Processing of C. abortus Pmp18D Figure 3. Schematic representation of Pmp18D cleavage products as revealed following proteomic analysis of protein fragments. doi:10.1371/journal.pone.0049190.g003 bacterium, comprising the passenger domain with a molecular mass of approximately 104 kDa and a 48 kDa protein comprising the M-domain and the carboxy-terminal b-barrel domain. The majority of these products appear to remain associated with the outer membrane of the organism, as only a small proportion of the cleaved 104 kDa molecule could be identified in the soluble fraction. Evidence from both Western blotting and proteomic methods has revealed further cleavage at the N-terminus of the 104 kDa protein. The Mid-Pmp18D antibody recognised an additional 3 protein bands, a single band of approximately 84 kDa and a doublet of 76 and 73 kDa that were not recognised by the N- terminal antibody. The smaller protein fragments cleaved from these proteins including the N-terminal 150aa polypeptide cleaved from the 104 kDa protein could not be subsequently identified in any cellular fraction. It has been previously hypothesised in C. trachomatis that some products of PmpD cleavage could act as soluble effector molecules [12]. These effectors could either impact on host transcription or cell lysis or be secreted from the host cell during the latter stages of the chlamydial developmental cycle. Certainly, the observed increase in the levels of the cleaved N- terminal proteins 72 h post-infection would fit with this hypothesis. However, while cleavage of the passenger domains appears to be a conserved feature of the PmpD molecule across Chlamydia spp., patterns of cleavage appear to be species-specific. Therefore, to understand whether the differences in cleavage patterns could be related to the presence of conserved regions or motifs within the molecule, an analysis of representative PmpD amino acid sequences was carried out to determine sequence similarity across species. Phylogenetic analysis of PmpD revealed a high degree of amino acid sequence heterogeneity between Chlamydia spp., particularly within the passenger domains. The identification of distinct clades of PmpD molecules comprising 3 clusters (Cluster 1: C. abortus, C. psittaci, C. caviae and C. felis; Cluster 2: C. pneumoniae and C. pecorum and Cluster 3: C. trachomatis, C. suis and C. muridarum), could indicate a different function for PmpD for the species belonging to these different clusters. Indeed, in addition to difference in amino acid sequence and proteolytic processing, Swanson and colleagues [12] highlighted the presence of putative RGD and NLS domains within specific cleavage products of C. trachomatis PmpD [12]. These motifs are maintained in C. suis PmpD, which has 100% similarity in terms of amino acid sequence to that of C. trachomatis serovar E. However, neither of these motifs were identified in Pmp18D of C. abortus, and of the other sequenced members of the Chlamydiaceae, the NLS motif could only be identified in C. felis, although in a different location within the protein. The differences in cleavage patterns and the absence of conserved motifs in PmpD across species suggests that the protein may play different roles in different species of Chlamydia. Cleavage of the PmpD molecule appears to be a conserved feature across the chlamydial species published to date. However, the patterns of proteolytic cleavage appear to be species-specific and phylogenetic analyses show large scale variation of PmpD PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49190