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.

A topical microbicide that women can use to prevent sexually transmitted diseases (STDs) is essential, and many microbicide candidates are being tested for activity against human immunodeficiency virus and other STDs, including Chlamydia trachomatis. Screening assays for assessing the activity of microbicides against C. trachomatis are typically done with laboratory-adapted strains, but it is possible that recent clinical isolates may have different susceptibilities to microbicides, as has been seen with Neisseria gonorrhoeae and Lactobacillus spp. (B. J. Moncla and S. L. Hillier, Sex. Transm. Dis. 32:491-494, 2005). We utilized three types of microbicides to help define this aspect of our assay to test microbicides against C. trachomatis in vitro. To simulate conditions of transmission, we used an assay that we previously developed in which we exposed chlamydial elementary bodies to microbicides prior to contact with epithelial cells. We first determined the toxicity of microbicides to the cells used to culture Chlamydia trachomatis in the assay and, if necessary, modified the assay to eliminate toxicity at the concentrations tested. We compared the sensitivities of recent clinical isolates of Chlamydia trachomatis versus laboratory strains of the same serovar and found major differences in sensitivity to nonoxynol-9 (non-9), but only minor differences were seen with the other microbicides. We thus conclude that when assessing activity of potential topical microbicides versus the obligate intracellular bacteria C. trachomatis, the use of recent clinical isolates may not be necessary to draw a conclusion about a microbicide's effectiveness. However, it is important to keep in mind that differences (like those seen with non-9) are possible and that clinical isolates could be included in later stages of testing.

Commonly used "inactive" pharmaceutical excipients were tested in a previously developed minimum cidal concentration assay to assess their ability to kill Chlamydia trachomatis topically. Sixteen excipients were evaluated in these studies under various conditions. A range of activities was found among the excipients that could be tested in our assay system.Promising topical antimicrobial agents that are active against sexually transmitted infection pathogens have been identified (1, 10, 11), but these agents must be delivered in an acceptable dose and formulation such as a cream, gel, or foam. These drug delivery systems contain excipients that serve multiple functions, including the enhancement of product stability, efficacy, and acceptability to the patient. Excipients can be classified into a number of categories, including preservatives, solvents, antioxidants, and gelling agents. Although excipients are important components of topical microbicide products and are also known to possess antimicrobial activity (4, 12), their activity against Chlamydia trachomatis have not yet been determined. Here we use the previously described (1, 10, 11) minimal cidal concentration (MCC) assay, designed to test the direct action of microbicides against the extracellular, infectious C. trachomatis elementary bodies (EBs), to measure the inherent antichlamydial activity of potential excipients for formulation of active agents into topical microbicide products.These studies used C. trachomatis serovar L2 (434/Bu). The bacterium was propagated in mouse McCoy fibroblasts (ATCC CRL 1696), purified on Renografin (6), and frozen at Ϫ70°C in a sucrose-phosphate-glutamate cryopreservative buffer (SPG) (8). The serotype of this strain was verified by a plate typing method (13). This strain was used because it is easier to grow and more sensitive to some topical microbicides than other strains (3).Excipients evaluated in these studies represented four principal categories. The antioxidants evaluated were vitamin E (Spectrum, Gardena, Calif.), EDTA (Sigma, St. Louis, Mo.), and butylated hydroxyanisole (Spectrum). The preservatives tested were propylparaben (Spectrum), methylparaben (Spectrum), sodium benzoate (Spectrum), potassium benzoate (Spectrum), benzalkonium chloride (BAK; Sigma), benzoic acid (Spectrum), sorbic acid (Spectrum), and PEG 400 (Spectrum). The acidifying agents were lactic acid (Spectrum) and citric acid (Sigma). Gelling agents studied were three carrageenan products, Viscarin 328, Gelcarin 812, and Seaspen (FMC, Newark, Del.). These excipients were evaluated at three concentrations (lower than the average, average, and higher than the average normal usage level in vaginal products) (9), two pH values (pH 5 and 7), and two time points (5 and 120 min). Most excipients were dissolved in SPG, while a few (sorbic acid, methylparaben, and benzoic acid) were solubilized in ethyl alcohol and then diluted in SPG. The concentration of ethyl alcohol used, however, was shown to have no activity against the chlamydial EBs (data n...