Several laboratories have confirmed the observation that T. cruzi infective forms from different strains display distinct infectivities towards cells and animals (Alves et al. 1986, Meirelles et al. 1982b, Melo and Brener 1978). Recent characterization of two major phylogenetic lineages of the parasite established that T. cruzi I strains are associated with the sylvatic cycle whereas T. cruzi II isolates are found mainly in patients and vectors in human dwellings (Souto et al. 1996). The comprehensive work by Nobuko Yoshida and co-workers (see below) established that metacyclic trypomastigotes from T. cruzi I (G strain) engage different signaling mechanisms to invade HeLa cells when compared to T. cruzi II (CL strain) (Neira et al. 2002).
When we initiated the studies of extracellular amastigote infection, it soon became apparent that these forms of the G strain (T. cruzi I) were usually much more infective than the corresponding metacyclic trypomastigotes. This was true not only for Vero and HeLa cells (Mortara 1991, Procópio et al. 1998, Procópio et al. 1999) but also for the sialic acid mutant Lec-2 cells (Stecconi-Silva et al. 2003). Of all target mammalian cells employed so far, only in the case of MDCK cells and the Rho transfectants, was metacyclic trypomastigotes infectivity higher than the corresponding extracellular amastigotes (see Table I and Fernandes and Mortara 2004).
When the infectivity of extracellular amastigotes derived from sylvatic type I strains was systematically compared to type II parasites, we always found that the former, particularly of the G strain, were much more infective (Barros 1996, Fernandes and Mortara 2004, Mortara et al. 1999). Interestingly, this higher infectivity trend followed the expression of a surface carbohydrate epitope defined by Mab 1D9 (Barros et al. 1997), that is correspondingly high in extracellular amastigotes of T. cruzi I strains and low in T. cruzi II isolates (Mortara et al. 1999, Verbisck et al. 1998). Moreover, the carbohydrate epitope defined by Mab 1D9 is present in the same protein that also contains another epitope designated Ssp-4, defined by Mab 2C2 (Andrews et al. 1987, Barros et al. 1997). Unlike 2C2 that is restricted to the surface of intracellular and extracellular amastigotes, the epitope defined by Mab 1D9 is also present in intracellular compartments such as cytoplasmic vesicles and Golgi apparatus (Barros et al. 1997). Consistent with the higher expression on the more infective T. cruzi I extracellular amastigotes, Mab 1D9 and its Fab fragments were also shown to specifically inhibit parasite invasion (Barros et al. 1993, Barros 1996). Unfortunately, due to the nature of the immunoglobulin (IgG3) that precipitated upon isolation, the identification of this epitope of extracellular amastigotes has so far not been possible.
Why extracellular amastigotes of highly infective strains such as Y and CL are poorly infective when compared to type I parasites, particularly G forms, showing the opposite behavior of the related trypomastigotes? This is a trend that we constantly found and that, so far we don't have a reasonable explanation. One highly speculative possibility is that subpatent infection caused by type I parasites (such as that found in experimental mice) could be at least in part sustained by the generation of infective extracellular amastigotes. Scharfstein and Morrot (1999) proposed that extracellular amastigotes (of either T. cruzi type) could also play a role by aggravating the pathology in the chronic phase of the disease. Possible differences in the expression of surface ligands required for cell invasion should also being considered (see below).
SIGNALLING MECHANISMS: ROLE OF CALCIUM IONS FROM ACIDOCALCISOMES OR IP3-DEPENDENT COMPARTMENTS
As indicated above, metacyclic trypomastigotes of the two major phylogenetic lineages use highly divergent signaling mechanisms to invade host cells. Using drugs to inhibit specific pathways, Yoshida and collaborators demonstrated that T. cruzi I trypomastigotes (the prototype being G strain) engage adenylate cyclase activation for cellular invasionwhereas CL strain parasites (T. cruzi II prototype) depend on tyrosine phosphorylation to accomplish this process (Neira et al. 2002). Also, G strain metacyclics appear to mobilize intracellular calcium from acidocalcisomes whereas CL strain parasites preferentially use (1,4,5-inositol-triphosphate, IP3-dependent) endoplasmic reticulum stores during invasion (Neira et al. 2002). Preliminary results from comparative studies between metacyclic trypomastigotes and extracellular amastigotes of the G strain, indicated that drugs that interfere with ER calcium mobilization (thapsigargin, A23187 ionophore) do not affect invasion of treated amastigotes (Stecconi-Silva et al. 2003). Further analysis with other IP3-interfering compounds (caffeine, neomycin and U73122) confirmed that calcium mobilization in the parasite through IP3 mobilization is not relevant for cellular invasion by extracellular amastigotes of either G or CL strains (Table I, Fernandes A.B., unpublished observations). Interestingly, drugs that interfere with calcium mobilization from acidocalcisomes (ionomycin, nigericin, NH4Cl) inhibit cell invasion by parasites of both strains (Fernandes A.B., unpublished observations), in contrast to the results of metacyclic trypomastigotes (Neira et al. 2002). From the host cell point of view, contact with TCT (Tardieux et al. 1994) or metacyclic trypomastigotes (Dorta et al. 1995), but not epimastigotes (Tardieux et al. 1994) give rise to transient calcium influxes. We have observed that cell extracts of extracellular amastigotes of both G and CL strains also induce calcium influxes in HeLa cells loaded with Fura-2 (Fernandes A.B., unpublished observations). These observations suggest that the distinct signaling pathways detected in metacyclics are not retained by extracellular amastigotes from the two phylogenetic lineages (Table I). A comprehensive study of these signaling routes is currently being carried out in our laboratory.