Recombinant Treponema Pallidum Protein Tp0965Activates Endothelial Cells And Increases The Permeability Of Endothelial Cell Monolayer

BackgroudSyphilis is a chronic systemic, sexually transmitted disease caused by the bacterial spirochete Treponema pallidum subsp. pallidum (T. pallidum). Many evidences demonstrated that activation and damage of endothelial cells may play an important role in pathogenic mechanism of T. pallidum infections. Previous investigations reported that T. pallidum was capable of activating directly host vascular endothelium, up-regulate the expression of adhesion molecules, and promote the adherence of T-lymphocytes to human dermal microvascular endothelial cells. Meanwhile several T. pallidum outer member proteins have been shown to regulate the expression of cell adhesion molecules and binding of T-lymphocytes to human dermal microvascular endothelial cells. T. pallidum is an obligate human pathogen and cannot be cultivated in vitro. This has severely impeded progress in understanding precise pathogenesis of this microbe. The availability of the T. pallidum genome sequence made it possible to examine predicted T. pallidum open reading frames (ORFs) for potential application as diagnostic or immunization tools. This approach permits identification of low-abundant T. pallidum antigens, since they may be expressed as recombinant proteins in much larger quantities. The Protein BLAST data revealed that Tp0965is a membrane fusion protein and is located on periplasm of T. pallidum. Previous studies suggest that Tp0965is reactive with sera from syphilitic individuals at all stages and shows strong immunoreactivity. However, reports concerning the role of Tp0965in the pathogenesis of syphilis are lacking. In the present study, we examined the effects of rTp0965on expression and mRNA levels of adhesion molecules in HUVECs. In addition, we examined the changes in permeability of HUVEC monolayers and transendothelial migration of monocytes. We also investigated effects of rTp0965on the reorganization of F-actin and expression of Claudin-1. The results indicated that rTp0965has the capability of triggering endothelial cell activation and regulates the function of the endothelial barrier. Objective To study the effects of recombinant T. pallidum membrane protein Tp0965on activating of endothelial cells and increasing the permeability of endothelial cell monolayer in vitro, in order to understand its roles in the immunopathogenesis of syphilis.MethodsThe gene of Tp0965was amplified by polymerase chain reaction (PCR) from T. pallidum genomic DNA and the nucleotide sequence was cloned into the expression plasmid pET28a. The new constructs were transformed into E. coli Rosetta (DE3) and the recombinant fusion proteins were purified on Ni-NTA chromatographic column. SDS-PAGE and immunoblot analysis using the anti-polyhistidine tag antibody were employed to identify the protein and assess its purity. Protein concentrations were determined using a BCA Protein Assay Kit. To remove LPS contamination, the recombinant protein was subsequently treated by polymyxin B-agarose and the LPS level was detected by the Limulus amebocyte lysate test kit.After co-culture of the rTp0965with HUVECs, the expression and mRNA transcription levels of ICAM-1and E-selectin were detected on HUVECs’ membrane and in HUVECs by cell enzyme-linked immunosobent assay (Cell ELISA) and fluorescent real-time quantitative PCR, respectively; meanwhile, the level of MCP-1in supernatants was determined by ELISA. To test the effects of rTp0965on monocyte adhesion to HUVECs, we pretreated confluent monolayers of HUVECs and then stimulated them with rTp0965, followed by incubation with THP-1cells and then the adhesion of monocytic THP-1cells was also observed by fluorescence microscopy. To examine the effects of rTp0965on HUVECs chemoattraction of monocytes, monocyte THP-1cells were added to the inserts of transwell systems that contained HUVECs (that had been pretreated with rTp0965) in the wells. Monocyte THP-1cells migration was monitored for2h by fluorescence microscopy.For endothelial permeability and transendothelial migration measurements, HUVECs were treated with rTp0965and then HRP was added on top of the HUVEC monolayers and50μl of samples were taken from the wells. The collected samples were analyzed for the flux of HRP with a TMB kit. For the transmigration assay, CCL-2was added into the wells as a chemoattractant. Monocyte THP-1cells stained with calcein AM were added on top of HUVEC monolayers. Next, the numbers of monocyte THP-1cells in the wells