analysis software (Bruker Daltonics) and searched against the C.abortus_NCBInr database utilising the Mascot (Matrix Science) search algorithm. The data was also searched specifically against the cognate pmp18D protein sequence using the Sequence-Editor function of BiotoolsTM analysis software (Bruker Daltonics). The interpretation of MS/MS data was performed in accordance with published guidelines [19]. To this end, fixed (carbamidomethyl C) and variable (deamidation N,Q and oxidation M) modifications were selected, and mass tolerance values for both MS and MS/MS were set at 60.5 Da allowing for one 13C isotope and a single missed cleavage. Molecular weight search (MOWSE) scores and percentage coverage values attained for individual protein identifications were inspected manually and considered significant only if a) two peptides were matched for each protein, and b) each peptide contained an unbroken ‘‘b’’ or ‘‘y’’ ion series of a minimum of four amino acid residues. Matrix-assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-ToF MS) Infected McCoy cell monolayers were disrupted with sterile glass beads, and was briefly sonicated, prior to centrifugation at 1000 rpm for 10 minutes in a JA-14 rotor (Beckman Coulter), to remove gross cellular debris. The supernatant was removed and centrifuged at 12000 rpm for 40 minutes at 4uC using a JA-16.250 rotor. The pellet was resuspended in 5 ml 2% sarcosyl/PBS by brief sonication before incubation at 37uC for 30 minutes. The suspension was then centrifuged at 120,0006 g using a SW55Ti rotor for 60 minutes at 4uC. The supernatant was removed and used for Pmp18D purification. Immunoprecipitation was carried out using Protein G DynabeadsH (Life Technologies) coupled to Pmp18D antibodies, as per the manufacturer’s instructions. The final antibody:protein complex was disassociated by the addition of Laemmli loading buffer and heating at 95uC. Proteins were resolved on NuPAGEH polyacrylamide gels and visualised using SimplyblueTM SafeStain (both Life Technologies). Gel slices were treated as described for LC-ESI-MS-MS analysis. Digests were analysed on a Bruker Ultraflex II MALDI-ToF-ToF mass spectrometer (Bruker Daltonics), scanning the 600 to 5000 dalton region in reflectron mode producing monoisotopic resolution. The spectra generated were mass calibrated using known standards. Masses obtained were then database searched using the MAS- COT search algorithm. Bioinformatic analysis of PmpD PmpD amino acid sequences were obtained from GenBank and NCBI conserved domain searches were performed to define passenger-, M- and autotransporter-domains. PmpD sequences from the passenger-, and combined M- and autotransporter- domains were aligned using MUSCLE [20], and checked by mapping YASPIN secondary structure predictions [21] before being joined into a single alignment. Regions which could not be unambiguously aligned were eliminated using GBlocks v 0.91b [22], resulting in 1032 positions being analyzed (59% of the original 1726 positions). Bayesian dendrograms were generated using MrBayes software [23], launched from the TOPALi v2.5 package [24] using the CPRev+I+G substitution model that was determined to be the model of best fit, based on the BIC criterion. Trees were generated using Markov chain Monte Carlo (MCMC) settings of 2 runs of 625,000 generations with a burn-in of 125,000 generations with trees sampled every 100 runs. Processing of C. abortus Pmp18D Results Expression of Pmp18D Western blot analysis using all 3 anti-Pmp18D antibodies demonstrated that expression of Pmp18D was time-dependent and correlated with an increase in Omp-1 levels over time (Figure 1). At any of the analyzed time points, using both the N-Pmp18D and Mid-Pmp18D antibodies, little if any intact 160 kDa molecular mass protein (which would correspond to the mature Pmp18D molecule) could be observed. The most highly immunoreactive protein among the identified bands was a molecule of approxi- mately 94 kDa that was recognised by both N-Pmp18D and Mid- Pmp18D (Figures 1a and 1b respectively) and a 50 kDa protein recognised by the C-Pmp18D (Figure 1c) antibody alone. Additional protein bands at approximately 84 kDa and a doublet of a 76 kDa and 73 kDa were also routinely recognised by the Mid-Pmp18D antibody, particularly by 72 h p.i. (Figure 1b). However none of these additional bands were recognised by the N-Pmp18D antibody (Figure 1a). Pmp18D solubility in infected McCoy cells To determine whether the Pmp18D passenger is secreted from C. abortus or retained on the bacterial surface, soluble and insoluble cell fractions were prepared from C. abortus-infected McCoy cells at Figure 1. Western blot demonstrating an increase in Pmp18D expression and proteolytic cleavage over the 72 h C. abortus developmental cycle using: a) anti-N-Pmp18D, b) anti-Mid- Pmp18D, c) anti-C-Pmp18D pAbs and d) anti-Omp-1 mAb 4/11. doi:10.1371/journal.pone.0049190.g001 PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49190