Signaling and host cell invasion by Trypanosoma cruzi

Signal transduction events triggered in mammalian host cells by the obligate intracellular parasite Trypanosoma cruzi are required for invasion. Infective T. cruzi trypomastigotes elicit Ca2+ signaling in mammalian host cells and activate transforming growth factor-beta receptor signaling pathways. The elevation of Ca2+ in T. cruzi, induced by host-cell contact, is also required for invasion, extending the concept of host-pathogen 'cross-talk' to invasive protozoan pathogens.     

Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.     

Trypanosoma cruzi: phagolysosomal fusion after invasion into non professional phagocytic cells

Parasite-containing endocytic vacuoles are formed during the process of in vitro interiorization of the trypomastigote forms of Trypanosoma cruzi by primary culture of mouse fibroblasts, heart and skeletal muscle cells. Fusion of these vacuoles with host cell lysosomes takes place. The process of T. cruzi-muscle cell interaction was analysed by ultrastructural cytochemistry. Two lysosomal enzymes, acid phosphatase and aryl sulphatase and the fusion of peroxidase-labeled secondary lysosomes with the parasitophorus vacuoles were studied. These finding indicate that the basic mechanism of interaction of T. cruzi with the so called non phagocytic cells is similar to that which occurs with phagocytic cells.     

Lysosome recruitment during host cell invasion by Trypanosoma cruzi

The protozoan parasite Trypanosoma cruzi uses an unusual mechanism to enter cells. Recent observations revealed that instead of trypanosomes being brought in to fuse with lysosomes, it is the lysosomes that migrate to the trypanosomes and actually participate in their internalization. Signalling events involving intracellular free Ca2+ occur upon contact of the parasites with host cells and may contribute to the regulation of this unusual process.     

Role of host lysosomal associated membrane protein (LAMP) in Trypanosoma cruzi invasion and intracellular development

Trypanosoma cruzi host cell entry depends on lysosomes for the formation of the parasitophorous vacuole. Lysosome internal surface is covered by two major proteins, highly sialilated, Lysosome Associated Membrane Proteins 1 and 2. T. cruzi, on the other hand, needs to acquire sialic acid from its host cell through the activity of trans-sialidase, an event that contributes to host cell invasion and later for parasite vacuole escape. Using LAMP1/2 knock out cells we were able to show that these two proteins are important for T. cruzi infection of host cells, both in entrance and intracellular development, conceivably by being the major source of sialic acid for T. cruzi.     

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.     

Trypanosoma cruzi utilizes the host low density lipoprotein receptor in invasion

Background

Trypanosoma cruzi, an intracellular protozoan parasite that infects humans and other mammalian hosts, is the etiologic agent in Chagas disease. This parasite can invade a wide variety of mammalian cells. The mechanism(s) by which T. cruzi invades its host cell is not completely understood. The activation of many signaling receptors during invasion has been reported; however, the exact mechanism by which parasites cross the host cell membrane barrier and trigger fusion of the parasitophorous vacuole with lysosomes is not understood.

Methodology/Principal Findings

In order to explore the role of the Low Density Lipoprotein receptor (LDLr) in T. cruzi invasion, we evaluated LDLr parasite interactions using immunoblot and immunofluorescence (IFA) techniques. These experiments demonstrated that T. cruzi infection increases LDLr levels in infected host cells, inhibition or disruption of LDLr reduces parasite load in infected cells, T. cruzi directly binds recombinant LDLr, and LDLr-dependent T. cruzi invasion requires PIP2/3. qPCR analysis demonstrated a massive increase in LDLr mRNA (8000 fold) in the heart of T. cruzi infected mice, which is observed as early as 15 days after infection. IFA shows a co-localization of both LDL and LDLr with parasites in infected heart.

Conclusions/Significance

These data highlight, for the first time, that LDLr is involved in host cell invasion by this parasite and the subsequent fusion of the parasitophorous vacuole with the host cell lysosomal compartment. The model suggested by this study unifies previous models of host cell invasion for this pathogenic protozoon. Overall, these data indicate that T. cruzi targets LDLr and its family members during invasion. Binding to LDL likely facilitates parasite entry into host cells. The observations in this report suggest that therapeutic strategies based on the interaction of T. cruzi and the LDLr pathway should be pursued as possible targets to modify the pathogenesis of disease following infection.     

Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi.

