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Infection and Immunity, January 2009, p. 508-516, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.01173-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Chlamydia trachomatis Polymorphic Membrane Protein D Is an Oligomeric Autotransporter with a Higher-Order Structure

Kena A. Swanson,^1, Lacey D. Taylor,^1, Shaun D. Frank,¹ Gail L. Sturdevant,¹ Elizabeth R. Fischer,² John H. Carlson,¹ William M. Whitmire,¹ and Harlan D. Caldwell^1*

Laboratory of Intracellular Parasites,¹ Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840²

Received 19 September 2008/ Returned for modification 22 October 2008/ Accepted 2 November 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chlamydia trachomatis is a mucosotropic obligate intracellular gram-negative pathogen that is a leading cause of sexually transmitted and ocular infections. Infection can result in serious sequelae such as infertility and blindness (54, 56) and an increased risk of human immunodeficiency virus infection and transmission (38). The pathophysiology of chlamydial infection is associated with the pathogen's propensities to cause persistent infection and to suppress host immunity (3). A vaccine is needed to control chlamydial diseases; however, progress toward this goal will not be forthcoming until more is known about the virulence factors that mediate persistence and immune evasion.

Chlamydiae are characterized by a unique biphasic developmental cycle that modulates between an extracellular, metabolically inactive, infectious elementary body (EB) and an intracellular, metabolically active, noninfectious reticulate body (RB) (34). Their obligate intracellular niche and the lack of a tractable genetic system present unique challenges in the study of chlamydial biology and pathogenesis. To overcome these hurdles, chlamydial genomes from a diverse spectrum of host-specific strains have been sequenced. Comparative genomics have shown considerable homology among various chlamydial species and have provided important insights into shared and species-specific virulence factors (7, 24, 41, 42, 46, 49).

The type V or autotransporter (AT) secretion pathway is the most widespread secretion mechanism employed by gram-negative bacteria to deliver virulence factors involved in initiating infection, disease progression, and immune evasion (reviewed in references 11 and 21). AT proteins are characterized by three domains, (i) a signal sequence (SS), (ii) a diverse N-terminal passenger domain (PD) that confers effector function, and (iii) a conserved C-terminal translocator domain (TD). The TD inserts into the outer membrane (OM) by assembling into a β-barrel pore that facilitates PD translocation to the bacterial surface. The PD remains tethered to the TD or is cleaved and either is released or remains noncovalently associated with the OM. Well-characterized examples of ATs found on the bacterial cell surface as monomers or oligomers are Neisseria meningitidis NalP (37) and Helicobacter pylori VacA (31), respectively.

C. trachomatis has a nine-member AT family (20), termed polymorphic membrane proteins (Pmps), whose role(s) in chlamydial pathogenesis has yet to be defined. The pmp paralogs (pmpA to pmpI) constitute 3.2% of the 1-Mb genome and are found at three chromosomal loci composed of two gene clusters (pmpA to pmpC and pmpE to pmpI) and the genetically isolated gene pmpD (46). Notably, PmpD is the second most highly conserved Pmp, exhibiting 99.2% amino acid identity among C. trachomatis serovars (16). Despite relatively low abundance in the chlamydial OM, Pmps are major immunogens and may be important virulence factors (29). C. trachomatis PmpD is a target of broadly cross-reactive neutralizing antibodies (Abs), which makes it an attractive vaccine candidate for the prevention of human infections (10).

