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Background Trichomonas 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. Methods The 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. Results Following 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. Conclusions Our 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. Graphical Abstract

Trichomoniasis is a common and curable sexually transmitted disease worldwide. The rapid, convenient, and accurate diagnosis of trichomoniasis is an important link in the prevention and treatment of the disease. The current detection methods of Trichomonas vaginalis are mainly wet mount microscopy, culture, nested PCR, and loop‐mediated isothermal amplification. However, these detection methods have some shortcomings. In this study, a recombinant enzyme polymerase amplification (RPA) assay had been conducted to detect T. vaginalis. The target gene and the corresponding primers were screened, and the reaction system and conditions were optimized in the assay of RPA. The sensitivity and specificity of this detection method were analyzed. The detection efficiency of wet mount microscopy, culture, nested PCR, and RPA was compared by testing 53 clinical samples from vaginal secretions. By screening, the actin gene of T. vaginalis could be used as a target gene for RPA detection of T. vaginalis, and the optimum reaction condition to amplify the actin gene by RPA was at 39°C for 30 min. The detection limit of T. vaginalis DNA using RPA was 1 pg, corresponding to a sensitivity of approximately five trophozoites. The RPA assay demonstrated high specificity for T. vaginalis, and there was no cross‐reactivity with Giardia lamblia, Escherichia coli, Lactobacillus, Toxoplasma gondii, Staphylococcus aureus, and Candida albicans. Of the 53 clinical samples, the positive rates of T. vaginalis detected by wet mount microscopy, culture, nested PCR and RPA were 50.9 4% (27/53), 71.7% (38/53), 71.7% (38/53), and 69.81% (37/53), respectively. Compared with culture which was used as the gold standard for diagnosing trichomoniasis, testing clinical samples by wet mount microscopy showed 71.05% sensitivity, 100% specificity, and moderate diagnostic agreement with the culture (K = 0.581, Z = 4.661, p < 0.001). The nested PCR showed 100% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 1, Z = 7.28, p < 0.001), while RPA displayed 97.37% sensitivity, 100% specificity, and excellent diagnostic agreement (K = 0.954, Z = 6.956, p < 0.001). At the present study, rapid amplification of actin gene by RPA could be used as a tool for detection of T. vaginalis. The detection method of RPA was more sensitive than wet mount microscopy and displayed excellent specificity. Moreover, RPA amplification of actin gene did not require a PCR instrument and the amplification time was shorter than that of ordinary PCR. Therefore, the RPA assay was proposed in this study as a point‐of‐care examination and a diagnostic method of T. vaginalis infection, which exhibited the potential value in the treatment and prevention of trichomoniasis.

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