The PmpD proteins of both C. trachomatis and C. pneumoniae are expressed throughout the chlamydial developmental cycle. Pmps are highly immunogenic and there has been much interest in their exploitation as vaccine and diagnostic candidates. Recently, attention has been focussed upon PmpD due to the ability of antibodies raised against it to neutralize the infectivity of both C. trachomatis and C. pneumoniae in vitro, and the level of seroconversion to the protein observed in C. trachomatis infected individuals. PmpD is cleaved, and it has been hypothesised that this may permit secretion of specific effector peptides into host cells or within the inclusion [12]. However, little is understood about the function or processing of the PmpD molecule in any other Chlamydia spp. This study focuses on C. abortus, the causative agent of ovine enzootic abortion (OEA), a disease of significant economic importance to the sheep rearing industry and of concern to human health, particularly pregnant women [13]. The pmp genes are grouped within specific families by their phylogenetic similarity with the originally identified C. trachomatis pmps [14]. However, due to the expansion in pmp gene number in C. abortus, the pmps have additionally been numbered sequentially by their position in genome, with the C. abortus PmpD protein being termed Pmp18D [8]. Given the potential conservation of roles of the PmpD molecules across chlamydial species studied to date and their potential as diagnostic or vaccine candidates, this study was carried out to investigate the structural features and processing of the C. abortus Pmp18D molecule. Materials and Methods C. abortus propagation McCoy cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and maintained in RPMI1640 medium supplemented with 10% heat inactivated fetal calf serum (PAA Laboratories Ltd, Yeovil, Somerset, UK). The C. abortus strain S26/3 was propagated in McCoy cells, according to a previously published protocol [15]. Antibodies and Western blotting Rabbit polyclonal antibodies were generated against C. abortus S26/3 Pmp18D peptides: N-Pmp18D (N terminal region of Pmp18D) EKPIHAQGPKKGETD (amino acids (aa) 67–81); Mid-Pmp18D (middle domain region of Pmp18D) DPNAKPTE- KIESPTS (aa 1052–1066) (both Eurogentec, Southampton, UK); C-Pmp18D (carboxy terminus region of Pmp18D) CQPNLGGSKGSWDSR (aa 1357–1370) (Genscript USA Inc., Piscataway, NJ, USA). In addition, the mouse anti-Omp-1 mAb 4/11 [16] was used for the detection of C.abortus Omp-1. Total cell lysates were prepared by scraping infected S26/3 infected McCoy monolayers and pelleting at 12,000 rpm in a microcentrifuge at 24, 48 or 72 h post-infection (p.i.). The resulting pellets were resuspended in 1 ml 16 Laemmli loading buffer [17]. After brief sonication and boiling for 5 mins, proteins were separated on 4– 12% NuPAGE gels (Life Technologies, Paisley, UK), transferred onto nitrocellulose and Western blotted. Detection was accom- plished using the ECL-advance system and results were visualized using the LAS400 Quantitative imaging system (both GE Healthcare, Chalfont St Giles, Buckinghamshire, UK). Molecular masses of recognized protein products were calculated using a standard curve, calculated from the Rf values obtained for SeeBlueH Plus2 markers (Life Technologies) and using Image- Quant TL 1D-PAGE analysis software (GE Healthcare). Processing of C. abortus Pmp18D Pmp18D solubility in infected McCoy cells To investigate if Pmp18D protein fragments are released or retained on the surface of the C. abortus outer membrane, soluble and insoluble protein fractions from C. abortus infected cells were prepared essentially as previously described [12]. Briefly, infected 225 cm2 flasks of McCoy cell monolayers were harvested at 48 h post-infection and washed with 5 ml hypotonic swelling buffer (15 mM KCl, 1.5 mM Mg(Ac)2 and 10 mM HEPES, pH 7.4) containing protease inhibitors (Sigma Aldrich, Dorset, UK; P9549). Cells were gently lysed by 20 strokes using a Dounce homogenizer. The homogenate was briefly centrifuged for 5 mins at 2006 g and the resulting supernatant further centrifuged at 125,0006 g (SW55Ti rotor Beckman Coulter). The supernatant (soluble material) was removed and the protein precipitated with 3 volumes of acetone at 220uC. The pellet (insoluble material) was washed twice with ice-cold PBS containing protease inhibitors before lysis in 1 ml 16 Laemmli loading buffer. The precipitated supernatant material was subsequently lysed in the same volume of 16 Laemmli buffer. Samples were analysed by Western blotting, as described above. Identification of major high molecular weight cleavage products Identification of the major passenger cleavage products was accomplished using 2 independent proteomic methods: Liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) For the initial identification of the cleavage products, replicate 100 ml samples of S26/3 infected total cell lysate were separated on a 7% 16 cm Slab gel (Hoefer SE600, GE Healthcare). The gel was cut into two equal pieces. One half was blotted onto nitrocellulose using a semi-dry blotting apparatus prior to Western blotting using the 3 antibodies directed against the different regions of Pmp18D. The second half of the gel was stained with SimplyblueTM SafeStain (Life Technologies). The position of the identified bands was determined and the gel slices excised from the gel. Proteins were destained and reductively alkylated using DTT and iodoacetamide. Gel slices were then digested overnight with trypsin (Porcine trypsin, Promega, Hants, UK) at 37uC. Samples were transferred to low-protein-binding HPLC sample vials and stored at 4uC until required for LC-ESI-MS/MS analysis. Liquid chromatography was performed using an Ultimate 3000 nano- UHPLC system (Dionex) comprising a WPS-3000 well-plate micro auto sampler, a FLM-3000 flow manager and column compartment, a UVD-3000 UV detector, an LPG-3600 dual- gradient micropump and an SRD-3600 solvent rack controlled by Chromeleon chromatography software (Dionex). A micro-pump flow rate of 246 ml/min was used in combination with a cap-flow splitter cartridge, affording a 1/82 flow split and a final flow rate of 3 ml/min through a 5 cm6200 mm ID monolithic reversed phase column (Dionex) maintained at 50uC. Samples of 4 ml were applied to the column by direct injection. Peptides were eluted by the application of a 15 min linear gradient from 8–45% solvent B (80% acetonitrile, 0.1% (v/v) formic acid) and directed through a 3 nl UV detector flow cell. LC was interfaced directly with a 3-D high capacity ion trap tandem mass spectrometer (amaZon ETD, Bruker Daltonics) via a low-volume (50 ml/min maximum) stainless steel nebuliser (Agilent, cat. no.G1946-20260) and ESI. Parameters for tandem MS analysis were set as previously described [18] with minor modifications as detailed below. Database Mining: Deconvoluted MS/MS data in Mascot generic file (mgf) format was imported into ProteinScapeTM PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49190