As mentioned earlier, an increasing number of both cellular and parasitic components that may be relevant for T. cruzi cell invasion have been identified over the last decades. A pragmatic analysis over the extensive and varied types of studies could lead the outsider to conclude that it is still not known precisely how T. cruzi invades host cells. For invasion to occur the parasite first has to attach, a process that can be separated from invasion by lowering temperature or fixing target cells (Andrews and Colli 1982, Meirelles et al. 1982a, Schenkman et al. 1991b). Several lines of evidence indicate, however, that motile trypomastigotes (both TCTs and metacyclics) promptly attach to fixed cells and invade live cells through an active (meaning parasite-dependent) mechanism that does not require intact host cell microfilaments (Schenkman et al. 1991b, Schenkman and Mortara 1992) but depends on parasite energy (Schenkman et al. 1991b). By contrast, extracellular amastigote attachment to fixed cells does not occur (Barros 1996) and invasion depends on functional host cell microfilaments (Mortara 1991, Procópio et al. 1998). A brief glance into these data immediately uncovers the complexity of the task.
Among the paradigmatic studies that laid new insights into the invasion mechanism is the description by the group of Norma Andrews that calcium-dependent lysosomal recruitment takes place during trypomastigote invasion (Tardieux et al. 1992, 1994). According to this model, TCTs engage signaling processes that culminate with the formation of parasitophorous vacuole (Burleigh and Andrews 1998, Burleigh and Woolsey 2002).
New evidences on the participation of components of the early endocytic traffic such as dynamin and Rab5 have indicated that the lysosomal process might be more elaborate and downstream of earlier events (Wilkowsky et al. 2002). We have also recently obtained evidence that about 20% of CL strain (T. cruzi II) metacyclic trypomastigotes may also recruit the early endosome antigen EEA-1 when invading Vero cells harboring the bacterium Coxiella burnetii (Andreoli and Mortara 2003a).
Using a more quantitative approach to identify the role of phosphatidyl-inositol 3-kinase (PI3-K) on the lysosomal pathway, Woolsey et al. (2003) were able to firmly confirm previous observations by Wilkowsky et al. (2001) that this cellular key component could be involved in a lysosome-independent T. cruzi internalization pathway by TCTs. Trypomastigotes that use this route mobilize phosphorylated inositides during the formation of the parasitophorous vacuole that then matures to become enriched in lysosomal marker LAMP-1. One important input of this work was that for the first time the relative contributions of each mode of entry, namely PI3-K (50%), lysosome (20%), and endosomal route (20%) were estimated (Woolsey et al. 2003).
The available information on the mechanisms of amastigote penetration is comparatively scarcer than for trypomastigote. In studies on the interaction with macrophages, it has been described that members of the transialidase-like surface antigens engage mannose receptors to enter the professional phagocytes (Kahn et al. 1995). In non- phagocytic cells we so far have been able to identify the previously mentioned carbohydrate epitope (defined by Mab 1D9) as one of the potential molecular candidates on extracellular amastigote surface that interact with cultured mammalian cells. The relative role of PI3-K, endosomal and the already described LAMP-1 (Procópio et al. 1998) pathways in extracellular amastigote invasion will be examined with the appropriate GFP constructs, described by Woolsey et al. (2003) that recently became available to us.
Once inside host cells, trypomastigotes are thought to secrete TcTOX, a complement 9 (C9) factor-related molecule that at low pH will destroy the PV membrane and lead the parasite to the cytosol (Andrews et al. 1990). This lytic activity is likely to be facilitated by the parasite transialidase activity on lumenal glycoproteins that protect the parasitophorous vacuole (Hall et al. 1992). Infective extracellular amastigotes also secrete TcTOX (Y and G strains) and transialidase (Andrews and Whitlow 1989, Ley et al. 1990, Stecconi-Silva et al. 2003, L'Abbate and Fernandes unpublished observations). In recent studies we compared how pH affected cellular invasion and intracellular traffic of metacyclic trypomastigotes and extracellular amastigotes. We had previously confirmed that recently internalized amastigotes and metacyclic trypomastigotes (G strain) can be found in LAMP-1- containing PVs (Procópio et al. 1998). Raising intracellular pH with weak bases affected metacyclic invasion and escape from the PV, that was substantially delayed (from 2 to about 10h). By contrast, the kinetics of amastigote invasion and escape was not affected (Stecconi-Silva et al. 2003). In agreement with the idea that glycosylation of lysosomal lumenal glycoproteins is relevant for the protection of the PV membrane, both parasite forms promptly escape from PVs formed in CHO cells deficient in sialylation (Stecconi-Silva et al. 2003).
So far we have been able to identify TcTOX activities in isolated extracellular amastigotes (Stecconi-Silva et al. 2003) and tissue-culture derived trypomastigotes (Andreoli and Mortara 2003a). In contrast, metacyclic trypomastigotes display very weak transialidase activity and undetectable TcTOX (Andreoli and Mortara 2003a, Stecconi-Silva et al. 2003). Therefore, whereas extracellular amastigotes display a somewhat predictable behavior regarding cell invasion and escape, at present we do not have a consistent model to understand how metacyclic trypomastigotes actually escape from their PVs. Using polyclonal antibodies to C9, we have recently been able to detect by immunofluorescence what appears to be a TcTOX-related component on intracellular amastigotes (Andreoli W.K., unpublished observations) and this tool may be useful to map this component throughout the intracellular traffic of the different infective forms. Another interesting observation regarding metacyclic trypomastigote traffic is that the acquisition of LAMP-1 molecules by the forming PV does not parallel its acidification, monitored in vivo by Lysotracker, a fluorescent probe for acidic intracellular compartments (Molecular Probes, OR, USA, Andreoli W.K., unpublished observations). This may indicate that the precise events that lead to PV maturation might be more elaborate than previously imagined.
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