The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa.

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 microm 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)(2)(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 assembly are still unclear, many details can be inferred from studies performed on the Salmonella SPI-1-encoded and enteropathogenic Escherichia coli T3S systems, which suggest that only upon full assembly of the basal structure can needle components be secreted and subsequently polymerize onto the surface of the cell (23,24). In Yersinia sp., the length of the needle is strictly controlled by YscP, a 50-kDa protein that acts as a molecular ruler (25,26).
In T3S virulence and flagellar systems, secretion-prone effectors are stabilized within the bacterial cytoplasm by small, acidic chaperones, that remain within the cytosol after effector secretion (27)(28)(29)(30). Interestingly, even though cognate molecules must be released from the chaperone prior to secretion, association constants between these complexes are in the nanomolar range (27,31,32). The mechanism of dissociation of these tight complexes is still unknown, but it is conceivable that the ATPase located on the cytosolic side of the base structure, which is believed to couple ATP hydrolysis and effector translocation through the T3S conduit, may also play the role of "docking platform" (22,33,34). To date, chaperones have been reported as being dedicated molecules, binding to one or, at most, two effectors or translocators (35). Chaperones that bind to building blocks of filament or flagellumforming proteins play the key role of preventing early polymerization of their cognate molecules (32,36). This is the case for FliC units, which polymerize at the distal end of a growing flagellum; within the bacterial cytoplasm, FliC is chaperoned by FliS, which traps it in a monomeric state (36). Likewise, CesA, a small acidic chaperone, prevents oligomerization of EspA, a protein that forms a filamentous structure prolonging the T3S needle, observed uniquely in enteropathogenic E. coli strains (32). However, the identity of chaperones recognizing the T3S needleforming subunits, as well as the mechanism behind T3S needle formation, have remained elusive.
Pseudomonas aeruginosa is an important opportunistic pathogen responsible for severe nosocomial infections as well as untreatable chronic disease in cystic fibrosis patients (37,38). The cytotoxicity of clinical isolates is strictly dependent on a functional type III injectisome that is able to translocate four distinct effectors/exoenzymes (ExoS, ExoT, ExoU, and ExoY), all of which play key roles in eukaryotic cell intoxication (39 -41). Genes required for assembly, regulation, and function of the T3S system in P. aeruginosa are located on the bacterial chromosome and clustered in five operons, one of which, exsDpscBC-DEFGHIJKL (Fig. 1A), encodes needle assembly components, among others. Notably, purified P. aeruginosa T3S needles are composed mostly of PscF, and a deletion mutant of this gene loses the ability to secrete effector proteins into culture supernatants or translocate molecules into the host cell cytoplasm. It is of interest that antibodies directed against YscF from Yersinia pestis have recently been shown to protect against bubonic plague in a mouse model, suggesting that needle-forming subunit proteins could also represent therapeutic targets (43).
In this work, we identify and characterize, for the first time, the macromolecules that participate in T3S needle biogenesis. We show that PscF from P. aeruginosa is able to form robust needle-like structures of different lengths when expressed on its own. Within the bacterial cytoplasm, PscF forms a stable, soluble complex with PscE and PscG in 1:1:1 stoichiometry, thus being trapped in a monomeric state. PscG and PscE are absolutely required for type III secretion and cytotoxicity of P. aeruginosa by influencing intrabacterial PscF levels. Moreover, overproduced PscF cannot be exported and assembled into the needle in PscE-and PscG-deficient mutant strains, suggesting that the formation of the PscE-PscF-PscG complex is required for proper targeting of PscF to the secretion. Circular dichroism melting temperature studies show that, as the number of macromolecules in the complex increases, so does thermodynamic stability, with the ternary PscE-PscF-PscG complex being more stable than its respective components. Thus, in the T3S needle biogenesis mechanism, two distinct macromolecules fulfill roles of preventing premature polymerization of the needle-forming subunit within the cytoplasm and maintaining it in a secretion-prone conformation.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions-Cytotoxic P. aeruginosa cystic fibrosis isolate CHA (44) was used as the parental strain, and all mutants are isogenic to it. P. aeruginosa strains were grown on Pseudomonas Isolation Agar (Difco) plates or in liquid Luria broth (LB) at 37°C with agitation. Carbenicillin was used at 500 g/ml for Pseudomonas Isolation Agar plates and 300 g/ml in LB. For type III induction, P. aeruginosa overnight cultures were diluted to an optical density at 600 nm (A 600 ) of 0.2 in LB containing 5 mM EGTA and 20 mM MgCl 2 . Incubation was prolonged for additional 3 h until the cultures reached A 600 values of 1.2-1.5. E. coli DH5␣ cells were used for standard cloning experiments, whereas E. coli BL21(DE3)Star (Invitrogen) was used for overproduction of all His-tagged recombinant proteins.
Construction of Expression Vectors-The pscEFGHI part of the exsD-pscBCDEFGHIJKL operon was amplified by standard PCR procedures from CHA genomic DNA and cloned into a TOPO vector (Invitrogen). During this work, we found that PscE contained two amino acid mutations when compared with the P. aeruginosa PAO1 sequence: C40G and H43R (45). 4 These changes were confirmed in several independent PCR reactions. Genes of interest were amplified by PCR with TOPO/EFGHI as a template and subsequently cloned into either pET-15b (Novagen) downstream from a hexahistidine tag, for pscE, pscG, and pscEFGHI, or pET-22b (Novagen) for pscF leading to the pscF-his6 fusion (see Table IS in supplemental material).
Expression and Purification of PscE, PscF, PscG, and PscE-PscF-PscG-Protein expression in E. coli BL21(DE3)Star was similar in all cases and was induced in Terrific broth (TB, Difco) with 1 mM isopropyl 1-thio-␤-Dgalactopyranoside at 37°C for 3 h. Cells were harvested by centrifugation and lysed by sonication in 25 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 2% glycerol. Supernatants were cleared by centrifugation and applied to a Ni 2ϩ -Sepharose column, pre-equilibrated in 25 mM Tris-HCl, pH 7.15, 0.2 M NaCl, 20 mM imidazole, 2% glycerol (except for PscE, in which 25 mM Tris-HCl, pH 8.0, was employed). Proteins were eluted with a step imidazole gradient and were subsequently eluted from a gel filtration column (Amersham Biosciences HR10/60) in 25 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA. 10 mM dithiothreitol was added to the samples after gel filtration. PscG was subsequently submitted to further purification on a Mono Q column in 25 mM Tris-HCl, pH 8.0, 1 mM EDTA and 5 mM dithiothreitol, being eluted with a NaCl gradient to 0.5 M.
Construction of Knock-out Mutants-The CHA⌬F strain is described elsewhere (42). PscE and PscG knock-out strains (CHA⌬E and CHA⌬G) were created by exchanging the wild-type copy of the gene by a deleted copy harbored on a non-replicative plasmid. The list of primers and constructed plasmids is provided in supplemental Table IS. The exchange was generated by a double recombination event obtained by negative selection procedure using the sacB gene (46,59). Selection for double recombination events was achieved by growing co-integrate strains on plates containing 5% sucrose. Carbenicillin-sensitive, sucrose-resistant strains were checked for correct replacement of the wild-type allele by the pscE-and pscG-deleted alleles by PCR and further verified by Southern blotting. All mutants were complemented in trans by a wild-type gene and checked for the restoration of the phenotype to ensure non-polarity of the mutation. All complementations were performed using pIApG in which the gfp cassette was replaced by the gene of interest, thus placing it under the control of the type III promoter ppcrG (pG), as described previously (47). The construction of pIApG/ pscF, which is able to complement the pscF deletion, is described elsewhere (42). The pscE and pscG genes were recovered from pET-15b/ pscE and pET-15b/pscG using NdeI-HindIII digestion and cloned into NdeI-HindIII-digested pIApG. In this case, the ribosome binding site from the gfp cassette that was left in the plasmid is used for the pscE and pscG translation. The his6-pscE and his6-pscG fusions were transferred to P. aeruginosa by introducing XbaI-HindIII fragments from pET-15b/ pscE and pET-15b/pscG into pIApG. Type III secretion capacity of all strains was checked systematically in vitro, by triggering the secretion in LB supplemented with 5 mM EGTA and 20 mM MgCl 2 and analyzing culture supernatants on SDS-PAGE (48).
Immunoblotting Analysis-Cell extracts were obtained from P. aeruginosa cells, which were broken by sonication and ultracentrifuged. Proteins in cleared lysates were resolved on SDS-18% PAGE run in Tris-Tricine buffer and transferred to nitrocellulose membrane. For batch purifications, P. aeruginosa-cleared lysates were incubated with Ni 2ϩ beads (HIS-select Nickel Affinity Gel, Sigma). Washing and elution steps were performed as recommended by the supplier. Anti-PscE and anti-PscG antibodies were raised in mouse at HybrIsère (Grenoble, France) using His 6 -tagged recombinant proteins. Anti-PcrV antibodies and anti-PscF antibodies were raised in rabbits (47). 4 Secondary antibodies were horseradish peroxidase-conjugated (Sigma). Membranes were developed with the ECL kit (Amersham Biosciences).
Infection Experiments and Cytotoxicity Assays-All cytotoxicity assays were performed as described previously (49) by using macrophage cell line J774. Cell death was determined with the use of a cytotoxicity detection kit (LDH; Roche Applied Science).
Circular Dichroism Measurements-Circular dichroism spectra were measured on a Jasco J-810 spectropolarimeter at 22°C in a 1-mm cell in 10 mM sodium phosphate, pH 7.2, 0.1 M NaCl at protein concentrations of 0.2 mg/ml for all samples. Subsequently, the thermodynamic stability was recorded at 222 nm by monitoring the circular dichroism signal in a range of 4 -96°C with scan rate of 1°C/min for increasing and decreasing temperature assays. The spectra were corrected against those of the buffer reference. The measured ellipticity was adjusted to the same starting value for all samples for a better visualization of the curves.
The equilibrium constant K(T) was determined using the equation where measured ellipticity values (y) were considered as linear combinations of the limit values of the folded state (y F ) and of the unfolded state (y U ) and y ϭ y F f F ϩ y U f U . The free energy curves were calculated using the equation, ⌬G(T) ϭ ϪRT ln(K(T)), where R is the gas constant (R ϭ 1.987 calories/degree/mole) and T is the absolute temperature (in Kelvin). The melting temperature (T m ) was determined at ⌬G(T m ) ϭ 0.
Mass Spectrometry-Samples at 1 M in water/acetonitrile (1/1, v/v) with 0.2% formic acid were infused at a flow rate of 5 l/min. Mass spectra were recorded in the 500-to 1800-m/z range in a Q-TOF Micro mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ion source operated with a needle voltage of 3 kV, with sample cone and extraction cone voltages of 45 and 2 V, respectively. Data were acquired in the positive mode, and calibration was performed using the multiply charged states produced by a separate injection of heart horse myoglobin dissolved in water/acetonitrile (1/1, v/v) with 0.2% formic acid. For native experimentation, sample cone and extraction cone voltages were of 150 and 7 V, respectively. The backing Pirani pressure was set at 4.4 mbar. Mass spectra were recorded in the 2000 -6100 mass-to-charge (m/z) range with sample concentrations at 20 M in 20 mM ammonium bicarbonate and continuously infused at a flow rate of 7 l/min. Data were acquired in the positive mode, and calibration was performed using a solution of 0.5 mg/ml CsI in water/isopropyl alcohol (1/1, v/v). Data were processed with MassLynx 4.0 (Micromass).
Electron Microscopy-Samples at a concentration of 0.15 mg/ml were applied to the clean side of carbon on mica (carbon/mica interface) and negatively stained with 1% phosphotungistic acid. A grid was placed on top of the carbon film, which was subsequently air dried. Micrographs were taken under low dose conditions with a Philips CM12 microscope operating at 120 kV and a nominal magnification of 45,000. major T3S needle component of Yersinia sp. (12), while harboring only 25% identity to T3S needle monomers of Salmonella and Shigella sp. (PrgI and MxiH, respectively, Fig. 1B). PscF is a naturally self-associating protein, which, when expressed in E. coli and purified by chromatography, elutes as a complex peak in the void volume of an analytical gel filtration column ( Fig. 2A). Negative-stained electron microscopy images of samples obtained from the forefront of the void volume peak reveal that PscF forms highly elongated needle-like fibers of Ͼ1 m in length (Fig. 2B), a value that is reminiscent of lengths of needles observed in Shigella strains, which overproduce MxiH (9) or lack a protein controlling needle length (50). The width of the PscF needle-like structures is ϳ8 nm, a value that is in agreement with that for T3S needles isolated directly from different pathogenic species (9, 11, 12) (Fig. 2B)). The latter part of the gel-filtration peak contains shorter needle-like structures (Fig. 2C), which may be representative of intermediary forms of the fully polymerized molecule.
The high propensity of PscF to generate elongated polymers suggests that, within the bacterial cytoplasm, its polymerization must be blocked by a macromolecular "bodyguard." To identify this molecule, we overexpressed and purified protein products from a polycistronic expression vector encoding the pscEFGHI section of the exsD-pscBCDEFGHIJKL P. aeruginosa operon (Fig. 1A) in an E. coli system, where the construct introduced a hexahistidine tag to the N terminus of PscE. This experiment yielded three co-purified polypeptides after Ni 2ϩ affinity chromatography and gel filtration, which were identified as His 6 -PscE (9,326 Da), PscF (9,153 Da), and PscG (12,451 Da) by mass spectrometry (data not shown). Elution of the ternary complex from a gel-filtration column revealed that it migrates much slower than PscF-His 6 on its own ( Fig.  2A), and its stoichiometry was found to be 1:1:1 by native mass spectrometry, which yielded a molecular mass of 30,933 Da (expected molecular mass 30,930 Da). It is of note that neither PscE nor PscG could rescue polymerized PscF when the proteins were expressed and purified separately, and formation of the soluble 1:1:1 complex required that all three proteins be expressed concomitantly from the same operon (data not shown). This result indicates that PscE and PscG trap PscF in a monomeric state within a ternary complex and thus act as inhibitors of PscF polymerization.
To demonstrate the existence of the PscE-PscF-PscG complex in the P. aeruginosa cytoplasm, cell extracts from a cytotoxic, P. aeruginosa wild-type strain (CHA) or PscE and PscG-deficient strains expressing either His 6 -PscE or His 6 -PscG, respectively, were batch-incubated with Ni 2ϩ affinity beads. Subsequently, the bound material was washed and eluted. Aliquots of eluted fractions were analyzed by immunoblotting using antibodies raised against recombinant PscF, PscE, and PscG. Eluted fractions obtained from P. aeruginosa cells synthesizing His 6 -PscE contained PscF and PscG, and from those synthesizing His 6 -PscG contained PscF and PscE (Fig. 3). Thus, in the P. aeruginosa cytoplasm, both PscE and PscG were able to associate to other members of the ternary complex.
PscE and PscG Are Required for Needle Biogenesis in P. aeruginosa-To examine the function of PscE and PscG in T3S and needle formation, we deleted the pscE and pscG genes from the chromosome of the cytotoxic P. aeruginosa strain CHA by a double recombination technique, generating strains CHA⌬E and CHA⌬G. The CHA⌬E strain was unable to secrete any of the type III proteins under in vitro inducing conditions (Ca 2ϩ depletion) and was non-cytotoxic on a macrophage cell line. Both phenotypes were restored in the mutant strain complemented with PscE or His 6 -PscE, showing that the mutation does not affect the expression of the downstream genes of the operon (Fig. 4A). Subse-  Batch Ni 2ϩ -affinity purifications were applied to cleared lysates from wild-type P. aeruginosa (WT) and mutant strains producing functional His 6 -PscE (⌬E/H 6 E) and His 6 -PscG proteins (⌬G/H 6 G). After washing steps, eluted aliquots were analyzed by immunoblotting with anti-PscG, anti-PscE, and anti-PscF antibodies. PcrV, which does not associate to the needle complex, was used as a control. quently, P. aeruginosa CHA and CHA⌬E strains were cultivated in T3Sinducing conditions (48), and crude extracts were analyzed by immunodetection. Notably, a deficiency in PscE resulted in the complete absence of PscF within the bacterial cytoplasm, strongly suggesting that, in the absence of PscE, PscF is rapidly degraded (Fig. 4B). It has been previously suggested that needle components are eliminated if they cannot be exported from the bacterium, as shown for MxiH and PrgI in secretion-deficient Shigella and Salmonella strains, respectively (23,51). Thus, PscF stability in the absence of secretion was tested in a secretion-deficient mutant strain, that lacks PscL, a protein suggested to participate in the control of the T3S ATPase (CHApscLTn5 (⌬L) (52)). The presence of PscF in the non-secretory strain at levels comparable to the wild-type levels suggests that its degradation in CHA⌬E (Fig. 4B) is directly related to the absence of PscE and not to unspecific degradation. To further examine the role of PscE, we introduced several copies of pscF in the CHA⌬E mutant strain in trans and analyzed transformed cells by Western blotting (⌬E/F, see Fig. 6). Notably, PscF was then easily detected in the P. aeruginosa cytoplasm, but was not able to rescue non-secretory and non-cytotoxic phenotypes (Fig. 4A), indicating that PscE is required for PscF export and/or needle formation.
To investigate the function of PscG, the third partner in the ternary complex, in needle biogenesis, a pscG knock-out strain was generated in a P. aeruginosa CHA background (CHA⌬G). CHA⌬G was non-cytotoxic on infected macrophages, and this phenotype was rescued by complementation with a wild-type pscG copy introduced in trans (Fig.  5A). Steady-state level analyses of PscF in CHA⌬G and complemented strains, performed by immunoblotting, revealed that PscF levels were severely diminished in the pscG knock-out (Fig. 5B). Of note, the pscG deletion also affected the levels of PscE. However, synthesis of PscE in trans was not sufficient to restore T3S functionality (Fig. 5). Similarly, in the CHA⌬E strain, no PscG protein could be detected, an indication that the two PscF partners co-stabilize each other (Fig. 6, cytoplasm).
As observed for PscF in the CHA⌬E strain, expression of pscF in trans in CHA⌬G, restored high levels of intracellular PscF, but did not permit the strain to regain secretion and cytotoxicity capabilities (Fig. 5A). Therefore, PscG and PscE seem to be required for the assembly of PscF into a functional, cell-surface-localized secretory channel.
Because PscF could be produced in CHA⌬E and CHA⌬G mutants, but the system was incapable of displaying T3S-dependent toxicity, we verified whether PscF is able to be exported by immunoanalyzing surface-detached appendices. Samples containing detached needles from wild-type P. aeruginosa obtained by gently washing the cells, followed by ultracentrifugation, displayed large amounts of PscF (Fig. 6). In the pscE and pscG knock-out strains producing PscF extrachromosomally, even though it was detected in the cytoplasm, no PscF was detected in semipurified samples of cellular appendages (Fig. 6). These results confirm that, in the absence of its partners, PscF, even when expressed in trans, is not able to be secreted and remains within the cytoplasmic compartment, possibly as a polymerized molecule. It is of interest that an immunolocalization assay of PscE and PscG (Fig. 6) revealed that they could not be detected in the PscF-needle fraction, indicating that they play their roles strictly within the cytoplasmic compartment.
PscE and PscG Block the Energetically Favorable Polymerization of PscF-To understand the role played by PscE and PscG in the stabilization process of PscF, we produced and purified both molecules individually as His-tagged fusions, and constructed pscE-pscF and pscF-pscG FIGURE 5. Role of PscG in type III cytotoxicity and PscF-needle assembly. A, the cytotoxicity of P. aeruginosa strains lacking PscG is reminiscent of that of strains lacking PscE, as shown in Fig. 4. B, cell extracts from indicated P. aeruginosa strains were analyzed as described for Fig. 4B, with anti-PscG, anti-PscE, anti-PscF, and anti-PcrV antibodies. Note the low levels of PscF in the PscG-deficient mutant (⌬G). Note also that the introduction of pscE and pscF in trans restored the intracellular levels of PscE and PscF proteins, but did not restore cytotoxicity. . PscE is required for cytotoxicity and needle assembly. A, macrophage cell line J774 was infected at multiplicity of infection of 5 with bacteria grown to mid-exponential phase. Cell death was assayed at 3 h post-infection by measuring the release of lactate dehydrogenase into cell supernatants. B, cell extracts from different P. aeruginosa strains were analyzed by Western blotting using antibodies raised against recombinant PscE, PscF, and PcrV, an unrelated T3S protein used as loading marker. PscF-deficient mutant strain (⌬F) as well as complemented strain (⌬F/F) were used as controls. The blot was revealed by chemiluminescence. Note the absence of PscF in the PscE-deficient mutant (⌬E).
