48 and 72 h p.i.. At both time points Omp-1 could be only identified in the insoluble fraction (Figure 2d), as could the 50 kDa band identified by the C-Pmp18D antibody (Figure 2c). The greater proportion of the protein bands recognised by both N- Pmp18D and Mid-Pmp18D antibodies was identified solely in the insoluble fraction. However, a small proportion of the 94 kDa product could also be detected in the soluble material (Figures 2a and 2b). Proteomic identification of Pmp18D products Proteomic analysis was carried out to identify the protein bands recognised by the different antibodies targeting the various regions of Pmp18D. The passenger domain was found to be readily soluble in 2% sarkosyl (Figure S1) and the protein identification of these N-terminal bands was successfully accomplished using two independent techniques, LC-ESI-MS-MS and MALDI ToF MS using both N-Pmp18D and Mid-Pmp18D antibodies. Immuno- precipitation prior to MALDI ToF MS using the C-Pmp18D antibody was not successful and so only LC-ESI-MS-MS could be used to identify bands recognised using this antibody. While 4 bands could be identified by Western blot using the Mid-Pmp18D antibody, only the protein sequence of the 2 most immunoreactive protein bands at approximately 94 kDa and 84 kDa could be determined by LC-ESI-MS-MS, and these were the only 2 proteins recovered by immunoprecipitation prior to MALDI-ToF MS analysis. These combined analyses gave broadly similar results, although peptide coverage with MALDI-ToF MS analysis appeared to be slightly greater. MALDI-ToF MS analysis identified the same peptide at the carboxy-terminus of both proteins, NAKPTEK1060 and this was confirmed by LC-ESI-MS- MS. The first detected N-terminal peptide sequence of the larger 94 kDa protein fragment, recognised by both N-Pmp18D and Mid-Pmp18D, was identified as 67EKPIHAQ by MALDI ToF MS and as 76KGETDQ by LC-ESI-MS-MS, giving a predicted molecular mass for this protein fragment of approximately 104 kDa based on sequence coverage (Figure S2a). Both methods identified the same initial peptide at the N-terminus of the predicted 84 kDa protein, with peptide coverage starting from 215LVDGCE, indicating that it is a product of the initial larger protein with cleavage at the N-terminus and the removal of approximately 150 amino acids (Figure S2b) giving an actual predicted molecular mass of 87.5 kDa. No protein could be recovered after immunoprecipitation using the carboxy antibody. However, LC-ESI-MS/MS analysis dem- onstrated sequence coverage of the 50 kDa fragment identified by the carboxy antibody from 1083TLADIN to LNCGMR1518 with a Figure 2. Western blot demonstrating the presence of the majority of Pmp18D in the insoluble material after sonication in PBS 48 h post-infection: a) anti-N-Pmp18D, b) anti-Mid- Pmp18D, c) anti-C-Pmp18D and d) anti-Omp-1 mAb 4/11. doi:10.1371/journal.pone.0049190.g002 Processing of C. abortus Pmp18D predicted molecular mass based on sequence coverage of 48 kDa (Figure S2c). A further cleavage product of the carboxy-terminal barrel was also identified with an initial peptide at 1083TLADIN with sequence coverage obtained to EHNYSR1445 (Figure S2d). The sizes of the predicted proteins remain approximate and are perhaps slightly underestimated as the peptides identified using both these techniques may not provide the actual first or last peptide sequences of the analysed proteins. A schematic diagram of the approximate cleavage sites for each of the identified PmpD protein fragments is shown in Figure 3. Bioinformatic analysis of PmpD To determine whether the cleavage products observed in C. abortus Pmp18D and other Chlamydia spp occurred in conserved regions or motifs of the PmpD molecule, we performed an alignment and clustering analysis of PmpD protein sequences from the nine Chlamydia spp. Clustering analysis of PmpD identified in the nine Chlamydia spp. showed that proteins group into three distinct lineages, each strongly supported with posterior probabilities of 1.00, consisting of cluster 1: C. abortus, C. felis, C. caviae and C. psittaci; cluster 2: C. pecorum and C. pneumoniae; and cluster 3: C. muridarum, C. trachomatis and C. suis (Figure 4). Within each lineage, PmpD clustered by species with the exception of C. suis which clustered with C. trachomatis serovar E strains. Mapping of N-terminal peptides identified in C. abortus onto PmpD sequence alignments demon- strated the M-domain cleavage site 1083TLADIN to occur in a species-variable region located between two flanking regions of high sequence conservation (Figure S3a). The cleavage site identified by peptide EHNYSR1445 occurred in a region of high sequence conservation between all Chlamydia spp., whereas peptides LNCGMR1518 and 215LVFDGCE, located in the barrel- and passenger domains respectively, occurred in semi-conserved regions flanked by regions of high sequence conservation (Figure S3b and c). The cleavage site identified in the passenger domain by peptide 67EKPIHAQ occurred in a region of unique sequence composition in C. abortus (Figure S2d). Discussion Although pmp gene carriage across Chlamydia spp. is variable through gene expansion and diversification in a number of pmp families, the genome of each species encodes only one pmpD gene. Our previous work has demonstrated that Pmp18D in C. abortus follows the same patterns of expression at both the transcript [25] and protein level [26] as PmpD of C. trachomatis. It was hypothesised that given these similarities in protein expression, the proteolytic processing of C. abortus Pmp18D would resemble that of the other previously studied PmpD molecules. The results from previous studies on C. trachomatis [12,27] and C. pneumoniae PmpD [28] together with those from this current study on C. abortus PmpD, demonstrate that there is a degree of heterogeneity in the processing of PmpD between species. At various time-points, full-length PmpD can be identified during the C. trachomatis developmental cycle. However, little if any full-length protein could be observed at any of the analysed time points in C. abortus, mirroring the observations made on the processing of the C. pneumoniae PmpD [28]. Due to differences in the timing of the chlamydial developmental cycle between species, direct compar- ison of specific time points is difficult and it cannot be discounted that full-length Pmp18D may have been observed with the analysis of additional time-points. However, the results from the current study suggest that virtually all of the Pmp18D in C. abortus is cleaved directly upon translocation to the outer surface of the PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49190