Processing of Chlamydia abortus Polymorphic Membrane Protein 18D during the Chlamydial Developmental Cycle Nick M. Wheelhouse1*, Michelle Sait1, Kevin Aitchison 1 , Morag Livingstone1, Frank Wright2, Kevin McLean3, Neil F. Inglis3, David G. E. Smith1,4, David Longbottom1 1 Moredun Research Institute, Edinburgh, Midlothian, United Kingdom, 2 Biomathematics and Statistics Scotland, Dundee, United Kingdom, 3 Moredun Proteomics Facility, Moredun Research Institute, Edinburgh, Midlothian, United Kingdom, 4 Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom Abstract Background: Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D). Methodology/Principal Findings: Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae. Conclusions/Significance: This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated. Citation: Wheelhouse NM, Sait M, Aitchison K, Livingstone M, Wright F, et al. (2012) Processing of Chlamydia abortus Polymorphic Membrane Protein 18D during the Chlamydial Developmental Cycle. PLoS ONE 7(11): e49190. doi:10.1371/journal.pone.0049190 Editor: David M. Ojcius, University of California Merced, United States of America Received August 27, 2012; Accepted October 5, 2012; Published November 8, 2012 Copyright: 2012 Wheelhouse et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Scottish Government Rural and Environment Science and Analytical Services Division (RESAS, http://www.scotland.gov. uk) and by grant no. BB/E018939/1 from the Biotechnology and Biological Sciences Research Council (http://www.bbsrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare that no competing interests exist that may have influenced the content of the manuscript. * E-mail: nick.wheelhouse@moredun.ac.uk. Introduction The family Chlamydiaceae are a diverse group of obligate intracellular Gram-negative bacteria that cause a range of pathogenic conditions in a wide variety of host species [1]. All known members share a similar and distinct biphasic develop- mental cycle, which is initiated with the entry of the infectious form of the organism, the elementary body (EB), into the host cell where it resides within a vacuole known as an inclusion. The EB undergoes conversion to the metabolically active reticulate body (RB), which replicates through binary fission. Towards the end of the cycle (48 to 72 hours following infection) the RBs re-condense to EBs before both the inclusion and host cell are lysed, allowing the release of the infective organisms to infect neighbouring cells [1]. The Type V or autotransporter (AT) secretion system comprises the largest family of proteins found across pathogenic Gram- negative bacteria. Classical AT structure is characterised by the presence of three separate functional domains; a cleavable N- terminal signal sequence; a passenger (effector) domain and a carboxy-terminal b-barrel translocator domain [2]. While the overall structure and organisation of ATs is similar across bacterial species, the function of the effectors vary. However, many of these proteins have been identified as virulence factors involved in bacterial pathogenesis. Chlamydia possess a unique family of proteins that have been identified as ATs (known as the Polymorphic membrane proteins (Pmps)) [3]. Pmps were first identified in Chlamydia abortus (C. abortus) due to their immuno- reactivity with convalescent sheep sera [4,5], and have now been identified in all of the pathogenic Chlamydia spp. Significant heterogeneity of Pmp gene carriage has been observed between chlamydial species. Genome sequencing of C. trachomatis has revealed the presence of 9 pmp genes (termed A-I) [6] while 21, 16, 18, 17, 21 and 20 pmps have been identified in C. pneumoniae, C. pecorum, C. abortus, C. caviae, C. psittaci and C. felis respectively [7– 11]. PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49190