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 alpha(L)/beta(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 alpha(L) domain and a yellow fluorescent protein-tagged beta(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 alpha(L) and beta(2) were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the alpha(L) and beta(2) subunits (K-d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin alpha(M), alpha(X), and beta(3) subunits (K-d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of alpha(L) and beta(2) show that LtxA binds membrane-proximal domain of alpha(L) and intermediate domain of beta(2).