Lipid Composition and Endosomal pH Regulates Anthrax Lethal Toxin Uptake and PA63 Channel Behavior

Abstract

The tight regulation of cellular pH is critical for the structure and function of cells and organelles. Besides, cells adjust bilayer biophysical parameters by controlling lipid composition and gradient across their plasma and intracellular organelle membranes. Both pH and membrane lipid composition change dramatically along the endocytic pathway, which is the internalization transport process that many bacterial toxins utilize to invade cells. In this study, we investigate the role of the lipid and pH environment in the uptake of the anthrax lethal toxin (composed of protective antigen (PA) and lethal factor (LF)). The anthrax toxin follows a multistep internalization process, which involves PA binding to its cellular receptors, proteolytic cleavage to PA63, oligomerization to form prepores, and LF binding followed by endocytosis. The acidic endosomal environment causes conformational changes in PA63 leading to its insertion into the early endosomal membrane and ion channel formation. The PA63-mediated delivery of the enzymatic LF to cytoplasm was suggested to take place later in the endocytic pathway via the back-fusion of intraluminal vesicles. Given the variations in lipid composition of the early endosome, the intraluminal vesicles, and the late endosomal limiting membrane, it is important to understand how these changes influence the anthrax toxin uptake. Using the planar lipid bilayer technique, we reconstituted single PA63 channels into neutral DOPC, DPhPC and DOPE and negatively-charged DOBMP, DPhPS membranes and varied the solution pH from 7.5 to 4.5 to mimic the changing physical environments of the endosomal pathway. The observed differences in the PA63 single-channel conductance, selectivity, and gating, and in LF binding and internalization were significant. These findings suggest that the anthrax toxin complex represents a robust universal model to study protein/lipid and protein/protein interactions in lipid membranes of different composition.