title: Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human β2 integrins and erythrocytes.
abstract: Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)β(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β(2) integrin. Cells expressing α(M)β(2) (CD11b/CD18) or α(X)β(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)β(2). Erythrocytes, which do not express β(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)β(2) and α(X)β(2) as novel receptors for LtxA.
authors.affiliation: Department of Dentistry, Aarhus University, Aarhus, Denmark. jr@microbiology.au.dk
authors.forename: Jesper
authors.initials: J
authors.lastname: Reinholdt
authors.forename: Knud
authors.initials: K
authors.lastname: Poulsen
authors.forename: Christel R
authors.initials: CR
authors.lastname: Brinkmann
authors.forename: Søren V
authors.initials: SV
authors.lastname: Hoffmann
authors.forename: Romualdas
authors.initials: R
authors.lastname: Stapulionis
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.forename: Uffe B
authors.initials: UB
authors.lastname: Jensen
authors.forename: Thomas
authors.initials: T
authors.lastname: Boesen
authors.forename: Thomas
authors.initials: T
authors.lastname: Vorup-Jensen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018821:CD18 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D015661:Pasteurellaceae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016871:Pasteurellaceae Infections / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D018821:CD18 Antigens; D005098:Exotoxins; C018579:leukotoxin