Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

Abstract

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while (31) P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.

Fig. 1

Localization of fluorescein-labelled LtxA on the surface of target cells. Top row: untreated control cells, Bottom row: LtxA-treated (30 min) cells. A and E. Phase contrast image. B and F. FITC (LtxA) stain. Arrows indicate LtxA clusters. C and G. WGA-AF594 (membrane) stain. D and H. FITC and WGA-AF-594 merge. Arrows indicate colocalization. Bar = 10 µm.

Fig. 2

LtxA mediates collapse of microvilli on Jn.9 target cells. The samples were prepared for conventional scanning electron microscopy of an untreated control (Fig. 2A_1–3) and experimental groups after 30 min (Fig. 2B_1–3) and 3 h (Fig. 2C_1–3) of incubation with LtxA (1 × 10⁻⁹ M). Images are representative of the unique phenotypes observed within each group in three independent experiments. All images were acquired at × 6000 and bars equal 2.5 µm. MV, microvilli; D, depression; C, cavity.

Fig. 3

SEM images of LtxA-treated Jn.9 cells. A. Jn.9 cell, which has not been exposed to LtxA. This control cell was sliced coronally with a Ga³⁺ FIB (A₁). B. Jn.9 cell exposed to LtxA (1 × 10⁻⁹ M) for 30 min. C. Jn.9 cell exposed to LtxA (1 × 10⁻⁹ M) for 3 h. The toxin-treated cells were serially sliced coronally with the FIB (B₂–B₄ and C₂–C₄). MV, microvilli; D, depression; C, cavity; T, tear. Bar = 5 µm.

Fig. 4

LtxA-induced leakage of calcein. A. Structure of the lipids used in the leakage study. B. Representative scans of leakage at a protein mole fraction of 0.0013 from liposomes composed of DPPC (100%) (●), DPPC/SPC (3:1) (▽) and DPPC/DMPE (3:1) (■). C. Leakage of calcein after 30 min of incubation with LtxA for liposomes composed of DPPC (100%) (●), DPPC/SPC (3:1) (▽) and DPPC/DMPE(−) (3:1) (■). D. LtxA-induced leakage of calcein at a protein mole fraction of 0.0010 for liposomes composed of DPPC (100%), DPPC/SPC (3:1), DPPC/DMPE (3:1), DPPC/DMPE/SPC (2:1:1) or DPPC/DMPE/CS (2:1:1). Results are statistically significant (P < 0.001) for differences between the control (DPPC/DMPE) and both DPPC/DMPE/SPC and DPPC/DMPE/CS.

Fig. 5

Effect of LtxA on bilayer-to-hexagonal phase transition of di16:1 PE. A. Raw DSC scans showing the phase transition temperature of liposomes treated with increasing concentrations of LtxA. B. T_H of each scan as a function of LtxA mol fraction.

Fig. 6

³¹P NMR spectra of N-methyl-DOPE liposomes. A. N-methyl-DOPE liposomes without LtxA. B. N-methyl-DOPE liposomes treated with an LtxA mole fraction of 0.0001. Spectra were collected at 10°C, 20°C, 30°C, 40°C, 50°C, 60°C and 70°C.

Fig. 7

Proposed model of H_II-phase membrane disruption. In this model, the acyl group of LtxA initially contacts the cell membrane, and the toxin undergoes a conformational change in which part of the toxin inserts into the membrane. This induces formation of an isotropic phase, resulting in a destabilization of the membrane.