We have shown here that different infective forms of T. cruzi exhibit distinct behavior throughout the process of cell invasion, parasitophorous vacuole formation and escape. Even though chloroquine inhibited metacyclic trypomastigote invasion (Fig. 3), the overall effect of raising cytoplasmic pH was not detectable in the kinetics of vacuole formation (Fig. 2) but dramatically affected their escape, increasing the mean residence by at least a factor of two, whereas not affecting the parameters of amastigote transit, suggesting that the mechanisms engaged for parasitophorous membrane disruption is stage-specific. Of the two known components involved, hemolysin and transialidase, (Andrews & Whitlow 1989, Andrews et al. 1990, Hall et al. 1992) only hemolysin activity could be detected in amastigotes whereas in metacyclic trypomastigotes none of the activities could be detected. Whereas hemolysin activity has been shown to be pH sensitive and hence inhabitable by pH elevation (Ley et al. 1990) this activity is apparently lacking in metacyclics of the G as well as of CL (Andreoli & Mortara 2003) strains. Unexpectedly, amastigotes of the G strain (this study) as well as of the Y strain (Ley et al. 1990) that express pH sensitive hemolysin are not affected by raising the cytoplasmic pH of the target cell (Fig. 2). These apparently contradictory findings thus suggest that other unknown mechanisms might either operate or be induced once the parasites are trapped within the parasitophorous vacuole. Among other factors, the tightness of this compartment might be a relevant one (Lopez et al. 2002). The kinetics of metacyclic trypomastigote escape from the parasitophorous vacuole (Fig. 2A, B) also suggests that there may be two distinct populations of parasites: a relatively fast escaping group that leaves the vacuole from 7 to 8 h and another set that remains in the vacuole for more that 5 h slowly leaving the vacuole between 12 to 15 h post invasion.
Although the presence of sialic acid on host cell glycoproteins appears to enhance trypomastigote invasion (Schenkman et al. 1993) and may also protect the parasitophorous membrane from the action of the above mentioned required factors for parasite escape (Hall et al. 1992), the invasion step by the two infective forms does not seem to be affected by host cell sialic acid (Fig. 4). This observation contrasts to the observation that Y (a type II strain) tissue-culture derived trypomastigotes appear to preferentially invade sialylated target cells (Schenkman et al. 1993). However, in salic acid deficient Lec-2 cells, the escape of both infective forms studied here is facilitated by sialic acid deficiency (Fig. 4), as previously described for Y strain tissue-culture derived trypomastigotes (Hall et al. 1992). Taken together, these findings reinforce the notion that there may be yet unknown mechanisms involved in the escape of these infective forms, particularly of metacyclic trypomastigotes, into the cytoplasm.
It has recently been shown that T. cruzi exists as at least two distinct phylogenetic lineages in nature (Souto et al. 1996). The G strain used in this study belongs to the sylvatic type I and it has recently been show that invasion mechanisms of metacyclic trypomastigotes of the two lineages are highly divergent (Neira et al. 2002). We have confirmed here that the requirement for host cell calcium mobilization is also dependent on the infective form since the invasion of Vero cells by amastigotes, in contrast to metacyclic trypomastigotes, is not affected by agents that modify host cell free calcium concentration (Fig. 5). We are currently undertaking studies to comprehensively compare the invasion and escape mechanisms used by infective forms of the two phylogenetic lineages.
In conclusion, we have shown that two distinct infective forms of the parasite display distinct behavior that probably reflect the engagement of specific mechanisms in the process of host cell invasion and parasitophorous vacuole escape.
Answers to all your Biology Questions
