Inhibition of RTX Toxin Activity by the Nuclear Stain, Draq5

Abstract

The repeats-in-toxin (RTX) family of proteins includes numerous toxins produced by Gram negative bacteria, including Bordetella pertussis (adenylate cyclase toxin), Escherichia coli (a-hemolysin), and Aggregatibacter actinomycetemcomitans (leukotoxin, LtxA). The mechanism by which LtxA recognizes and kills target cells involves multiple steps, including recognition of a b2 integrin receptor (LFA-1), binding to cholesterol/lipid rafts, and membrane bilayer destabilization. In a confocal imaging experiment, we found that LtxA rapidly becomes internalized in Jn.9 cells, and colocalizes with lysosomes. However, when the cells were pretreated with Draq5, a common nuclear stain, LtxA was found to be associated with the membrane, but not inside the cell. This inhibition of internalization was correlated with a decrease in the activity of the toxin. We hypothesized that upon crossing the plasma membrane, Draq5 alters the packing of the bilayer in a manner that inhibits the interaction of LtxA with the membrane, thus decreasing its activity. We therefore conducted a series of membrane biophysical experiments to determine what changes this dye induces in the membrane structure and their effects on LtxA activity. Using the water-sensitive dye, laurdan, we determined that Draq5 decreases the fluidity of both gel- and liquid-phase membranes; however, differential scanning calorimetry indicated that the dye does not alter the transition temperature of the lipid. Draq5 does not inhibit LtxA association with membranes, but does inhibit the ability of LtxA to disrupt the bilayer, a process we have shown previously to be required for activity. Together, these results suggest that the Draq5-mediated decrease in membrane fluidity inhibits LtxA penetration of (but not binding to) the plasma cell membrane. This work will guide us as we seek less toxic molecules that have a similar effect on membrane structure and toxin activity for the treatment of bacterial infections.