chlamydia trachomatis polymorphic membrane protein d is an ... · conserved among c. trachomatis...

INFECTION AND IMMUNITY, Jan. 2009, p. 508–516 Vol. 77, No. 10019-9567/09/$08.00�0 doi:10.1128/IAI.01173-08

Chlamydia trachomatis Polymorphic Membrane Protein D Is anOligomeric 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,2John H. Carlson,1 William M. Whitmire,1 and Harlan D. Caldwell1*

Laboratory of Intracellular Parasites,1 Research Technologies Branch,2 Rocky Mountain Laboratories, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840

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

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause ofsexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preven-tative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it isconserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We showhere 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 theoligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 pas-senger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependentproteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed intoa p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interactingfunctional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findingsshow that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-likestructure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulencefactor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control ofhuman chlamydial infections.

Chlamydia trachomatis is a mucosotropic obligate intracellulargram-negative pathogen that is a leading cause of sexually trans-mitted and ocular infections. Infection can result in serious se-quelae such as infertility and blindness (54, 56) and an increasedrisk of human immunodeficiency virus infection and transmission(38). The pathophysiology of chlamydial infection is associatedwith the pathogen’s propensities to cause persistent infection andto suppress host immunity (3). A vaccine is needed to controlchlamydial diseases; however, progress toward this goal will notbe forthcoming until more is known about the virulence factorsthat mediate persistence and immune evasion.

Chlamydiae are characterized by a unique biphasic develop-mental cycle that modulates between an extracellular, meta-bolically inactive, infectious elementary body (EB) and an in-tracellular, metabolically active, noninfectious reticulate body(RB) (34). Their obligate intracellular niche and the lack of atractable genetic system present unique challenges in the studyof chlamydial biology and pathogenesis. To overcome thesehurdles, chlamydial genomes from a diverse spectrum of host-specific strains have been sequenced. Comparative genomicshave shown considerable homology among various chlamydialspecies and have provided important insights into shared andspecies-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-neg-ative bacteria to deliver virulence factors involved in initiatinginfection, disease progression, and immune evasion (reviewedin references 11 and 21). AT proteins are characterized bythree 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 assemblinginto a �-barrel pore that facilitates PD translocation to thebacterial surface. The PD remains tethered to the TD or iscleaved and either is released or remains noncovalently asso-ciated with the OM. Well-characterized examples of ATsfound on the bacterial cell surface as monomers or oligomersare Neisseria meningitidis NalP (37) and Helicobacter pyloriVacA (31), respectively.

C. trachomatis has a nine-member AT family (20), termedpolymorphic membrane proteins (Pmps), whose role(s) inchlamydial pathogenesis has yet to be defined. The pmp para-logs (pmpA to pmpI) constitute 3.2% of the �1-Mb genomeand are found at three chromosomal loci composed of twogene clusters (pmpA to pmpC and pmpE to pmpI) and thegenetically isolated gene pmpD (46). Notably, PmpD is thesecond most highly conserved Pmp, exhibiting 99.2% aminoacid identity among C. trachomatis serovars (16). Despite rel-atively low abundance in the chlamydial OM, Pmps are majorimmunogens and may be important virulence factors (29). C.trachomatis PmpD is a target of broadly cross-reactive neutral-izing antibodies (Abs), which makes it an attractive vaccinecandidate for the prevention of human infections (10).

Previous reports have described proteolytic processing of C.pneumoniae and C. trachomatis PmpD (25, 52). Furthermore,

* Corresponding author. Mailing address: Laboratory of Intracellu-lar Parasites, Rocky Mountain Laboratories, National Institute of Al-lergy and Infectious Diseases, National Institutes of Health, 903 South4th St., Hamilton, MT 59840. Phone: (406) 363-9333. Fax: (406) 363-9380. E-mail: [email protected].

† These authors contributed equally to this work.� Published ahead of print on 10 November 2008.

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recombinant C. pneumoniae PmpD has been suggested tofunction as an adhesin capable of inducing proinflammatorycytokine production (35, 52). Nothing is known about the na-tive structure of C. trachomatis PmpD or the potential signif-icance of its structure to chlamydial pathogenesis. Here weshow that C. trachomatis PmpD is present on the organism’ssurface as an oligomer with a higher-order flower-like struc-ture. Moreover, we describe novel infection-dependent proteo-lytic processing of PmpD that produces soluble fragments withpredicted eukaryotic motifs, implying a multifunctional proteinimportant to chlamydial pathogenesis.

MATERIALS AND METHODS

Chlamydiae, Abs, and Western blotting. C. trachomatis serovar L2/LGV-434and 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. ForPmpD purification and solubility experiments, L2 was grown in L929 cell sus-pension cultures and harvested by 30% density gradient centrifugation as de-scribed previously (6). Rabbit polyclonal Abs were generated against C. tracho-matis 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 ofPmpD), ALFASEDGDLSPESS (aa 761 to 775); C�-PmpD (C�-terminal regionof 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), rabbitanti-L2 EB, and mouse anti-chlamydial protease-like activity factor (CPAF) (13).Purified EBs or RBs were solubilized in Laemmli buffer, electrophoresed on 4 to15% Criterion gels (Bio-Rad), transferred to a polyvinylidene difluoride mem-brane, and Western blotted.

