title: Heterologous expression of Bartonella adhesin A in Escherichia coli by exchange of trimeric autotransporter adhesin domains results in enhanced adhesion properties and a pathogenic phenotype.
abstract: Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model.
authors.affiliation: Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmidgen
authors.affiliation: Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Patrick O
authors.initials: PO
authors.lastname: Kaiser
authors.affiliation: Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Wibke
authors.initials: W
authors.lastname: Ballhorn
authors.affiliation: Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Franz
authors.affiliation: Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Goethe-Universität, Frankfurt am Main, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Göttig
authors.affiliation: Max-Planck-Institut für Entwicklungsbiologie, Abteilung Proteinevolution, Tübingen, Germany Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Dirk
authors.initials: D
authors.lastname: Linke
authors.affiliation: Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Klinikum der Goethe-Universität, Frankfurt am Main, Germany volkhard.kempf@kgu.de.
authors.forename: Volkhard A J
authors.initials: VA
authors.lastname: Kempf
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology; D018416:Bartonella henselae / Q000378:metabolism*; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D007814:Larva / Q000382:microbiology; D009036:Moths / Q000382:microbiology; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins
chemical_list: D018829:Adhesins, Bacterial; D011994:Recombinant Proteins; D003094:Collagen