Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue

In this study, we isolated and characterized a chromosomal locus of Helicobacter pylori previously identified by transposon shuttle mutagenesis as being involved in the adhesion of the pathogen to gastric epithelial cells. Two closely homologous genes were identified, designated as alpA and alpB, encoding outer membrane (OM) proteins of 518 amino acids each. They are members of the outer membrane protein supergene family identified in the H. pylori 26695 complete genome sequence. AlpA carries a functional lipoprotein signal sequence. AlpB carries a putative standard N‐terminal signal sequence and shows a strong amino‐acid sequence identity to AlpA. Transposon insertion mutagenesis, immunoblotting and primer extension studies indicate that both genes are organized in an operon, but no obvious consensus promoter sequence was found upstream of the transcriptional start site. The C‐terminal portion of both proteins is predicted to form a porin‐like β‐barrel in the outer membrane, consisting of 14 transmembrane amphipathic β‐strands. Adhesion experiments with defined isogenic mutants indicate that both proteins are necessary for specific adherence of H. pylori to human gastric tissue. The pattern of AlpAB‐dependent adherence of H. pylori to the gastric epithelial surface shows a clear difference to the BabA2‐mediated adherence to Lewisb, suggesting that a different receptor is involved.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  L. Engstrand,et al.  Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging. , 1998, Science.

[3]  Mark Borodovsky,et al.  The complete genome sequence of the gastric pathogen Helicobacter pylori , 1997, Nature.

[4]  S. Falkow,et al.  Induction of host signal transduction pathways by Helicobacter pylori. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  R. Haas,et al.  Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain , 1996, Journal of bacteriology.

[6]  A. Blum,et al.  Adhesion of Helicobacter pylori to polarized T84 human intestinal cell monolayers is pH dependent , 1996, Infection and immunity.

[7]  R. Haas,et al.  Optimized BlaM‐transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence , 1996, Molecular microbiology.

[8]  S. Falkow,et al.  Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Meyer,et al.  An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions. , 1995, Gene.

[10]  P. O’Toole,et al.  The putative neuraminyllactose-binding hemagglutinin HpaA of Helicobacter pylori CCUG 17874 is a lipoprotein , 1995, Journal of bacteriology.

[11]  F. Jähnig,et al.  Common structural features of IgA1 protease‐like outer membrane protein autotransporters , 1995, Molecular microbiology.

[12]  R. Hancock,et al.  Isolation and characterization of a family of porin proteins from Helicobacter pylori , 1995, Infection and immunity.

[13]  P. O’Toole,et al.  Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori , 1994, Journal of bacteriology.

[14]  S. Falkow,et al.  A Haemophilus influenzae IgA protease‐like protein promotes intimate interaction with human epithelial cells , 1994, Molecular microbiology.

[15]  R. Haas,et al.  Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein , 1994, Molecular microbiology.

[16]  R. Curtiss,et al.  Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain , 1994, Infection and immunity.

[17]  S. Normark,et al.  Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. , 1993, Science.

[18]  T. Meyer,et al.  A plasmid system for high-level expression and in vitro processing of recombinant proteins. , 1993, Gene.

[19]  R. Schmitt,et al.  TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis. , 1993, Gene.

[20]  R. Rappuoli,et al.  Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[21]  T. Meyer,et al.  Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis , 1993, Molecular microbiology.

[22]  J. Gordon,et al.  An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[23]  D. Evans,et al.  Cloning, nucleotide sequence, and expression of a gene encoding an adhesin subunit protein of Helicobacter pylori , 1993, Journal of bacteriology.

[24]  C. Lingwood,et al.  Comparison of Helicobacter pylori and attaching-effacing Escherichia coli adhesion to eukaryotic cells , 1993, Infection and immunity.

[25]  T. Trust,et al.  The Helicobacter pylori 19.6-kilodalton protein is an iron-containing protein resembling ferritin , 1993, Journal of bacteriology.

[26]  R. Haas,et al.  Cloning and genetic characterization of a Hellcobacter pylori flagellin gene , 1992, Molecular microbiology.

[27]  M. Schmidt,et al.  AIDA‐I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule , 1992, Molecular microbiology.

[28]  C. Lingwood,et al.  The glycerolipid receptor for Helicobacter pylori (and exoenzyme S) is phosphatidylethanolamine , 1992, Infection and immunity.

[29]  M. Kostrzynska,et al.  Production of a conserved adhesin by the human gastroduodenal pathogen Helicobacter pylori , 1992, Journal of bacteriology.

[30]  M. Schell,et al.  Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum , 1992, Journal of bacteriology.

[31]  P. O’Toole,et al.  High-affinity binding of the basement membrane proteins collagen type IV and laminin to the gastric pathogen Helicobacter pylori , 1991, Infection and immunity.

[32]  P. Sherman,et al.  Adherence of Helicobacter pylori to human gastric epithelial cells in vitro. , 1991, Journal of medical microbiology.

[33]  F. Jähnig,et al.  Structure predictions of membrane proteins are not that bad. , 1990, Trends in biochemical sciences.

[34]  A. Axon,et al.  Bacterial adhesion and disease activity in Helicobacter associated chronic gastritis. , 1990, Gut.

[35]  D. Graham,et al.  Receptor-mediated adherence of Campylobacter pylori to mouse Y-1 adrenal cell monolayers , 1989, Infection and immunity.

[36]  R. Munson,et al.  Lipoproteins of Haemophilus influenzae type b , 1988, Journal of bacteriology.

[37]  F. Jähnig,et al.  Models for the structure of outer-membrane proteins of Escherichia coli derived from raman spectroscopy and prediction methods. , 1986, Journal of molecular biology.

[38]  E. Chen,et al.  Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Levine,et al.  Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines , 1983, Infection and immunity.

[40]  R. Doolittle,et al.  A simple method for displaying the hydropathic character of a protein. , 1982, Journal of molecular biology.

[41]  W. Rutter,et al.  Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. , 1979, Biochemistry.

[42]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[43]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[44]  H. Boyer,et al.  A complementation analysis of the restriction and modification of DNA in Escherichia coli. , 1969, Journal of molecular biology.

[45]  V. Braun,et al.  Chapter 14 Lipoproteins, structure, function, biosynthesis and model for protein export , 1994 .

[46]  P. Falk,et al.  ヒトの胃の上皮細胞でのHelicobacter pylori細胞の系統特異的な親和性を表すことを示すin vitroでの付着解析 , 1993 .