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The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33).

ABSTRACT: Trichomoniasis is caused by Trichomonas vaginalis (T. vaginalis), which is a widespread and serious sexually transmitted pathogen in humans. The procedure of T. vaginalis adherence to the host cell is the precondition for T. vaginalis parasitism and pathogenicity. The AP33 adhesin of T. vaginalis (TvAP33) plays a key role in the process of adhesion. In this study, the specific primers for polymerase chain reaction (PCR) were designed based on the sequence of TvAP33 (GenBank Accession No. U87098.1) to amplify the open reading frame (ORF), and the ORF was inserted into pET-32a (+) to produce recombinant TvAP33 (rTvAP33). The sequence analysis indicated that the TvAP33 gene encoded a protein of 309 amino acids with 32.53 kDa, and the protein was predicted to have a high antigen index. Western blotting assay showed rTvAP33 was successfully recognized by the sera of mice experimentally infected with T. vaginalis, while native TvAP33 in the somatic extract of T. vaginalis trophozoite was as well detected by sera from rats immunized with the rTvAP33. Immunofluorescence analysis using an antibody against rTvAP33 demonstrated that the protein was expressed and located on the surface of T. vaginalis trophozoites. The recombinant protein was emulsified in Freund's adjuvant and used to immunize BALB/C mice three times at days 0, 14, and 28. The result of animal challenge experiments revealed the levels of IgG, IgG1, and IgG2a, and IL-4, IL-10, and IL17 among rTvAP33 vaccinated animals were integrally increased. Moreover, the rTvAP33 vaccinated animals were apparently prolonged survival time (26.45 ± 4.10) after challenge infection with this parasite. All these results indicated that TvAP33 could be used as vaccine candidate antigen to induce cell-mediated and humoral immunity.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC7338309 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33).

Zhang Zhenchao Z Li Yuhua Y Wang Shuai S Hao Lixia L Zhu Yunqing Y Li Haoran H Song Xiaoxiao X Duan Yujuan Y Sang Yuhui Y Wu Pucheng P Li Xiangrui X

Frontiers in microbiology 20200630

Trichomoniasis is caused by Trichomonas vaginalis (T. vaginalis), which is a widespread and serious sexually transmitted pathogen in humans. The procedure of T. vaginalis adherence to the host cell is the precondition for T. vaginalis parasitism and pathogenicity. The AP33 adhesin of T. vaginalis (TvAP33) plays a key role in the process of adhesion. In this study, the specific primers for polymerase chain reaction (PCR) were designed based on the sequence of Tv ...[more]

PMID: 32695085

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Project description:BackgroundTrichomonas vaginalis is a widespread and important sexually transmitted pathogen. Adherence to the surface of the host cell is the precondition for the parasitism and pathogenicity of this parasite. Trichomonas vaginalis adhesion protein 33 (TvAP33) plays a key role in the process of adhesion, but how this protein mediates the adhesion and pathogenicity of T. vaginalis to host cells is unclear.MethodsThe expression of TvAP33 in trophozoites was knocked down by small interfering RNA. VK2/E6E7 cells and mice infected with T. vaginalis were used to evaluate the pathogenicity of T. vaginalis. We constructed a complementary DNA library of VK2/E6E7 cells and screened the protein molecules interacting with TvAP33 by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 (Bcl-2 interacting protein 3) was analyzed by co-immunoprecipitation and colocalization.ResultsFollowing knockdown of TvAP33 expression, the number of T. vaginalis trophozoites adhering to VK2/E6E7 cells decreased significantly, and the inhibition of VK2/E6E7 cell proliferation and VK2/E6E7 cell apoptosis and death induced by T. vaginalis were reduced. Animal challenge experiments showed that the pathogenicity of trophozoites decreased following passive immunization with TvAP33 antiserum or blocking of the TvAP33 protein. Immunofluorescence analysis revealed that TvAP33 could bind to VK2/E6E7 cells. Eighteen protein molecules interacting with TvAP33 were identified by the yeast two-hybrid system. The interaction between TvAP33 and BNIP3 was further confirmed by co-immunoprecipitation and colocalization. When the expression of both TvAP33 and BNIP3 in trophozoites was knocked down by small RNA interference, the number of T. vaginalis adhering to VK2/E6E7 cells and the inhibition of VK2/E6E7 cell proliferation were significantly lower compared to trophozoites with only knockdown of TvAP33 or only BNIP3. Therefore, the interaction of TvAP33 and BNIP3 in the pathogenesis of T. vaginalis infecting host cells is not unique and involves other molecules.ConclusionsOur study showed that the interaction between TvAP33 and BNIP3 mediated the adhesion and pathogenicity of T. vaginalis to host cells, providing a basis for searching for drug targets for T. vaginalis as well as new ideas for the prevention and treatment of trichomoniasis.

Project description:Ornithine decarboxylase (ODC), the lead enzyme in polyamine biosynthesis, was partially purified from Trichomonas vaginalis and its kinetic properties were studied. The enzyme appears to be of special significance in this anaerobic parasite, since the arginine dihydrolase pathway generates ATP as well as putrescine from arginine. ODC from T. vaginalis had a broad substrate specificity, decarboxylating ornithine (100%), lysine (1.0%) and arginine (0.1%). The enzyme had a pH optimum of 6.5, a temperature optimum of 37 degrees C and was pyridoxal 5'-phosphate-dependent. Attempts to separate ornithine- from lysine-decarboxylating activity by thermal-stability and pH-optima curves were not successful. Although Km values for ornithine and lysine were 109 and 91 microM respectively, and the Vmax values for these substrates were 1282 and 13 nmol/min per mg of protein respectively, the most important intracellular substrate is ornithine, since intracellular ornithine levels are 3.5 times those of lysine and extracellular putrescine levels are 7.5 times those of cadaverine. Ornithine was also an effective inhibitor of lysine-decarboxylating activity (Ki 150 microM), whereas lysine was relatively ineffective as inhibitor of ornithine-decarboxylating activity (Ki 14.5 mM). Crude ODC activity was localized (86%) in the 43,000 g supernatant and 3303-fold purification was obtained by (NH4)2SO4 salting and DEAE-Sephacel, agarose-gel and hydroxyapatite chromatography steps. The enzyme bound difluoro[3H]methylornithine ([3H]DFMO) with a ratio of drug bound to activity of 2500 fmol/unit, where 1 unit corresponds to 1 nmol of CO2 released from ornithine/min. The enzyme had a native M(r) of 210000 (gel filtration), with a subunit M(r) of 55,000 (by SDS/PAGE), suggesting that the trichomonad enzyme is a tetramer. From the subunit M(r) and binding ratio of DFMO, there is about 137 ng of ODC per mg of T. vaginalis protein (0.013%). The significant amount of ODC protein present supports the view that putrescine synthesis in T. vaginalis plays an important role in the metabolism of the parasite.

Dataset Information

The Molecular Characterization and Immunity Identification of Trichomonas vaginalis Adhesion Protein 33 (AP33).

Publications

The Molecular Characterization and Immunity Identification of <i>Trichomonas vaginalis</i> Adhesion Protein 33 (AP33).

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