T. cruzi invasion of cells transfected with cytoskeletal elements, carbohydrates or regulatory Rho-GTPases
In the course of our studies we used a number of available cell lines with altered expression of distinct components. Cytoskeletal mutants of a melanoma cell line expressing varying amounts of actin binding protein 280, ABP280 (Cunningham et al. 1992), a microfilament cross-linking protein, as well as NIH mouse fibroblasts expressing variable amounts of the actin assembly regulator protein gelsolin (Cunningham et al. 1991) display distinct susceptibility towards metacyclic trypomastigotes or extracellular amastigotes of the G strain (Procópio et al. 1998). We also observed that CHO cells with poorly sialylated proteins (Lec-2 cells, Deutscher et al. 1984) are slightly more susceptible to extracellular amastigotes, when compared to the normal controls (Stecconi-Silva et al. 2003).
The observation that amastigotes and trypomastigotes become associated with distinct actin-rich projections upon cell invasion prompted us to examine the role of regulatory Rho GTPases in this process. Constitutively activated (GTPase activity deficient) mutants of RhoA and Rac1 were found to induce the assembly of contractile actin and myosin filaments (stress fibers) and actin rich surface protrusions (lamellipodia), respectively (Hall 1994, Ridley et al. 1992, Ridley and Hall 1992). Later, Cdc42 was shown to promote the formation of actin-rich, finger like membrane extensions (filopodia) (Kozma et al. 1995, Nobes and Hall 1995). Thus, RhoA, Rac1, and Cdc42 regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct filamentous actin structures. In order to evaluate the relative importance of RhoA GTPases in host cell invasion by different T. cruzi infective forms of distinct strains, we used MDCK cells transfectants that express variants of RhoA, Rac1 and Cdc42 proteins (Jou and Nelson 1998). We demonstrated that metacyclic trypomastigotes from strains of T. cruzi I presented lower infectivity than T. cruzi II parasites for the different target cells, with no apparent specific requirement for GTPases (Fernandes and Mortara 2004). As previously noted, regardless of the strain analyzed, intracellular amastigotes were not only susceptible to complement lysis but also showed very low infectivity towards the different transfectants. Extracellular amastigotes from G strain infected transfected MDCK cells more efficiently than the other strains. Invasion was particularly high in Rac1V12 cells and was specifically reduced in the corresponding dominant negative line Rac1N17 suggesting a key role for Rac in this invasion process (Fernandes and Mortara 2004).