Membrane association and destabilization by Aggregatibacter actinomycetemcomitans leukotoxin requires changes in secondary structures

Abstract

SUMMARY Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis in children, and localized aggressive periodontitis. A. actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and/or destabilization. In this study, we tested this hypothesis by analyzing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies thus provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans.

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli

“…S2 in the supplemental material). LtxA is an RTX toxin, and it is still debated whether it forms actual pores in biological membranes (50)(51)(52)(53). Peculiarly, we could demonstrate that LtxA releases ATP from phospholipid-1-palmitoyl-2-oleoyl-phosphatidyl choline (POPC) vesicles with kinetics quite similar to those of HlyA.…”

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

“…Asolectin is a mixture of lipids from soy beans containing roughly equal proportions of phosphatidylcholine (PC, about 25%), phosphatidylethanolamine (PE), and phosphatidylinositol along with minor amounts other phospholipids and typical plant lipids, such as mono- and digalactosylglycerides. Interestingly, it has been previously shown that A. actinomycetemcomitans LtxA forms pores preferentially in liposomal membranes composed of lipids containing PE that is able to form inverted hexagonal (nonbilayer) phases in the bilayer membrane [59, 60]. It is also possible that specific hydrophobic interactions occur between the toxin and only certain phospholipids in asolectin membrane that are able to cause conformational changes necessary to facilitate the toxin binding to the membrane.…”

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…CD spectroscopy analysis was performed on a Jasco J-810 spectropolarimeter, as discussed in our previous studies (13). The samples were analyzed at 25°C in a 0.2-mm-path length quartz cell using the step-scanning mode from 260 to 200 nm, with a 1-nm wavelength step, 10-s averaging time, and 1-nm bandwidth.…”

“…Neither the water-soluble nor the membrane-embedded structures of these toxins (and LtxA in particular) have been solved; however, it has been proposed that this family of toxins undergoes a conformational change upon membrane association (12). We have recently shown that LtxA undergoes distinct lipid-specific secondary structure changes upon binding to or disruption of lipid bilayers (13).…”