis and by serology, havebeen described before (strain MO19 [4] and serotype O6[1b]), and likely strains B1.5, B2.1, B3.1, and 1b aresimilar to MO19 and serotype O6 (Fig.1). The single, Lewisy-expressing band (lanes 2 in Fig.4a and5b) likely represents asingle Lewis y unit, covalently linked to the core. In contrast to thereadily detected reversible phase variation Lewis x to i antigen, theswitch from Lewis y to Lewis x was nondetectable in mutants B1.5 andvariant B3.1 and with only one observed event of backswitching instrain 1b (Table2). Again the similarity of the mutant and thevariants is striking, and it is possible that those strains that weclassified as mutants because they were isolated after UV irradiationsimply are spontaneous phase variants.

In variant 1b, the switch from polylactosamine with multimeric Lewis xto a single, Lewis y-carrying lactosamine unit appears to be due to acomplete turn off of a chain-elongating GlcNAcT, as no activity wasfound with a substrate specific for such an enzyme. In mammals, theGlcNAcTs that link GlcNAc residues to the core regions of protein- andlipid-linked glycans differ from the aglycon-nonspecific β3-GlcNAcTthat is involved in the elongation of lactosamine chains (26,27). Because in variant 1b only one lactosamine unit is present,it is likely that also inH. pylori the attachment of aGlcNAc to thed-glycero-α-d-manno-heptoseunit of the LPS core is catalyzed by a GlcNAcT that differs from theelongating enzyme. Likely, the high expression of Lewis y by variant 1bis caused by the absence of the GlcNAcT, which may compete with theα2-FucT for linking a glycosyl group to galactose. In addition, apreferred action of the α2-FucT on short lactosamine chains ratherthan elongated ones is possible.

Third, a switch from Lewis x to Lewis x plus Lewis y was observed(strain 1c [Table1; Fig.4]). This variant expressed polymeric Lewisx, forming a ladder structure like its parent strain but with a muchstronger expression of Lewis y; i.e., terminal α1,2-linked fucose isstrongly increased. Strains with this phenotype, as determined bystructural analysis and serology, have been described before (strainP466 [4] and serotype O3 [1b]). Wedid not observe backswitches of 1c to the parent phenotype. Themechanism involved may be a switching from low to high levels ofexpression of α2-FucT. It is known that inH. influenzaeLPS, phase variation may involve both off-to-on switches and switchesfrom low to high levels of expression (10).

The substrate used to assay FucT activity[Galβ1→4GlcNAcβ-O-(CH2)8-COOCH3]did not allow us to discriminate between α2- and α3-FucT.Previously it has been shown that using the same acceptor substrate theFucT in several strains ofH. pylori only catalyzed thetransfer of Fuc to GlcNAc in α1→3 linkage to yield the Lewis xstructure (6). The high FucT activity in variant 1c might,however, in part be due to a strongly enhanced activity of an α2-FucTleading to the high expression of Lewis y. No such transfer could bedemonstrated in assays using a substrate(Galβ-O-para-nitrophenol) that is known as a specificacceptor for mammalian α2-FucTs (data not shown). It has, however,been suggested that in the formation of Lewis y inH.pylori, α2-fucosylation may have to precede α3-fucosylation(16). Thus, it is conceivable that the high FucT activity invariant 1c reflects the consecutive transfers of Fuc residues inα1→3 and α1→2 linkage. Thus, α2-FucT may be involved in theswitch from parental type to variant 1c.H. pylori FucTgenes have been identified (8,16,23) and expressed(8,16); these genes contain poly(C) tracts. Interestingly,in strain NCTC 11637 (expressing Lewis x), the reported FucT genecontains a C9 tract (gene off, truncated enzyme), while in strain826695 (expressing Lewis xy [1a]), C13 is present(gene on, full-length enzyme). These data suggest the possibility thatthe expression of serotypes is regulated on the genetic level by meansof variable-length oligonucleotide repeats. They also suggest that themechanism of phase variation inH. pylori may mimic that ofH. influenzae. When screened with anti-Lewis y MAbs, most ofthe colonies of strain NCTC 11637 stained only faintly, and wedemonstrated that those faintly staining colonies (that also stronglyexpress Lewis x) are the ones that switch to the variant with a highexpression of Lewis y (i.e., variants 1b and 1c [Fig.2]).

Finally, variant 3a that expressed LPS that was less reactive or notreactive with MAb 4D2 did not yield the characteristic LPS ladderpattern. As yet, we have no indication as to the structural change thathas taken place in this variant.

Phase variation contributes to the heterogeneity ofH.pylori and may explain the finding that from one patient, severalhighly related yet different isolates may be obtained (28,32). Whether phase variation-induced heterogeneity is functionalor merely an epiphenomenon has not yet been assessed. The phenomenonoccurred not only in the laboratory strain NCTC 11637, which hasundergone many in vitro passages, but also in clinical isolates thathad been passaged only a few times. Phase variation in LPS ofN.gonorrhoeae orH. influenzae serves a biological role,one variant being more adequate in one situation (e.g., adherence tomucosal cells) and the other one being more resistant to killing bycomplement. It would be interesting to investigate if variants of onestrain can be isolated differentially from different gastric sites ofone patient or experimental animal (14) and to investigatewhether certain variants adhere better. Further studies are required toassess the molecular mechanism of phase variation inH.pylori LPS and its biological relevance.

ACKNOWLEDGMENTS

We thank S. L. Martin (GlaxoWellcome, Stevenage, England),D. E. Taylor (University of Alberta, Edmonton, Alberta, Canada),and G. O. Aspinall (York University, North York, Ontario, Canada)for providing unpublished data. We thank R. Negrini (Brescia, Italy)and G. Van Dam (Leiden, The Netherlands) for providing MAbs.

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