Insights into the Membrane-Interaction Properties of the PSEUDOMONAS Aeruginosa Type III Secretion Translocator Popd

Abstract

In order to infect human cells, pathogenic Gram-negative bacteria depend on their ability to secrete toxins into the cytoplasm of an adjacent eukaryotic host cell. A conserved and efficient delivery mechanism, called the type III secretion (T3S) system, acts as a syringe to inject these toxic proteins called “effectors” which need to translocate through the eukaryotic plasma membrane. In Pseudomonas aeruginosa PopB and PopD are essential for effector translocation. Substantial genetic and biochemical data support a model with PopB and PopD inserting into the target membrane and forming an oligomeric translocon pore, however virtually nothing is known about this critical transmembrane complex. Our primary goal is to determine the arrangement of PopD in membrane assembled translocons. What segments of PopD interact with the membrane? Does PopB influence the PopD/membrane interaction? Using membrane binding assays, conductivity measures in planar lipid bilayers, site-directed fluorescence labeling, and multiple fluorescence approaches we have analyzed the interaction of PopD with model membranes and the pore formation activity of the translocator in the presence and absence of PopB. Despite both proteins can individually form pores in membranes, we found that PopD oligomerizes with PopB and forms pores with an inner diameter of approximately 30Å. Our analysis also showed that the PopD predicted transmembrane segment (residues L119-V137), does not adopt a transmembrane orientation. Our data support a model that consists in the binding of the unfolded translocators to the membrane, oligomerization, and formation of discrete transmembrane pores.