Previous reports have described proteolytic processing of C. pneumoniae and C. trachomatis PmpD (25, 52). Furthermore, recombinant C. pneumoniae PmpD has been suggested to function as an adhesin capable of inducing proinflammatory cytokine production (35, 52). Nothing is known about the native structure of C. trachomatis PmpD or the potential significance of its structure to chlamydial pathogenesis. Here we show that C. trachomatis PmpD is present on the organism's surface as an oligomer with a higher-order flower-like structure. Moreover, we describe novel infection-dependent proteolytic processing of PmpD that produces soluble fragments with predicted eukaryotic motifs, implying a multifunctional protein important to chlamydial pathogenesis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chlamydiae, Abs, and Western blotting. C. trachomatis serovar L2/LGV-434 and D/UW-3 EBs and RBs were propagated and purified as previously described (5), except that the band at the 40 to 44% interface was harvested for RBs. For PmpD purification and solubility experiments, L2 was grown in L929 cell suspension cultures and harvested by 30% density gradient centrifugation as described previously (6). Rabbit polyclonal Abs were generated against C. trachomatis serovar A/HAR-13 PmpD peptides (Quality Controlled Biochemicals; Fig. 1A; see also Fig. 5C) as follows: N-PmpD (N-terminal region of PmpD), RLIVGDPSSFQEKDADTL (amino acids [aa] 76 to 93); M-PmpD (middle region of PmpD), ALFASEDGDLSPESS (aa 761 to 775); C'-PmpD (C'-terminal region of PmpD), QQGHAISKPEAEIESSSE (aa 1041 to 1058); and C-PmpD (C-terminal region of PmpD), KNEAKVPLMSFVASGDEA (aa 1136 to 1153). Other Abs used were mouse anti-major OM protein (MOMP; L2I-45), rabbit anti-L2 EB, and mouse anti-chlamydial protease-like activity factor (CPAF) (13). Purified EBs or RBs were solubilized in Laemmli buffer, electrophoresed on 4 to 15% Criterion gels (Bio-Rad), transferred to a polyvinylidene difluoride membrane, and Western blotted.

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FIG. 1. Structural features and proteolytic processing of PmpD. (A) C. trachomatis PmpD (aa 1 to 1530) contains GGA(I/L/V) and FXXN tetrapeptide repeats and cysteine residues (C) concentrated in the N-terminal half of the protein. An RGD sequence (red) is located within a predicted β helix adjacent to a putative NLS (yellow). Rabbit anti-PmpD polyclonal Abs were generated against the amino acids indicated by dashed lines. (B) C. trachomatis serovar L2 EBs were prepared for SDS-PAGE and CBB stained or immunoblotted with anti-PmpD Abs. The mature protein (p155) and two major processed forms, N-terminal p73 and C-terminal p82, were detected. The values on the left are molecular sizes in kilodaltons.

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FIG. 5. PmpD is proteolytically processed to soluble forms late in infection. Western blot assays of insoluble (A) and soluble (B) fractions of C. trachomatis-infected L929 cells at various times postinfection were probed with N-PmpD, M-PmpD, C'-PmpD, and C-PmpD Abs. p155, p82, and p73 remained organism associated, while p111, p73, and p30 were detected at 30 to 36 hpi in the soluble fractions. Monoclonal Abs against MOMP and CPAF were used as controls. The values on the left are molecular sizes in kilodaltons. (C) Stick drawing summarizing the immunoreactivity of PmpD Abs to primary (insoluble) and secondary (soluble) forms of PmpD. Upon secondary cleavage, the RGD and NLS motifs are separated into p73 and p30, respectively.

Immunoaffinity purification of PmpD. The M-PmpD Ab was coupled to Affi-Gel 10 (Bio-Rad) according to the manufacturer's protocol. The column was pretreated with the elution buffers 50 mM glycine-HCl, pH 2.3, and 3 M NaI. For PmpD purifications, EB pellets were resuspended at 10¹⁰ inclusion-forming units/ml in ice-cold 1% octyl-β-D-glucopyranoside (OGP) in phosphate-buffered saline (PBS), pH 7.4, containing Complete protease inhibitor cocktail (Roche); incubated for 30 min at 37°C; and ultracentrifuged at 100,000 x g for 1 h at 4°C. The C-PmpD-containing extract was passed over the column twice and washed with 0.1% OGP-PBS, pH 8.0, containing increasing concentrations of NaCl. PmpD was acid eluted with 0.1% OGP-50 mM glycine-HCl, pH 2.3, containing protease inhibitors and directly neutralized with 1 M Tris-0.15 M NaCl, pH 8.0, or eluted with 0.1% OGP-3 M NaI containing protease inhibitors and dialyzed into 0.01% OGP-PBS, pH 7.4.

Sequence analysis. Immunoaffinity-purified PmpD (40 µg) was pooled, precipitated with trichloroacetic acid, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with GelCode Blue, excised, and submitted for sequence analysis (Harvard Microchemistry and Proteomics Analysis Facility, Boston, MA). Samples were digested with chymotrypsin, GluC, and elastase, followed by microcapillary reverse-phase high-performance liquid chromatography nano-electrospray tandem mass spectrometry (µLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer. Edman degradation for N-terminal sequencing was performed by the Research Technologies Branch, NIAID, NIH, Bethesda, MD.

