Immunocompetence And Function Potential Research Of Treponema Pallidum Tp0751 Binding Adhesin

Syphilis, caused by Treponema pallidum subsp. Pallidum, is a serious, multistage sexually transmitted infection with diverse and wide-ranging manifestations. Although effective drug treatment regimes exist, syphilis remains a pandemic disease. As this disease also contributes to the spread of human immunodeficiency virus (HIV), the development of an effective vaccine against syphilis has remained an important research and public health goal. Understanding of the pathogenesis of this disease and how host-pathogen interactions influence the course of disease progression is crucial to controlling and preventing syphilis. However, study of the disease has been restricted by the limitations that the organism cannot be grown in vitro, and that, as an exclusively human pathogen, inferences made from animal models are of limited applicability.In the absence of cytotoxins and other known virulence factors, inflammation caused by T. pallidum and the ensuing adaptive immune response to T. pallidum may cause the tissue destruction characteristic of syphilis infection. T. pallidum membrane proteins are thought to be potent inducers of inflammation during early syphilis infection. There are a number of potential candidates among the T. pallidum membrane proteins. For example, TpN47 has been shown to induce the production of tumor necrosis factor-a (TNF-a), human interleukin-1β(IL-1β), and cytokine interleukin-6 (IL-6) by activating nuclear factor kappa-B (NF-κB) through a toll-like receptor 2 (TLR2) and CD 14 signaling pathway. However, no other T. pallidum proteins have yet been characterized that might also induce the inflammatory reaction. The molecular mechanisms by which they might trigger and sustain the inflammatory cascades also remain obscure. Therefore, screening a cohort of T. pallidum membrane proteins for inflammatory responses is crucial for clarifying the full progression of disease pathogenesis.Tp0751 is an adhesion protein from the T. pallidum membrane that has recently been related to pathogen invasion by virtue of its potential for attachment to the host cells. Tp0751 is also one of the first T. pallidum proteins to come into contact with host cells. Tp0751 may have other functions in T. pallidum pathogenesis, for example, in stimulating inflammatory reactions, but its full role in the host immunity has yet to be explored.This study was carried out to investigate the immunocompetence, cytotoxicity and induction of proinflammatory reactivity of Tp0751 laminin-binding adhesin of T. pallidum. This study was very important to further understand the pathogenicity of T. pallidum at the molecular level.The Tp0751 ORF without upstream non-coding region was chosen by computer analysis, was amplified from T. pallidum complete genome by polymerase chain reactions (PCR), was ligased into the expression vector pET-28a(+) to generate recombinant plasmid pET-28a(+)/Tp0751, expressed in E.coli R2566, and analyzed by SDS-PAGE and Western blot.The fusion protein was purified with Ni-NTA affinity chromatography, Purified protein was analyzed by SDS-PAGE, and protein concentration was determined by the BCA protein assay kit.The immunoreactivity of fusion protein was evaluated by Western-blot. Zealand rabbits were immunized with the fusion protein, antibodies to anti-Tp0751 in sera were detected with indirected ELISA.In order to study the cytotoxicity of Tp0751 in THP-1 cells, Endotoxin was removed by endotoxin removal gel. The skin changes in the injection site were observed by subcutaneous injection in New Zealand white rabbits. Lactate dehydrogenase leakage rate and NO release of macrophages after Tp0751 treatment were detected to study the cytotoxicity in cells.THP-1 cells were stimulated with different concentrations of Tp0751 for suitable periods, further cells were stimulated with suitable concentration of Tp0751 for different time. The cytokines'(TNF-a, IL-1βand IL-6) concentration was measured using the quantitative "sandwich" ELISA technique.For inhibitor experiments, the cell culture were treated respectively 30 min with mouse anti-TLR2 mAb, mouse anti-CD 14 mAb, PD98059, SP600125, SB203580(Three-MAPK specific inhibitors, SP600125, PD98059 and SB203580 can selectively inhibit SAPK/JNK, ERK and p38 signaling pathways without affecting the others) and pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor) before Tp0751 stimulation. The activation of NF-κB and MAPKs after Tp0751 treatment in THP-1 cells was detected by Western blot.The size of PCR amplification product was about 600 bp. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene were Tp0751, compared with gene reported by GenBank, it had 100% similarity. and the results of BLAST confirmed that the sequence was Tp0751. A fusion protein with molecular weight about 26 kDa was attained after expression and purification, the concentration of Tp0751 recombinant protein was 1.2 mg/mLWestern blot proved that the recombinant protein can specifically react with T. pallidum IgG positive sera. Specific humoral response were elicited by recombinant protein in zealand rabbit and the specific antibody titer was above 1:10 240 after immunization for 4 times.Tp0751 stimulated the New Zealand rabbit to show fever red skin in leg, disappeared after six days to ten days, and delayed-type hypersensitivity was positive. THP-I were stimulated into macrophages by PMA and the macrophage cells stimulated by the target protein were collected after 48 h and cell supernatant. LDH leakage rate and NO concentration compared with the control group were unsignificantly.Cytokine production by Tp0751 treated THP-1 cells was found to be dose-dependent in a concentration range from 0.5μg/mL to 10.0μg/mL The level of cytokines decreased above a concentration of 5μg/mL Cytokine production by Tp0751 treated THP-1 cells was also found to be time-dependent in a time range from 6 hours to 72 hours. The amount of TNF-a and IL-1βreached peak levels at 48 hours after stimulation, but The peak amount of IL-6 occurred at 24 hours after stimulation.The expression of Tp0751-induced proinflammatory cytokines was significantly decreased by pretreatment with anti-TLR2 Ab, anti-CD 14 antibodies, SB203580 (p38 signaling pathways specific inhibitor) and PDTC (NF-κB signaling pathways specific inhibitor). PD98059 (ERK signaling pathways specific inhibitor) slightly inhibited TNF-a production, but the amounts of IL-1p, IL-6 were not affected. SP600125 (SAPK/JNK signaling pathways specific inhibitor) could not inhibit the production of TNF-a, IL-1βand IL-6. The phosphorylated SAPK/JNK, ERK and p38 increased in a time dependent manner, the peak occurred at 45 min and then decreased. The duration of MAPK/P38 and SAPK/JNK pathways were longer than SAPK/JNK pathway, they could last 90 min post induction. Western blot found that NF-κB translocation was obviously inhibited after treatment with PDTC (25μmol/L).1. Prokaryotic expression vector pET-28a(+)/Tp0751 was constructed successfully and Tp0751 recombinant protein with molecular weight near 26 kDa was well attained.2. Tp0751 recombinant protein showed excellent immunongenicity and immunoreactivity, can specifically react with T. pallidum IgG positive sera, and also can induce the humoral responses in zealand rabbits efficiently.3. Tp0751 recombinant protein can induce delayed-type hypersensitivity in New zealand rabbits.4. The leakage rate of LDH and NO release were not increased after Tp0751 recombinant protein stimulated the macrophages, which indicated no cytotoxicity effect in cells.5. Tp0751 stimulated THP-1 cells'production of proinflammatory cytokines, and may be relative to the inflammatory reaction after T. pallidum infection.6. TLR2, CD 14 might play important roles in the the activation of monocytes/macrophages by Tp0751.7. Tp0751 induced MAPKs and NF-κB activation in THP-1 cells.