Chagas disease is a neglected infectious disease caused by the protozoan Trypanosoma cruzi, primarily transmitted by triatomine vectors, and it threatens approximately seventy-five million people worldwide. This parasite undergoes a complex life cycle, transitioning between hosts and shifting from extracellular to intracellular stages. To ensure its survival in these diverse environments, T. cruzi undergoes extreme morphological and molecular changes. The metacyclic trypomastigote (MT) form, which arises from the metacyclogenesis (MTG) process in the triatomine hindgut, serves as a crucial link between the insect and human hosts and can be considered the starting point of Chagas disease. This review provides an overview of the current knowledge regarding the parasite\'s life cycle, molecular pathways, and mechanisms involved in metabolic and morphological adaptations during MTG, enabling the MT to evade the immune system and successfully infect human cells.

Na+/H+ exchanger isoform 1 (NHE1), a member of a large family of integral membrane proteins, plays a role in regulating the cortical actin cytoskeleton. Trypanosoma cruzi, the agent of Chagas disease, depends on F-actin rearrangement and lysosome mobilization to invade host cells. To determine the involvement of NHE1 in T. cruzi metacyclic trypomastigote (MT) internalization, the effect of treatment in cells with NHE1 inhibitor amiloride or of NHE1 depletion was examined in human epithelial cells. MT invasion decreased in amiloride-treated and NHE1-depleted cells. The phosphorylation profile of diverse protein kinases, whose activation is associated with remodeling of actin fibers, was analyzed in amiloride-treated and NHE1-depleted cells. In amiloride-treated cells, the phosphorylation levels of protein kinase C (PKC), focal adhesion kinase (FAK) and Akt were similar to those of untreated cells, whereas those of extracellular signal-regulated protein kinases (ERK1/2) increased. In NHE1-deficient cells, with marked alteration in the actin cytoskeleton architecture and in lysosome distribution, the levels of phospho-PKC and phospho-FAK decreased, whereas those of phospho-Akt and phospho-ERK1/2 increased. These data indicate that NHE1 plays a role in MT invasion, by maintaining the activation status of diverse protein kinases in check and preventing the inappropriate F-actin arrangement that affects lysosome distribution.

In recent decades, the oral infection of Trypanosoma cruzi has gathered increased attention due to frequent outbreaks that can lead to more severe clinical signs than those usually found in the areas of vector transmission. This study addresses the main routes of infection using metacyclic trypomastigotes (MT) and blood trypomastigotes (BT). Herein, BALB/c mice were infected with the Colombian (TcI) strain via intraperitoneal (IP), oral, intragastric (IG), ocular (OC) and cutaneous (CT) routes with 106 culture-derived MT or BT. Parasitemia was intermittent and low in animals inoculated with MT, in contrast, high parasitemia levels were found in BT-mice. A tropism for the muscles was observed in oral or IG infection with BT. Differently, the parasite was widely distributed in the tissues of mice infected with MT. However, the intensity of the inflammation infiltrating the tissues was higher in oral or IG infection with BT. Animals inoculated with BT via the IG route had similar serum levels of IFN-γ and smaller IL-10 compared to those infected with MT via the IG route. TNF-α levels were higher in the serum from BT-animals, which could explain the higher intensity of heart inflammation in these animals. Our results suggest that the infective form and the route of infection differentially modulated the outcome of Trypanosoma cruzi mice infection.

Cellular invasion by Trypanosoma cruzi metacyclic trypomastigotes (MTs) or tissue culture trypomastigotes (TCTs) is a complex process involving host-parasite cellular and molecular interactions. Particularly, the involvement of host cell actin cytoskeleton during trypomastigote invasion is poorly investigated, and still, the results are controversial. In the present work, we compare side by side both trypomastigote forms and employ state-of-the-art live-cell imaging showing for the first time the dynamic mobilization of host cell actin cytoskeleton to MT and TCT invasion sites. Moreover, cytochalasin D, latrunculin B, and jasplakinolide-pretreated cells inhibited MT and TCT invasion. Furthermore, our results demonstrated that TCT invasion decreased in RhoA, Rac1, and Cdc-42 GTPase-depleted cells, whereas MT invasion decreased only in Cdc42-and RhoA-depleted cells. Interestingly, depletion of the three studied GTPases induced a scattered lysosomal distribution throughout the cytosol. These observations indicate that GTPase depletion is sufficient to impair parasite invasion despite the importance of lysosome spread in trypomastigote invasion. Together, our results demonstrate that the host cell actin cytoskeleton plays a direct role during TCT and MT invasion.

Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-β-cyclodextrin (MβCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MβCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MβCD-treated parasites. CM from untreated and MβCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MβCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.

The surface molecule gp82 of metacyclic trypomastigote (MT) forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca2+ signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca2+-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca2+ from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.

Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.

The involvement of β-adrenergic receptor (β-AR) in host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is not known. We examined whether isoproterenol, an agonist of β-AR, or nonselective β-blocker propranolol affected MT internalization mediated the stage-specific surface molecule gp82. Treatment of HeLa cells with propranolol significantly inhibited MT invasion whereas isoproterenol had no effect. Propranolol, but not isoproterenol, also inhibited the lysosome spreading required for gp82-dependent MT invasion. The effect of propranolol in inhibiting MT internalization was not due to the prevention of gp82 interaction with β-AR. It was mainly associated with its ability to impair lysosome spreading.