Thomas Vorup-Jensen

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisReviewForskningpeer review

Standard

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes. / Reinholdt, Jesper; Poulsen, Knud; Brinkmann, Christel Rothe; Hoffmann, Søren Vrønning; Stapulionis, Romualdas; Enghild, Jan Johannes; Jensen, Uffe Birk; Boesen, Thomas; Vorup-Jensen, Thomas.

I: BBA General Subjects, Bind 1834, Nr. 2, 02.2013, s. 546-558.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisReviewForskningpeer review

Harvard

APA

CBE

MLA

Vancouver

Author

Bibtex

@article{9ef8f4129b9845989a2763cae33d9d09,
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 αLβ2 (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 β2 integrin. Cells expressing αMβ2 (CD11b/CD18) or αXβ2 (CD11c/CD18) were killed as efficiently as cells expressing αLβ2. Erythrocytes, which do not express β2 integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β2 chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins αMβ2 and αXβ2 as novel receptors for LtxA.",
author = "Jesper Reinholdt and Knud Poulsen and Brinkmann, {Christel Rothe} and Hoffmann, {S{\o}ren Vr{\o}nning} and Romualdas Stapulionis and Enghild, {Jan Johannes} and Jensen, {Uffe Birk} and Thomas Boesen and Thomas Vorup-Jensen",
year = "2013",
month = "2",
doi = "10.1016/j.bbapap.2012.12.004",
language = "English",
volume = "1834",
pages = "546--558",
journal = "B B A - General Subjects",
issn = "0304-4165",
publisher = "Elsevier BV",
number = "2",

}

RIS

TY - JOUR

T1 - Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

AU - Reinholdt, Jesper

AU - Poulsen, Knud

AU - Brinkmann, Christel Rothe

AU - Hoffmann, Søren Vrønning

AU - Stapulionis, Romualdas

AU - Enghild, Jan Johannes

AU - Jensen, Uffe Birk

AU - Boesen, Thomas

AU - Vorup-Jensen, Thomas

PY - 2013/2

Y1 - 2013/2

N2 - 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 αLβ2 (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 β2 integrin. Cells expressing αMβ2 (CD11b/CD18) or αXβ2 (CD11c/CD18) were killed as efficiently as cells expressing αLβ2. Erythrocytes, which do not express β2 integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β2 chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins αMβ2 and αXβ2 as novel receptors for LtxA.

AB - 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 αLβ2 (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 β2 integrin. Cells expressing αMβ2 (CD11b/CD18) or αXβ2 (CD11c/CD18) were killed as efficiently as cells expressing αLβ2. Erythrocytes, which do not express β2 integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β2 chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins αMβ2 and αXβ2 as novel receptors for LtxA.

U2 - 10.1016/j.bbapap.2012.12.004

DO - 10.1016/j.bbapap.2012.12.004

M3 - Review

C2 - 23234758

VL - 1834

SP - 546

EP - 558

JO - B B A - General Subjects

JF - B B A - General Subjects

SN - 0304-4165

IS - 2

ER -