Abstract
BackgroundAs 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.ResultsCK18 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.ConclusionThe 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.
Highlights
As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells
RNA interference (RNAi) knockdown of endogenous Cytokeratin 18 (CK18) in cultured HeLa cells Earlier studies have suggested that CK18 plays a role during the interaction between trypomastigotes of T. cruzi and non-phagocytic mammalian cells [23,26]
Following the transfection of cultured HeLa cells with CK18 RNAi, we evaluated the levels of CK 18-specific transcripts with the aid of quantitative real-time PCR
Summary
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. We addressed whether RNA interference (RNAi)mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas' disease, which chronically affects more than 10 million individuals in the Americas, with an annual death toll of approximately 45,000 people [1]. Based on these poor short term prospects, studies aimed at understanding the requirements for parasite invasion and multiplication of T. cruzi are still relevant
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