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 . In spite of the significant reduction in transmission over the past twenty years in countries such as Argentina, Brazil, Chile and Uruguay, Chagas' disease is still a major health problem for many Latin American countries. A recent epidemiological study in dogs also revealed a dangerously widespread epidemic in the state of Texas .
The efficacy of conventional chemotherapy with nifurtimox or benznidazole is low and varies greatly according to the infection status of the patients (acute or chronic) and the parasite strains. Although new drugs could potentially solve the problem, Chagas' disease is one of the most neglected diseases in terms of drug development with a single new compound being tested in phase 1/2 trials (reviewed in ref. ). Based on these poor short term prospects, studies aimed at understanding the requirements for parasite invasion and multiplication of T. cruzi are still relevant.
Invasion and multiplication within a variety of host cells are critical features for the survival of T. cruzi. The invasion process of mammalian non-phagocytic cells has attracted a great deal of interest in the past 20 years and notable advances have been obtained. Different routes of invasion described by researchers have been mediated by distinct cell surface receptors, secondary messengers and transcription factors (recently reviewed in refs. [4–6]). In one of the mechanisms described, the infective forms of T. cruzi (trypomastigotes) take advantage of a host cell mechanism for puncture repair and exocytosis of lysosomes to recruit these organelles to the parasite-cell junction. After a Ca2+ dependent fusion with the cell membrane, lysosomes become part of the intracellular parasitophorous vacuole. Alternatively, parasites can use a lysosome-independent pathway. In this case, the host cell plasma membrane accounts for the parasitophorus vacuole. Ultimately, these organelles fuse with lysosomes, a step that recently has been shown to be critical for parasite retention and productive infection . Among the parasite surface molecules that might be relevant for the attachment, signaling and penetration are members of a family of mucin-like glycoproteins, members of a trans-sialidases family, and proteases (reviewed in ref. [8–10]).
Once inside the host cells, T. cruzi manages to disrupt the parasitophorus vacuole, an event that could be mediated by lytic proteins and the enzyme trans-sialidase [11–14]. During the escape, trypomastigotes transform into amastigotes and express on its surface some stage-specific molecules [15–17]. In the host cell cytoplasm, GPI-deficient amastigotes arrest replication and subsequently failed to differentiate into trypomastigotes . The molecular events required for amastigote transformation into trypomastigotes and their release from the infected cells are completely unknown.
The accessibility of the new reverse genetic tools such as RNAi have allowed investigators to search for host molecules that might be important for the T. cruzi invasion as well as intracellular parasite survival and multiplication. Villalta's group demonstrated that the silencing of laminin-γ1 expression, a protein belonging to the extracellular matrix and regulated by the parasite, by cultured human coronary artery smooth muscle cells rendered them significantly more resistant to the binding and penetration of parasites (reviewed in [5, 6, 19]). This observation supports the hypothesis that the binding to laminin is an important step for the early process of T. cruzi infection. Also, it suggests that laminin-binding proteins expressed on the surface of T. cruzi are important virulence factors during the infection of human smooth muscle cells [5, 6, 19, 20]. Utilizing a similar approach, the same group demonstrated that stable interference of thrombospondin-1 expression, also a protein belonging to the extracellular matrix, in cultured HeLa cells in vitro caused an increase in the cell resistance to T. cruzi invasion [5, 6, 21]. These studies demonstrated the effectiveness of the RNAi strategy when studying the host cell factors critical for parasite survival and eventually identifying potential targets for new therapies.
One of the laminin-binding proteins, a member of the family of the trans-sialidases of T. cruzi (Tc-85-11), was shown to bind to host cytokeratin 18 (CK18). The CK18 is an intermediate filament protein of the acid cytokeratin family belonging to type I, expressed in the internal epithelia cell cytoplasm. This protein together with CK8 exhibit resistance features in response to other forms of stress and to apoptosis . This interaction through a C-terminal peptide ligand denominated FLY domain (VTVXNVFLYNR, ) was shown to be relevant for parasite invasion as anti-cytokeratin antibodies inhibited the infection of epithelial cells by T. cruzi. The FLY motif is not only present in proteins expressed on the surface of trypomastigotes but also on a protein that is abundant on the surface of amastigotes denominated Amastigote Surface Protein-2 (ASP-2, [24, 25]). Based on this observation, it was our intention to further investigate whether the host cell CK18 expression was indeed critical for T. cruzi invasion and/or subsequent parasite growth in vitro. For this purpose we used HeLa cells in which we transiently down-regulated the host CK18 using the RNAi strategy. Upon infection, we evaluated parasite attachment, penetration, lysosome recruitment, phagolysosomal escape and cytoplasm replication in cultured HeLa cells. RNAi-mediated knock-down of CK18 did not interfere with trypomastigotes mediated events. Once amastigotes from two distinct strains of T. cruzi were in the cytoplasm of CK18 RNAi-treated cells, their development was severely inhibited.