Arroyo R, Alderete J F
Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758.
Infect Immun. 1989 Oct;57(10):2991-7. doi: 10.1128/iai.57.10.2991-2997.1989.
The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated. Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T. vaginalis to recognize and bind to epithelial cells. Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors, also decreased T. vaginalis cytadherence. Parasites incubated with TLCK and washed extensively still did not adhere to cells at levels equal to those seen for control trichomonads treated with phosphate-buffered saline or culture medium alone. Exposure of TLCK-treated organisms with other cysteine proteinases restored cytadherence levels, indicating that proteinase action on the parasite surface is prerequisite for host cell attachment. Concentrations of TLCK which inhibited cytadherence did not alter the metabolism of T. vaginalis, as determined by metabolic labeling of trichomonad proteins; the protein patterns of T. vaginalis in the presence and absence of TLCK were identical. Kinetics of TLCK-mediated inhibition of cytadherence of other T. vaginalis isolates with different levels of epithelial-cell parasitism were similar to the concentration-dependent inhibition seen for isolate NYH 286. Incubation of TLCK-treated, washed organisms in growth medium resulted in regeneration of adherence. Finally, treatment of T. vaginalis organisms with proteinase inhibitors for abrogation of cytadherence effectively rendered the trichomonads unable to kill host cells, which is consistent with the contact-dependent nature of host cytotoxicity. These data show for the first time the involvement of T. vaginalis cysteine proteinases in parasite attachment to human epithelial cells. These results have implications for future pharmacologic intervention at a key step in infection.
评估了半胱氨酸蛋白酶在阴道毛滴虫NYH 286黏附HeLa细胞和人阴道上皮细胞中的作用。仅用半胱氨酸蛋白酶抑制剂N-α-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）预处理滴虫，而非上皮细胞，会大大降低阴道毛滴虫识别和黏附上皮细胞的能力。亮抑酶肽和L-1-对甲苯磺酰胺-2-苯乙基氯甲基酮等其他半胱氨酸蛋白酶抑制剂也会降低阴道毛滴虫的细胞黏附。用TLCK孵育并充分洗涤后的寄生虫，其黏附细胞的水平仍不及单独用磷酸盐缓冲盐水或培养基处理的对照滴虫。用其他半胱氨酸蛋白酶处理经TLCK处理的生物体可恢复细胞黏附水平，这表明蛋白酶对寄生虫表面的作用是宿主细胞附着的先决条件。抑制细胞黏附的TLCK浓度不会改变阴道毛滴虫的代谢，这通过对滴虫蛋白质进行代谢标记来确定；有无TLCK时阴道毛滴虫的蛋白质模式相同。TLCK介导的对其他具有不同上皮细胞寄生水平的阴道毛滴虫分离株细胞黏附抑制的动力学，与NYH 286分离株所见的浓度依赖性抑制相似。在生长培养基中孵育经TLCK处理并洗涤后的生物体，会导致黏附能力再生。最后，用蛋白酶抑制剂处理阴道毛滴虫以消除细胞黏附，有效地使滴虫无法杀死宿主细胞，这与宿主细胞毒性的接触依赖性性质一致。这些数据首次表明阴道毛滴虫半胱氨酸蛋白酶参与寄生虫与人上皮细胞的附着。这些结果对感染关键步骤的未来药物干预具有启示意义。
