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. 2012;7(1):e30220.
doi: 10.1371/journal.pone.0030220. Epub 2012 Jan 17.

Structural characterization of a novel Chlamydia pneumoniae type III secretion-associated protein, Cpn0803

Chris B Stone  1 , Seiji Sugiman-Marangos, David C Bulir, Rob C Clayden, Tiffany L Leighton, Jerry W Slootstra, Murray S Junop, James B Mahony
Affiliations

Structural characterization of a novel Chlamydia pneumoniae type III secretion-associated protein, Cpn0803

Chris B Stone et al. PLoS One. 2012.

Abstract

Type III secretion (T3S) is an essential virulence factor used by gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts. JWS is an employee of PepScan Presto and performed the author's epitope mapping experiments. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Cpn0803 interacts with type III secretion components in vitro.
A. A glutathione plate assay was applied to screen for interactions of Cpn0803 between either CdsN, CdsQ or CdsF. His-Cpn0803 was applied to GST-CdsN, -CdsQ and -CdsF immobilized on glutathione plates, washed three times with PBS, and monitored using a colorimetric assay. Data is represented on the graph as the mean ± standard deviation for each interaction. A significant interaction was considered to be two standard deviations above the negative control (GST alone against Cpn0803). As a positive control, we screened GST-Cpn0803 against His-Cpn0803, as well as GST-Lcrh-2 against His-CopN. Cpn0803 interacted significantly with CdsN, CdsQ, CdsF, and Cpn0803. while it did not interact with GST alone. GST-Lcrh-2 and His-CopN also had a significant interaction. B. We applied GST pull-down assays to corroborate the interactions found with the glutathione plate assay. GST-CdsN, -CdsQ, –CdsF or GST alone immobilized on glutathione beads were incubated with an E. coli lysate over-expressing His-Cpn0803 and washed with 500 mM NaCl. GST-CdsN, -CdsQ, and CdsF co-purified with Cpn0803 under 500 mM NaCl conditions while GST alone did not.
Figure 2
Figure 2. Cpn0803 interacts with type III secretion components in vivo.
C. pneumoniae EB lysates were incubated with recombinant GST-CdsN, -CdsF, -CdsQ or -Cpn0803. Glutathione agarose beads were incubated with the lysates overnight, collected, and washed with 500 mM NaCl. The protein on the beads was analyzed by SDS PAGE and Western blot with anti-Cpn0803 antibody. Native Cpn0803 co-purified with GST-CdsN, -CdsF, and -CdsQ. As a positive control, Cpn0803 also co-purified with GST-Cpn0803, but not with GST alone.
Figure 3
Figure 3. Stereo image of Cpn0803 monomer and dimer.
The structure of full-length Cpn0803 was determined by SAD phasing of an anomalous data set collected from crystals of SeMet derivatized protein. The overall structure of Cpn0803 has a unique fold and no structural orthologs on the DALI server. A. Cartoon representation of the Cpn0803 monomer. Secondary structure elements are colored as follows; α-helices in blue and β-strands in red. B. Cartoon representation of the Cpn0803 dimer colored by chain.
Figure 4
Figure 4. Stereo image of a predicted Cpn0803 hexamer colored by chain.
We evaluated Cpn0803 for its ability to form multimers by analysis with the PISA server. Based on crystal contacts and buried surface area, the biologically active unit of Cpn0803 was predicted to be a hexamer formed by a trimer of dimers. The individual monomeric units are shown in different colours.
Figure 5
Figure 5. Stereo image of the hydrophobic pocket in Cpn0803.
Dimerization of Cpn0803 results in the formation of a cone-shaped hydrophobic pocket between the two 3-stranded β-sheets, closed off at the ‘top’ by α-6 from both chains A and B, and stoppered at the ‘bottom’ by F129. The amino acid residues lining the interior of the pocket are represented in stick form.
Figure 6
Figure 6. Cpn0803 interacts with phosphatidylinositol and phosphatidic acid.
His-Cpn0803 was incubated with membrane lipid strips containing purified eukaryotic membrane components and visualized by anti-his antibody and ECL reagents. Cpn0803 interacted with phosphatidylinositol and phosphatidic acid, but none of the other molecules evaluated. His-CdsL, as a negative control, did not interact with any lipid components.
Figure 7
Figure 7. Cpn0803 exists in a hexameric and dimeric state in solution.
A. Gel filtration chromatography was used to analyze purified His-Cpn0803. We found two dominant peaks, one corresponding to a hexamer and one corresponding to a dimer. The elution volume of the protein standards are shown on the x-axis. B. Peak fractions in the elution volumes were collected and analyzed by anti-His Western blot. Cpn0803 was present in the peak fractions.
Figure 8
Figure 8. Pepscan mapping of the Cpn0803 binding regions shown in stereo.
A. Pepscan epitope mapping against a Cpn0803 peptide library was performed to determine the residues of Cpn0803 responsible for mediating its interactions with CdsN, CdsF and CdsQ. Recombinant CdsN, CdsF and CdsQ was reacted against the Cpn0803 peptide library and monitored for the corresponding interacting regions. The corresponding surfaces are color-coded as follows: CdsN in blue (residues 22–26), CdsF in purple (residues 109–128), and CdsQ in orange (residues 153–161).
Figure 9
Figure 9. Corroboration of the Cpn0803 binding regions identified by Pepscan using GST pull-downs.
To corroborate the Pepscan results, we expressed 50 amino acid fragments of Cpn0803 encompassing the CdsN and CdsQ binding domains and performed GST pull-down assays against recombinant CdsN or CdsQ. GST-Cpn0803120–180, containing the CdsQ binding domain, co-purified with His-CdsQ under high salt conditions. GST-Cpn08031–50, which does not contain the CdsQ binding domains, did not co-purify with CdsQ. GST-Cpn0803120–180, which does not contain the CdsN binding domain, did not co-purify with CdsN.
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