Triggering of endocytosis

Following binding to and recognition of the parasite by the host cell surface, a series of cell signaling processes takes place, which culminate in the invasion of the host cell. The available evidence indicates that the T. cruzi trypomastigotes use several mechanisms of invasion, including the following: (1) phagocytosis/macropino- cytosis, an actin-mediated process in which the cells emit pseudopods. In professional phagocytes such as macrophages, the activation of tyrosine kinase proteins occurs, followed by the recruitment of PI-3 kinase and actin filaments (a process that has been associated with the mechanism of phagocytosis) at the trypomastigote entry site⁹⁷ (Fig. 18.14); (2) endocytosis, without the emission of pseudopods but with the participation of actin filaments; and (3) invagination of the membrane, without the participation of actin filaments. This latter process has been regarded as an active mechanism for parasite entry that wastes energy.⁹⁸

Figure 18.14 Schematic representation of T. cruzi entry in host cells.

In the initial moments of the recognition between T. cruzi and the host cell, a transient increase of cytoplasmic levels of calcium occurs in both the parasite and the host cell.^3,99,100 If this transient increase in cytoplasmic calcium is blocked (by treatment with thapsigargin, for example), there is a reduction in parasite invasion.¹⁰¹ Additionally, in nonprofessional phagocytic cells, there is a recruitment of lysosomes to the place of parasite invasion, although this phenomenon occurs in only about 20% of parasites that enter. Recently, Cortez and Yoshida¹⁰² observed that the participation of lysosomes in the site of entry into host cells may be related to the origin of the trypomastigote used. When nonphagocytic host cells are deprived of nutrients, metacyclic trypomastigotes (released by the insect vector) recruit lysosomes to penetrate. Cultured trypomastigotes use a lysosome- independent pathway. This difference occurs primarily because metacyclic trypo- mastigotes stimulate the biogenesis/dispersion of lysosomes after gp82-mediated parasite adhesion to the host. The lysosome-dependent pathway is initiated by targeted Ca²¹-regulated exocytosis of lysosomes at the plasma membrane. Another pathway used for parasite internalization in nonphagocytic cells is the lysosome- independent pathway. In this model, parasites enter cells through plasma membrane invaginations that accumulate PIP3, the major product of class I PI3K activation. As a result of this mode of entry, around 50% of total internalized parasites are contained in vacuoles enriched in plasma membrane markers, and about 20% are in early endosomes (EEA1 labeled) at 10 min postinfection. In this case, the immature vacuole becomes filled with lysosomes within 60 min.¹⁰³ Several studies using host cells treated with cytochalasins D or B (agents that depolymerize actin filaments) before the process of interaction with trypomastigotes produced controversial data.

Some authors say that the treatment inhibits the entry of trypomastigote forms,^104-106 while others describe a sharp increase in entry,¹⁰⁷ and still others report almost no effect. Participation of actin cytoskeleton has been discussed in the retention of trypomastigotes in the cytoplasm of the host cell.^106,108,109 Woolsey and Burleigh described that cytochalasin-treated host cells showed a reduction in the number of parasites inside them, confirming earlier data¹⁰¹ that showed a decrease in infection of cytochalasin pretreated host cells after 40 min of interaction with try- pomastigotes.^14,105 More recently, Teixeira et al.¹⁰⁹ also demonstrated using amasti- gotes that actin participates in T. cruzi entry. This participation is followed by the accumulation of Arf6 and Anexin 2 around the parasitophorous vacuole. Actin is also involved in a process known as macropinocytosis. Recently, Barrias and coworkers¹⁰⁹ demonstrated that macropinocytosis is involved in T. cruzi entry, as raban- kirin, PAK1, actin, and PI3K are recruited to the entry site of the parasite. In addition, host cell treatment with classic inhibitors of the macropinocytosis pathway impairs parasite entry. The entry of the trypomastigote activates signaling processes in the host cell and parasite that lead to parasite invasion. In professional phagocytes, tyrosine kinase activation and recruitment of PI-3 kinase and actin to the site of parasite entry also occur.^99,105

Host cell plasma membrane microdomains were also shown to be involved in T. cruzi entry both in nonphagocytic and phagocytic cells.^111,112 Both groups demonstrated that cholesterol, major membrane raft components, and microdomain molecular markers like flotillin1 colocalize with T. cruzi entry site suggesting the microdomains’ participation in internalization (Fig. 18.15).

Another mechanism involved in trypomastigote internalization into host cells is trogocytosis. Trogocytosis is a phenomenon involved in the bidirectional transfer of molecules between interacting cells or to cells to which the cell is conjugated via the exchange of plasma membrane fragments. The transferred membrane and associated molecules become part of the recipient cell.¹¹³