| Background and Objective: Syphilis, the most frequent sexually transmitted diseases(STDs) in recent years, is a worldwide public health problem that caused by the Treponema pallidum subsp. pallidum(T.pallidum) and it could increase the risks of HIV infection significantly.Accurate diagnosis is important for the prevention and treatment of syphilis. However, the pathogenesis and virulence factors of Treponema pallidum are still unclear. Flagellin is a potent immunogen that promotes the invasion of the pathogens and induces inflammation. Keratinocytes are the immune sentinels that involved in skin inflammation. Therefore,we propose the hypothesis that flagellins of T. pallidum could enhance the inflammatory responses and invasion by activating the keratinocytes to release matrix metalloproteinases(MMPs) for the degradation of extracellular matrix. In this study, the flagellin Fla B1, Fla B2 and Fla B3 of T. pallidum were cloned, expressed in E. coli and purified. We determined the immunogenicity and immunoreactivity of the flagellin and evaluated the diagnostic potential of Fla B1(Fla B2 and Fla B3) based ELISA for serodiagnosis of syphilis. We also explored the interaction of flagellins and keratinocytes to determine the role of flagellin in the pathogenesis of T. pallidum infection, and identify the signaling transduction pathway of T. pallidum flagellins induce the keratinocytes to generate MMPs. This study will provide new insight to investigate the pathogenesis of T. pallidum and will be helpful for the prevention of syphilis.Methods:1. The regions encoding recombinant proteins Fla B1, Fla B2 and Fla B3 were amplified by PCR from T. pallidum genomic DNA and cloned into the expression vector p ET28 a. The resulting plasmid was confirmed by DNA sequencing and then transformed into E. coli strain BL21(DE3).2. Protein expression was induced with 0.2 m M IPTG after OD600 reached0.8. His-tagged proteins were purified by affinity chromatography using Ni2+-NTA beads. The concentrations of recombinant proteins were estimated by using BCA protein assay kit.3. To determine whether the flagellin Fla B1, Fla B2 or Fla B3 of T.pallidum is expressed in vivo and is a target of the immune response in infected hosts, the rabbit model of syphilitic infection was utilized.Rabbits were infected with two T. pallidum(Nichols and Chicago) strains and two clinical isolates of T. pallidum(nhgz-01 and nhgz-02), and sera were collected postinfection(dpi). Western Blot analysis the specific antibody response in sera from infected rabbits and patients with syphilis.4. Four adult male New Zealand White rabbits were immunized three times at 2-weeks intervals with 200μg of purified r Ndk emulsified in the Freund’s adjuvant(complete adjuvant at the first time and incomplete adjuvant at other times) and antisera were collected at 7, 14, 21, 28, 35,42 days post immunized. The immunogenicities of recombinant flagellins were determined by Western Blot and ELISA.5. The recombinant proteins were tested against a well characterized panel of serum samples from syphilis patients to determine their sensitivities for the detection of Ig G and Ig M antibodies. To further evaluate the specificities of the recombinant proteins, sera obtained from uninfected controls(n = 30) and potentially cross-reactive diseases, including Lyme disease(n = 30), Leptospirosis(n = 5) and Hepatitis B(n= 30) were tested.6. Ha Ca T were stimulated with PBS(control) or 10 μg/m L Fla B1(Fla B2 or Fla B3) for 48 h. Culture supernatants were collected, pooled(n=3/group), and screened for MMP proteins.7. Ha Ca T cells were stimulated with Fla B1, Fla B2 or Fla B3 at concentrations of 0.1, 1, 10 μg/m L for 24 h. The cellular expression levels of MMP-9, MMP-13 m RNA and GAPDH were analyzed by semi-quantitative RT-PCR. Cell culture medium was isolated for zymography. Cells were isolated for detection of MMP-9 protein expression by Western Blot. The expression levels of MMP-13 in culture media were analyzed by ELISA.8. Ha Ca T cells were transfected with interference vector targeting for TLR5, TLR2, TLR4 or My D88 or control vector for 24 h, and then treated with 10μg/m L of Fla B1, Fla B2 or Fla B3 for 48 h. Cells were isolated for detection of MMP-9 protein expression by Western Blot. The expression levels of MMP-13 in culture media were analyzed by ELISA.9. After the cells were treated with 10 μg/m L of Fla B1, Fla B2 or Fla B3 for different time periods, protein were extracted and analyzed the expression levels of phosphorylated and total JNK, ERK, p38 by Western Blot.10. After the cells were treated with 10 μg/m L of Fla B1, Fla B2 or Fla B3 for different time periods, protein was extracted, and the expressions of phosphorylated and total IκBα were analyzed by Western Blot. Ha Ca T cells were stimulated with 10 μg/m L of Fla B1, Fla B2 or Fla B3 for 2h.Cells were fixed and then stained with 4’-6-diamidino-2-phenylindole(DAPI) or anti-NF-κB p65 antibody, followed by incubation with Cy3-conjugated antimouse antibody. Images were obtained using a fluorescence microscope.11. Ha Ca T cells were pretreated with ERK inhibitor(10μM PD98059),JNK