and beneath the HUVEC monolayers were counted using a fluorescence microscope. In some experiments, HUVEC monolayers were pre-incubated with the ROCK inhibitor Y-27632for30mins before the endothelial permeability and transendothelial migration assays were performed as described above.To assess the effects of rTp0965on expression of tight junction proteins of HUVECs, HUVECs were treated with rTp0965and then harvested for detection of Claudin-1expression by Western blot, In some experiments, HUVECs were pre-incubated with Y-27632for30mins before HUVECs were harvested and Western blot was performed. To analyze F-actin distribution, HUVECs were treated with rTp0965then were fixed in4%buffered paraformaldehyde and stained with rhodamine-phalloidin. Finally, the coverslips were examined using a confocal laser scanning microscope system. In some experiments, HUVECs monolayers were preincubated with Y-27632for30mins before F-actin distribution was tested as described above.ResultsAfter expression and purification, the concentrations of rTp0965was210μg/mL and the final LPS level was lower than2.0EU/mL, which is an amount that did not stimulate proinflammatory cytokine production by itself.Real-time RT-PCR study showed that the mRNA transcription levels of ICAM-1and E-selectin were increased significantly after incubation with rTp0965(800ng/ml)(P<0.05) compared to controls. Cell ELISA analysis showed similar results. The rTp0965induced a remarkable increase of ICAM-1and E-selectin, compared to the controls. Adherence assay results showed that rTp0965stimulated an increase in adherence of THP-1cells to HUVECs (48.5±7.5%versus23.3±7.9%, P<0.05).ELISA data showed that the amount of soluble MCP-1was increased significantly after incubation with rTp0965(800ng/ml) compared with the control (P<0.05). Real-time RT-PCR analysis showed similar results. A remarkable increase of MCP-1mRNA, compared to the control, was induced by rTp0965. The chemotaxis assay showed that some monocyte THP-1cells migration was evoked by HUVECs in control group, but significantly more monocyte THP-1cells migrated towards the HUVECs pretreated with rTp0965.To analyze the effects of rTp0965on the permeability of HUVECs monolayers the flux of HRP was calculated. After HRP was added to the transwell inserts for4h, the permeability in rTp0965group was1.2±0.11, and that in control was0.52±0.06(P<0.05). In an in vitro static assay of transendothelial migration, addition of rTp0965(800ng/ml) to confluent monolayers of HUVECs cultured in transwell inserts resulted in an approximately two-fold increase in the migration of monocyte THP-1cells at the4h time point (35.3%versus12.7%, P<0.001). The ROCK inhibitor Y-27632partially blocked the increase in migration of monocyte THP-1cells after24h of rTp0965exposure compared with the group treated with rTp0965but without Y-27632(19.0%versus35.3%, P<0.05).To evaluate the effect of rTp0965on F-actin reorganization in HUVECs, we preincubated HUVECs with rTp0965and then stained the HUVECs with rhodamine-phalloidin. The results showed that in the absence of rTp0965, a rim of F-actin staining was present at the margins of the treated cells, with a few randomly disoriented stress fibers within the cytoplasm. However, in the presence of rTp0965, F-actin rapidly formed organized filamentous networks. Y-27632partially prevented the F-actin reorganization and redistribution after treatment with rTp0965. Western blot proved that after treatment with rTp0965for24h, the level of Claudin-1was markedly decreased in HUVECs and the effect was significantly prevented by the ROCK inhibitor Y-27632.Conclusion1. T. pallidum membrane protein Tp0965could be successfully expressed through E. coli expression system; the protein could be used for the study of the pathogenesis of syphilis.2. The protein Tp0965could increase the levels of ICAM-1, E-selectin, and MCP-1mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1cells) to HUVECs preincubated with rTp0965. The protein Tp0965may activate endothelial cells.3. The protein Tp0965induced reorganization of F-actin and decreased expression of Claudin-1in HUVECs and inhanced higher endothelial permeability. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced reorganization of F-actin and expression of Claudin-1as well as transendothelial migration of monocytes, indicating that the Rho signaling was involved in the dysfunction of endothelial barrier induced by rTp0965.4. The Tp0965protein may play a certain role in the immunopathogenesis of syphilis via activation as well as demage of vasicular endothelial cells.