title: Self-association motifs in the enteroaggregative Escherichia coli heat-resistant agglutinin 1.
abstract: The heat-resistant agglutinin 1 (Hra1) is an integral outer membrane protein found in strains of Escherichia coli that are exceptional colonizers. Hra1 from enteroaggregative E. coli strain 042 is sufficient to confer adherence to human epithelial cells and to cause bacterial autoaggregation. Hra1 is closely related to the Tia invasin, which also confers adherence, but not autoaggregation. Here, we have demonstrated that Hra1 mediates autoaggregation by self-association and we hypothesize that at least some surface-exposed amino acid sequences that are present in Hra1, but absent in Tia, represent autoaggregation motifs. We inserted FLAG tags along the length of Hra1 and used immune-dot blots to verify that four in silico-predicted outer loops were indeed surface exposed. In Hra1 we swapped nine candidate motifs in three of these loops, ranging from one to ten amino acids in length, to the corresponding sequences in Tia. Three of the motifs were required for Hra1-mediated autoaggregation. The database was searched for other surface proteins containing these motifs; the GGXWRDDXK motif was also present in a surface-exposed region of Rck, a Salmonella enterica serotype Typhimurium complement resistance protein. Cloning and site-specific mutagenesis demonstrated that Rck can confer weak, GGXWRDDXK-dependent autoaggregation by self-association. Hra1 and Rck appear to form heterologous associations and GGXWRDDXK is required on both molecules for Hra1-Rck association. However, a GGYWRDDLKE peptide was not sufficient to interfere with Hra1-mediated autoaggregation. In the present study, three autoaggregation motifs in an integral outer membrane protein have been identified and it was demonstrated that at least one of them works in the context of a different cell surface.
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Glaubman
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hofmann
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Bonney
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Sumin
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Thomas
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Bashkim
authors.initials: B
authors.lastname: Kokona
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Laura I
authors.initials: LI
authors.lastname: Ramos Falcón
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Yoonjie K
authors.initials: YK
authors.lastname: Chung
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Fairman
authors.affiliation: Department of Biology, Haverford College, Haverford, PA, USA.
authors.forename: Iruka N
authors.initials: IN
authors.lastname: Okeke
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion / Q000502:physiology*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012486:Salmonella typhimurium / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C090012:HRA1 protein, E coli; C450844:enterotoxigenic invasive protein A, E coli