PmpD solubility. L2-infected L929 suspension cultures were harvested by centrifugation, washed in PBS and sensitized with hypo-osmotic swelling medium (15 mM KCl, 1.5 mM magnesium acetate tetrahydrate and 10 mM HEPES-KOH, pH 7.4) containing Complete protease inhibitor cocktail (Roche). Cells were gently lysed by 20 strokes of a 0.05-mm clearance pestle in a Dounce homogenizer. Insoluble and soluble fractions were prepared as previously described (14).

Confocal microscopy. L2-infected HeLa 229 cells were methanol fixed at 28 h postinfection (hpi) and sequentially labeled with rabbit anti-PmpD, Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG; Invitrogen), mouse anti-MOMP, and Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen). Monolayers were extensively washed between steps, and DNA was stained with DRAQ5 (Alexis). Images were acquired with a Carl Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss Micro Imaging) equipped with a 63x 1.4 numerical aperture oil immersion objective and processed with Adobe Photoshop CS2 (Adobe Systems Inc.).

2D BN-SDS-PAGE and electroelution. Two-dimensional (2D) blue native (BN)-SDS-PAGE was performed as described previously (55), with the following modifications. BN-PAGE samples were electrophoresed on 4 to 15% Criterion XT Bis-Tris gels (Bio-Rad) at a voltage of 100 V, which was increased to 500 V after 30 min with a 40-mA threshold, with anode buffer (25 mM imidazole, pH 7.0) and cathode buffer (50 mM Tricine, 7.5 mM imidazole, 0.02% Coomassie brilliant blue [CBB], pH 7.0). Electrotransfer to polyvinylidene difluoride was performed in 50 mM Tricine-7.5 mM imidazole, pH 7.0, with 75-V and 200-mA maximum thresholds for 3 h at 4°C. For 2D BN-SDS-PAGE, a single lane was excised from the BN-PAGE gel, boiled in 1% SDS-1% 2-mercaptoethanol, and loaded into a 4 to 15% Criterion gel (Bio-Rad) prep well underlaid with 0.5% agarose. Second-dimension SDS-PAGE and immunoblotting were performed as described above. To electroelute oligomers, BN-PAGE and Western blotting were performed. An 850-kDa immunoreactive band was excised from an overlaid, unstained duplicate gel, and proteins were eluted with a model 422 electroeluter (Bio-Rad) for 14 h at 2 mA/elution chamber with 0.01% OGP-25 mM Tricine-3.75 mM imidazole-5 mM 6-aminohexanoic acid buffer.

Electron microscopy. Electroeluted PmpD oligomers were adsorbed to carbon-coated Formvar 200-mesh copper grids and negatively stained with 2% (wt/vol) aqueous ammonium molybdate. Purified L2 EBs were adsorbed onto copper grids, immunolabeled in a Pelco 3451 laboratory microwave oven (Ted Pella Inc.) (40) with M-PmpD Ab and 5-nm colloidal gold (BBInternational) as the secondary Ab, and negatively stained. Purified D EBs and RBs were pelleted; fixed in 4% paraformaldehyde-2.5% glutaraldehyde-0.1 M sodium cacodylate buffer, pH 7.2; and sectioned. Images were acquired on a Philips CM-10 transmission electron microscope (FEI Company) at 60 or 80 kV. Focused and defocused images were acquired and analyzed with a bottom mount AMT digital camera system (ATM). More than 100 purified oligomers were measured for two independent experiments to determine the diameter. Statistical analysis was performed with a two-tailed Student t test. Scanning electron microscopy (SEM) was performed as previously described (10). All images were processed with Adobe PhotoShop CS2 (Adobe Systems Inc.).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Structural features and proteolytic processing of PmpD. The structural features of C. trachomatis PmpD are shown in Fig. 1A. PmpD shows many of the characteristics found in ATs, including a relatively large size (1,530 aa, 160.5 kDa), N-terminal GGA(I/L/V) and FXXN tetrapeptide repeats (19), an integrin-binding RGD motif (aa 698 to 670) (47), and a putative nuclear localization signal (NLS; aa 783 to 798). The NLS is a bipartite motif defined by two clusters of basic residues separated by a stretch of 9 aa (KRRX₉KRVR) (2).

