Chlamydia trachomatis Polymorphic Membrane Protein D Is an Oligomeric Autotransporter with a Higher-Order Structure

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.Chlamydia trachomatis is a mucosotropic obligate intracellular gram-negative pathogen that is a leading cause of sexually transmitted and ocular infections. Infection can result in serious sequelae such as infertility and blindness (54, 56) and an increased risk of human immunodeficiency virus infection and transmission (38). The pathophysiology of chlamydial infection is associated with the pathogen''s propensities to cause persistent infection and to suppress host immunity (3). A vaccine is needed to control chlamydial diseases; however, progress toward this goal will not be forthcoming until more is known about the virulence factors that mediate persistence and immune evasion.Chlamydiae are characterized by a unique biphasic developmental cycle that modulates between an extracellular, metabolically inactive, infectious elementary body (EB) and an intracellular, metabolically active, noninfectious reticulate body (RB) (34). Their obligate intracellular niche and the lack of a tractable genetic system present unique challenges in the study of chlamydial biology and pathogenesis. To overcome these hurdles, chlamydial genomes from a diverse spectrum of host-specific strains have been sequenced. Comparative genomics have shown considerable homology among various chlamydial species and have provided important insights into shared and species-specific virulence factors (7, 24, 41, 42, 46, 49).The type V or autotransporter (AT) secretion pathway is the most widespread secretion mechanism employed by gram-negative bacteria to deliver virulence factors involved in initiating infection, disease progression, and immune evasion (reviewed in references 11 and 21). AT proteins are characterized by three domains, (i) a signal sequence (SS), (ii) a diverse N-terminal passenger domain (PD) that confers effector function, and (iii) a conserved C-terminal translocator domain (TD). The TD inserts into the outer membrane (OM) by assembling into a β-barrel pore that facilitates PD translocation to the bacterial surface. The PD remains tethered to the TD or is cleaved and either is released or remains noncovalently associated with the OM. Well-characterized examples of ATs found on the bacterial cell surface as monomers or oligomers are Neisseria meningitidis NalP (37) and Helicobacter pylori VacA (31), respectively.C. trachomatis has a nine-member AT family (20), termed polymorphic membrane proteins (Pmps), whose role(s) in chlamydial pathogenesis has yet to be defined. The pmp paralogs (pmpA to pmpI) constitute 3.2% of the ∼1-Mb genome and are found at three chromosomal loci composed of two gene clusters (pmpA to pmpC and pmpE to pmpI) and the genetically isolated gene pmpD (46). Notably, PmpD is the second most highly conserved Pmp, exhibiting 99.2% amino acid identity among C. trachomatis serovars (16). Despite relatively low abundance in the chlamydial OM, Pmps are major immunogens and may be important virulence factors (29). C. trachomatis PmpD is a target of broadly cross-reactive neutralizing antibodies (Abs), which makes it an attractive vaccine candidate for the prevention of human infections (10).Previous reports have described proteolytic processing of C. pneumoniae and C. trachomatis PmpD (25, 52). Furthermore, recombinant C. pneumoniae PmpD has been suggested to function as an adhesin capable of inducing proinflammatory cytokine production (35, 52). Nothing is known about the native structure of C. trachomatis PmpD or the potential significance of its structure to chlamydial pathogenesis. Here we show that C. trachomatis PmpD is present on the organism''s surface as an oligomer with a higher-order flower-like structure. Moreover, we describe novel infection-dependent proteolytic processing of PmpD that produces soluble fragments with predicted eukaryotic motifs, implying a multifunctional protein important to chlamydial pathogenesis.