Immunoaffinity purification of PmpD. The M-PmpD Ab was coupled to Affi-Gel 10 (Bio-Rad) according to the manufacturer’s protocol. The column waspretreated with the elution buffers 50 mM glycine-HCl, pH 2.3, and 3 M NaI. For

FIG. 2. PmpD is extracted from chlamydiae by nonionic detergents. C. trachomatis serovar L2 EBs were incubated in a 1% solution of TritonX-100 (TX-100), Igepal CA630 (NP-40), OGP, or PBS for 30 min and centrifuged at 100,000 � g for 1 h. Detergent-treated EBs (P) (1 � 108)and an equivalent volume of detergent extracts (S) were separated by SDS-PAGE and either CBB stained (A) or immunoblotted with the M-PmpDAb (B). PmpD was efficiently extracted with nonionic detergents, especially OGP, as most of the protein was found in the supernatant (S) and notin the pellet (P). The values on the left are molecular sizes in kilodaltons.

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

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PmpD purifications, EB pellets were resuspended at 1010 inclusion-formingunits/ml in ice-cold 1% octyl-�-D-glucopyranoside (OGP) in phosphate-bufferedsaline (PBS), pH 7.4, containing Complete protease inhibitor cocktail (Roche);incubated for 30 min at 37°C; and ultracentrifuged at 100,000 � g for 1 h at 4°C.The C-PmpD-containing extract was passed over the column twice and washedwith 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, containingprotease 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 dialyzedinto 0.01% OGP–PBS, pH 7.4.

Sequence analysis. Immunoaffinity-purified PmpD (�40 �g) was pooled, pre-cipitated with trichloroacetic acid, separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), stained with GelCode Blue, ex-cised, and submitted for sequence analysis (Harvard Microchemistry andProteomics Analysis Facility, Boston, MA). Samples were digested with chymo-trypsin, GluC, and elastase, followed by microcapillary reverse-phase high-per-formance liquid chromatography nano-electrospray tandem mass spectrometry(�LC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer. Edman degra-dation for N-terminal sequencing was performed by the Research TechnologiesBranch, NIAID, NIH, Bethesda, MD.

PmpD solubility. L2-infected L929 suspension cultures were harvested bycentrifugation, 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). Cellswere gently lysed by 20 strokes of a 0.05-mm clearance pestle in a Douncehomogenizer. Insoluble and soluble fractions were prepared as previously de-scribed (14).

Confocal microscopy. L2-infected HeLa 229 cells were methanol fixed at 28 hpostinfection (hpi) and sequentially labeled with rabbit anti-PmpD, Alexa Fluor488-conjugated goat anti-rabbit immunoglobulin G (IgG; Invitrogen), mouseanti-MOMP, and Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen).Monolayers were extensively washed between steps, and DNA was stained withDRAQ5 (Alexis). Images were acquired with a Carl Zeiss LSM 510 confocallaser scanning microscope (Carl Zeiss Micro Imaging) equipped with a 63� 1.4numerical aperture oil immersion objective and processed with Adobe Photo-shop 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

FIG. 3. Immunoaffinity purification of native PmpD. (A) PmpDwas extracted from L2 EBs with 1% OGP and purified by immunoaf-finity chromatography with the M-PmpD Ab. Column fractions wereseparated by SDS-PAGE and CBB stained or processed for immuno-blotting with the M-PmpD Ab. S, OGP supernatant; FT, flowthrough;V, column void volume. (B) Pooled and concentrated eluates wereexamined by Western blot assay and silver staining. The major prod-ucts were p155, p82, and p73. M-PmpD was weakly reactive with an�100-kDa polypeptide that was identified by mass spectrometry asPmpD. All of the Abs recognized a 50-kDa polypeptide that wasidentified as the rabbit IgG heavy chain. (C) Identification of PmpDproteolytic processing sites (black arrows). Cleavage of the signal se-quence (gray) between 52A and 53V was identified by immunoprecipi-tation of PmpD from OGP-extracted L2 EBs and N-terminal sequenc-ing. To identify the cleavage site that produced the N-terminal p73 PD(white) and the C-terminal p82 TD (black), immunoaffinity-purifiedPmpD was trichloroacetic acid precipitated, separated by SDS-PAGE,stained with CBB, and excised. Mass spectrometry analysis was per-formed as described in Materials and Methods. The cleavage site wasidentified between 761A and 762L. The values on the left are molecularsizes in kilodaltons.

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

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modifications. BN-PAGE samples were electrophoresed on 4 to 15% CriterionXT Bis-Tris gels (Bio-Rad) at a voltage of 100 V, which was increased to 500 Vafter 30 min with a 40-mA threshold, with anode buffer (25 mM imidazole, pH7.0) and cathode buffer (50 mM Tricine, 7.5 mM imidazole, 0.02% Coomassiebrilliant blue [CBB], pH 7.0). Electrotransfer to polyvinylidene difluoride wasperformed in 50 mM Tricine–7.5 mM imidazole, pH 7.0, with 75-V and 200-mAmaximum thresholds for 3 h at 4°C. For 2D BN–SDS-PAGE, a single lane wasexcised from the BN-PAGE gel, boiled in 1% SDS–1% 2-mercaptoethanol, andloaded into a 4 to 15% Criterion gel (Bio-Rad) prep well underlaid with 0.5%agarose. Second-dimension SDS-PAGE and immunoblotting were performed asdescribed above. To electroelute oligomers, BN-PAGE and Western blottingwere performed. An �850-kDa immunoreactive band was excised from an over-laid, unstained duplicate gel, and proteins were eluted with a model 422 elec-troeluter (Bio-Rad) for 14 h at 2 mA/elution chamber with 0.01% OGP–25 mMTricine–3.75 mM imidazole–5 mM 6-aminohexanoic acid buffer.