We generated Abs specific to peptides located in the N-terminal (N-PmpD), middle (M-PmpD), and C-terminal (C-PmpD) regions of PmpD (Fig. 1A) to study proteolytic processing of the protein. CBB-stained gels of EB lysates exhibited a relatively low abundance of PmpD compared to the 40-kDa MOMP. Western blot assays with PmpD Abs showed specific immunoreactivity with the mature protein (p155) and two predominant lower-molecular-weight proteins, p82 and p73 (Fig. 1B). Based on these results, we concluded that p73 was the N-terminal PD and p82 was the C-terminal TD. The N-terminal half of PmpD is cysteine rich, suggesting a potential for disulfide bond interactions. We found no evidence for intermolecular PmpD disulfide bonding, as no supramolecular complexes were observed in samples solubilized without 2-mercaptoethanol. However, a subtle but reproducible increase in the electrophoretic mobility of the p73 PD was observed, implying intramolecular disulfide bonding within this fragment (data not shown).

Immunoaffinity purification of native PmpD. We screened a panel of nonionic detergents for the ability to extract PmpD from EBs to purify the native protein by immunoaffinity chromatography. CBB-stained gels indicated that a small fraction of the total chlamydial proteins was solubilized in nonionic detergents (Fig. 2A). Western blot assays with M-PmpD Ab show that C. trachomatis PmpD is unique as it was efficiently extracted from EBs in a soluble form by nonionic detergents (Fig. 2B). This is in contrast to other Pmps that are retained in the Sarkosyl-insoluble OM fraction (48). We used OGP to extract PmpD from EBs and purified the protein by immunoaffinity chromatography (Fig. 3A). A single 155-kDa polypeptide (Fig. 3A, arrowhead) was observed in the eluted fractions following CBB staining. Western blot analysis with the M-PmpD Ab detected mature p155 and the p82 TD. Elution fractions were pooled, concentrated, and analyzed by Western blot assay and silver staining (Fig. 3B). Consistent with PmpD from whole EB lysates, immunoaffinity-purified PmpD was composed predominantly of mature p155, N-terminal p73, and C-terminal p82. The 50-kDa polypeptide was identified by mass spectrometry as the rabbit IgG heavy chain. We concluded that p73 and p82 were the naturally processed PD and TD, respectively, a finding in agreement with previously published reports (25, 52).

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FIG. 2. PmpD is extracted from chlamydiae by nonionic detergents. C. trachomatis serovar L2 EBs were incubated in a 1% solution of Triton X-100 (TX-100), Igepal CA630 (NP-40), OGP, or PBS for 30 min and centrifuged at 100,000 x g for 1 h. Detergent-treated EBs (P) (1 x 108) and an equivalent volume of detergent extracts (S) were separated by SDS-PAGE and either CBB stained (A) or immunoblotted with the M-PmpD Ab (B). PmpD was efficiently extracted with nonionic detergents, especially OGP, as most of the protein was found in the supernatant (S) and not in the pellet (P). The values on the left are molecular sizes in kilodaltons.

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FIG. 3. Immunoaffinity purification of native PmpD. (A) PmpD was extracted from L2 EBs with 1% OGP and purified by immunoaffinity chromatography with the M-PmpD Ab. Column fractions were separated by SDS-PAGE and CBB stained or processed for immunoblotting with the M-PmpD Ab. S, OGP supernatant; FT, flowthrough; V, column void volume. (B) Pooled and concentrated eluates were examined by Western blot assay and silver staining. The major products were p155, p82, and p73. M-PmpD was weakly reactive with an 100-kDa polypeptide that was identified by mass spectrometry as PmpD. All of the Abs recognized a 50-kDa polypeptide that was identified as the rabbit IgG heavy chain. (C) Identification of PmpD proteolytic processing sites (black arrows). Cleavage of the signal sequence (gray) between 52A and 53V was identified by immunoprecipitation of PmpD from OGP-extracted L2 EBs and N-terminal sequencing. To identify the cleavage site that produced the N-terminal p73 PD (white) and the C-terminal p82 TD (black), immunoaffinity-purified PmpD was trichloroacetic acid precipitated, separated by SDS-PAGE, stained with CBB, and excised. Mass spectrometry analysis was performed as described in Materials and Methods. The cleavage site was identified between 761A and 762L. The values on the left are molecular sizes in kilodaltons.

