It has become increasingly evident that several intracellular pathogens specialized in subverting host cell pathways to their benefit. This is particularly well characterized for invasive bacteria such as Shigella, Listeria and enteropathogenic Escherichia coli (EPEC) (Bourdet-Sicard et al. 2000, Cossart 1997, Dramsi and Cossart 1998, Frischknecht and Way 2001, Goosney et al. 2000). Interaction between EPEC and HeLa cells involved aggregation of surface microvilli at the points where the bacterium attached to the dorsal surface of cultured HeLa cells (Silva et al. 1989). In order to promote the interaction, the bacteria were centrifuged onto the cells and actin aggregation was monitored by staining cells with fluorescently labeled phalloidin, using what is now known as the FAS (fluorescent actin staining) assay (Silva et al. 1989). Earlier data in the literature indicated that amastigotes (or amastigote-like forms) could be generated by the extracellular differentiation of trypomastigotes and these forms were capable of invading cultured cells (Behbehani 1973, Hudson et al. 1984, Ley et al. 1988, Nogueira and Cohn 1976). Using the EPEC-derived protocol, we then centrifuged extracellular amastigotes of the G strain onto HeLa cells and observed that they promptly aggregate actin filaments by attaching to dorsal surface microvilli (Mortara 1991). Microvillus aggregation was followed by the formation of cup-like structures underneath the parasite (Figure 1), that resemble the pedestals formed during EPEC attachment/effacing (Rosenshine and Finlay 1993). Extracellular amastigote invasion can be easily detected by several techniques, including freeze-fracture replicas of recently-infected HeLa cells (Figure 2).
By contrast, trypomastigotes enter HeLa cells by penetrating at their borders (Mortara 1991), a behavior that had been described by Schenkman et al. (1988). Interestingly, the invasion of HeLa cells by trypomastigotes induced the formation of previously undescribed actin-rich pseudopodial protrusions around the parasites (Figure 3, Schenkman and Mortara 1992). This phenomenon was not inhibited by cytochalasin D indicting that the major driving force derived from the parasite, and was detected in HeLa but not in MDCK cells (Schenkman and Mortara 1992). Later on, other investigators found similar pseudopodial extensions around trypomastigotes invading cadiomyocytes (Barbosa and Meirelles 1995).
We further investigated the entry of extracellular amastigotes into HeLa and Vero cells using the centrifugation protocol and compared the results with metacyclic trypomastigotes. First, we confirmed that the mechanisms of cell invasion used by the two forms is distinct, in line with results of Schenkman et al. (1991a) that observed no competition towards cell binding between the two forms. We then treated the mammalian target cells withcytochalasin D and nocodazole to evaluate the role of actin and tubulin mobilization. Cytochalasin D always inhibited amastigote invasion indicating that unlike what was observed for trypomastigotes, amastigotes had a more passive role in the entry process. Nevertheless, the main conclusion was that the effect of a particular drug was specific and unpredictable (inhibitory or stimulatory) for a parasite infective form and a particular host cell (Procópio et al. 1998).
Besides the formation of the surface cups in HeLa cells, we also noticed a remarkable response to extracellular amastigotes when they invade Vero cells. In this fibroblastic cell line devoid of surface microvilli, protrusive lamellae formed at the sites of amastigote invasion (Procópio et al. 1999) were markedly similar to shown by attaching Shigella flexneri (Bourdet-Sicard et al. 2000, Clerc and Sansonetti 1987, Tran et al. 2000). In figure 4 the co-localization of different actin-binding proteins and actin filaments in the membrane projections involved in T. cruzi invasion of HeLa and Vero cells is illustrated. In all the actin-rich membrane extensions formed around invading amastigotes or trypomastigotes, accumulation of cytoskeletal elements, integrins or matrix elements could be detected, with some variability observed between the infective forms and target cells (Procópio et al. 1999). These results were again consistent with the notion that each parasite-host cell pair mobilizes specific interacting components (see Table I).