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Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA.

ABSTRACT: Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA(25-460)) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA(25-460) were collected to 2.25?Å resolution from a crystal that belonged to space group P21, with unit-cell parameters a = 50.96, b = 131.41, c = 123.40?Å, ? = 90.0, ? = 94.8, ? = 90.0°, and which was predicted to contain two BabA(25-460)-nanobody complexes per asymmetric unit.

SUBMITTER: Subedi S

PROVIDER: S-EPMC4259228 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA.

Subedi Suresh S Moonens Kristof K Romão Ema E Lo Alvin A Vandenbussche Guy G Bugaytsova Jeanna J Muyldermans Serge S Borén Thomas T Remaut Han H

Acta crystallographica. Section F, Structural biology communications 20141114 Pt 12

Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the ...[more]

PMID: 25484214

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Project description:UNLABELLED:Helicobacter pylori undergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin of H. pylori is BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed paired H. pylori isolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons of babA identified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with a babA gene amplified from the binding strain, H. pylori strains with mosaic babA genes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane ?-barrel domain, plays an essential role in the biogenesis of this protein. IMPORTANCE:Helicobacter pylori causes a chronic infection of the human stomach that can lead to ulcers and cancer. The bacterium can bind to gastric epithelial cells with specialized outer membrane proteins. The best-studied protein is the BabA adhesin which binds to the Lewis b blood group antigen. Since H. pylori is a bacterium with very high genetic variability, we asked whether babA evolves during chronic infection and how mutations or recombination in babA affect binding. We found that BabA-mediated adherence was stable in most individuals but observed a complete loss of binding or reduced binding in 22% of individuals. One strain pair in which binding was lost was used to generate babA sequences that were mosaics of a functional allele and a nonfunctional allele, and the mosaic sequences were used to identify amino acids critically involved in binding of BabA to Lewis b.

Dataset Information

Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA.

Publications

Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA.

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