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Nonphagocytic cell invasion by T. cruzi

Trypomastigotes are capable of invading a wide variety of mammalian cells, through a process that differs from classical phagocytosis in that no pseudopods occur during entry and it is not prevented by actin filament depolymerization.23,24 Different research groups have characterized at least three processes involved in T. cruzi trypo- mastigote invasion of nonprofessional phagocytic cells, all of which converge in the lysosomal compartment of the infected host cell (reviewed by Caradonna et al.9). While initially presented as mechanistically distinct pathways, an invasion model proposing that the three invasion routes are aspects of the same overall process reconciles historical and emerging information. Below, experimental evidence supporting different T. cruzi trypomastigote invasion routes/aspects is introduced in separate sections before discussing a unified model of invasion.

Lysosome exocytosis invasion route

Evidence for lysosome involvement in the process cell of invasion by T. cruzi originally arose in the early 1990s. Lysosomes on the host cell periphery were shown to be recruited toward the parasite attachment site and fuse with the plasma membrane during invasion of nonphagocytic cells by T. cruzi.25,26 Experimental conditions which facilitate movement of lysosomes toward the host cell surface lead to increased parasite invasion, whereas those which deplete cells from peripheral lyso- somes or prevent their fusion with the plasma membrane reduce invasion.25 For example, parasite entry can be impaired by lysosome agglutination through microinjection of antibodies against lysosomal proteins or by chemical disruption of the microtubules required for lysosome migration.26

Trypomastigotes are able to trigger signaling cascades through host cell trimeric guanine nucleotide-binding protein (G-protein) receptors, activating cyclic adenosine monophosphate (cAMP)-dependent pathways and inducing cytosolic Ca21 concentration fluxes in the host cell cytoplasm. , Liberation of Ca from intracellular stores was suggested to activate the phospholipase C/inositol-3-phos- phate signaling pathway and results in transient actin fiber reorganization in the host cells,29 which is known to facilitate parasite entry.25 Synaptotagmin VII serves as a Ca21 sensor in lysosome—plasma membrane fusion,30 and blocking its activity causes a significant reduction (~50%) of T. cruzi invasion.31

T. cruzi appears to have evolved redundant mechanisms to elicit Ca21 signaling in host cells. A parasite-derived cytosolic serine endopeptidase, termed oligopeptidase-B, triggers Ca transients over a variety of mammalian cells. , This enzyme has recently been shown to form dimers in solution,34 and is believed to act by producing a Ca21 agonist through proteolysis in the cytoplasm of the parasite. The agonist, which has not been identified to date, is proposed to act by initiating signaling cascades resulting in Ca transients in mammalian cells. , Parasites carrying oligopeptidase-B gene deletions show defects in cell invasion in vitro and are less infective in the murine model as well.35 Furthermore, they are defective in the initiation of Ca21 transients in fibroblasts, myoblasts and HeLa cells, an activity which is restored by addition of recombinant oligopeptidase-B, showing that this enzyme plays a key role in invasion of mammalian cells by the

parasite.35

However, even in parasites carrying a double KO for oligopeptidase-B, a residual capacity to induce Ca21 transients and to infect cells persists; reinforcing the notion that the parasite possesses redundant mechanism involved in the generation of Ca21 transients.35 In fact, several other T. cruzi proteins have been shown to be able to initiate Ca21 signaling in nonphagocytic cells, including cruzipain,36 members of the gp85/transialidase family (reviewed by Maeda et al.8), a novel family of T. cruzi surface membrane proteins (TcSMP),37 the variant of trypomastigote small surface antigen present in T. cruzi lineage VI (TSSA VI),38,39 and T. cruzi serine-, alanine-, and proline-rich proteins (SAP).40 Among these, the mechanisms of action of cruzipain and that of metacyclic trypomastigotes surface glycoproteins are the best characterized, and will be discussed in further detail below.

The major T. cruzi protease, cruzipain, initiates signaling through kinin generation. Kinins are short-lived peptidic hormones involved in circulatory homeostasis which signal through the B2 bradykinin receptor (B2R).41 Scharfstein et al.36 showed that purified cruzipain triggers robust Ca21 responses in umbilical vein endothelial cells and B2R-expressing Chinese hamster ovary (CHO) cells. HOE, a specific, and E-64, an irreversible inhibitor of cruzipain, were shown to block such Ca21 transients. Furthermore, live tissue culture-derived trypomastigotes induced Ca21 transients in B2R-expressing CHO but not in mock transfected cells, and addition of purified kininogens or physiological concentrations of bradykinin to the culture medium increased parasite entry into B2R-expressing CHO cells.36,42 These findings imply that cruzipain is capable of initiating Ca21 transients in the host cell by proteolytically cleaving host cell-bound kininogens to generate kinins in umbilical vein endothelial cells and B2R expressing CHO cells.36 Since protease inhibitors which block cruzipain activity, such as cystatin-C or E-64, do not block parasite entry into the host cells, it has been proposed that the kininogen lysis by cruzipain requires close contact between the cell membranes of the host cell and the parasite,36,42 although this has not been demonstrated experimentally.

The work by Yoshida and collaborators regarding the signaling of surface glycoproteins in metacyclic trypomastigotes is also compelling. It has been postulated that tissue culture-derived trypomastigotes (equivalent to blood stream forms) and metacyclic trypomastigotes induce Ca21 transients by different mechanisms (reviewed by Maeda et al.8). While tissue culture-derived trypomastigotes would induce Ca21 flux through oligopeptidase B and cruzipain, metacyclics of the CL strain are believed to signal through gp82, a gp85/transialidase family member, activating phospholipase C, the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K), and resulting in Ca21 transients in HeLa cells.843 The less infective G-strain is believed to signal through the gp35/50 surface mucin and to generate Ca21 transients in a cyclic AMP-dependent fashion.43

The relative expression of gp82, gp35/50, and gp90 (a nonsignaling member of the gp85/trans-sialidase family) has been proposed to govern the infectivity of T. cruzi stocks, where immunoprecipitation, immunoblotting, and FACS analysis with monoclonal antibodies revealed that the poorly infective strains (including G strain) express relatively high levels of gp35/50 and gp90, while highly infective strains (including the CL strain) expressed high levels of gp82 and negligible gp90 on its surface.44 These three metacyclic trypomastigote-specific surface proteins interact with receptors in the surface of mammalian cells, where gp82 induces a robust Ca21 response, while the response induced by gp35/50 is weaker and that of gp90 is negligible.44 Experimentally induced reduction of gp90 expression through antisense nucleotides was shown to increase parasite infectivity,45 and it is believed that gp90 negatively regulate T. cruzi infectivity.46 Furthermore, parasite isolates expressing gp90 variants that are sensitive to gastric peptidases are infective through the oral route in vivo despite showing poor infectivity in vitro. High infec- tivity in vitro can be induced in such isolates by gp90 degradation though exposure to gastric juice.47

 
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