Departmental Papers (Dental)

Document Type

Journal Article

Date of this Version

3-2019

Volume

21

Issue

3

Start Page

e12967

DOI

10.1111/cmi.12967

Abstract

Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin α L /β 2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic α L domain and a yellow fluorescent protein-tagged β 2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both α L and β 2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α L and β 2 subunits (K d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin α M , α X , and β 3 subunits (K d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α L and β 2 show that LtxA binds membrane-proximal domain of α L and intermediate domain of β 2 . © 2018 John Wiley & Sons Ltd

Keywords

integrin; leukotoxin (LtxA); LFA-1; microbial pathogenesis; RTX toxin; surface plasmon resonance (SPR)

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Date Posted: 08 December 2022

This document has been peer reviewed.