Mammalian cell invasion by the intracellular protozoan parasite Trypanosoma cruzi is mediated by recruitment and fusion of host cell lysosomes, an unusual process that has been proposed to be dependent on the ability of parasites to trigger intracellular free calcium concentration ([Ca2+]i) transients in host cells. Previous work implicated the T.cruzi serine hydrolase oligopeptidase B in the generation of Ca2+-signaling activity in parasite extracts. Here we show that deletion of the gene encoding oligopeptidase B results in a marked defect in host cell invasion and in the establishment of infections in mice. The invasion defect is associated with the inability of oligopeptidase B null mutant trypomastigotes to mobilize Ca2+ from thapsigargin-sensitive stores in mammalian cells. Exogenous recombinant oligopeptidase B reconstitutes the oligopeptidase B-dependent Ca2+ signaling activity in null mutant parasite extracts, demonstrating that this enzyme is responsible for the generation of a signaling agonist for mammalian cells.     

Features of host cell invasion by different infective forms of Trypanosoma cruzi

Through its life cycle from the insect vector to mammalian hosts Trypanosoma cruzi has developed clever strategies to reach the intracellular milieu where it grows sheltered from the hosts' immune system. We have been interested in several aspects of in vitro interactions of different infective forms of the parasite with cultured mammalian cells. We have observed that not only the classically infective trypomastigotes but also amastigotes, originated from the extracellular differentiation of trypomastigotes, can infect cultured cells. Interestingly, the process of invasion of different parasite infective forms is remarkably distinct and also highly dependent on the host cell type.     

Trypanosoma cruzi trans-sialidase and cell invasion

Trypanosoma cruzi does not synthesize sialic acid but does contain a trans-sialidase, an enzyme capable of transferring sialic acid between host glycoconjugates and the parasite. Sialic acids are negatively charged carbohydrates attached to the terminal non-reducing end of glycoproteins and glycolipids, and their presence can dramatically influence many cell-surface recognition processes. Since sialic acids have been implicated in several ligand-receptor interactions, including the interaction of pathogenic viruses, bacteria and protozoans with their hosts, the expression of trans-sialidase and the acquisition of sialic acid by T. cruzi may be relevant to the interaction of the parasite with the host, and consequently may influence the pathobiology of Chagas disease. In this review, Sergio Schenkman and Daniel Eichinger discuss recent data about the structure and function of T. cruzi trans-sialidase.     

Cell signalling and Trypanosoma cruzi invasion

Mammalian cell invasion by the protozoan pathogen Trypanosoma cruzi is critical to its survival in the host. To promote its entry into a wide variety of non-professional phagocytic cells, infective trypomastigotes exploit an arsenal of heterogenous surface glycoproteins, secreted proteases and signalling agonists to actively manipulate multiple host cell signalling pathways. Signals initiated in the parasite upon contact with mammalian cells also function as critical regulators of the invasion process. Whereas the full spectrum of cellular responses modulated by T. cruzi is not yet known, mounting evidence suggests that these pathways impinge on a number of cellular processes, in particular the ubiquitous wound-repair mechanism exploited for lysosome-mediated parasite entry. Furthermore, differential engagement of host cell signalling pathways in a cell type-specific manner and modulation of host cell gene expression by T. cruzi are becoming recognized as essential determinants of infectivity and intracellular survival by this pathogen.     

Trypanosoma cruzi: mechanisms for entry into host cells

Infective trypomastigote stages of the obligate intracellular protozoan parasite Trypanosoma cruzi are capable of entering virtually any mammalian cell in vitro. Entry is a complex process, involving initial parasite attachment to surface moieties of the target cell, internalization of the parasite via formation of a vacuole, and finally disruption of the vacuolar membrane to permit access of the parasite to the host cell cytoplasm. Attachment requires parasite metabolic energy. At sites of parasite entry recruitment of host cell lysosomes may occur, and lysosomal membrane components contribute prominently to formation of the parasitophorous vacuole. Parasite escape from the vacuole depends upon vacuolar acidification and is mediated by the coordinated action of a parasite-derived neuramindase/trans-sialidase that is capable of desialylating host-derived vacuolar membrane constituents, and a parasite-derived trans-membrane pore-forming protein. Dissection of the entry process at both the organellar and molecular level is providing fundamental and complementary insights into microbial pathogenesis and cell biology.     

Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi

The cholesterol content of human erythrocyte membranes has been modified by incubation of intact cells with sonicated egg phosphatidylcholine/cholesterol vesicles and with egg phosphatidylcholine vesicles. (Na+ + K+)-ATPase ATP hydrolyzing activity was measured as a function of membrane cholesterol content. High membrane cholesterol inhibits the ATPase activity of the enzyme and low membrane cholesterol activates that enzyme activity. The most likely mechanism of inhibition is suggested to comprise direct cholesterol-protein interactions which lead to a low activity conformation. Ouabain binding studies show that the inhibition is not due to a loss of enzyme from the membrane.     

Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.     

