Cell signaling is an essential requirement for mammalian cell invasion by Trypanosoma cruzi. Depending on the parasite strain and the parasite developmental form, distinct signaling pathways may be induced. In this short review, we focus on the data coming from studies with metacyclic trypomastigotes (MT) generated in vitro and tissue culture-derived trypomastigotes (TCT), used as counterparts of insect-borne and bloodstream parasites, respectively. During invasion of host cells by MT or TCT, intracellular Ca2+ mobilization and host cell lysosomal exocytosis are triggered. Invasion mediated by MT surface molecule gp82 requires the activation of mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) in the host cell, associated with Ca2+-dependent disruption of the actin cytoskeleton. In MT, protein tyrosine kinase, PI3K, phospholipase C, and PKC appear to be activated. TCT invasion, on the other hand, does not rely on mTOR activation, rather on target cell PI3K, and may involve the host cell autophagy for parasite internalization. Enzymes, such as oligopeptidase B and the major T. cruzi cysteine proteinase cruzipain, have been shown to generate molecules that induce target cell Ca2+ signal. In addition, TCT may trigger host cell responses mediated by transforming growth factor β receptor or integrin family member. Further investigations are needed for a more complete and detailed picture of T. cruzi invasion.

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A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently described. Here we characterized a T. cruzi strain from this new genetic group, which could be a potential source of infection to humans. Metacyclic trypomastigotes (MT) of this strain, herein designated BAT, were compared to MT of well characterized CL and G strains, as regards the surface profile and infectivity toward human epithelial HeLa cells. BAT strain MT expressed gp82, the surface molecule recognized by monoclonal antibody 3F6 and known to promote CL strain invasion by inducing lysosomal exocytosis, as well as mucin-like molecules, but lacked gp90, which functions as a negative regulator of invasion in G strain. A set of experiments indicated that BAT strain internalization is gp82-mediated, and requires the activation of host cell phosphatidylinositol 3-kinase, protein kinase C and the mammalian target of rapamycin. MT of BAT strain were able to migrate through a gastric mucin layer, a property associated with p82 and relevant for oral infection. Gp82 was found to be a highly conserved molecule. Analysis of the BAT strain gp82 domain, containing the cell binding- and gastric mucin-binding sites, showed 91 and 93% sequence identity with G and CL strains, respectively. Hela cell invasion by BAT strain MT was inhibited by purified mucin-like molecules, which were shown to affect lysosome exocytosis required for MT internalization. Although MT of BAT strain infected host cells in vitro, they were less effective than G or CL strains in infecting mice either orally or intraperitoneally.

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insectstage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-␤1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. E xtracellular matrix (ECM) proteins, which serve as substrates for diverse adhesion molecules and are involved in many important physiological processes, also may mediate cell attachment and/or invasion of pathogenic microorganisms. Among these molecules, fibronectin (FN) has been reported to play a role in adherence to and invasion of host cells by bacteria such as Staphylococcus aureus, Streptococcus pyogenes, and Campylobacter jejuni (1-6). The interaction of fibronectin with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, also has been described. T. cruzi infection is initiated by metacyclic trypomastigote (MT) from the insect vector. This parasite form is responsible for the initial T. cruzi-host cell interaction upon entering the mammalian host through the skin or by the oral route. Following MT internalization in a membrane-bound vacuole and escape to the cytoplasm, the parasite differentiates into amastigote form. After several rounds of replication, amastigotes transform into trypomastigotes that are released in the circulation upon host cell rupture. Experiments with tissue culture trypomastigote (TCT), which corresponds to the bloodstream trypomastigote, revealed the involvement of fibronectin in target cell adhesion/invasion. The treatment of 3T3 fibroblasts or rat peritoneal macrophages, or TCT, with human plasma FN increased parasite-cell association (7), and binding ...

BackgroundOutbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil.MethodsTrypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were incubated with metacyclic forms and the number of internalized parasites was counted.ResultsAll TcI and TcIV T. cruzi strains were poorly infective by the oral route. Parasites were either undetectable or were detected in small numbers in the mouse stomach four days post oral administration. Replicating parasites were found in the stomach and/or in the heart 30 days post-infection. As compared to TcI lineage, the migration capacity of TcIV parasites through the gastric mucin-coated filter was higher but lower than that exhibited by TcVI metacyclic forms previously shown to be highly infective by the oral route. Expression of pepsin-resistant gp90, the surface molecule that downregulates cell invasion, was higher in TcI than in TcIV parasites and, accordingly, the invasion capacity of TcIV metacyclic forms was higher. Gp90 molecules spontaneously released by TcI metacyclic forms inhibited the parasite entry into host cells. TcI parasites exhibited low intracellular replication rate.ConclusionsOur findings indicate that the poor capacity of TcI lineage, and to a lesser degree of TcIV parasites, in invading gastric epithelium after oral infection of mice may be associated with the inefficiency of metacyclic forms, in particular of TcI parasites, to migrate through the gastric mucus layer, to invade target epithelial cells and to replicate intracellularly.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1455-z) contains supplementary material, which is available to authorized users.

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Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Therefore, intracellular iron accumulation must be tightly regulated, by balancing uptake with storage or export. Iron uptake in Leishmania is mediated by the coordinated action of two plasma membrane proteins, the ferric iron reductase LFR1 and the ferrous iron transporter LIT1. However, how these parasites regulate their cytosolic iron concentration to prevent toxicity remains unknown. Here we characterize Leishmania Iron Regulator 1 (LIR1), an iron responsive protein with similarity to membrane transporters of the major facilitator superfamily (MFS) and plant nodulin-like proteins. LIR1 localizes on the plasma membrane of L. amazonensis promastigotes and intracellular amastigotes. After heterologous expression in Arabidopsis thaliana, LIR1 decreases the iron content of leaves and worsens the chlorotic phenotype of plants lacking the iron importer IRT1. Consistent with a role in iron efflux, LIR1 deficiency does not affect iron uptake by L. amazonensis but significantly increases the amount of iron retained intracellularly in the parasites. LIR1 null parasites are more sensitive to iron toxicity and have drastically impaired infectivity, phenotypes that are reversed by LIR1 complementation. We conclude that LIR1 functions as a plasma membrane iron exporter with a critical role in maintaining iron homeostasis and promoting infectivity in L. amazonensis.