Proton-Driven Molecular Ratchet Iteratively Activated by Microfluidics

Abstract

Anthrax toxin (AT), the key virulence factor secreted by Bacillus anthracis, injects lethal enzymes into cells using pH and voltage transmembrane gradients. Anthrax toxin is comprised of three proteins: protective antigen (PA, 83 kDa), the pore-forming protein which delivers the other two toxic enzymes - edema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa) - into the cell cytosol. The mechanism of AT translocation is dependent on a pH gradient, but exactly how this works is not yet clear. Here, we use a droplet-interface bilayer(DIB)-based fluid exchange device that allows reagents to be introduced to the model membrane in rapid sequence. We are able to switch proton gradient across the membrane on and off and reload AT proteins repeatedly. To support the putative Brownian-ratchet mechanism of translocation, the AT translocation was induced using a gradient and subsequently paused. By cycling the gradient on and off, the translocation can be started and stopped repeatedly as shown in our electrical recordings. This method may be of utility in the study of other AB toxins that are believed to operate using a proton gradient for energy.