Expression of the BabA adhesin during experimental infection with Helicobacter pylori

Abstract

The Helicobacter pylori babA gene encodes an outer membrane protein that mediates binding to fucosylated ABH antigens of the ABO blood group. We recently demonstrated that BabA expression is lost during experimental infection of rhesus macaques with H. pylori J166. We sought to test the generality of this observation by comparison of different H. pylori strains and different animal hosts. Challenge of macaques with H. pylori J99 yielded output strains that lost BabA expression, either by selection and then expansion of a subpopulation of J99 that had a single-base-pair mutation that encoded a stop codon or by gene conversion of babA with a duplicate copy of babB, a paralog of unknown function. Challenge of mice with H. pylori J166, which unlike J99, has 5' CT repeats in babA, resulted in loss of BabA expression due to phase variation. In the gerbil, Leb binding was lost by replacement of the babA gene that encoded Leb binding with a nonbinding allele that differed at six amino acid residues. Complementation experiments confirmed that change in these six amino acids of BabA was sufficient to eliminate binding to Leb and to gastric tissue. These results demonstrate that BabA expression in vivo is highly dynamic, and the findings implicate specific amino acid residues as critical for binding to fucosylated ABH antigens. We hypothesize that modification of BabA expression during H. pylori infection is a mechanism to adapt to changing conditions of inflammation and glycan expression at the epithelial surface.

FIG. 1.

Leb adherence ratio for individual H. pylori colonies isolated from two rhesus macaques (30364 and 31105) 1 to 18 weeks after inoculation with strain H. pylori J99. H. pylori attachment to Leb decreased significantly with time after inoculation (Kruskal-Wallis test, two tailed, P < 0.001).

FIG. 2.

Percentage of H. pylori output colonies with an ORF for babA (▪) and babB (░⃞) 2 weeks postinoculation with strain J166 in three strains of mice (n = 4 to 6 mice/strain). Percentages were calculated as a fraction of total colonies (13 to 32 per strain) isolated from each mouse strain. An ORF was present if DNA sequencing demonstrated the presence of eight CT dinucleotide repeats in the 5′ end of babA. Out of frame sequences had seven or nine CT repeats, which resulted in a stop codon at bp 78 (addition of CT) or bp 48 (loss of CT).

FIG. 3.

Verification of expression and Leb adherence in H. pylori J166 with a babA ORF. Immunoblot of representative single-colony isolates from mouse output strains with an in frame (ORF+) or out of frame (ORF−) babA gene probed with antibody to BabA (top panel). Wild-type H. pylori J166 (J166WT) and an isogenic babA knockout (J166rbabA) served as controls. Only strains with the babA ORF expressed protein. Analysis of Leb binding (bottom panel) of H. pylori J166 single-colony mouse output strains showed that those with a babA ORF showed significantly greater attachment to Leb (Mann-Whitney U test, two tailed, P < 0.001).

FIG. 4.

Schematic representation of the gene conversion event that occurred in H. pylori strain 7.13 after 12 weeks of colonization in gerbils. Input strain 7.13 has an expressed BabA2 and a silent BabA1, which has one additional adenine residue in a polynucleotide tract (7A versus 6A) that begins immediately after the ATG start codon and results in a stop codon at bp 61. Shown are the 11 amino acid residues (single-letter designations numbered according to the mature protein after removal of the signal peptide) for BabA1 that differ from those predicted in BabA2 if the protein were expressed. Gerbil output strain 8.31 has undergone gene conversion of babA2 by babA1. The recombination occurred so as to retain the 6A polynucleotide tract, so BabA2 is expressed, but the replacement of 6 amino acids from BabA2 with those from BabA1 (shaded) is sufficient to eliminate binding to Leb.

FIG. 5.

Gastric histosections from gerbils that were uninfected (A to D) or infected (E to H) with H. pylori. Hematoxylin and eosin stained sections (A and E) demonstrate the marked cellular infiltrate and loss of normal epithelial architecture that is characteristic of H. pylori gastritis. Incubation of uninfected tissue sections with FITC-labeled bacteria showed that H. pylori 7.13, which binds Leb, also attached to gastric tissue (B), whereas strains 8.31 (C) and 7.13 expressing the 8.31 babA (D) did not. Interestingly, none of the strains bound appreciably to infected gerbil tissue (F to H), suggesting that H. pylori infection reduces expression of the BabA receptor. The percentage of epithelium with bound bacteria was determined as described in Materials and Methods and is shown in the upper right of each panel.