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

RESULTS

Structural features and proteolytic processing of PmpD.The structural features of C. trachomatis PmpD are shown inFig. 1A. PmpD shows many of the characteristics found inATs, 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 aputative nuclear localization signal (NLS; aa 783 to 798). TheNLS is a bipartite motif defined by two clusters of basicresidues 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 pro-cessing of the protein. CBB-stained gels of EB lysates exhibiteda relatively low abundance of PmpD compared to the 40-kDaMOMP. Western blot assays with PmpD Abs showed specificimmunoreactivity with the mature protein (p155) and two pre-dominant lower-molecular-weight proteins, p82 and p73 (Fig.1B). Based on these results, we concluded that p73 was theN-terminal PD and p82 was the C-terminal TD. The N-termi-nal half of PmpD is cysteine rich, suggesting a potential fordisulfide bond interactions. We found no evidence for inter-molecular PmpD disulfide bonding, as no supramolecular com-plexes were observed in samples solubilized without 2-mercap-toethanol. However, a subtle but reproducible increase in theelectrophoretic mobility of the p73 PD was observed, implyingintramolecular disulfide bonding within this fragment (data notshown).

Immunoaffinity purification of native PmpD. We screened apanel of nonionic detergents for the ability to extract PmpDfrom EBs to purify the native protein by immunoaffinity chro-matography. CBB-stained gels indicated that a small fractionof the total chlamydial proteins was solubilized in nonionic

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



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detergents (Fig. 2A). Western blot assays with M-PmpD Abshow that C. trachomatis PmpD is unique as it was efficientlyextracted from EBs in a soluble form by nonionic detergents(Fig. 2B). This is in contrast to other Pmps that are retained inthe Sarkosyl-insoluble OM fraction (48). We used OGP toextract PmpD from EBs and purified the protein by immuno-affinity chromatography (Fig. 3A). A single 155-kDa polypep-tide (Fig. 3A, arrowhead) was observed in the eluted fractionsfollowing CBB staining. Western blot analysis with the M-PmpD Ab detected mature p155 and the p82 TD. Elutionfractions were pooled, concentrated, and analyzed by Westernblot assay and silver staining (Fig. 3B). Consistent with PmpDfrom whole EB lysates, immunoaffinity-purified PmpD wascomposed predominantly of mature p155, N-terminal p73, andC-terminal p82. The 50-kDa polypeptide was identified bymass spectrometry as the rabbit IgG heavy chain. We con-cluded that p73 and p82 were the naturally processed PD andTD, respectively, a finding in agreement with previously pub-lished reports (25, 52).

We performed N-terminal sequence analysis of p155 andp82 fragments (Fig. 3C). Edman degradation of p155 identified53VLLLDQ as the mature N terminus. Sequence analysis ofp82 identified the N terminus as 762LFASEDGDLS. Theseresults are the first to describe the precise location of PmpDproteolytic cleavage sites that define the mature PD and TD.They also indirectly define the PmpD signal sequence as anextended 52-aa sequence; similarly large signal sequences havebeen described for other type V ATs (11).

PmpD exhibits unique staining properties. PmpD was ex-amined by confocal microscopy of C. trachomatis-infectedHeLa cells immunolabeled with PmpD- and MOMP-specificAbs (Fig. 4). M-PmpD Ab exhibited strong OM staining, sim-

ilar to MOMP staining, but also appeared as diffuse intrain-clusion staining and as bright punctate clusters (Fig. 4A). Theresolution limits of light microscopy precluded us from differ-entiating whether these punctate structures were on the organ-ism’s surface or free within the inclusion. C-PmpD Ab in-tensely stained the OM but reacted weakly with the punctatestructures (Fig. 4B). C-PmpD Ab did not exhibit the diffuseintrainclusion staining found with both the M-PmpD and N-PmpD Abs (Fig. 4 and data not shown). We did not detectPmpD beyond the inclusion lumen in infected cells, consistentwith previous studies (25, 52). These findings suggest thatPmpD exists as multiple structures in infected cells, includingorganism-associated and soluble forms generated by possiblesecondary cleavage events.

PmpD is proteolytically processed to soluble forms late ininfection. To more thoroughly examine PmpD expression, C.trachomatis-infected cells were harvested at various timespostinfection, Dounce homogenized, and subjected to ultra-centrifugation to obtain soluble and insoluble fractions. Weanalyzed the fractions by Western blot assay with our panel ofPmpD Abs (Fig. 5). Anti-MOMP and -CPAF Abs were used aspositive controls for insoluble and soluble chlamydial proteins,respectively. As expected, both proteins were first detected atmid-growth cycle, with MOMP being present only in the insol-uble fraction and CPAF being present in both the insolubleand soluble fractions. Full-length p155, the p82 TD, and thep73 PD were detected in the insoluble fractions beginning at 24hpi (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 organismassociated (Fig. 5A). Interestingly, the N-PmpD Ab detected a111-kDa fragment (p111) and p73 in the soluble fractions late

