Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-Å x-ray crystal structure of the PscE-PscF 55-85 -PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus-or flagellum-forming molecules. Disruption of specific PscG-PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials.bacterial pathogenicity ͉ chaperones ͉ x-ray crystallography

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...