Aggregatibacter Actinomycetemcomitans Leukotoxin Disrupts Membranes By Inducing the Formation of An Inverted Hexagonal Lipid Phase

Abstract

The leukotoxin (LtxA) secreted by Aggregatibacter actinomycetemcomitans is a member of the repeats-in-toxin (RTX) family, and like the other members of the family, is a virulence protein which destroys host cells. LtxA exhibits specificity to human white blood cells, thereby allowing A. actinomycetemcomitans to flourish in the human upper aerodigestive track. Scanning electron micrographs of LtxA-treated human immune cell lines showed that LtxA induces the formation of large pores, which appear to be caused by membrane bending. To understand the molecular mechanism of this interaction, we analyzed the behavior of LtxA-membrane interactions in model membranes. Freeze-fracture transmission electron microscopy revealed the presence of unique structures, such as nanotubules, in multilamellar liposomes treated with LtxA. Formation of these structures indicated that the toxin acts by bending the membrane, possibly by inducing a bilayer-to-nonbilayer transition. This phase transition was quantified using differential scanning calorimetry, and it was found that LtxA is a potent inverted hexagonal (HII) phase promoter. The relationship between HII phase induction and membrane disruption was determined with a calcein leakage assay. LtxA-induced leakage from calcein-encapsulating liposomes composed of lipids with negative curvature, favoring HII phase formation, was significantly enhanced compared to leakage from liposomes composed of lipids with neutral curvature, favoring bilayer formation. Addition of lipids with positive curvature, inhibiters of HII phase formation, or cholesterol sulfate, a bilayer stabilizer, completely eliminated calcein leakage. It appears that LtxA causes membrane disruption by inducing the formation of the HII phase, and its toxic effect is therefore highly dependent on lipid curvature. Supported by NIH DE09517.