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



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in the developmental cycle (Fig. 5B). M-PmpD Ab recognizedthe same p111 fragment but also reacted with a novel 30-kDapolypeptide (p30). The p111 and p73 soluble forms are likelythe previously reported 120- and 67-kDa polypeptides (25).However, to our knowledge, this is the first description of thesoluble p30 polypeptide. Western blot analysis with the C�-PmpD Ab, which lies upstream of C-PmpD, showed reactivityagainst both p111 and p30 (Fig. 5B). This indicates that p111is generated by cleavage between aa 1058 and 1136 and thatp30 originates from the C-terminal end of p111 (Fig. 5C).Collectively, these findings demonstrate that PmpD undergoesmultiple proteolytic cleavage events, i.e., (i) primary processingto produce the organism-associated p73 PD and p82 TD and(ii) late infection-dependent secondary processing to generatea soluble extended PD (p111) that is further cleaved to pro-duce 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 ana-lyzed by BN-PAGE and transmission electron microscopy(TEM). BN-PAGE relies on CBB to impose a negative chargeshift on detergent-extracted membrane proteins under neutralpH and nondenaturing conditions (55). BN-PAGE Westernblot assays of PmpD revealed a complex pattern of high-mo-lecular-size oligomers ranging from �250 to 1,050 kDa, withstronger reactivity observed at �530 and �850 kDa (Fig. 6Aand B). Complexes of �530 and �850 kDa correspond tooligomers composed of four or five and five or six subunits,respectively. The PD was also detected as a single �100-kDaband (Fig. 6A, top panel). The composition of the oligomerswas defined by 2D BN-SDS-PAGE and Western blotting witheither N-PmpD (Fig. 6A) or C-PmpD (Fig. 6B) Ab. Theseresults showed that oligomers were composed of full-lengthp155, the p73 PD, and the p82 TD. Purified PmpD oligomersdid not contain detectable p111 or p30 fragments (Fig. 6A anddata not shown) that were found in the soluble fractions ofinfected cells (Fig. 5B).

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 pop-ulation of particulate structures that exhibited a 23 � 3-nm(P � 0.0001) ring surrounded by five or six symmetricallyarrayed petals to form a flower-like structure (Fig. 6D). Thesecharacteristics are remarkably similar to the rosettes describedby Matsumoto following freeze-deep-etching of chlamydial or-ganisms (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 tolocalize to one pole or distinct locations in the bacterial OM(23, 44). Interestingly, EM of anti-PmpD immunogold-labeledEBs showed asymmetric localization of PmpD oligomers (Fig.7C and F). This was in contrast to the abundant and uniformstaining observed following anti-EB or -MOMP labeling (Fig.7A, B, D, and E). The polarization of PmpD oligomers wasobserved on the majority of EBs examined and is consistentwith the bright punctate structures observed by confocal mi-croscopy (Fig. 4A). This clustering is suggestive of the asym-metric hemispheric projections described on the surface ofboth C. psittaci and C. trachomatis EBs (17, 32). PmpD distri-bution on EBs was compared to purified RBs. C. trachomatisRBs and EBs were purified to relative homogeneity by density

gradient centrifugation (Fig. 8A). Western blot assays andSEM showed that PmpD oligomers were more abundant onpurified RB than on purified EB (Fig. 8B and C). Also, a morehomogeneous distribution of PmpD was observed on RBs, incontrast to a more polarized localization on EBs (Fig. 8B).

DISCUSSION

In contrast to other gram-negative organisms, C. trachomatishas a disproportionately large number of AT genes relative toits genome size, suggesting that the chlamydial type V secre-tion proteins play an important role in pathogenesis. We reporthere that C. trachomatis PmpD is an oligomeric AT that un-dergoes multiple proteolytic processing steps to produce or-ganism-associated and soluble forms. Primary proteolytic pro-cessing of PmpD results in membrane-associated oligomerscomposed of mature p155, p73 PD, and p82 TD. Biochemical

FIG. 7. Polarized PmpD distribution on the EB surface. (A to C) L2EBs 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 andlabeled as described for panels A to C, except that 10-nm gold was usedand EBs were fixed postlabeling and imaged by SEM. In contrast tohomogeneous MOMP distribution, PmpD was sparse and appeared inpolarized clusters (arrowheads) on the OM. One cluster is shown at ahigher magnification (F, inset). The concentric distribution of gold parti-cles suggests oligomeric forms. Scale bars: A to F, 50 nm; F inset, 25 nm.



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characterization of immunoaffinity-purified PmpD oligomersdemonstrated that they were �23-nm flower-like moleculesasymmetrically positioned on the EB surface. These structuresare remarkably similar to the surface projections that localizein hexagonal clusters on C. psittaci EBs (33). The surface pro-jections or rosettes described by Matsumoto and others (8, 17,45) are speculated to be part of the chlamydial type III secre-tion apparatus (1). However, we show here that the PmpDoligomer size, flower-like structure, abundance on RBs, andasymmetric localization on EBs are all features shared by Mat-sumoto’s surface projections. Thus, our data suggest that thepreviously described surface projections could be PmpD oligo-mers but do not exclude the possibility that other proteinscould form similar structures. As the other eight pmp familymembers are all expressed during infection (18, 36), any of thePmps could serve as candidates. The questions remain whetherthese Pmps exist as monomers or oligomers and if they interactwith PmpD in the OM.

