The oral bacterium Aggregatibacter actinomycetemcomitans produces multiple virulence factors, including a repeats-in-toxin (RTX) leukotoxin (LtxA). This toxin has been shown to kill human white blood cells in a lymphocyte function-associated antigen-1 (LFA-1, integrin αL/β2)-dependent manner, although the mechanism for this interaction has not been identified. We used confocal microscopy to determine that LtxA is internalized only in cells expressing LFA-1 and that internalized LtxA resides close to the plasma membrane.
The functional significance of LtxA internalization was explored with FRET microscopy using a cell line that expresses LFA-1 with a CFP-tagged cytosolic αL domain and an YFP-tagged β2 domain. PMA activation of LFA-1 caused transient cytosolic domain separation, as expected. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared to the inactive state. Unlike activation, this effect was not transient, lasting more than 30 mins. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and β2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and β2 subunits (KD = 15 nM and 4.2 nM, respectively) and a significantly lower affinity for the β3 cytoplasmic domain (KD = 230 nM), used as a control. Peptide fragments of αL and β2 were used to identify the regions of the tails where LtxA binds (membrane-proximal domain of αL, intermediate domain of β2).
These findings give new insight into the mechanism by which LtxA kills immune cells. Supported by DE09517.