We performed N-terminal sequence analysis of p155 and p82 fragments (Fig. 3C). Edman degradation of p155 identified ⁵³VLLLDQ as the mature N terminus. Sequence analysis of p82 identified the N terminus as ⁷⁶²LFASEDGDLS. These results are the first to describe the precise location of PmpD proteolytic cleavage sites that define the mature PD and TD. They also indirectly define the PmpD signal sequence as an extended 52-aa sequence; similarly large signal sequences have been described for other type V ATs (11).

PmpD exhibits unique staining properties. PmpD was examined by confocal microscopy of C. trachomatis-infected HeLa cells immunolabeled with PmpD- and MOMP-specific Abs (Fig. 4). M-PmpD Ab exhibited strong OM staining, similar to MOMP staining, but also appeared as diffuse intrainclusion staining and as bright punctate clusters (Fig. 4A). The resolution limits of light microscopy precluded us from differentiating whether these punctate structures were on the organism's surface or free within the inclusion. C-PmpD Ab intensely stained the OM but reacted weakly with the punctate structures (Fig. 4B). C-PmpD Ab did not exhibit the diffuse intrainclusion staining found with both the M-PmpD and N-PmpD Abs (Fig. 4 and data not shown). We did not detect PmpD beyond the inclusion lumen in infected cells, consistent with previous studies (25, 52). These findings suggest that PmpD exists as multiple structures in infected cells, including organism-associated and soluble forms generated by possible secondary cleavage events.

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FIG. 4. PmpD exhibits unique staining properties. C. trachomatis-infected HeLa cells were methanol fixed at 28 hpi and immunolabeled with M-PmpD (A) or C-PmpD (B) and MOMP-specific Abs. PmpD (green) localized to chlamydial OMs (filled arrows) and to punctate clusters within the inclusion lumen (arrowheads). Diffuse intrainclusion staining detected with M-PmpD Ab (open arrows) was not seen with C-PmpD Ab. A single confocal plane is shown. MOMP is labeled red, and DNA (blue) is shown in the overlay. Scale bars, 5 µm.

PmpD is proteolytically processed to soluble forms late in infection. To more thoroughly examine PmpD expression, C. trachomatis-infected cells were harvested at various times postinfection, Dounce homogenized, and subjected to ultracentrifugation to obtain soluble and insoluble fractions. We analyzed the fractions by Western blot assay with our panel of PmpD Abs (Fig. 5). Anti-MOMP and -CPAF Abs were used as positive controls for insoluble and soluble chlamydial proteins, respectively. As expected, both proteins were first detected at mid-growth cycle, with MOMP being present only in the insoluble fraction and CPAF being present in both the insoluble and soluble fractions. Full-length p155, the p82 TD, and the p73 PD were detected in the insoluble fractions beginning at 24 hpi (Fig. 5A). Consistent with our microscopy data, the C-PmpD Ab was immunoreactive with the insoluble fractions, suggesting that the C terminus of PmpD remains organism associated (Fig. 5A). Interestingly, the N-PmpD Ab detected a 111-kDa fragment (p111) and p73 in the soluble fractions late in the developmental cycle (Fig. 5B). M-PmpD Ab recognized the same p111 fragment but also reacted with a novel 30-kDa polypeptide (p30). The p111 and p73 soluble forms are likely the previously reported 120- and 67-kDa polypeptides (25). However, to our knowledge, this is the first description of the soluble p30 polypeptide. Western blot analysis with the C'-PmpD Ab, which lies upstream of C-PmpD, showed reactivity against both p111 and p30 (Fig. 5B). This indicates that p111 is generated by cleavage between aa 1058 and 1136 and that p30 originates from the C-terminal end of p111 (Fig. 5C). Collectively, these findings demonstrate that PmpD undergoes multiple proteolytic cleavage events, i.e., (i) primary processing to produce the organism-associated p73 PD and p82 TD and (ii) late infection-dependent secondary processing to generate a soluble extended PD (p111) that is further cleaved to produce soluble p73 PD and p30 (Fig. 5C).

