Pseudomonas aeruginosa efficiently intoxicates eukaryotic cells through the activity of the type III secretiontranslocation system (TTSS). Gene deletions within the translocation operon pcrGVH-popBD abolish poreforming activity of P. aeruginosa strains with macrophages and TTSS-dependent hemolysis. Here we investigated the requirements for PcrV, PopB, and PopD in pore formation by analyzing specific mutants using red blood cells (RBCs) and fibroblasts expressing green fluorescent protein fused to actin. Simultaneous secretion of three proteins, PopB, PopD, and PcrV, was required to achieve wild-type hemolysis and effector translocation. Deletion of pcrV in a cytotoxic strain did not affect secretion of PopB and PopD but abolished hemolytic activity and translocation of effectors into fibroblasts. Notably, the PcrV-deficient mutant was not capable of inserting PopD into host cell membranes, whereas PopB and PopD, but not PcrV, were readily found within membranes of wild-type-infected RBCs. Immunoprecipitation experiments performed by using a liposome model of pore assembly revealed a direct interaction between PopD and PopB but not between PopD and PcrV. Consequently, PcrV is necessary for the functional assembly of the PopB/D translocon complex but does not interact directly with pore-forming Pop proteins.

Type III secretion (T3S) systems play key roles in pathogenicity of many Gram-negative bacteria and are employed to inject toxins directly into the cytoplasm of target cells. They are composed of over 20 different proteins that associate into a basal structure that traverses both inner and outer bacterial membranes and a hollow, needle-like structure through which toxins travel. The PscF protein is the main component of the Pseudomonas aeruginosa T3S needle. Here we demonstrate that PscF, when purified on its own, is able to form needle-like fibers of 8 nm in width and >1 m in length. In addition, we demonstrate for the first time that the T3S needle subunit requires two cytoplasmic partners, PscE and PscG, in P. aeruginosa, which trap PscF in a ternary, 1:1:1 complex, thus blocking it in a monomeric state. Knock-out mutants deficient in PscE and PscG are non-cytotoxic, lack PscF, and are unable to export PscF encoded extrachromosomally. Temperature-scanning circular dichroism measurements show that the PscE-PscF-PscG complex is thermally stable and displays a cooperative unfolding/refolding pattern. Thus, PscE and PscG prevent PscF from polymerizing prematurely in the P. aeruginosa cytoplasm and keep it in a secretion prone conformation, strategies which may be shared by other pathogens that employ the T3S system for infection. Type III secretion (T3S)3 systems are present in most Gram-negative bacteria and are devoted to the secretion and injection of bacterial toxins (effectors) directly into the target cell cytoplasm. Such nanomachines, which play key roles in functions such as virulence and symbiosis, are composed of ϳ20 distinct proteins, and translocated effectors possess diverse enzymatic activities and target major cellular processes ranging from actin assembly to apoptosis (1-3). Macromolecular components of T3S injectisomes from a variety of pathogens display high levels of sequence similarity and are assembled in supramolecular structure, reminiscent of the bacterial flagellar apparatus, and imbedded within the two bacterial membranes (4, 5). The basal portion of the injectisome consists of two sets of protein rings to which is associated a needle-like structure, which may protrude up to 80 nm from the bacterial surface (6 -13). The needle is essential for secretion and translocation and is the hollow conduit through which effectors travel to reach the target cell (9,14,15). At the level of the host cell membrane, the translocation process is initiated by the insertion of a proteinaceous pore whose components are themselves transported through the needle-like conduit, which gives access to the target cell cytosol to other T3S-translocated effectors (16 -20). Effectors and translocators are thought to travel in partially unfolded states through the needle, which, in all bacteria studied to date, is mostly composed of one polymerized small molecular mass protein (PrgI in Salmonella sp., MxiH in Shigella sp., and YscF in Yersinia sp.) (7,8,12,21,22). Although the precise steps involved in needle assembl...