Importantly, we present new findings showing a late infec-tion-dependent secondary PmpD processing step resulting insoluble p111, p73, and p30 fragments. p111 and p30 were notdetected in immunoaffinity-purified oligomers but were ob-served in the inclusion lumen by confocal microscopy. How-ever, we cannot exclude the possibility that they are secretedinto the host cytosol, as shown for other chlamydial proteins(12, 26, 28, 57). Bacterial effectors secreted into host cells atlow levels are undetectable by immunofluorescence (27), sug-gesting that limited quantities of PmpD may exist in the hostcytosol. Soluble forms of PmpD are not likely structural but are

possible effectors that function either in the host cell duringlate stages of infection or on neighboring cells after release ofthe inclusion into the extracellular environment (22, 50).

The cleavage of soluble p111 results in a p73 PD and a novelC-terminal p30 fragment. It is not known if p111 processing isdependent on chlamydial or host proteases or if soluble p73and p30 remain stably associated after p111 cleavage. It isnoteworthy, however, that p111 proteolysis separates the eu-karyotic RGD and NLS motifs into the p73 and p30 fragments,respectively, implying distinct effector functions for the twopolypeptides. H. pylori VacA and the Neisseria IgA proteaseare examples of multifunctional ATs that undergo proteolyticprocessing to generate distinct functional subunits. VacA issecreted as a mature 88-kDa toxin that is cleaved into p33 andp55, which form flower-like oligomers that function in toxininternalization and cytotoxicity (51). The secreted PD of theNeisseria IgA protease is processed to an NLS-containing -protein that localizes to the host cell nucleus (39). Similar tothe -protein, the NLS of PmpD p30 could target this fragmentto the nucleus. The p73 PD integrin-binding RGD motif issimilar to the PDs of Bordetella pertussis pertactin and Esche-richia coli Ag43, which function as adhesins (53). These struc-tural similarities suggest that soluble forms of PmpD interactwith receptors on epithelial cells or lymphocytes to producepleiotropic effects important in pathogenicity. There are fasci-nating parallels between the structures of H. pylori VacA andC. trachomatis PmpD and their host-pathogen relationships.Both are primarily human mucosotropic pathogens that causechronic inflammatory disease and actively suppress host immu-

FIG. 8. Differential distribution and abundance of PmpD on C. trachomatis EBs and RBs. (A) Purified C. trachomatis serovar D RBs and EBswere 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 stainingis more evenly distributed for the RB than for the EB. (C) CBB staining and Western blot assays of equivalent amounts of purified serovar D RBand EB proteins (15 �g) 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.



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nity. H. pylori VacA oligomers have T-cell suppressive activity(15). Interestingly, chlamydiae suppress the development ofmemory CD8� T cells through an unknown mechanism (30). Itis tempting to speculate that PmpD may play a similar role inT-cell suppression.

A working model of C. trachomatis PmpD structure andfunction is shown in Fig. 9. The model is based on the exper-imental findings presented here together with known proper-ties of bacterial ATs. We propose two unique PmpD functionsin the interaction of chlamydiae with its host. First, we suggestthat EB surface-associated oligomers function in early host cellinteractions to promote either chlamydial attachment or entry.The most logical mechanism for this function would bethrough the p73 PD RGD motif binding to its cognate hostintegrin receptor(s). Polarized PmpD oligomers on the EBsurface (Fig. 5) would be multivalent and could enhance thisinteraction. The ability of anti-PmpD Abs to inhibit chlamydialinfection (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 p30fragment, respectively. We speculate that these secretedeffectors have downstream targets important to chlamydialpathogenicity. For example, through unknown mechanismschlamydiae actively inhibit apoptosis early and promote pro-grammed cell death late during infection to escape from thehost cell (4). PmpD secreted into the host cytosol late ininfection could be targeted to the nucleus by the eukaryoticNLS in p30 to regulate host gene expression. Following releasefrom 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 apoptosisof neighboring uninfected cells (43). Moreover, chlamydialinfections have potent T-cell suppressive activity (30). Themolecular mechanism(s) for these pathogenic activities is un-known but could be mediated by soluble PmpD peptide frag-ments.

A more complete understanding of PmpD oligomeric struc-ture and its soluble peptide fragments is important, as thisinformation will be fundamentally critical in the design of aPmpD-based vaccine. The results of this study represent animportant advancement toward this end. For example, nativePmpD oligomers could represent key structures that elicit con-formation-dependent broadly neutralizing Abs. Moreover, def-inition of the natural proteolytic cleavage sites of solublePmpD fragments will be useful in the construction and expres-sion of biologically relevant recombinant proteins. A highlyefficacious PmpD vaccine might require the incorporation ofmultiple protein targets capable of inducing Abs that neutral-ize a broad spectrum of M-PmpDediated biological activities.

ACKNOWLEDGMENTS

We thank Robert Heinzen and Leigh Knodler for critical reading ofthe 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 ofthe NIH, NIAID.