inhibitor(20μM SP600125), p38 MAPK inhibitor(10μM SB203580),NF-κB inhibitor(10μM BAY117082) or DMSO as control for 1 h, and then treated with 10μg/m L of Fla B1, Fla B2 or Fla B3 for 48 h. Cells were isolated for detection of MMP-9 protein expression by Western Blot. The expression levels of MMP-13 in culture media were analyzed by ELISA.Results:1. The 861bp(Fla B1), 861bp(Fla B2) and 858bp(Fla B3) ORFs were successfully amplified from the T. pallidum Nichols strain genome, and then cloned into expression vector p ET28 a.2. After transformation into E. coli BL21(DE3) and induction with IPTG,The recombinant proteins were expressed as His-tagged proteins in E.coli BL21(DE3) and purified from cell-free supernatants by affinity chromatography using Ni2+-NTA beads. The yield of purified Fla B1,Fla B2 or Fla B3 was approximate 2.0 mg/ml of induced bacterial culture.3. Rabbits were infected with two T. pallidum subsp. pallidum(Nichols and Chicago) strains and two clinical isolates of T. pallidum(nhgz-01 and nhgz-02), and sera were collected at 0, 7, 14, 21, 28, 56 and 90 days postinfection(dpi). Western Blot analysis revealed that a specific antibody response to flagellin(Fla B1, Fla B2 and Fla B3) was detectable in rabbit infected with the T. pallidum Nichols and Chicago strains, as well as clinical isolates, whereas no antibodies to flagellin were present in preinfection(day 0) sera, specific single band was observed in sera from patients with primary, secondary, and early latent syphilis, whereas no reactivity against sera from uninfected controls and potentially cross-infections, including Lyme disease, leptospirosis and Hepatitis B were tested.4. The ELISA results demonstrated that the specific anti-flagellin(Fla B1,Fla B2 and Fla B3) Ig G antibody appeared at 7 days after the first immunization and remained elevated throughout the immunization. At42 days post immunized, anti-sera were collected and determined with a high titer 1:51200.5. For the Ig G ELISA, the overall sensitivity of Ig G ELISA was 95.4%for Fla B1, 92.6% for Fla B2, 95.1% for Fla B3. The overall specificity of Ig G ELISA was 98.9% for Fla B1, 95.8% for Fla B2, 95.8% for Fla B3, For the Ig M ELISA, the flagellins exhibited excellent performances for detection of Ig M antibody of primary and congenital syphilis, with sensitivities of 76.8% and 83.1% for Fla B1, 72.0% and 87.7% for Fla B2,74.4% and 89.2% for Fla B3, respectively, and the specificities were96.8%, 97.9% and 95.8%, respectively.6. Ha Ca T were stimulated with PBS(control) or 10 μg/ml Fla B1(Fla B2 and Fla B3) for 48 h. MMP antibody assay revealed that Fla B1, Fla B2 and Fla B3 stimulated secretion of the MMP-13, TIMP-1and TIMP-2, whereas,only Fla B1 stimulated secretion of the MMP-9.7. To clarify whether Fla B1(Fla B1, Fla B2 and Fla B3 regulated MMP-13)regulated MMP-9 activity at the transcriptional and translational levels,we examined MMP-9 m RNA and protein expression in Ha Ca T cells and found that Fla B1 dose-dependently increased MMP-9 m RNA and protein expression.8. After Ha Ca T cells were transfected with interference vectors targeting for TLR5, or My D88 for 24 h, Fla B1 induced protein expression level of MMP-9 were reduced significantly, as well as Fla B1, Fla B2 and Fla B3 induced protein expression levels of MMP-13. In contrast, Ha Ca T cells were transfected with interference vector targeting for TLR2, or TLR4 for24 h were not resulted in significantly reduced secretion of MMP-9 and MMP-13.9. After Ha Ca T cells were treated with 10 μg/m L of Fla B1, Fla B2 or Fla B3 for different time periods, we found that 10 μg/m L of Fla B1, Fla B2 or Fla B3 induced time-dependent JNK, p38, and ERK phosphorylation.10. After Ha Ca T cells were treated with 10 μg/m L of Fla B1, Fla B2 or Fla B3 for different time periods, we found that HMGB1 induced NF-κB activation by phosphorylation of inhibitor of kappa B(IκB), degradation of IκB. After Ha Ca T cells were treated with 10 μg/m L of Fla B1, Fla B2 or Fla B3 for 2h, we found that NF-κB p65 translocation into nucleus.11. After Ha Ca T cells were pretreated with ERK inhibitor(10 μM PD98059), JNK inhibitor(20 μM SP600125), p38 MAPK inhibitor(10μM SB203580), NF-κB inhibitor(10μM BAY117082) or DMSO as control for 1 h, Fla B1-induced protein expression levels of MMP-9 was reduced significantly in Ha Ca T cells. In addition, after Ha Ca T cells were pretreated p38 MAPK inhibitor(10μM SB203580) for 1 h, the protein expression levels of MMP-13 induced by Fla B1, Fla B2 or Fla B3 were not significantly reduced.Conclusions:1. The flagellin Fla B1, Fla B2 and Fla B3 of T. pallidum exhibited excellent immnoreactivity and be able to evoke specific immune responses in the host. They could be as diagnostic candidates for serodiagnosis of syphilis.2. The flagellin Fla B1, Fla B2 and Fla B3 of T. pallidum induced MMP-9and MMP-13 production in Ha Ca T cells were mediated by TLR5 and My D88 dependent.3. The flagellin Fla B1, Fla B2 and Fla B3 of T. pallidum induced MMP-9and MMP-13 production in Ha Ca T cells through activation of MAPK and NF-κB signal pathways. |
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