Native PmpD is an oligomer with a flower-like structure. Immunoaffinity-purified PmpD from EBs (Fig. 3B) was analyzed by BN-PAGE and transmission electron microscopy (TEM). BN-PAGE relies on CBB to impose a negative charge shift on detergent-extracted membrane proteins under neutral pH and nondenaturing conditions (55). BN-PAGE Western blot assays of PmpD revealed a complex pattern of high-molecular-size oligomers ranging from 250 to 1,050 kDa, with stronger reactivity observed at 530 and 850 kDa (Fig. 6A and B). Complexes of 530 and 850 kDa correspond to oligomers composed of four or five and five or six subunits, respectively. The PD was also detected as a single 100-kDa band (Fig. 6A, top panel). The composition of the oligomers was defined by 2D BN-SDS-PAGE and Western blotting with either N-PmpD (Fig. 6A) or C-PmpD (Fig. 6B) Ab. These results showed that oligomers were composed of full-length p155, the p73 PD, and the p82 TD. Purified PmpD oligomers did not contain detectable p111 or p30 fragments (Fig. 6A and data not shown) that were found in the soluble fractions of infected cells (Fig. 5B).

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FIG. 6. Native PmpD is a flower-like oligomer composed of full-length and processed forms. Western blot assays of 2D BN-SDS-PAGE gels of immunoaffinity-purified PmpD were performed with either N-PmpD (A) or C-PmpD (B) Abs. First-dimension (1-D) BN-PAGE showed that PmpD migrated from 250 to 1,050 kDa and as a discrete band at 100 kDa. PmpD oligomers were dissociated by second-dimension SDS-PAGE into p155, p82, and p73. The values on the left are molecular sizes in kilodaltons. (C) TEM of negatively stained electroeluted 850-kDa PmpD oligomers. Scale bar, 100 nm. (D) A magnified oligomer (arrowhead in panel C) displayed a distinct flower-like structure with a central core and symmetrically arrayed petals. Scale bar, 25 nm.

PmpD oligomers (850 kDa) were excised, electroeluted, negatively stained, and examined by TEM (Fig. 6C and D). Imaging showed that the oligomers were a homogeneous population of particulate structures that exhibited a 23 ± 3-nm (P < 0.0001) ring surrounded by five or six symmetrically arrayed petals to form a flower-like structure (Fig. 6D). These characteristics are remarkably similar to the rosettes described by Matsumoto following freeze-deep-etching of chlamydial organisms (33) and the secreted PD of H. pylori VacA (9).

Localization of PmpD oligomers on the chlamydial surface. ATs that perform critical pathogenic functions are known to localize to one pole or distinct locations in the bacterial OM (23, 44). Interestingly, EM of anti-PmpD immunogold-labeled EBs showed asymmetric localization of PmpD oligomers (Fig. 7C and F). This was in contrast to the abundant and uniform staining observed following anti-EB or -MOMP labeling (Fig. 7A, B, D, and E). The polarization of PmpD oligomers was observed on the majority of EBs examined and is consistent with the bright punctate structures observed by confocal microscopy (Fig. 4A). This clustering is suggestive of the asymmetric hemispheric projections described on the surface of both C. psittaci and C. trachomatis EBs (17, 32). PmpD distribution on EBs was compared to purified RBs. C. trachomatis RBs and EBs were purified to relative homogeneity by density gradient centrifugation (Fig. 8A). Western blot assays and SEM showed that PmpD oligomers were more abundant on purified RB than on purified EB (Fig. 8B and C). Also, a more homogeneous distribution of PmpD was observed on RBs, in contrast to a more polarized localization on EBs (Fig. 8B).

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FIG. 7. Polarized PmpD distribution on the EB surface. (A to C) L2 EBs were fixed in 4% paraformaldehyde; labeled with anti-L2 EB, anti-MOMP, or anti-M-PmpD and secondary Ab conjugated to 5-nm gold; and imaged by TEM. (D to F) L2 EBs were adsorbed to silicon chips and labeled as described for panels A to C, except that 10-nm gold was used and EBs were fixed postlabeling and imaged by SEM. In contrast to homogeneous MOMP distribution, PmpD was sparse and appeared in polarized clusters (arrowheads) on the OM. One cluster is shown at a higher magnification (F, inset). The concentric distribution of gold particles suggests oligomeric forms. Scale bars: A to F, 50 nm; F inset, 25 nm.

