Current Fluctuation Analysis of the PopB and PopD Translocon Components of the Pseudomonas aeruginosa Type III Secretion System

Pseudomonas aeruginosa is a major agent of hospital-acquired infections, and a pathogen of immunocompromised, cystic fibrosis and burn patients. It uses a type III secretion system for the injection of toxins directly into host cells through a translocon assembled in the host cell membrane. The hydrophobic translocator subunits of this system, PbpB and PopD, have membrane permeabilizing activity based on previous dye leakage experiments, but little is known about the mechanism of assembly and the pore properties of this translocon. Using electrophysiology, we have observed that an equimolar mixture of PopB and PopD induces current fluctuations in planar lipid bilayers, with a unitary conductance of 57 pS in 1 M KCl and numerous larger conductance levels. The activity depends on voltage magnitude and polarity, and increases with protein concentration and the duration of the voltage step. PopB alone is sufficient for producing current fluctuations.. PopD rarely displays any transitions, but accelerates PopB onset of activity. The effects of pH, ionic-strength, and lipid composition have also been explored. Our data provide new, to our knowledge, insights into the behavior of PopB and PopD by highlighting similarities, with secreted pore-forming peptides, and by suggesting that PopB/PopD may form channels via the toroidal pore model. We believe that the events we report here represent the initial steps of insertion and assembly of, these translocators in the membrane.