A type 3 secretion system is used by many bacterial pathogens to inject proteins into eukaryotic cells. Pathogens insert a translocon complex into the target eukaryotic membrane by secreting two proteins known as translocators. How these translocators form a translocon in the lipid bilayer and why both proteins are required remains elusive. Pseudomonas aeruginosa translocators PopB and PopD insert pores into membranes forming homo-or hetero-complexes of undetermined stoichiometry. Single-molecule fluorescence photobleaching experiments revealed that PopD formed mostly hexameric structures in membranes, whereas PopB displayed a bi-modal distribution with 6 and 12 subunits peaks. However, individually the proteins are not functional for effector translocation. We have found that when added together, the translocators formed distinct heterocomplexes containing 8 PopB and 8 PopD molecules. Thus, the interaction between PopB and PopD guide the assembly of a unique hetero-oligomer in membranes.The transport of proteins across membranes is essential at many stages of pathogen infection and colonization of human cells. This process typically involves the discharge of proteins from the pathogen (secretion) and the introduction of these secreted toxins/effectors into the cytosol of the target cell (translocation). Many pathogens, including the Shigella, Salmonella, Yersinia, and Pseudomonas species, exploit a sophisticated and efficient mechanism of protein secretion and translocation known as type III secretion (T3S) 3 system (1, 2). The T3S system is a syringe-like macromolecular machine formed by more than 20 different proteins organized in three major structures to span: (i) the inner bacterial membrane, the periplasmic space, and the outer bacterial membrane (the secreton); (ii) the extracellular space (the needle); and (iii) the host cellular membrane (the translocon) (3-6).A phylogenetic analysis of bacterial T3S systems based on conservation of their basal body ATPase indicates the presence of at least 7 families of T3S machines. The Pseudomonas aeruginosa genome encodes a single T3S system grouped within the Ysc family, named after the Yersinia spp. T3S system (the archetypical T3S system in this family) (7). The Ysc family includes pathogens like Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Bordetella pertussis, Vibrio parahemeolyticus, and P. aeruginosa, among others. The Ysc family shares structural similarity with the Inv-Mxi-Spa family of T3S systems, which includes the secretion systems used by Salmonella enterica and Shigella spp. (8).Great progress has been made in the structural characterization of the secreton and the needle for different T3S system families (9). However, little is known about how T3S-secreted proteins are translocated across the plasma membrane of the target cell to alter the normal function of the host (4). Two T3S-secreted proteins, known as the T3S translocators, insert into the target membrane to facilitate effector translocation. P. aeruginosa translocators PopB/PopD...

Translocation of bacterial toxins or effectors into host cells using the Type III Secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH, and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH dependant manner as observed by two independent assays, time-resolved FRET and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH lower than 5.0, suggesting that acidic-residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.

Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics.