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Journal of Bacteriology, October 2008, p. 6580-6588, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00761-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the Putative Type III Secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae

Chris B. Stone, Dustin L. Johnson, David C. Bulir, Jodi D. Gilchrist, and James B. Mahony^*

M. G. DeGroote Institute for Infectious Disease Research, Faculty of Health Sciences and Department of Pathology and Molecular Medicine, McMaster University, and Father Sean O'Sullivan Research Centre, St. Joseph's Healthcare, Hamilton, Ontario, Canada

Received 28 May 2008/ Accepted 1 August 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P_i from ATP at a rate of 0.55 ± 0.07 µmol min^–1 mg^–1 in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.

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Type III secretion (T3S) systems (T3SS) are essential virulence factors utilized by a variety of gram-negative bacterial pathogens such as Salmonella, Pseudomonas, Yersinia, and Escherichia coli to facilitate the invasion of host cells (12, 13, 15). The T3SS facilitates the injection of effector proteins from the cytoplasm of the bacteria directly into the host cell cytoplasm across the inner membrane, periplasmic space, and outer membrane of the bacteria. Bacterial effector proteins exert a number of effects on the host cell, facilitating bacterial uptake by manipulating the actin cytoskeleton and, subsequently, growth by providing an environmentally friendly niche (20). The distinguishing feature of the T3SS is a needle-like nanomachine, or injectisome, which protrudes from the bacterial outer membrane and interacts with lipid rafts in the host cell membrane, through which effector proteins are transported (9).

C. pneumoniae is an obligate intracellular pathogen that has been associated with pneumonia, bronchitis, and atherosclerosis. The developmental cycle of C. pneumoniae begins when an elementary body (EB), a metabolically attenuated developmental form, attaches to the host cell cytoplasmic membrane (21). Bacterial uptake is induced by an as-yet-unknown mechanism that involves the activation of the host MEK-extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase pathways (6, 8, 38), possibly mediated by T3S effector proteins. The injection of one effector, translocated actin-recruiting protein (TARP), is believed to play a key role in the activation of a signaling cascade that recruits and remodels actin at the site of EB attachment, facilitating chlamydial entry into the cell (7, 26). Once inside the cell, EBs exist in a parasitophorous membrane-bound vesicle known as an inclusion, where they differentiate into metabolically active, noninfectious reticulate bodies (RBs). Inside the inclusion, RBs are associated with the inclusion membrane injecting Inc proteins into the membrane and other proteins into the host cell cytoplasm, possibly using the T3S injectisome, although this has not yet been shown. RBs divide by binary fission until an unknown signal triggers asynchronous differentiation into infectious EBs that exit the host cell either by lysis of the inclusion and host cell or by a packaged release mechanism known as extrusion, which leaves the host cell intact (21, 29). The differentiation of RBs into EBs may be triggered by chlamydial T3S (18, 34, 37).

Although C. pneumoniae contains all the genes coding for a functional T3SS, a systematic study of the injectisome proteins has not been undertaken, and little is known about individual proteins (11, 19, 32, 36). Unlike other bacterial species, where the genes encoding T3S proteins are organized into one or two distinct operons, or on virulence plasmids, the T3S genes of C. pneumoniae are scattered throughout the genome in several fragmented operons (16). Reports on the initial characterization of some T3SS proteins in C. pneumoniae have recently appeared. Johnson et al. have shown that CdsD, a unique protein homologous to YscD that contains two forkhead-associated (FHA) domains, interacts with the predicted C. pneumoniae ATPase tethering protein CdsL and CdsQ, a cytosolic component of the inner membrane that presumably forms the bulk of the T3S C ring (23). Betts et al. also recently characterized CdsF, the needle filament protein of the C. pneumoniae T3SS (4). C. trachomatis CopN, a homolog of the negative regulator of T3S in other species (that blocks the injectisome channel and is a known effector protein), has recently been shown to be translocated through heterologous T3S systems and may be an effector protein in C. pneumoniae (10, 17). Two important T3S genes, however, have not yet been identified in Chlamydia. These include the needle sensor protein, an LcrV homolog, which is important for sensing the host cell and initiating pore formation in the host cell membrane. The gene encoding the ruler protein, a YscP homolog, which is thought to act as a molecular ruler controlling the length of the needle, also has not been identified in C. pneumoniae (24). Cpn0705 in C. pneumoniae is the putative ruler protein based on sequence homology and its location in the genome, but no further studies have been completed with this protein.

T3S ATPase proteins are assembled as a dodecameric ring at the basal body of the injectisome in the bacterial cytosol and are believed to play a role in the delivery of effector proteins through the injectisome. Several T3S ATPases have been characterized, including EscN from E. coli (3, 41), YscN from Yersinia (5, 40), and InvC from Salmonella (2). These proteins have been shown to have significant sequence homology to the β subunit of the F_oF₁ ATPase and have been shown to hydrolyze ATP. Not only are these ATPases important for providing energy for protein transport, but they are also believed to play a role in unfolding the effector proteins before translocation, which may be accomplished by removing the chaperone from its cognate effector protein (1). In Yersinia, the ATPase tethering protein YscL localizes the ATPase to the inner membrane (5, 31). YscL has recently been shown to play a role in the regulation of ATPase activity by downregulating enzymatic function (5, 28). T3S ATPases oligomerize as a dodecamer at the inner membrane, and this oligomeric state has been associated with enhanced enzymatic activity (30, 33). The predicted C. pneumoniae T3S ATPase CdsN (Cpn0707) has significant sequence similarity to other T3S ATPases such as InvC, YscN, and EscN. The C. trachomatis homolog of CdsN has been shown to localize to the inner membrane by immunofluorescent staining (27). Here, we report an initial characterization of C. pneumoniae CdsN and show that it possesses ATPase activity and interacts with CdsD, CdsQ, and CdsL, three putative components of the C. pneumoniae T3S apparatus, but not Cpn0705, the putative ruler protein. We also show that CdsN interacts with the effector and negative regulator of T3S, CopN, implying a role for CdsN in effector selection before secretion through the injectisome. The importance of these protein interactions is discussed in the context of T3S injectisome assembly and function in C. pneumoniae.

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Expression plasmids. C. pneumoniae CWL029 (ATCC VR1310) (GenBank accession number AE001363) was the strain used to isolate genomic DNA for cloning and protein expression. Full-length CdsN, CdsL, CdsD, CopN, CdsQ, Cpn0705, and Cpn0706 were amplified from CWL029 using AttB-containing primers (Gateway; Invitrogen). The amplified products were cloned into pDONR₂₀₁ (Gateway; Invitrogen) to generate pENT vectors. The pENT vectors were then used in LR reactions (Gateway; Invitrogen) to produce pEX vectors containing the genes of interest. A C-terminal truncation of CdsN at amino acids 1 to 405 (CdsN_1-405) was amplified in the same manner. We used either pEX₁₇ (His-tagged) or pEX₁₅ (glutathione S-transferase [GST]-tagged) vectors for our protein expression. Table 1 outlines the final constructs used for protein expression. All constructs were confirmed by sequencing at Mobix Labs (McMaster University).

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TABLE 1. Plasmids used in this study

The bacterial two-hybrid system was used to identify protein-protein interactions (25). Genes of interest were cloned into pT18 and pT25, each vector expressing a different fragment of adenylate cyclase (AC). If the proteins of interest interact, the two subunits of adenylate cyclase will be brought together and produce cyclic AMP (cAMP). Increases in cAMP levels results in increased levels of transcription of the β-galactosidase gene that can be monitored using β-galactosidase activity assays. pT18 and pT25 were digested with KpnI (New England Biolabs) as well as genes amplified from CWL029 (CopN, CdsD, CdsQ, CdsL, Cpn0706, Cpn0705, and PknD) that had a KpnI site designed into the primers. For a positive control, the FHA-2 domain of CdsD (CdsD-FHA-2) was also cloned to interact with PknD. Ligation was performed overnight at 16°C using T4 ligase (Invitrogen), the resulting mixture was used to transform E. coli XL-1 cells, and transformants were selected on Luria-Bertani (LB) plates with 100 µg/µl ampicillin and 34 µg/µl chloramphenicol. Plasmids were prepared using the GenElute Plasmid Miniprep kit (Sigma). Table 1 summarizes the final constructs used in the bacterial two-hybrid system.

Protein expression. All constructs were expressed in E. coli BL21(DE3) cells with the exception of CdsL, which was expressed in E. coli BL21(DE3)(pLysS) cells to reduce the basal expression of the protein. Expression plasmids were used to transform E. coli BL21(DE3) and plated onto LB plates containing 100 µg/ml ampicillin. LB broth (750 ml) containing antibiotics was then inoculated with 5 ml of a culture grown overnight, and cultures were grown at 37°C until they reached an optical density at 600 nm (OD₆₀₀) of approximately 0.8. Cultures were then cooled on ice to 20°C and induced with 0.2 mM of isopropyl-β-D-thiogalactopyranoside (IPTG). Cultures were then incubated at 23°C for 3 h, and bacteria were harvested by centrifugation at 6,500 x g for 20 min in a Sorvall RC-5B centrifuge and washed with ice-cold phosphate-buffered saline (PBS). Bacteria containing His-tagged protein were resuspended in binding buffer (50 mM potassium phosphate [pH 7.2], 150 mM KCl, 1 mM MgCl₂), while the bacteria containing GST-tagged proteins were resuspended in PBS and stored at –20°C until further use.

Purification of recombinant proteins. E. coli pellets containing overexpressed proteins were thawed on ice and sonicated using a Fischer Scientific Sonic Dismembrator model 100, followed by centrifugation at 20,000 x g for 30 min to remove insoluble material. Supernatants containing His-tagged protein were stored at 4°C for use in GST pull-down assays, while the GST-tagged protein supernatants were filtered through 0.2-µm Acrodisc filters (Pall Corporation) and incubated overnight at 4°C with 300 µl of glutathione-agarose beads (Sigma). For GST pull-down assays, beads were blocked overnight in Tris-buffered saline with 0.1% Tween and 4% bovine serum albumin and stored at 4°C until use. For ATPase activity measurements, glutathione beads were washed on a column with PBS plus 0.1% Tween until the flowthrough had an OD₂₈₀ of less than 0.005. GST-tagged protein was then eluted off the column using 1.5 µg/µl reduced glutathione (Sigma) and dialyzed into activity buffer (50 mM Tris-HCl [pH 7.0], 5 mM MgCl₂, 10 mM KCl). Purity was confirmed using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

ATPase activity. ATP hydrolysis by GST-CdsN_1-405 purified from glutathione-agarose beads was measured using a malachite green assay (R&D Systems). For all experiments, the specific activity was determined using the equation of a standard line generated from a phosphate standard (R&D Systems). Reaction mixtures contained 600 ng of GST-CdsN_1-405, 4 mM ATP, 50 mM Tris-HCl (pH 7.0), 5 mM MgCl₂, and 10 mM KCl. The reaction mixture (1 ml) was incubated at 37°C for 1 h, and 50 µl of the mixture was taken for inorganic phosphate determination at various time points. The reaction was stopped by the addition of 10 µl of malachite green reagent A followed by 10 µl of malachite green reagent B to the mixture, and the mixture was incubated at room temperature for 1 min before an OD₆₁₀ reading was taken, according to the manufacturer's instructions. For the negative control, purified CdsN was digested for 10 min at 37°C using proteinase K (Invitrogen). ATPase activity was expressed as µmol phosphate released min^–1 mg^–1 of protein, and all experiments were performed in triplicate.

Oligomerization assay. In order to determine whether CdsN and CdsL form oligomers, formaldehyde fixation and nondenaturing PAGE were used. GST-CdsN_1-405 was purified from glutathione-agarose beads, dialyzed against PBS, and concentrated using Amicon 10-kDa (Millipore) concentrators to a final concentration of 5 ng/µl. Formaldehyde was added to purified GST-CdsN_1-405 to a final concentration of 10%, and fixation was allowed to continue for 10 min. Samples containing 100 ng of GST-CdsN_1-405 were electrophoresed on an 8% nondenaturing PAGE gel and visualized by Western blotting using anti-GST antibody (Sigma). His-CdsL was purified from Ni-nitrilotriacetic acid beads and utilized in the same manner, with the following difference: formaldehyde was not necessary to visualize the dimerization of CdsL, and anti-His antibody (Sigma) was used. As a control for the presence of the GST and His tags, GST-CopN and His-CopN, a protein not known to form oligomers, were also formaldehyde fixed and run on a nondenaturing PAGE gel to test for oligomerization.

GST pull-down assays. To examine the interaction of CdsN with other T3S components, GST pull-down assays were performed as described previously by Johnson et al., with the following modifications (23). Briefly, glutathione-agarose beads (30 µl) bound to 50 ng of GST-tagged CdsN or CdsN_1-405 protein was used in the assay. The beads were incubated overnight at 4°C with the E. coli lysate expressing the His-tagged proteins. The beads were collected by centrifugation and washed with 0.1% Triton X-100 and increasing concentrations of NaCl to eliminate spurious protein interactions. All proteins were eluted from the glutathione beads and electrophoresed on an 11% SDS-PAGE gel before being probed for His-tagged protein.

Bacterial two-hybrid assay. The bacterial two-hybrid assay uses protein-protein interactions to bring two fragments of the adenylate cyclase catalytic domain together to produce cAMP, stimulating β-galactosidase activity. β-Galactosidase activity is therefore a representation of the protein interaction. This protocol was performed as described previously by Karimova et al. (25). Briefly, E. coli DHP-1 cells (an adenylate cyclase-deficient cell line) were transformed using pT18-CdsN and either pT25-CopN, pT25-CdsD, pT25-CdsQ, pT25-CdsL, pT25-Cpn0706, pT25-Cpn0705 (putative ruler protein), or pT25-CdsN and selected with 100 µg/µl ampicillin and 34 µg/µl chloramphenicol. Three individual colonies were selected from each plate and grown overnight in 3.0 ml of LB broth at 30°C in the presence of 0.5 mM IPTG plus appropriate antibiotics. A culture grown overnight (200 µl) was diluted 1 in 5 into a solution containing 70 mM Na₂HPO₄-H₂O, 30 mM NaHPO₄-H₂O, 1 mM MgSO₄, and 0.2 mM MnSO₄ (PM2 buffer), and the OD₆₀₀ was recorded. The cells were permeabilized using 0.01% toluene and 0.01% SDS. For the reaction, 50 µl of the permeabilized cells was diluted into 450 µl of LB broth. The diluted cells were then added to 500 µl of PM2 buffer containing 100 mM β-mercaptoethanol. The reaction was initiated by adding 250 µl of 12 mg/ml ortho-nitrophenyl-β-galactoside to the mixture and allowed to continue for 15 s at 28°C. The reaction was stopped by the addition of 500 µl of 1.0 M Na₂CO₃ to the mixture. The absorbance was measured at 420 nm, and the β-galactosidase activity was expressed as units of β-galactosidase activity per milligram of bacteria. Empty pT18 and pT25 vectors were transformed into E. coli DHP1 cells as a negative control, and pT18-PknD and pT25-CdsD-FHA-2, which is the FHA-2 domain of CdsD, were used as a positive control (22). The cutoff for a positive interaction (677 units of activity/mg bacteria) was determined as the mean plus 2 standard deviations of the negative control values obtained from 20 assays.

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CdsN shares significant similarity with other T3S ATPases. CdsN (Cpn0707) is 443 amino acids in length, with a predicted molecular mass of 48.1 kDa and an acidic pI of 6.10. CdsN has significant sequence similarity with the β subunit of the F_oF₁ ATPase as well as with other T3S ATPases including EscN from E. coli and YscN from Yersinia (Fig. 1A). CdsN has a nonconserved N-terminal region, which is possibly important for oligomerization (amino acids 1 to 155), a catalytic domain with Walker A and B domains (amino acids 155 to 403), and a C-terminal domain of unknown function (amino acids 403 to 443). Based on the similarity of CdsN to EscN, the structure of the catalytic domain is most likely a mixed /β Rossmann fold, typical of the catalytic domain of EscN (41). Using NCBI BLAST, the expect value of the CdsN catalytic domain with the YscN catalytic domain is 9e^–95, and that with the EscN catalytic domain is 2e^–81, demonstrating a highly conserved catalytic domain. The catalytic domain of CdsN is 54% identical to the catalytic domains of EscN and YscN.

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FIG. 1. Sequence conservation of CdsN with other T3S ATPases. (A) Sequence alignment of the putative catalytic domain of CdsN (amino acids 155 to 403) with the active domains of YscN from Yersinia and EscN from E. coli. Asterisks refer to identical amino acids, double dots refer to conserved substitutions, and single dots refer to semiconserved substitutions. (B) Domain organization of CdsN based on structural similarity to EscN and YscN. CdsN contains Walker A and B domains and a highly conserved catalytic domain, most likely as an /β Rossmann fold. The expected value is shown for similarity between the CdsN catalytic subunit and the catalytic subunit of EscN and YscN, representing the similarity between the species.

CdsN_1-405 and CdsL oligomerize in solution. The GST-tagged C-terminal truncation of CdsN, GST-CdsN_1-405, was overexpressed in E. coli BL21(DE3) cells and purified using glutathione-agarose beads. Figure 2A (left) shows that GST-CdsN_1-405 was essentially free of contaminating proteins by SDS-PAGE followed by Coomassie blue staining. To determine whether CdsN was capable of forming oligomers, we first used a bacterial two-hybrid system to assess the interactions between CdsN monomers. CdsN monomers exhibited levels of interaction that were significantly above that seen with the background (empty vectors). The β-galactosidase activities were 431.0 ± 122.7, 1,019.3 ± 52.2, and 799.4 ± 45.1 units/mg bacteria for pT18 and pT25 (negative control), pT18-PknD and pT25-CdsD-FHA-2 (positive control), and pT18-CdsN and pT25-CdsN (positive interactions), respectively. CdsN was cloned into both pT18 and pT25 for association studies using the bacterial two-hybrid system. Both vectors were transformed into an E. coli adenylate cyclase knockout (DHP1) and grown for 24 h at 30°C. The bacterial two-hybrid assay was performed in triplicate as described in Materials and Methods. The cutoff for a positive interaction (677 units of activity/mg protein) is the mean of the negative control values (empty pT18 plus pT25) plus 2 standard deviations obtained from 20 assays. To confirm this observation and analyze the presence of higher-molecular-weight oligomers, we allowed CdsN to form oligomers in solution and then fixed the protein with formaldehyde and ran the sample on a nondenaturing PAGE gel. Due to the insolubility of full-length GST-CdsN, we utilized the C-terminal truncation GST-CdsN_1-405. The predicted molecular mass of GST-CdsN_1-405 is 64 kDa, and CdsN monomers migrated in the gel at a position corresponding to the 64-kDa molecular mass marker (Fig. 3A). Multiple high-molecular-mass bands corresponding to 120-kDa and 180-kDa complexes were also seen, demonstrating oligomer formation of CdsN (Fig. 3A). Smaller bands were also seen on the gel, possibly representing N-terminal fragments oligomerizing in solution. N-terminal fragments were visible when purified GST-CdsN_1-405 was analyzed by Western blotting (Fig. 2A, right). This suggests that the oligomerization domain of CdsN is near the N-terminal region since the GST tag is also present on the N terminus of CdsN. Some GST-tagged protein was also seen at the top of the gel, possibly representing high-molecular-weight oligomers. The high-molecular-weight bands could be consistent with the formation of dodecamers, which have been described for other T3S ATPases (30, 33). Since YscL homologs have been shown to dimerize in other bacteria, CdsL was assessed for its ability to form dimers (31). His-tagged CdsL was overexpressed in E. coli BL21(DE3)(pLysS) cells; analyzed by SDS-PAGE, Coomassie blue staining (Fig. 2B, left), and anti-His Western blot analysis (Fig. 2B, right); and then purified on Ni-nitrilotriacetic acid beads and analyzed for its ability to form oligomers using nondenaturing PAGE gels and Western blotting. Purified His-CdsL monomers migrated with a molecular mass of 25.9 kDa, as shown in Fig. 3B. The presence of CdsL dimers is indicated by a band migrating with a molecular mass of approximately 50 kDa (Fig. 3B). To demonstrate that GST and His tags alone had no effect on the oligomerization of these proteins, GST-CopN and His-CopN were run as controls, and neither tagged protein formed oligomers (Fig. 3C).

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FIG. 2. Purification of soluble GST-CdsN1-405 and CdsL analyzed by Coomassie blue staining and Western blotting. (A, left) GST-CdsN1-405 (0.1 µg) electrophoresed on an 11% SDS-PAGE gel and stained with Coomassie blue migrating at slightly slower than the 64-kDa molecular marker. GST-CdsN1-405 (0.05 µg) electrophoresed on an 11% SDS-PAGE gel and analyzed by anti-GST Western blotting. (Right) The presence of C-terminal breakdown products is visible migrating at smaller sizes. Ladder markers are shown on the left of each figure. (B, left) E. coli BL21(DE3)(pLysS), either induced with IPTG or not induced, overexpressing His-CdsL, stained with Coomassie blue. CdsL, in the induced sample, can be seen migrating at approximately 26 kDa. (Right) The induced and uninduced samples were also probed by anti-His Western blotting. His-CdsL is seen migrating at 26 kDa in the induced sample. Ladder markers are shown on the left.

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FIG. 3. Oligomerization of CdsN and CdsL by Western blot analysis. (A) GST-CdsN1-405 monomers can be seen on a nondenaturing PAGE gel probed by anti-GST Western blotting with a molecular mass of 64 kDa, and the formation of dimers and trimers can be seen at 120 and 180 kDa, respectively. Smaller GST-tagged bands are also seen migrating faster than the 115-kDa and 180-kDa markers, possibly corresponding to N-terminal fragments of CdsN oligomerizing. (B) His-CdsL monomers can be seen on a nondenaturing PAGE gel probed by anti-His Western blotting migrating with a molecular mass of 25.9 kDa, and the formation of a dimer can be seen at approximately 50 kDa. (C) GST-CopN, after formaldehyde fixation and being run on a nondenaturing PAGE gel, probed by anti-GST Western blotting. (Left) GST-CopN monomers can be seen migrating at approximately 70 kDa as a monomer, and free GST can be seen at approximately 26 kDa. (Right) His-CopN, after formaldehyde fixation, being run on a nondenaturing PAGE gel and probed by anti-His Western blotting, can be seen running in its monomeric form at approximately 45 kDa.

ATPase activity of CdsN. Purified GST-CdsN_1-405 was assessed for its ability to hydrolyze ATP using the malachite green binding assay. GST-CdsN_1-405 hydrolyzed ATP in a linear, time-dependent manner (Fig. 4A, triangles), with phosphate release occurring at a rate of 0.55 ± 0.07 µmol min^–1 mg^–1. This activity is comparable with those of other T3S ATPases such as InvC, HrcN, and EscN (3, 5). Also, ATPase activity seemed to decrease at higher protein concentrations (Fig. 4B, triangles) and may be indicative of positive cooperativity, which is characteristic of other T3S ATPases (3, 5). Proteinase K-treated CdsN_1-405 (squares) displayed no ATPase activity, as well as purified GST-CopN as a negative control.

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FIG. 4. Time course and dose response of CdsN ATPase activity. (A) Time course of GST-CdsN1-405 ATP hydrolysis (triangles) and proteinase K-digested GST-CdsN1-405 ATP hydrolysis (squares). All experiments were performed in triplicate. (B) Inorganic phosphate released at different concentrations of GST-CdsN1-405 (triangles) or proteinase K-digested GST-CdsN1-405 (squares). All experiments were performed in triplicate.

CdsN interacts with CdsD, CdsL, CdsQ, and CopN. We have used the bacterial two-hybrid system described by Karimova et al. to screen for interactions between CdsN and other chlamydial T3SS proteins (25). CdsN showed significant interactions with CdsD, CdsL, CdsQ, and CopN (Table 2). CdsN was also screened against Cpn0705, the YscP "ruler protein" homolog, but no interaction was detected. Following screening with the bacterial two-hybrid assay, protein interactions were further evaluated using a GST pull-down assay. GST pull-down assays were used to confirm the interactions of CdsN with other chlamydial T3S proteins under both low- and high-salt conditions in the presence of 0.1% Triton X-100 to dissociate spurious protein-protein interactions. GST-CdsN interacted with His-CdsD, His-CdsL, His-CdsQ, and His-CopN (Fig. 5A), while GST alone, used as a negative control, did not pull down any of the T3S proteins under any salt concentrations (lane 1 for each sample). The interactions were very specific, and there were no contaminating bands on the Western blots. GST-CdsN, however, did not interact with Cpn0705, the putative ruler protein. To determine whether the C terminus of CdsN is important for mediating interactions with these T3S proteins, pull-down assays were performed with a GST-CdsN_1-405 fragment. GST-CdsN_1-405 interacted with CdsD, CdsL, CdsQ, and CopN, suggesting that the C-terminal 38 amino acids of CdsN are not involved in these protein-protein interactions (Fig. 5B). All interactions were stable in the presence of 500 mM NaCl (Fig. 5, lane 4), suggesting that CdsN forms stable interactions with CdsD, CdsQ, CdsL, and CopN. Consistent with full-length CdsN, the GST-CdsN_1-405 protein also did not interact with Cpn0705 under any salt conditions.

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TABLE 2. Interaction between CdsN and other T3S proteins using the bacterial two-hybrid systema

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FIG. 5. Interaction between CdsN and other T3SS proteins. (A) Full-length GST-CdsN was bound to glutathione beads and was used to pull down His-tagged protein from E. coli lysates. Beads were harvested by centrifugation; washed with 0 mM (lane 2), 200 mM (lane 3), and 500 mM (lane 4) NaCl; and probed for His-tagged protein by Western blotting using anti-His antibody. GST-CdsN pulled down CdsD, CdsL, CdsQ, and CopN, while GST alone did not pull down any of these proteins (lane 1). GST-CdsN also could not pull down Cpn0705. (B) GST-CdsN1-05 bound to glutathione beads and used to pull down CdsD, CdsL, CdsQ, and CopN from E. coli lysates. As a negative control, Cpn0705 was used, which GST-CdsN1-405 could not pull down.

Interaction of CdsN with a putative chaperone, Cpn0706. The Cpn0706 gene encodes a 169-amino-acid protein with a predicted molecular mass of 19.8 kDa and a pI of 9.70. The Cpn0706 open reading frame is located directly upstream of the T3S ATPase CdsN (Cpn0707 gene). The bacterial two-hybrid assay and GST pull-down assays were used to test for interactions between Cpn0706 and CdsN. CdsN interacts with recombinant Cpn0706 in the bacterial two-hybrid assay. β-Galactose activities of 203.0 ± 75.7, 1,019.3 ± 52.2, and 683.2 ± 36.2 units/mg bacteria were found for pT18 and pT25 (positive control), pT18-PknD and pT25-CdsD-FHA-2, and pT18-CdsN and pT25-0706, respectively. CdsN was cloned into vector pT18, while Cpn0706 was cloned into vector pT25. The bacterial two-hybrid system was performed as described in Materials and Methods. The cutoff for a positive interaction (677 units of activity/mg protein) is the mean of the negative control values (empty pT18 plus pT25) plus 2 standard deviations obtained from data for 20 assays. To corroborate the interaction seen in the bacterial two-hybrid system, a GST pull-down assay was used. An interaction was seen when the full-length GST-CdsN beads were used to interact with Cpn0706 under low-salt conditions in the presence of 0.1% Triton X-100, but no interaction was detected between GST-CdsN and Cpn0706 at 200 mM or 500 mM NaCl (Fig. 6A, top). When GST-CdsN_1-405 was used to pull down Cpn0706, no interaction was seen, suggesting that Cpn0706 interacts with the C-terminal 38 amino acids of CdsN (Fig. 6A). Since T3S effectors and their chaperones have been shown to form dimers, we tested Cpn0706 for its ability to dimerize. Purified His-Cpn0706 was electrophoresed on a nondenaturing gel and visualized using Western blotting. As shown in Fig. 6B, Cpn0706 monomers migrate at their predicted molecular mass of approximately 19.8 kDa, and dimers can be seen migrating with an apparent molecular mass of 40 kDa.

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FIG. 6. Interaction of CdsN with a putative chaperone, Cpn0706. (A) Full-length GST-CdsN was bound to glutathione beads and used to pull down His-tagged Cpn0706. The beads were washed with 0.1% Triton X-100 alone (lane 1), 0.1% Triton X-100 plus 200 mM NaCl (lane 2), or 500 mM NaCl (lane 3) and probed by anti-His Western blotting. GST alone did not interact with Cpn0706 (lane 4). Full-length CdsN interacted with Cpn0706 under low-salt conditions. C-terminally truncated GST-CdsN1-405 was not able to interact with Cpn0706 under any salt conditions. As a negative control, full-length CdsN could not pull down Cpn0705. (B) Cpn0706 monomers, which have a predicted molecular mass of 19.8 kDa, migrate slightly slower than the 19-kDa molecular marker. Cpn0706 forms dimers, which migrate at an apparent molecular mass of 40 kDa, as shown by anti-His Western blotting of soluble Cpn0706 run on a nondenaturing SDS-PAGE gel.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although C. pneumoniae contains all the genes coding for a T3SS, only a fraction of these have been biochemically characterized. Here, we report a preliminary characterization of CdsN, the putative T3S ATPase of C. pneumoniae. We have demonstrated that CdsN possesses ATPase activity, hydrolyzing ATP in a time- and dose-dependent manner. CdsN also forms oligomers in solution and interacts with other putative T3S components including CdsD, CdsQ, CdsL, and CopN. We also identified an interaction with Cpn0706, a small protein located directly upstream from CdsN, which may be a CdsN chaperone. Collectively, these data suggest that CdsN is the ATPase of the C. pneumoniae T3SS.

CdsN hydrolyzes ATP in a linear, time-dependent manner at a rate of 0.55 ± 0.06 µmol phosphate min^–1 mg^–1, which is typical of rates for other T3S ATPases (3, 5). At higher protein concentrations, ATPase activity decreased, suggesting a positive cooperativity of CdsN, which was previously described for other T3S ATPases (3, 5, 30). We have shown that both CdsN and the tethering protein CdsL form dimers and, in the case of CdsN, higher-molecular-weight oligomers. The presence of high-molecular-weight forms of CdsN may correspond to dodecamers that were reported previously for other T3S ATPases (30).

Interactions between specific proteins of the T3SS in various bacteria have been reported, but studies of chlamydial T3SS proteins are limited. We previously showed that the putative T3S structural protein CdsD interacts with the putative CdsN tethering protein CdsL and CdsQ, a T3S structural component localized to the inner membrane (23). In the present report, we extend these studies and show that CdsN interacts with CdsL, CdsD, and CdsQ. These interactions likely form a tetrameric or higher-order complex at the inner membrane of C. pneumoniae. The interaction between CdsN and CdsD is a novel finding not previously shown for other bacteria. This interaction appears to be specific since it is not disrupted by high salt (500 mM). It is possible that CdsD acts as a docking platform at the chlamydial inner membrane to allow the ATPase to dock and form a dodecamer during injectisome assembly. CdsQ, which has been shown to interact with the effector protein CopN and which resides in both the inner membrane and cytosol of C. pneumoniae (R. Toor et al., unpublished data), may play a role in shuttling chaperone/effector complexes to the ATPase at the basal body of the injectisome. An important observation is that the C-terminally truncated CdsN protein, lacking the C-terminal 38 amino acid residues, is capable of interacting with CdsL, CdsD, and CdsQ, indicating that the binding site for these proteins is not located in the C-terminal 38 amino acid residues. It is tempting to speculate that the interactive domains of CdsN for CdsL, CdsD, and CdsQ resides in the N-terminal 155 amino acids of the protein since the catalytic domain encompasses the remainder of the protein. This is presently under investigation.

We have also shown that CdsN interacts with the putative effector and plug protein CopN using both a bacterial two-hybrid assay and a GST pull-down assay. CopN is known to negatively regulate T3S by physically blocking the injectisome in Yersinia, but its role in C. pneumoniae T3S has not yet been elucidated. CopN from C. trachomatis has, however, been shown to be secreted by heterologous T3S systems, suggesting that it is an effector in C. pneumoniae (10). The fact that CopN interacts strongly with CdsN, even in the presence of 500 mM NaCl, suggests that CdsN may be important for effector protein recognition, as has been seen in other bacteria (14, 35). CopN interacts with both full-length CdsN and CdsN_1-405, indicating that the binding site for CopN is located within the first 405 amino acids of CdsN and likely within the first 155 amino acids preceding the catalytic domain. In other bacteria, the ATPases have been shown to interact with chaperones of effector proteins (14, 39), often via the C-terminal region (41), but this has not been demonstrated for CdsN in Chlamydia. Since we have shown that CdsN interacts with the effector CopN, it is likely that it interacts with other effectors or their chaperones, as was shown previously for other bacteria (14).

Cpn0706, a previously unannotated open reading frame, encodes a small protein with a predicted molecular mass of 19.8 kDa and a pI of 9.7. Cpn0706 lies adjacent to CdsN (Cpn0707), suggesting that it may be a CdsN chaperone. We have shown that Cpn0706 interacts with CdsN using both GST pull-down and bacterial two-hybrid assays. The binding sites for effector chaperones on other characterized T3S ATPases are thought to be on the C-terminal end of the protein (41). The CdsN_1-405 protein did not interact with Cpn0706 (even when washed with low-salt buffer), while the full-length CdsN did, suggesting that the binding site for Cpn0706 lies within the C-terminal 38 amino acids of CdsN. Since nondenatured CdsN was used for this assay, and chaperones are known to interact with both folded and unfolded proteins, it could be expected that any interaction between CdsN and its potential chaperone, Cpn0706, would be weak and transient. This may explain why the interactions seen in the GST pull-down assay were disrupted by high salt. More corroborating experiments will be required before Cpn0706 can be designated a CdsN chaperone.

Bacterial T3S is a virulence factor that has been shown to play a crucial role in the invasion of host cells for a number of bacteria. Although this has not yet been demonstrated for Chlamydophila, the presence of T3S genes in both C. pneumoniae and C. trachomatis, as well as the secretion of the chlamydial effector proteins TARP, CopN, and Pkn5 through heterologous systems, is highly suggestive of a functional T3S apparatus (17, 26). In this study, we have shown that CdsN is a functional ATPase and interacts with other key proteins of C. pneumoniae T3SS. We have also shown that CdsN interacts with a putative chaperone, Cpn0706, and with CopN, the putative plug and effector protein. These observations add to the accumulating evidence that C. pneumoniae contains a structurally intact and functional T3SS. In the absence of a functional T3S assay or a genetic transformation system for the chlamydiae, direct evidence for a functional T3SS continues to elude us. Further studies of protein interactions, together with genetic complementation and injectisome reconstruction in heterologous bacteria, may provide tools to investigate the role of T3S in Chlamydia pathogenesis.

 
We thank Justin Nodwell for vectors pT18 and pT25 as well as E. coli strain DHP-1 for use in the bacterial two-hybrid assay. We thank Brian Coombes for his critical review of the manuscript.

C.B.S. and D.L.J. are both recipients of a Father Sean O'Sullivan Research Center studentship. This research was funded by a Canadian Institute of Health Research grant to J.B.M.

 
* Corresponding author. Mailing address: Regional Virology and Chlamydiology Laboratory, St. Joseph's Hospital, 50 Charlton Avenue East, Hamilton, Ontario L8N 4A6, Canada. Phone: (905) 521-6021. Fax: (905) 521-6083. E-mail: mahonyj{at}mcmaster.ca

Published ahead of print on 15 August 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, October 2008, p. 6580-6588, Vol. 190, No. 20
0021-9193/08/$08.00+0     doi:10.1128/JB.00761-08
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