Claser Carla, Curcio Marli, de Mello Samanta M, Silveira Eduardo V, Monteiro Hugo P, Rodrigues Mauricio M
Centro Interdisciplinar de Terapia Gênica (CINTERGEN), Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Mirassol, 207, São Paulo-SP 04044-010, Brazil.
BMC Cell Biol. 2008 Dec 17;9:68. doi: 10.1186/1471-2121-9-68.
As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18) is among the host molecules that have been suggested as a mediator of important events during T. cruzi-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi)-mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence.
CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different T. cruzi strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18.
The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of T. cruzi in HeLa cells, but not trypanosome binding and invasion.
作为一种专性细胞内寄生虫，恰加斯病的病原体克氏锥虫必须在哺乳动物细胞内侵入并繁殖。细胞角蛋白18（CK18）是宿主分子之一，已被认为是克氏锥虫与宿主细胞相互作用过程中重要事件的介导因子。基于这种可能性，我们研究了RNA干扰（RNAi）介导的CK18基因下调是否会在体外干扰寄生虫的生命周期。用CK18-RNAi瞬时转染的HeLa细胞中CK18转录本水平可忽略不计，通过免疫印迹或免疫荧光测定，CK18蛋白表达水平显著降低。
不同克氏锥虫菌株的锥鞭毛体对CK18阴性或阳性的HeLa细胞的侵入能力相同。此外，在CK18阴性或阳性细胞中，寄生虫募集宿主细胞溶酶体并同样顺利地从寄生泡中逸出。此后，Y株或CL-Brener株无鞭毛体在CK18 RNAi处理的细胞中的生长被显著抑制。48小时后，与对照细胞相比，CK18 RNAi处理的细胞中无鞭毛体的数量低几倍。寄生虫与CK18的同时染色显示，在感染Y株的HeLa细胞中两者共定位。尽管无鞭毛体表面蛋白-2含有先前描述的与CK18结合的VTVXNVFLYNR结构域，但经过多次尝试，我们未能检测到重组蛋白与CK-18的结合。
该研究表明，通过瞬时RNAi沉默CK18可抑制克氏锥虫Y株和CL株在HeLa细胞中的细胞内增殖，但不影响锥虫的结合和侵入。
