Mundodi V, Kucknoor A S, Alderete J F
Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.
BMC Microbiol. 2007 Jul 3;7:64. doi: 10.1186/1471-2180-7-64.
Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.
We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum.
These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis.
阴道毛滴虫对宿主的寄生作用较为复杂。其对阴道上皮细胞（VECs）的黏附由表面蛋白介导。我们之前表明，黏附素AP65表达的反义下调会减少蛋白质的量，从而降低阴道毛滴虫对VECs的黏附水平。我们现在对ap33基因的表达进行反义下调，以评估和确认AP33在阴道毛滴虫黏附中的作用。我们还使用了一种已建立的转染系统，用于在胎儿三毛滴虫中异源表达AP33，作为另一种验证方法。
我们成功地筛选出了带有正义（S）和反义（AS）质粒的稳定滴虫。逆转录聚合酶链反应（RT-PCR）证实，AS转染的寄生虫中ap33 mRNA的量减少，与野生型（wt）滴虫相比，AP33量的减少对生长和活力没有影响。与wt和S转染的滴虫相比，AS转染体蛋白质的免疫印迹显示，能够与宿主细胞结合的功能性AP33量显著减少。正如预期的那样，AS转染体对VECs的黏附水平较低，这与AP33表面表达的减少有关。通过免疫印迹和荧光证实了阴道毛滴虫AP33::HA融合蛋白在胎儿三毛滴虫中的稳定表达。游离表达的表面AP33::HA融合蛋白增加了滴虫对人VECs的黏附，抗AP33血清可消除这种黏附。
这些使用基因表达的反义抑制、AP33合成以及AP33在胎儿三毛滴虫中的异源表达的结果，证实了该蛋白作为阴道毛滴虫黏附素的作用。
