Genotypic profile of the outer membrane proteins BabA and BabB in clinical isolates of Helicobacter pylori.
ABSTRACT: Helicobacter pylori BabA is the ABO blood group antigen binding adhesin, which has a closely related paralogue (BabB) whose function is unknown. PCR and DNA sequence analysis showed extensive genotypic diversity in babA and babB across different strains, as well as within a strain colonizing an individual patient. We hypothesize that diverse profiles of babA and babB reflect selective pressures for adhesion, which may differ across different hosts and within an individual over time.
Project description:Most Helicobacter pylori strains express the BabA adhesin, which binds to ABO/Leb blood group antigens on gastric mucin and epithelial cells and is found more commonly in strains that cause peptic ulcers or gastric cancer, rather than asymptomatic infection. We and others have previously reported that in mice, gerbils, and rhesus macaques, expression of babA is lost, either by phase variation or by gene conversion, in which the babB paralog recombines into the babA locus. The functional significance of loss of babA expression is unknown. Here we report that in rhesus monkeys, there is independent selective pressure for loss of babA and for overexpression of BabB, which confers a fitness advantage. Surprisingly, loss of babA by phase variation or gene conversion is not dependent on the capacity of BabA protein to bind Leb, which suggests that it may have other, unrecognized functions. These findings have implications for the role of outer membrane protein diversity in persistent H. pylori infection.
Project description:Helicobacter pylori strains show both geographic and disease-associated allelic variation. We investigated the diversity present in two genes, babA and babB, which are members of a paralogous family of outer membrane proteins. Eleven family members within a single H. pylori strain, predicted to encode proteins with substantial N- and C-terminal similarity to each other, were classified as babA paralogues. In their central regions, most are less than 54% related to one another. Examining the babA and babB central regions in 42 H. pylori strains from different geographic locales, we identified five different allele groups of babA (AD1 to AD5) and three different allele groups of babB (BD1 to BD3). Phylogenetic analysis revealed that the allelic groupings of babA and babB are independent of one another and that, for both, geographic variation is present. Analysis of synonymous and nonsynonymous substitutions in these regions showed that babA is more diverse, implying an earlier origin than that of the same region of babB, but that the babA diversity region may have more functional constraints. Although recombination has been central to the evolution of both genes, with babA and babB showing low mean compatibility scores and homoplasy ratios of 0.71 and 0.67, respectively, recombination is not sufficient to obscure evidence of clonal descent. Despite the involvement of babA in binding to the host blood group antigen Lewis B, neither the presence of different babA allele groups nor that of different babB allele groups is a determining factor in Lewis B binding of H. pylori strains.
Project description:Heterogeneity among Helicobacter pylori strains in gastric epithelial adherence is postulated to contribute to pathogen fitness in the physiologically diverse human population. H. pylori adherence to ABO and Lewis b (Leb) blood group antigens in the human stomach is mediated by the blood group antigen-binding adhesin BabA. Approximately 70% of Swedish and U.S. H. pylori clinical isolates exhibit Leb binding, but here we show that the babA gene is present in each of 10 Leb-nonbinding strains. Fluorescence microscopy identified occasional bacterial cells with a Leb-binding phenotype in populations of Leb-nonbinding strains. Thus, nonbinding seemed to be a metastable phenotype. To model metastable transition into the virulence-associated Leb-binding mode, Leb-binding clones were isolated from nonadherent strains by panning with Leb-magnetic beads and characterized. Strain 17875 has two babA genes, babA1 (silent) and babA2 (expressed). We found that a babA2-cam derivative of strain 17875 regained Leb binding by recombination of the formerly silent babA1 gene into the expressed and partially homologous babB locus. The chimeric BabB/A adhesin binds Leb with an affinity similar to that of wild-type BabA adhesin, but its expression level was lower and was subject to phase variation through slipped-strand mispairing. Equivalent results were obtained with strain NCTC11638. We propose that adhesin metastability and heterogeneity contributes to bacterial fitness and results in some clones having potential for periodic activation and deactivation of virulence appropriate for intensity of the host response to infection.
Project description: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.