L-arginine metabolism during interaction of Trypanosoma cruzi with host cells

Trypanosoma cruzi invades a diversity of nucleated cells in the mammalian host. Macrophages are among the first cells to be parasitized and, after activation by inflammatory stimuli, they participate in the control of infection. However, some parasites manage to evade the immune response and establish a chronic infection in differentiated cells. L-arginine is located at the crossroads of divergent routes that produce metabolites, including nitric oxide and polyamines, which influence the outcome (i.e. resolution or progression) of infection. This article discusses the fate and actions of L-arginine-derived biomolecules formed both in the host and in the parasite during T. cruzi-host-cell interactions.     

Interaction with host factors exacerbates Trypanosoma cruzi cell invasion capacity upon oral infection

Outbreaks of severe acute Chagas’ disease acquired by oral infection, leading to death in some cases, have occurred in recent years. Using the mouse model, we investigated the basis of such virulence by analyzing a Trypanosoma cruzi isolate, SC, from a patient with severe acute clinical symptoms, who was infected by oral route. It has previously been shown that, upon oral inoculation into mice, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium by engaging the stage-specific surface glycoprotein gp82, whereas the surface molecule gp90 functions as a down-modulator of cell invasion. We found that, when orally inoculated into mice, metacyclic forms of the SC isolate, which express high levels of gp90, produced high parasitemias and high mortality, in sharp contrast with the reduced infectivity in vitro. Upon recovery from the mouse stomach 1 h after oral inoculation, the gp90 molecule of the parasites was completely degraded, and their entry into HeLa cells, as well as into Caco-2 cells, was increased. The gp82 molecule was more resistant to digestive action of the gastric juice. Host cell invasion of SC isolate metacyclic trypomastigotes was augmented in the presence of gastric mucin. No alteration in infectivity was observed in T. cruzi strains CL and G which were used as references and which express gp90 molecules resistant to degradation by gastric juice. Taken together, our findings suggest that the exacerbation of T. cruzi infectivity, such as observed upon interaction of the SC isolate with the mouse stomach components, may be responsible for the severity of acute Chagas’ disease that has been reported in outbreaks of oral T. cruzi infection.     

Starvation and rapamycin differentially regulate host cell lysosome exocytosis and invasion by Trypanosoma cruzi metacyclic forms

The molecular mechanisms of host cell invasion by T. cruzi metacyclic trypomastigotes (MT), the developmental forms that initiate infection in the mammalian host, are only partially understood. Here we aimed at further identifying the target cell components involved in signalling cascades leading to MT internalization, and demonstrate for the first time the participation of mammalian target of rapamycin (mTOR). Treatment of human epithelial HeLa cells with mTOR inhibitor rapamycin reduced lysosomal exocytosis and MT invasion. Downregulation of phosphatidylinositol 3-kinase and protein kinase C also impaired exocytosis and MT internalization. The recombinant protein based on gp82, the MT surface molecule that mediates cell adhesion/invasion, induced exocytosis in HeLa cells. Such an effect has not previously been attributed to any T. cruzi surface molecule. Rapamycin treatment diminished gp82 binding as well. Cell invasion assays under conditions that promoted lysosome exocytosis, such as 1 h incubation in starvation medium PBS(++) , increased MT invasion, whereas pre-starvation of cells for 1-2 h had an opposite effect. In contrast to MT, invasion of tissue culture trypomastigotes (TCT) increased upon host cell pre-starvation or treatment with rapamycin, a novel finding that discloses quite distinctive features of the two infective forms in a key process for infection.     

Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization of components of the host cell plasma membrane during infection by Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi attach to the macrophage surface and are internalized with the formation of a membrane bounded vacuole, known as the parasitophorous vacuole (PV). In order to determine if components of the host cell membrane are internalized during formation of the PV we labeled the macrophage surface with fluorescent probes for proteins, lipids and sialic acid residues and then allowed the labeled cells to interact with the parasites. The interaction process was interrupted after 1 hr at 37 masculineC and the distribution of the probes analyzed by confocal laser scanning microscopy. During attachment of the parasites to the macrophage surface an intense labeling of the attachment regions was observed. Subsequently labeling of the membrane lining the parasitophorous vacuole containing epimastigote and trypomastigote forms was seen. Labeling was not uniform, with regions of intense and light or no labeling. The results obtained show that host cell membrane lipids, proteins and sialoglycoconjugates contribute to the formation of the membrane lining the PV containing epimastigote and trypomastigote T. cruzi forms. Lysosomes of the host cell may participate in the process of PV membrane formation.     

Purification of Trypanosoma cruzi surface proteins involved in adhesion to host cells

We have identified four surface 83 kDa proteins of pI values 6.3, 6.4, 6.5 and 6.6 in T. cruzi trypomastigotes which specifically bind to rat heart myoblasts. These proteins were purified by isoelectric focusing and anion-exchange chromatography in an FPLC system. These 83 kDa proteins inhibit the attachment of trypomastigotes to myoblasts in a concentration-dependent manner, indicating that these trypomastigote proteins mediate the attachment of trypomastigotes to heart myoblasts.