BackgroundWhile families of polymorphic membrane protein (pmp) genes have been identified in several Chlamydia species, their function remains mostly unknown. These proteins are of great interest, however, because of their location in the outer membrane and possible role in chlamydial virulence.Methodology/Principal FindingWe analyzed the relative transcription of the pmpD gene, a member of the pmp gene family in C. trachomatis serovar L2, and its protein product translation and processing during the chlamydial developmental cycle. By real-time reverse transcription polymerase chain reaction, the pmpD gene was found to be upregulated at 16 to 24 four hours after infection. Using polyclonal antibodies generated against the predicted passenger domain of PmpD, we demonstrated that it is initially localized on the surface of reticulate bodies, followed by its secretion outside Chlamydia starting at 24 hours after infection. In elementary bodies, we found a ≈157 kDa PmpD only inside the cell. Both events, the upregulation of pmpD gene transcription and PmpD protein processing and secretion, are coincidental with the period of replication and differentiation of RBs into EBs. We also demonstrated that, in the presence of penicillin, the cleavage and secretion of the putative passenger domain was suppressed.Conclusion/SignificanceOur results are in agreement with the general concept that PmpD is an autotransporter protein which is post-translationally processed and secreted in the form of the putative passenger domain outside Chlamydia at mid- to- late point after infection, coinciding with the development of RBs into EBs.

BackgroundThe polymorphic membrane protein D (PmpD) in Chlamydia is structurally similar to autotransporter proteins described in other bacteria and may be involved in cellular and humoral protective immunity against Chlamydia. The mechanism of PmpD post-translational processing and the role of its protein products in the pathogenesis of chlamydial infection have not been very well elucidated to date.Methodology/Principal FindingsHere we examined the expression and post-translational processing of the protein product of the pmpD gene during the life cycle of C. trachomatis serovars A, D, and L2. Each of these three serovars targets different human organs and tissues and encodes a different pmpD gene nucleotide sequence. Our quantitative real-time reverse transcription polymerase chain reaction results demonstrate that the pmpD gene is up-regulated at 12–24 hours after infection regardless of the Chlamydia serovar. This up-regulation is coincidental with the period of exponential growth and replication of reticulate bodies (RB) of Chlamydia and indicates a probable similarity in function of pmpD in serovars A, D, and L2 of Chlamydia. Using mass spectrometry analysis, we identified the protein products of post-translational processing of PmpD of C. trachomatis serovar L2 and propose a double pathway model for PmpD processing, with one cleavage site between the passenger and autotransporter domains and the other site in the middle of the passenger domain. Notably, when Chlamydia infected culture cells were subjected to low (28°C) temperature, PmpD post-translational processing and secretion was found to be uninhibited in the resulting persistent infection. In addition, confocal microscopy of cells infected with Chlamydia confirms our earlier hypothesis that PmpD is secreted outside Chlamydia and its secretion increases with growth of the chlamydial inclusion.Conclusion/SignificanceThe results of this current study involving multiple Chlamydia serovars support the general consensus that the pmpD gene is maximally expressed at mid infection and provide new information about PmpD as an autotransporter protein which is post-translationally processed and secreted outside Chlamydia during normal and low temperature induced persistent chlamydial infection.

Topical microbicides for prevention of sexually transmitted diseases (STDs) would be especially useful for women who are not able to persuade their partner(s) to take precautions. Many topical microbicides are in various stages of development, based on a variety of active ingredients. We investigated the in vitro activity of an engineered antimicrobial peptide (WLBU2) and a lipid (3-O-octyl-sn-glycerol [3-OG]) which could potentially be used as active ingredients in such a product. Using commercially available cytotoxicity reagents [Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH)], we first determined the toxicity of WLBU2 and 3-OG to the host cells in our assay procedure and excluded toxic concentrations from further testing. To determine activity against Chlamydia trachomatis, we used an assay previously developed by our laboratory in which chlamydial elementary bodies (EBs) were exposed to microbicides prior to contact with epithelial cells: the minimum (microbi)cidal concentration (MCC) assay. To further simulate conditions of transmission, we carried out the same assay in the presence of a simulated vaginal fluid, a simulated seminal fluid, human serum albumin, and a range of pH values which might be found in the human vagina at the time of exposure. Last, we tested WLBU2 and 3-OG in combination to determine if adding them together resulted in synergistic activity. We found that WLBU2 and 3-OG both have excellent activity in vitro against C. trachomatis and significantly more activity when added together. The simulated fluids reduced activity, but the synergy seen is good evidence that they would be effective when combined in a microbicide formulation.

Chlamydia trachomatis is the most common sexually transmitted organism in industrialized countries. Nucleic acid amplification testing, using non-invasively collected specimens, is considered to be the method of choice for diagnosis of chlamydial infections of the urethra and the lower genital tract. Serological testing has the potential to circumvent the problem of specimen sampling in invasive C. trachomatis infections of the upper genital tract. However, only a few defined chlamydial antigens have been used in a standardized diagnostic assay format. In this study, we used serological two-dimensional proteomic analysis to broaden the spectrum of diagnostically relevant C. trachomatis proteins. The genes encoding an assortment of already known chlamydial antigens, as well as immunogenic proteins that have not been described before, were cloned, and the recombinant proteins were purified in order to compare their diagnostic usefulness in parallel with a newly developed line immunoassay. With 189 sera collected from patients with and without C. trachomatis infection, recombinant major outer membrane protein (MOMP), chlamydial protease-like activity factor (CPAF), outer membrane protein 2 (OMP2), translocated actin-recruiting protein, and polymorphic membrane protein D (PmpD) showed the highest level of diagnostic sensitivity and specificity. In patients suffering from ascending and invasive C. trachomatis infections, such as pelvic inflammatory disease and lymphogranuloma venereum, the sensitivity reached with these proteins ranged between 71% (PmpD) and 94% (OMP2), and the specificity ranged between 82% (PmpD) and 100% (MOMP and OMP2). Recombinant thio-specific antioxidant peroxidase, ribosomal protein S1 (RpsA) and hypothetical protein 17 showed lower sensitivity but comparably high specificity, ranging from 94% to 100%. The novel line immunoassay based on defined recombinant antigens has promise for improved serodiagnosis in severe and invasive C. trachomatis infections.

Heparin preparations used in clinics of our country (produced in Minsk, Kaunass, Baku, Hungary and Czechoslovakia) differ in their biological activity. Heparin is a variable molecular weight polymer of about 17,000 daltons. Its monomer unit consists of a-glucuronic acid esterified with another sulfuric acid at carbon 2, glucoseamine esterified with sulfuric acid at carbon 6 and at the amino group of carbon 2, and L-iduronic acid. The remains of glucuronic acid and glucosamine, iduronic acid and glucosamine are linked by glycosidic a (1 - 4) bonds.