title: Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1.
abstract: Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.
authors.affiliation: Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Paul-Ehrlich-Strasse 40, 60596 Frankfurt, Germany.
authors.forename: Corinna
authors.initials: C
authors.lastname: Siegel
authors.forename: Johanna
authors.initials: J
authors.lastname: Schreiber
authors.forename: Katrin
authors.initials: K
authors.lastname: Haupt
authors.forename: Christine
authors.initials: C
authors.lastname: Skerka
authors.forename: Volker
authors.initials: V
authors.lastname: Brade
authors.forename: Markus M
authors.initials: MM
authors.lastname: Simon
authors.forename: Brian
authors.initials: B
authors.lastname: Stevenson
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wallich
authors.forename: Peter F
authors.initials: PF
authors.lastname: Zipfel
authors.forename: Peter
authors.initials: P
authors.lastname: Kraiczy
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D025065:Borrelia burgdorferi / Q000378:metabolism*; D003180:Complement C3b Inactivator Proteins; D017242:Complement Factor H / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; C493495:CFH protein, human; C517621:CFHR1 protein, human; D003180:Complement C3b Inactivator Proteins; D008024:Ligands; D008565:Membrane Proteins; C440651:complement regulator-acquiring surface proteins, Borrelia burgdorferi; D017242:Complement Factor H