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FIG. 8. Differential distribution and abundance of PmpD on C. trachomatis EBs and RBs. (A) Purified C. trachomatis serovar D RBs and EBs were pelleted, sectioned, and imaged by TEM to demonstrate the homogeneity and purity of the chlamydial preparations. Scale bar, 500 nm. (B) SEM of a serovar D RB and EB immunolabeled with anti-M-PmpD Abs and 10-nm colloidal gold. Scale bar, 50 nm. The anti-PmpD staining is more evenly distributed for the RB than for the EB. (C) CBB staining and Western blot assays of equivalent amounts of purified serovar D RB and EB proteins (15 µg) were probed with the C-PmpD Ab. Consistent with a higher density of PmpD on the RB surface shown in panel B, immunoblots demonstrate more PmpD in RBs compared to EBs. The values on the left are molecular sizes in kilodaltons.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In contrast to other gram-negative organisms, C. trachomatis has a disproportionately large number of AT genes relative to its genome size, suggesting that the chlamydial type V secretion proteins play an important role in pathogenesis. We report here that C. trachomatis PmpD is an oligomeric AT that undergoes multiple proteolytic processing steps to produce organism-associated and soluble forms. Primary proteolytic processing of PmpD results in membrane-associated oligomers composed of mature p155, p73 PD, and p82 TD. Biochemical characterization of immunoaffinity-purified PmpD oligomers demonstrated that they were 23-nm flower-like molecules asymmetrically positioned on the EB surface. These structures are remarkably similar to the surface projections that localize in hexagonal clusters on C. psittaci EBs (33). The surface projections or rosettes described by Matsumoto and others (8, 17, 45) are speculated to be part of the chlamydial type III secretion apparatus (1). However, we show here that the PmpD oligomer size, flower-like structure, abundance on RBs, and asymmetric localization on EBs are all features shared by Matsumoto's surface projections. Thus, our data suggest that the previously described surface projections could be PmpD oligomers but do not exclude the possibility that other proteins could form similar structures. As the other eight pmp family members are all expressed during infection (18, 36), any of the Pmps could serve as candidates. The questions remain whether these Pmps exist as monomers or oligomers and if they interact with PmpD in the OM.

Importantly, we present new findings showing a late infection-dependent secondary PmpD processing step resulting in soluble p111, p73, and p30 fragments. p111 and p30 were not detected in immunoaffinity-purified oligomers but were observed in the inclusion lumen by confocal microscopy. However, we cannot exclude the possibility that they are secreted into the host cytosol, as shown for other chlamydial proteins (12, 26, 28, 57). Bacterial effectors secreted into host cells at low levels are undetectable by immunofluorescence (27), suggesting that limited quantities of PmpD may exist in the host cytosol. Soluble forms of PmpD are not likely structural but are possible effectors that function either in the host cell during late stages of infection or on neighboring cells after release of the inclusion into the extracellular environment (22, 50).

The cleavage of soluble p111 results in a p73 PD and a novel C-terminal p30 fragment. It is not known if p111 processing is dependent on chlamydial or host proteases or if soluble p73 and p30 remain stably associated after p111 cleavage. It is noteworthy, however, that p111 proteolysis separates the eukaryotic RGD and NLS motifs into the p73 and p30 fragments, respectively, implying distinct effector functions for the two polypeptides. H. pylori VacA and the Neisseria IgA protease are examples of multifunctional ATs that undergo proteolytic processing to generate distinct functional subunits. VacA is secreted as a mature 88-kDa toxin that is cleaved into p33 and p55, which form flower-like oligomers that function in toxin internalization and cytotoxicity (51). The secreted PD of the Neisseria IgA protease is processed to an NLS-containing - protein that localizes to the host cell nucleus (39). Similar to the -protein, the NLS of PmpD p30 could target this fragment to the nucleus. The p73 PD integrin-binding RGD motif is similar to the PDs of Bordetella pertussis pertactin and Escherichia coli Ag43, which function as adhesins (53). These structural similarities suggest that soluble forms of PmpD interact with receptors on epithelial cells or lymphocytes to produce pleiotropic effects important in pathogenicity. There are fascinating parallels between the structures of H. pylori VacA and C. trachomatis PmpD and their host-pathogen relationships. Both are primarily human mucosotropic pathogens that cause chronic inflammatory disease and actively suppress host immunity. H. pylori VacA oligomers have T-cell suppressive activity (15). Interestingly, chlamydiae suppress the development of memory CD8⁺ T cells through an unknown mechanism (30). It is tempting to speculate that PmpD may play a similar role in T-cell suppression.