REFERENCES

1. Bavoil, P. M., and R. C. Hsia. 1998. Type III secretion in Chlamydia: a caseof deja vu? Mol. Microbiol. 28:860–862.

2. Boulikas, T. 1993. Nuclear localization signals (NLS). Crit. Rev. Eukaryot.Gene Expr. 3:193–227.

3. Brunham, R. C., and J. Rey-Ladino. 2005. Immunology of Chlamydia infec-tion: implications for a Chlamydia trachomatis vaccine. Nat. Rev. Immunol.5:149–161.

4. Byrne, G. I., and D. M. Ojcius. 2004. Chlamydia and apoptosis: life and deathdecisions of an intracellular pathogen. Nat. Rev. Microbiol. 2:802–808.

5. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purification andpartial characterization of the major outer membrane protein of Chlamydiatrachomatis. Infect. Immun. 31:1161–1176.

6. Caldwell, H. D., C. C. Kuo, and G. E. Kenny. 1975. Antigenic analysis ofchlamydiae by two-dimensional immunoelectrophoresis. I. Antigenic hetero-geneity between C. trachomatis and C. psittaci. J. Immunol. 115:963–968.

7. Carlson, J. H., S. F. Porcella, G. McClarty, and H. D. Caldwell. 2005.Comparative genomic analysis of Chlamydia trachomatis oculotropic andgenitotropic strains. Infect. Immun. 73:6407–6418.

8. Chang, J. J., K. Leonard, T. Arad, T. Pitt, Y. X. Zhang, and L. H. Zhang.1982. Structural studies of the outer envelope of Chlamydia trachomatis byelectron microscopy. J. Mol. Biol. 161:579–590.

9. Cover, T. L., P. I. Hanson, and J. E. Heuser. 1997. Acid-induced dissociationof VacA, the Helicobacter pylori vacuolating cytotoxin, reveals its pattern ofassembly. J. Cell Biol. 138:759–769.

10. Crane, D. D., J. H. Carlson, E. R. Fischer, B. Bavoil, R. C. Hsia, C. Tan, C. C.Kuo, and H. D. Caldwell. 2006. Chlamydia trachomatis polymorphic mem-brane protein D is a species-common pan-neutralizing antigen. Proc. Natl.Acad. Sci. USA 103:1894–1899.

FIG. 9. Model of C. trachomatis PmpD structure and function. Wepropose two distinct PmpD functions in the interaction of chlamydiaewith host cells. The first involves the EB surface oligomers. We suggestthat these higher-order structures are important in chlamydial entry, ahypothesis supported by the ability of anti-PmpD Abs to block infec-tion (10, 52). The second function involves the soluble forms of pro-cessed PmpD present only late in the developmental cycle. The pro-cessing of p111 (dashed lines) segregates the RGD (red) and putativeNLS (yellow) motifs into p73 and p30, respectively, which may remainstably associated or exist separately. The soluble peptides could belimited to the inclusion lumen or access the host cell cytosol (dashedarrow). The NLS in the p30 fragment suggests that it translocates tothe nucleus to effect host transcription. The biological activity of in-clusion-restricted PmpD could depend on release into the extracellularenvironment after cell lysis or inclusion egression. We speculate thatthe p111 PD is similar to the H. pylori VacA PD (51), which dissociatesafter binding host receptors to deliver the NLS to uninfected or by-stander cells and thus regulates host gene expression. This interactioncould result in the induction of apoptosis in uninfected epithelial cellsand T-cell suppression.



.org/D

ownloaded from

http://iai.asm.org/

11. Dautin, N., and H. D. Bernstein. 2007. Protein secretion in gram-negativebacteria via the autotransporter pathway. Annu. Rev. Microbiol. 61:89–112.

12. Dong, F., R. Flores, D. Chen, J. Luo, Y. Zhong, Z. Wu, and G. Zhong. 2006.Localization of the hypothetical protein Cpn0797 in the cytoplasm of Chla-mydia pneumoniae-infected host cells. Infect. Immun. 74:6479–6486.

13. Dong, F., J. Sharma, Y. Xiao, Y. Zhong, and G. Zhong. 2004. Intramoleculardimerization is required for the chlamydia-secreted protease CPAF to de-grade host transcriptional factors. Infect. Immun. 72:3869–3875.

14. Fan, T., H. Lu, H. Hu, L. Shi, G. A. McClarty, D. M. Nance, A. H. Greenberg,and G. Zhong. 1998. Inhibition of apoptosis in Chlamydia-infected cells:blockade of mitochondrial cytochrome c release and caspase activation. J.Exp. Med. 187:487–496.

15. Gebert, B., W. Fischer, E. Weiss, R. Hoffmann, and R. Haas. 2003. Helico-bacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science301:1099–1102.

16. Gomes, J. P., A. Nunes, W. J. Bruno, M. J. Borrego, C. Florindo, and D.Dean. 2006. Polymorphisms in the nine polymorphic membrane proteins ofChlamydia trachomatis across all serovars: evidence for serovar Da recom-bination and correlation with tissue tropism. J. Bacteriol. 188:275–286.

17. Gregory, W. W., M. Gardner, G. I. Byrne, and J. W. Moulder. 1979. Arraysof hemispheric surface projections on Chlamydia psittaci and Chlamydiatrachomatis observed by scanning electron microscopy. J. Bacteriol. 138:241–244.

18. Grimwood, J., L. Olinger, and R. S. Stephens. 2001. Expression of Chlamydiapneumoniae polymorphic membrane protein family genes. Infect. Immun.69:2383–2389.

