(656a) Induction of a Lipid Phase Change In the Host Cell Membrane by a Bacterial Toxin, Aggregatibacter Actinomycetemcomitans leukotoxin (LtxA)

Pathogenic bacteria have developed numerous strategies to evade the host immune response, one of which is the secretion of protein toxins and other virulence factors to produce an environment within the host that is more conducive to their survival.  The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans performs this task by selectively killing human white blood cells, allowing the bacteria to flourish in the host and disease progression (localized aggressive periodontitis) to occur.  This toxin is a member of the repeats-in-toxin (RTX) family of proteins, each of which is produced by a virulent Gram-negative bacterium, including Escherichia coli and Bordetella pertussis (the causative agent of whooping cough).  It has long been established that the RTX toxins are cytolytic; however, recent evidence has shown that the interactions of these toxins with cell membranes may be more complex than simple pore-formation.  We therefore initiated this study to determine the mechanism of this early but important step in the pathogenicity of the bacterium. 

To characterize the interaction of LtxA with membranes, we used a variety of biochemical and biophysical techniques, including scanning electron microscopy (SEM), fluorescence spectroscopy, differential scanning calorimetry (DSC), ³¹P nuclear magnetic resonance (³¹P NMR), and surface plasmon resonance (SPR).  Most of this work was performed with model membranes (liposomes) to determine the lipid-specific interactions that occur during the toxin-membrane interaction.

Using fluorescence spectroscopy, we showed that LtxA disrupts membranes only when they contain a nonlamellar lipid, such as phosphoethanolamine (PE).  These types of lipids are involved in the formation of nonlamellar lipid phases, including inverted hexagonal (H_II) phases. This membrane disruption was inhibited by cholesterol sulfate and a single-chained phospholipid (lysoPC), both of which inhibit H_II phase formation, suggesting that the toxin may promote the formation of this nonlamellar lipid phase. DSC demonstrated that the toxin lowers the bilayer-to-H_II phase transition temperature (T_H) of nonlamellar lipids, indicating that LtxA does promote the formation of the nonbilayer (H_II) phase. ³¹P NMR verified this promotion of the H_II phase and demonstrated that this process occurs through the formation of an isotropic intermediate lipid structure.

We compared these results to standard models of pore formation, including the barrel stave pore, the toroidal pore, and the carpet mechanism, and determined that these results represent a previously undescribed mechanism of membrane disruption. Importantly, several RTX toxins, as well as other bacterial and eukaryotic toxins, have been shown, to some extent, to interact with nonlamellar lipids, suggesting that this newly detailed mechanism may, in fact, be widely conserved among protein toxins.

In this work, we have determined a new example of a mechanism by which a pathogen hijacks certain properties of the lipids in the host cell membrane to facilitate its attack and cause disease.  Specifically, we have shown that LtxA cytotoxicity occurs through a direct interaction of the toxin with the lipids of the membrane, resulting in a lipid phase change from a stable bilayer phase to a weaker nonlamellar phase.  This mechanism of membrane damage may be well-conserved and therefore represents an important target of future therapeutic approaches.