The PscE-PscF-PscG Complex Controls Type III Secretion Needle Biogenesis in Pseudomonas aeruginosa

Abstract

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...

Molecular model of a type III secretion system needle: Implications for host-cell sensing

“…MxiH also has structural similarity to the Yersinia T3SS protein YscE (21) (Fig. 1E), which has recently been proposed to be the chaperone for YscF, the Yersinia needle subunit (22,23). The strong structural similarity between a needle subunit, MxiH, and a needle-subunit chaperone, YscE, suggests that the subunit-chaperone interactions might mimic the subunit-subunit interactions in the assembled needle to block further polymerization.…”

Structure of the heterotrimeric complex that regulates type III secretion needle formation

“…P. aeruginosa strains that lack chromosomally encoded pscE are incapable of producing PscG stably; the same occurs in pscG-deleted strains, which lack PscE and, in both cases, strains are noncytotoxic. In addition, PscE and PscG can be purified in vitro as a 1:1 complex that is more stable than isolated PscE or PscG (16). PscE folds into two antiparallel helices, Hb and Hc, that are preceded by a short independent helix (Ha).…”

“…PscF is found in the bacterial cytoplasm uniquely within a soluble heterotrimeric complex with two partners, PscE and PscG; clinical isolates mutated to lack either one of these two molecules are viable but noncytotoxic (16). Interestingly, homologs of PscE and PscG exist in a variety of pathogens (Fig.…”

Bioinformatic and Biochemical Evidence for the Identification of the Type III Secretion System Needle Protein ofChlamydia trachomatis

“…It has also been demonstrated that Yersinia YscF and Pseudomonas PscF require two distinct chaperones, YscE/YscG and PscE/PscG, respectively, to maintain stability and prevent their premature polymerization within the bacterial cytosol (10,44,45). Mutational analysis of PscE and PscG has demonstrated that the formation of heterotrimeric PscE-PscF-PscG is absolutely required for efficient secretion of PscF and full cytotoxicity.…”