19. Grimwood, J., and R. S. Stephens. 1999. Computational analysis of thepolymorphic membrane protein superfamily of Chlamydia trachomatis andChlamydia pneumoniae. Microb. Comp. Genomics 4:187–201.

20. Henderson, I. R., and A. C. Lam. 2001. Polymorphic proteins of Chlamydiaspp.—autotransporters beyond the proteobacteria. Trends Microbiol. 9:573–578.

21. Henderson, I. R., F. Navarro-Garcia, M. Desvaux, R. C. Fernandez, and D.Ala’Aldeen. 2004. Type V protein secretion pathway: the autotransporterstory. Microbiol. Mol. Biol. Rev. 68:692–744.

22. Hybiske, K., and R. S. Stephens. 2007. Mechanisms of host cell exit by theintracellular bacterium Chlamydia. Proc. Natl. Acad. Sci. USA 104:11430–11435.

23. Jain, S., P. van Ulsen, I. Benz, M. A. Schmidt, R. Fernandez, J. Tommassen,and M. B. Goldberg. 2006. Polar localization of the autotransporter family oflarge bacterial virulence proteins. J. Bacteriol. 188:4841–4850.

24. Kalman, S., W. Mitchell, R. Marathe, C. Lammel, J. Fan, R. W. Hyman, L.Olinger, J. Grimwood, R. W. Davis, and R. S. Stephens. 1999. Comparativegenomes of Chlamydia pneumoniae and C. trachomatis. Nat. Genet. 21:385–389.

25. Kiselev, A. O., W. E. Stamm, J. R. Yates, and M. F. Lampe. 2007. Expression,processing, and localization of PmpD of Chlamydia trachomatis serovar L2during the chlamydial developmental cycle. PLoS ONE 2:e568.

26. Kleba, B., and R. S. Stephens. 2008. Chlamydial effector proteins localized tothe host cell cytoplasmic compartment. Infect. Immun. 76:4842–4850.

27. Kujat Choy, S. L., E. C. Boyle, O. Gal-Mor, D. L. Goode, Y. Valdez, B. A.Vallance, and B. B. Finlay. 2004. SseK1 and SseK2 are novel translocatedproteins of Salmonella enterica serovar Typhimurium. Infect. Immun. 72:5115–5125.

28. Li, Z., D. Chen, Y. Zhong, S. Wang, and G. Zhong. 2008. The chlamydialplasmid-encoded protein pgp3 is secreted into the cytosol of Chlamydia-infected cells. Infect. Immun. 76:3415–3428.

29. Longbottom, D., M. Russell, S. M. Dunbar, G. E. Jones, and A. J. Herring.1998. Molecular cloning and characterization of the genes coding for thehighly immunogenic cluster of 90-kilodalton envelope proteins from theChlamydia psittaci subtype that causes abortion in sheep. Infect. Immun.66:1317–1324.

30. Loomis, W. P., and M. N. Starnbach. 2006. Chlamydia trachomatis infectionalters the development of memory CD8� T cells. J. Immunol. 177:4021–4027.

31. Lupetti, P., J. E. Heuser, R. Manetti, P. Massari, S. Lanzavecchia, P. L.Bellon, R. Dallai, R. Rappuoli, and J. L. Telford. 1996. Oligomeric andsubunit structure of the Helicobacter pylori vacuolating cytotoxin. J. Cell Biol.133:801–807.

32. Matsumoto, A. 1982. Electron microscopic observations of surface projec-tions on Chlamydia psittaci reticulate bodies. J. Bacteriol. 150:358–364.

33. Matsumoto, A. 1982. Surface projections of Chlamydia psittaci elementarybodies as revealed by freeze-deep-etching. J. Bacteriol. 151:1040–1042.

34. Moulder, J. W. 1991. Interaction of chlamydiae and host cells in vitro.Microbiol. Rev. 55:143–190.

35. Niessner, A., C. Kaun, G. Zorn, W. Speidl, Z. Turel, A.-S. Christiansen, S.Pederson, S. Birkelund, A. Simon, A. Georgopoulos, W. Graninger, R. deMartin, J. Lipp, B. R. Binder, G. Maurer, K. Huber, and J. Wojta. 2003.

Polymorphic membrane protein (PMP) 20 and PMP 21 of Chlamydia pneu-moniae induce proinflammatory mediators in human endothelial cells invitro by activation of the nuclear factor-�B pathway. J. Infect. Dis. 188:108–113.

36. Nunes, A., J. P. Gomes, S. Mead, C. Florindo, H. Correia, M. J. Borrego, andD. Dean. 2007. Comparative expression profiling of the Chlamydia tracho-matis pmp gene family for clinical and reference strains. PLoS ONE 2:e878.

37. Oomen, C. J., P. van Ulsen, P. van Gelder, M. Feijen, J. Tommassen, and P.Gros. 2004. Structure of the translocator domain of a bacterial autotrans-porter. EMBO J. 23:1257–1266.

38. Plummer, F. A., J. N. Simonsen, D. W. Cameron, J. O. Ndinya-Achola, J. K.Kreiss, M. N. Gakinya, P. Waiyaki, M. Cheang, P. Piot, and A. R. Ronald.1991. Cofactors in male-female sexual transmission of human immunodefi-ciency virus type 1. J. Infect. Dis. 163:233–239.