A working model of C. trachomatis PmpD structure and function is shown in Fig. 9. The model is based on the experimental findings presented here together with known properties of bacterial ATs. We propose two unique PmpD functions in the interaction of chlamydiae with its host. First, we suggest that EB surface-associated oligomers function in early host cell interactions to promote either chlamydial attachment or entry. The most logical mechanism for this function would be through the p73 PD RGD motif binding to its cognate host integrin receptor(s). Polarized PmpD oligomers on the EB surface (Fig. 5) would be multivalent and could enhance this interaction. The ability of anti-PmpD Abs to inhibit chlamydial infection (10, 52) indirectly supports this function. Second, soluble p111 produced late in the infection cycle possesses RGD and NLS motifs that associate with the p73 PD and p30 fragment, respectively. We speculate that these secreted effectors have downstream targets important to chlamydial pathogenicity. For example, through unknown mechanisms chlamydiae actively inhibit apoptosis early and promote programmed cell death late during infection to escape from the host cell (4). PmpD secreted into the host cytosol late in infection could be targeted to the nucleus by the eukaryotic NLS in p30 to regulate host gene expression. Following release from the inclusion into the extracellular environment (22, 50), they could act on additional cellular targets. C. trachomatis-infected cells (>40 hpi) have been shown to induce apoptosis of neighboring uninfected cells (43). Moreover, chlamydial infections have potent T-cell suppressive activity (30). The molecular mechanism(s) for these pathogenic activities is unknown but could be mediated by soluble PmpD peptide fragments.

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FIG. 9. Model of C. trachomatis PmpD structure and function. We propose two distinct PmpD functions in the interaction of chlamydiae with host cells. The first involves the EB surface oligomers. We suggest that these higher-order structures are important in chlamydial entry, a hypothesis supported by the ability of anti-PmpD Abs to block infection (10, 52). The second function involves the soluble forms of processed PmpD present only late in the developmental cycle. The processing of p111 (dashed lines) segregates the RGD (red) and putative NLS (yellow) motifs into p73 and p30, respectively, which may remain stably associated or exist separately. The soluble peptides could be limited to the inclusion lumen or access the host cell cytosol (dashed arrow). The NLS in the p30 fragment suggests that it translocates to the nucleus to effect host transcription. The biological activity of inclusion-restricted PmpD could depend on release into the extracellular environment after cell lysis or inclusion egression. We speculate that the p111 PD is similar to the H. pylori VacA PD (51), which dissociates after binding host receptors to deliver the NLS to uninfected or bystander cells and thus regulates host gene expression. This interaction could result in the induction of apoptosis in uninfected epithelial cells and T-cell suppression.

A more complete understanding of PmpD oligomeric structure and its soluble peptide fragments is important, as this information will be fundamentally critical in the design of a PmpD-based vaccine. The results of this study represent an important advancement toward this end. For example, native PmpD oligomers could represent key structures that elicit conformation-dependent broadly neutralizing Abs. Moreover, definition of the natural proteolytic cleavage sites of soluble PmpD fragments will be useful in the construction and expression of biologically relevant recombinant proteins. A highly efficacious PmpD vaccine might require the incorporation of multiple protein targets capable of inducing Abs that neutralize a broad spectrum of M-PmpDediated biological activities.

 
We thank Robert Heinzen and Leigh Knodler for critical reading of the manuscript; Gaungming Zhong for the kind gift of anti-CPAF Abs; Naomi Crane for technical support; and Kelly Matteson, Anita Mora, and Gary Hettrick for editorial and graphic assistance.

This research was supported by the Intramural Research Program of the NIH, NIAID.

 
* Corresponding author. Mailing address: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840. Phone: (406) 363-9333. Fax: (406) 363-9380. E-mail: hcaldwell{at}niaid.nih.gov

Published ahead of print on 10 November 2008.

Editor: S. R. Blanke

These authors contributed equally to this work.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, January 2009, p. 508-516, Vol. 77, No. 1
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