
Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function‐associated antigen 1
Author(s) -
Nygren Patrik,
Balashova Nataliya,
Brown Angela C.,
Kieba Irene,
Dhingra Anuradha,
BoeszeBattaglia Kathleen,
Lally Edward T.
Publication year - 2019
Publication title -
cellular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.542
H-Index - 138
eISSN - 1462-5822
pISSN - 1462-5814
DOI - 10.1111/cmi.12967
Subject(s) - förster resonance energy transfer , cytosol , biology , cytoplasm , microbiology and biotechnology , antigen , effector , biophysics , biochemistry , fluorescence , immunology , physics , quantum mechanics , enzyme
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 .