bicistronic vectors to attempt to produce the binary complexes in E. coli. Individually prepared PscE and PscG displayed low solubility (Ͻ5 mg/ml), and neither binary complex could be isolated (data not shown). Notably, a PscE-PscG complex could be isolated by gel-filtration techniques by mixing equimolar quantities of each of the purified proteins, indicating that these two macromolecules interact directly. This complex was more soluble than its individual components (up to ϳ20 mg/ml). Interestingly, the PscE-PscF-PscG ternary complex could be concentrated to Ͼ100 mg/ml, suggesting that all three proteins are required for optimal solubility. Circular dichroism spectroscopy measurements showed that PscG, PscE, and the PscE-PscG and PscE-PscF-PscG complexes are all predominantly helical (not shown). Subsequent thermal denaturation experiments performed by following the change in ellipticity at 222 nm revealed sigmoidal, cooperative, two-state compatible folding transitions for all four molecules/complexes, with transition temperatures of 52°C (PscG), 53°C (PscE), 62°C (PscE-PscG), and 64°C (PscE-PscF-PscG) (Fig. 7). The less precise sigmoidal nature of the curves for PscG and PscE suggests that, when on their own, they may be less stable than the binary or ternary complexes (Fig. 7). These results indicate that, as the number of partners in the complex increases, so does macromolecular stability. Notably, unfolding and refolding curves, as well as circular dichroism spectra before and after heating, were superimposable in all four cases. This suggests not only that PscG, PscE, PscE-PscG, and PscE-PscF-PscG are able to fold autonomously, but also that their respective unfolding and refolding pathways are identical.
In contrast, although PscF could also be shown to be mostly helical, no clear endotherm indicative of a cooperative melting behavior could be observed (Fig. 7). Considering these results in conjunction with those described above, it is possible to conclude that PscF, once released from the ternary complex, adopts a lower energetic state by polymerizing into a robust, highly stable structure, whose polymeric nature cannot be modified either by physical stress or by binding to PscE or PscG.

DISCUSSION
The T3S needle is an essential structure that is believed to serve as a secretion channel/conduit for bacterial effectors from the cytoplasm into the target cell cytosol. Although many of the proteins that are conserved within T3S systems of different bacteria also display sequence similarity to macromolecules that make up the basal body of the flagellum, this is not the case for the needle subunits, which display no sequence similarity to flagellar building blocks (4,53). In all species studied to date, the T3S needle is believed to be formed by a single polymerized molecule. In this work, we have demonstrated that the major component of the T3S needle in P. aeruginosa, PscF, polymerizes mostly into elongated (Ͼ1 m) needle-like structures of 8 nm in diameter when expressed on its own; in addition, it can also form shorter fibrous intermediates (Fig. 2), which can either represent forms whose polymerization was interrupted, or intermediate forms generated by sonication during the purification process. Because the length of the PscF needle has been reported as being ϳ80 nm (42), this work shows that in the presence of a large amount of PscF subunits and in the absence of an adjuvant molecule, which could regulate needle length, the size of the PscF polymers cannot be controlled. Loss of needle length control has been reported in Shigella mutants that overexpress MxiH or lack Spa32, as well as in Salmonella mutants that lack InvJ (9,21,50). Spa32 and InvJ have been likened to the flagellar protein FliK, which acts as a controller of the length of the flagellar hook structure in the flagellum hook/basal-body complex (53,54). In Yersinia, the function of "molecular ruler" is played by YscP, which regulates the length of the YscF needle (26). Tight control of T3S needle length is crucial for cytotoxicity and infectivity (25), and it is conceivable that, in the P. aeruginosa system, needle length control is regulated by the homologous PscP protein, as suggested recently by Cornelis and co-workers (55).