Project description:Helicobacter pylori BabA adhesin metastability could yield variants with potential for periodic activation and deactivation of their mediated adherence. babA/B or babB/A chimeras could play an important role in translational regulation. We investigated the frequency of different bab gene profiles in paired isolates from antrum and corpus recovered from patients with chronic gastritis. Isolates from 174 biopsies from 34 patients were included, and bab genes at the three common chromosomal loci were investigated. Inter-micro-niche variation was found in 1/4 patients, counting duplicate copies of babA or babB, babB/A or babA/B chimeras, opposite location of babA and babB or babC and babB, and absence of babB ATG translational codon. Truncated BabA was identified in 2/34 patients without inter-micro-niche variation. Isolates from 12/34 patients harbored babA/B or babB/A chimeras -either in one, several or all micro-niches indicating that chimera formation is a common mechanism to control BabA expression. To note, babA gene was absent in 11/34 patients, and in this population, babA/B chimeras which lack expression predominated over babB/A, able to exhibit Le(b) binding phenotype.
Project description:The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.
Project description:The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an approximately 75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.
Project description:Chronic infection of Helicobacter pylori in the stomach mucosa with translocation of the bacterial cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV secretion system (TFSS) into host epithelial cells are major risk factors for gastritis, gastric ulcers, and cancer. The blood group antigen-binding adhesin BabA mediates the adherence of H. pylori to ABO/Lewis b (Le(b)) blood group antigens in the gastric pit region of the human stomach mucosa. Here, we show both in vitro and in vivo that BabA-mediated binding of H. pylori to Le(b) on the epithelial surface augments TFSS-dependent H. pylori pathogenicity by triggering the production of proinflammatory cytokines and precancer-related factors. We successfully generated Le(b)-positive cell lineages by transfecting Le(b)-negative cells with several glycosyltransferase genes. Using these established cell lines, we found increased mRNA levels of proinflammatory cytokines (CCL5 and IL-8) as well as precancer-related factors (CDX2 and MUC2) after the infection of Le(b)-positive cells with WT H. pylori but not with babA or TFSS deletion mutants. This increased mRNA expression was abrogated when Le(b)-negative cells were infected with WT H. pylori. Thus, H. pylori can exploit BabA-Le(b) binding to trigger TFSS-dependent host cell signaling to induce the transcription of genes that enhance inflammation, development of intestinal metaplasia, and associated precancerous transformations.
Project description:Clinical isolates of Helicobacter pylori show marked diversity, which may derive from genomic changes that occur during the often lifelong association of the bacterium with its human host. We used the rhesus macaque model, together with DNA microarrays, to examine genomic changes in H. pylori that occur early during experimental infection. Microarray analysis showed that H. pylori recovered from challenged macaques had deleted babA, a member of a large family of paralogous outer membrane proteins (OMPs) that mediates attachment of H. pylori to the Lewis B blood group antigen on gastric epithelium. In some cases the babA gene was replaced by babB, an uncharacterized OMP that is closely related to babA. In other cases the babA gene was present but was not expressed because of alteration in dinucleotide CT repeats in the 5' coding region. In either case, strains lacking babA did not adhere to Lewis B, which is expressed on macaque gastric epithelium. Absence of babA and duplication of babB was also seen in H. pylori isolates derived from human clinical samples, suggesting that this gene conversion event is not unique to experimentally infected rhesus monkeys. These results demonstrate in real time with a relevant animal model that H. pylori regulates OMP expression in vivo by using both antigenic variation and phase variation. We suggest that changes in babA and babB after experimental infection of macaques represent a dynamic response in the H. pylori outer membrane that facilitates adherence to the gastric epithelium and promotes chronic infection.
Project description:Expression of the Helicobacter pylori blood group antigen binding adhesin A (BabA) is more common in strains isolated from patients with peptic ulcer disease or gastric cancer, rather than asymptomatic colonization. Here we used mouse models to examine host determinants that affect H. pylori BabA expression. BabA expression was lost by phase variation as frequently in WT mice as in RAG2-/- mice that do not have functional B or T cells, and in MyD88-/-, TLR2-/- and TLR4-/- mice that are defective in toll like receptor signaling. The presence of other bacteria had no effect on BabA expression as shown by infection of germ free mice. Moreover, loss of BabA expression was not dependent on Le<sup>b</sup> expression or the capacity of BabA to bind Le<sup>b</sup>. Surprisingly, gender was the host determinant most associated with loss of BabA expression, which was maintained to a greater extent in male mice and was associated with greater bacterial load. These results suggest the possibility that loss of BabA expression is not driven by adaptive immunity or toll-like receptor signaling, and that BabA may have other, unrecognized functions in addition to serving as an adhesin that binds Le<sup>b</sup>.