39. Pohlner, J., U. Langenberg, U. Wolk, S. C. Beck, and T. F. Meyer. 1995.Uptake and nuclear transport of Neisseria IgA1 protease-associated alpha-proteins in human cells. Mol. Microbiol. 17:1073–1083.

40. Rangell, L. K., and G. A. Keller. 2000. Application of microwave technologyto the processing and immunolabeling of plastic-embedded and cryosections.J. Histochem. Cytochem. 48:1153–1159.

41. Read, T. D., R. C. Brunham, C. Shen, S. R. Gill, J. F. Heidelberg, O. White,E. K. Hickey, J. Peterson, T. Utterback, K. Berry, S. Bass, K. Linher, J.Weidman, H. Khouri, B. Craven, C. Bowman, R. Dodson, M. Gwinn, W.Nelson, R. DeBoy, J. Kolonay, G. McClarty, S. L. Salzberg, J. Eisen, andC. M. Fraser. 2000. Genome sequences of Chlamydia trachomatis MoPn andChlamydia pneumoniae AR39. Nucleic Acids Res. 28:1397–1406.

42. Read, T. D., G. S. Myers, R. C. Brunham, W. C. Nelson, I. T. Paulsen, J. F.Heidelberg, E. Holtzapple, H. Khouri, N. B. Federova, H. A. Carty, L. A.Umayam, D. H. Haft, J. Peterson, M. J. Beanan, O. White, S. L. Salzberg, R.Hsia, G. McClarty, R. G. Rank, P. M. Bavoil, and C. M. Fraser. 2003.Genome sequence of Chlamydiophilia caviae (Chlamydia psittaci GPIC):examining the role of niche-specific genes in the evolution of the Chlamy-diaceae. Nucleic Acids Res. 31:2134–2147.

43. Schoier, J., K. Ollinger, M. Kvarnstrom, G. Soderlund, and E. Kihlstrom.2001. Chlamydia trachomatis-induced apoptosis occurs in uninfected McCoycells late in the developmental cycle and is regulated by the intracellularredox state. Microb. Pathog. 31:173–184.

44. Shapiro, L., H. H. McAdams, and R. Losick. 2002. Generating and exploitingpolarity in bacteria. Science 298:1942–1946.

45. Soloff, B. L., R. G. Rank, and A. L. Barron. 1982. Ultrastructural studies ofchlamydial infection in guinea-pig urogenital tract. J. Comp. Pathol. 92:547–558.

46. Stephens, R. S., S. Kalman, C. Lammel, J. Fan, R. Marathe, L. Aravind, W.Mitchell, L. Olinger, R. L. Tatusov, Q. Zhao, E. V. Koonin, and R. W. Davis.1998. Genome sequence of an obligate intracellular pathogen of humans:Chlamydia trachomatis. Science 282:754–759.

47. Takada, Y., X. Ye, and S. Simon. 2007. The integrins. Genome Biol. 8:215.48. Tanzer, R. J., D. Longbottom, and T. P. Hatch. 2001. Identification of

polymorphic outer membrane proteins of Chlamydia psittaci 6BC. Infect.Immun. 69:2428–2434.

49. Thomson, N. R., M. T. Holden, C. Carder, N. Lennard, S. J. Lockey, P.Marsh, P. Skipp, C. D. O’Conner, I. Goodhead, H. Norbertzake, B. Harris,D. Ormond, R. Rance, M. A. Quail, J. Parkhill, R. S. Stephens, and I. N.Clarke. 2008. Chlamydia trachomatis: genome sequence analysis of lympho-granuloma venereum isolates. Genome Res. 18:161–171.

50. Todd, W. J., and H. D. Caldwell. 1985. The interaction of Chlamydia tracho-matis with host cells: ultrastructural studies of the mechanism of release ofa biovar II strain from HeLa 229 cells. J. Infect. Dis. 151:1037–1044.

51. Torres, V. J., S. E. Ivie, M. S. McClain, and T. L. Cover. 2005. Functionalproperties of the p33 and p55 domains of the Helicobacter pylori vacuolatingcytotoxin. J. Biol. Chem. 280:21107–21114.

52. Wehrl, W., V. Brinkmann, P. R. Jungblut, T. F. Meyer, and A. J. Szczepek.2004. From the inside out—processing of the chlamydial autotransporterPmpD and its role in bacterial adhesion and activation of human host cells.Mol. Microbiol. 51:319–334.

53. Wells, T. J., J. J. Tree, G. C. Ulett, and M. A. Schembri. 2007. Autotrans-porter proteins: novel targets at the bacterial cell surface. FEMS Microbiol.Lett. 274:163–172.

54. Whitcher, J. P., M. Srinivasan, and M. P. Upadhyay. 2001. Corneal blind-ness: a global perspective. Bull. W. H. O. 79:214–221.

55. Wittig, I., H. P. Braun, and H. Schagger. 2006. Blue native PAGE. Nat.Protoc. 1:418–428.

56. World Health Organization. 2001. Global prevalence and incidence of se-lected curable sexually transmitted infections: overview and estimates. WorldHealth Organization, Geneva, Switzerland.

57. Zhong, G., P. Fan, H. Ji, F. Dong, and Y. Huang. 2001. Identification of achlamydial protease-like activity factor responsible for the degradation ofhost transcription factors. J. Exp. Med. 193:935–942.

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