Although other fiber-forming molecules whose sequences are not related to that of the T3S needle also require cytoplasmic stabilization to prevent early polymerization, such as flagellin, FimC, and EspA, these molecules remain in monomeric form in the presence of a single chaperone (32,36,56). In the T3S system of P. aeruginosa, two distinct bodyguards, PscE and PscG, are essential for stabilizing PscF, as demonstrated by co-purification experiments and phenotypic analyses of knock-out mutants. PscE, PscF, and PscG form a stable, 1:1:1 complex, as verified by mass spectrometry, gel filtration, and circular dichroism melting scans. PscE has counterparts in Yersinia sp. (YscE) and Salmonella sp. (SsaE), although no obvious candidates could be identified in Shigella (Fig. 1B). Sequence alignments of PscG reveal that this acidic molecule shares 47% similarity with YscG from Yersinia sp., but sequence homologues could not be find in either Salmonella or Shigella sp. However, T3S loci in those pathogens harbor several small open reading frames of unknown function, which could fulfill the roles played by PscE and PscG.
PscE and PscG are absolutely essential for PscF functionality in vivo. P. aeruginosa PscG and PscE knock-out strains both lack PscF and are  consequently non-cytotoxic; both cytotoxicity and PscF levels are re-established upon complementation with the wild-type pscG and pscE genes introduced in trans. In addition, PscF export from the P. aeruginosa cytoplasm toward the bacterial surface cannot be accomplished in the absence of PscE and PscG in mutants where PscF is encoded in trans (Fig. 6). In this case, it is conceivable that free PscF polymerizes within the cytoplasmic compartment, thus not being able to be exported through the T3S basal body. Interestingly, PscE and PscG form a stable complex, which can be purified by gel filtration and whose thermodynamic stability is greater than that of the individual macromolecules (Fig. 7). This suggests that the PscE-PscG binome could function as a "PscF-stabilizing unit" in the P. aeruginosa cytoplasm. In addition, PscE and PscG seem to co-stabilize each other, because PscE was absent in the pscG-deleted mutant strain and vice versa. It is of note that an interaction between Y. pestis YscE and YscG (Fig. 1B) was identified by twohybrid technology (57), suggesting that this complex is also present in the Yersinia system.
The PscF needle-like polymer is thermodynamically stable, because no clear T m value could be identified for the polymerized molecule and a clear helical signal could be measured before unfolding and after refolding of PscF (Fig. 7). Thus both PscE and PscG capture PscF subunits within the cytoplasm with the goal of blocking an energetically favorable, but highly disadvantageous, intracytoplasmic polymerization of PscF. The fact that PscE-PscF-PscG is more stable than PscE-PscG (Fig. 7) suggests that, within the complex structure, PscF is at least partly folded. It is of interest that in the case of the adhesive type 1 pilus, chaperone-subunit complexes have been suggested as being highly unstable in solution, with chaperones acting as folding platforms and trapping subunits in a molten globule-like conformation (58). The thermodynamic stability of the PscE-PscF-PscG complex presented here argues against a molten globule-like structure. In fact, the reversibility and cooperative nature of unfolding/refolding temperature-scanning curves for PscE-PscF-PscG indicate that, once the ternary complex is unfolded, refolding is able to occur through the same pathway, and that, in this case, PscF remains associated to PscE and PscG. Taken together, these observations suggest that, in P. aeruginosa, once PscF is stably trapped by PscE and PscG, an external event must take place to aid it in becoming dissociated from its bodyguards; this event could include interaction with another T3S protein located within the basal body, as is the case of flagellar axial proteins, which are targeted to the FliI-FliH flagellar export complex prior to secretion (34). This is also the case for the CesT chaperone of enteropathogenic E. coli, which brings its substrate, Tir, to the ATPase (EscN), located at the base of the T3S machinery (33). Future genetic and biochemical studies should determine if either of the two ternary complex partners is responsible for interaction of PscF with the basal body of the P. aeruginosa secretion apparatus.