RB Stecconi-Silva; WK Andreoli; RA Mortara1
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu 862, 6o andar, 04023-062 São Paulo, SP, Brasil
In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.
Keywords: Trypanosoma cruzi – infective forms – parasitophorous vacuole – escape – invasion – kinetics
In order to complete its life cycle, Trypanosoma cruzi, the protozoan ethiological agent of Chagas disease must invade mammalian cells, escape the parasitophorous vacuole and then transform into amastigotes in the cytoplasm in order to grow. In this process, trypomastigotes, the classical infective forms originated from the insect vector or infected cells, and also amastigotes, derived from the extracellular transformation of trypomastigotes or by the premature release from infected cells, may invade mammalian cells in vitro as well as in vivo (Behbehani 1973, Pan 1978, Hudson et al. 1984, Ley et al. 1988, Mortara 1991).
Previous studies from our laboratory have shown that the parameters controlling the invasion of HeLa and Vero cells are highly dependent on the infective form as well as the target cell used (Procópio et al. 1998). However, both recently internalized extracellular amastigotes and metacyclic trypomastigotes could be found in compartments bearing the lysosomal marker LAMP-1 (Procópio et al. 1998). It has been shown that Ca2+ mobilization is involved in the signaling pathway leading to parasitophorous vacuole formation during try-pomastigote invasion (Burleigh & Andrews 1995). Moreover, the combined activities of hemolysin and transialidase, activated within the acidified trypomastigote parasitophorous vacuole, are believed to be crucial in providing the means for these forms to escape into the cytoplasm, that is facilitated in cells lacking proper sialylation of lysosomal glycoproteins (Andrews & Whitlow 1989, Andrews et al. 1990, Hall et al. 1992). By contrast, little is known on the signals triggered during amastigote invasion, and the available data (Procópio et al. 1998) indicate that parasitophorous vacuole formation probably involves lysosomal recruitment. Furthermore, the mechanism by which amastigotes escape from this compartment seems to depend mostly on hemolysin (Ley et al. 1990). In the present work we have: (i) studied the kinetics of parasitophorous vacuole formation and escape of both infective forms in Vero and HeLa cells; (ii) determined the hemolytic and transialidase activities of both parasite forms; (iii) examined the effect of host cell sialic acid on parasite invasion and escape, (iv) studied the effect of compounds that interfere with calcium mobilization on the initial invasion step.
We have found significant differences between extracellular amastigotes and metacyclic trypomastigotes in relation to several of the parameters studied here.