Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Claser
Carla
C
Centro Interdisciplinar de Terapia Gênica (CINTERGEN), Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Mirassol, 207, São Paulo-SP 04044-010, Brazil. carla_claser@immunol.a-star.edu.sg
Curcio
Marli
M
de Mello
Samanta M
SM
Silveira
Eduardo V
EV
Monteiro
Hugo P
HP
Rodrigues
Mauricio M
MM
D000818:Animals; D002448:Cell Adhesion / Q000235:genetics; D014355:Chagas Disease / Q000235:genetics / Q000378:metabolism* / Q000469:parasitology*; D004261:DNA Replication / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D015536:Down-Regulation; D006367:HeLa Cells; D054884:Host-Pathogen Interactions / Q000235:genetics*; D006801:Humans; D053538:Keratin-18 / Q000235:genetics* / Q000378:metabolism; D008247:Lysosomes / Q000235:genetics; D010587:Phagocytosis / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013045:Species Specificity; D014162:Transfection; D014349:Trypanosoma cruzi / Q000472:pathogenicity* / Q000502:physiology; D014774:Virulence / Q000235:genetics
D016054:DNA, Protozoan; D053538:Keratin-18; D034741:RNA, Small Interfering