Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function-associated antigen 1

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 /β₂ )-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 β₂ 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 β₂ were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α_L and β₂ 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 β₃ subunits (K_d  = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α_L and β₂ show that LtxA binds membrane-proximal domain of α_L and intermediate domain of β₂ .