Lectin receptors in Trypanosoma cruzi an N-acetyl-d-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the ¹³¹I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent M_r 85 000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unnafected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}$ inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}\text{-}\text{specific}$ receptor activity.     

Role of surface N-acetylglucosamine residues on host cell infection by Trypanosoma cruzi

We studied the role of surface GlcNAc residues on the surface of invasive (mouse-blood and insect-derived trypomastigotes) and non-invasive amastigote forms of Trypanosoma cruzi on parasite association with (i.e., surface binding plus internalization) macrophages and heart myoblasts. Removal of GlcNAc from the three forms of the parasite with β-N-acetylglucosaminidase markedly increased the number of organisms per 100 cells and caused the organisms to associate with a greater percentage of host cells. N-Acetylglucosaminidase did not produce this effect after heat-inactivation and a substrate of the enzyme, N,N′-diacetylchitobiose, reduced it when it was present during the enzymatic treatment. The N-acetylglucosaminidase effect on T. cruzi was reversible after 2.5 h. When macrophages or myoblasts were treated with N-acetylglucosaminidase, their capacities to associate with blood or insect-derived trypomastigotes was reduced. Since removal of GlcNAc residues from the parasite surface increased their association with the host cells, GlcNAc would appear to interfere with the association process. On the other hand, GlcNAc residues on the host cell appear to favor the association.     

Biochemical Requirements for Intracellular Invasion by Trypanosoma cruzi: Protein Synthesis1

The effects of irreversible inhibition of protein synthesis by pactamycin in either infective forms of Trypanosoma cruzi or mammalian host cells on cellular invasion by this human pathogen were investigated. Treatment of bloodstream forms of T. cruzi with pactamycin markedly reduced their ability to bind either fibroblast-like cells of monkey origin or myoblasts of rat origin. The number of amastigote forms that could be established intracellularly was also significantly decreased with respect to control values obtained when mock-treated (medium alone) trypomastigotes were incubated with the cells. Pactamycin treatment also reduced the infectivity of T. cruzi trypomastigotes for mice as evidenced by both significantly reduced parasitemia levels and mortality rates when compared with those of control mice infected with mock-treated parasites. Inhibition of protein synthesis in the host cells neither prevented cell infection by untreated trypomastigotes nor altered the percentages of infected cells or the magnitude of the infection in vitro. These results indicate that protein synthesis is a requirement for cell invasion by T. cruzi and that the parasite can establish itself and replicate within cells relying on its own protein synthesis ability.     

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.     

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.     

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co‐opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host–parasite interaction.     

Ceramide and inositol content of the lipopeptidophosphoglycan from Trypanosoma cruzi.

Inositol (2.5%), 17-methyl-sphinganine (4.8%) and sphinganine (1.5%) have been identified as constituents of the lipopeptidophospholycan isolated from whole cells of epimastigote forms of $\text{Trypanosoma cruzi}$. The branched chain base was characterized by combined gas chromatography — mass spectrometry.     

The distribution of (Na++K+)-ATPase along the villus crypt-axis in the rabbit small intestine

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na⁺-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of around 10^?7 M. Both enzyme assay and ouabain-binding measurements suggest that a 2–3-fold increase in the number of Na⁺-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.     

Association of R6K plasmid replicative intermediates with the folded chromosome of Escherichiacoli

When $\text{Escherichia}\text{coli}$ strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [³H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome.     

Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages

Kierszenbaum F., Lima M. F. and Wirth J. J. 1985. Effects of antiserum to Trypanosoma cruzi on the uptake and rate of killing of vector-borne, metacyclic forms of the parasite by macrophages. International Journal for Parasitology15: 409–413. The contribution of phagocytic function to host defense against infection with metacyclic forms of Trypanosoma cruzi isolated from insect vectors was investigated in mice passively transferred with anti-T. cruzi serum. The protective effect resulting from the passive transfers was significantly reduced by administration of either silica or cobra venom factor (CVF). A more pronounced curtailment of the protective effect was seen when both silica and CVF were administered to the mice. This effect was greater than that calculated by adding the effects produced by silica and CVF alone. In in vitro experiments, presence of anti-T. cruzi antibodies enhanced the capacity of mouse macrophages to take up the metacyclic organisms and increased the proportion of macrophages associating with the parasites. Increased macrophage-parasite association was also seen when either the flagellates or the macrophages were preincubated with the antiserum. Antibody-treated metacyclic forms of T. cruzi were more rapidly cleared by untreated macrophages than parasites pretreated with normal mouse serum. These results support a role for macrophages in host defense against the form of T. cruzi responsible for natural infections and emphasize the role played by anti-T. cruzi antibodies. The combined effect of the silica and CVF treatments suggests that C activity may contribute to the protective action of antibodies through its opsonic properties, though a concomitant role for C-dependent immune lysis cannot be ruled out. These results highlight the protective role of antibodymediated mechanisms against infection with the form of T. cruzi responsible for natural infections.     

t-Butyl hydroperoxide-induced changes in the physicochemical properties of human erythrocytes

Treatment of human erythrocytes with micromolar concentrations of $\text{t-}\text{butyl}$ hydroperoxide causes a variety of changes in the physical properties of the cells. Red cells exposed to concentrations of $\text{t-}\text{butyl}$ hydroperoxide of less than 750 μM for 15 min exhibited significant decreases in cellular and membrane deformability, increases in membrane-associated protein crosslinking, osmotic fragility and the viscosity of the intracellular hemoglobin solution. No changes in the volume or density of the cells were observed. Changes in cellular deformability are probably attributable solely to changes in the mechanical properties of the cell membrane. Conversely, when red cells are exposed to $\text{t-}\text{butyl}$ hydroperoxide concentrations in excess of 750 μM for 15 min they exhibited decreases in cellular deformability which may be related to increases in cell volume as well as membrane rigidity.     

Trypanosoma cruzi uses macropinocytosis as an additional entry pathway into mammalian host cell

Several intracellular pathogens are internalized by host cells via multiple endocytic pathways. It is no different with Trypanosoma cruzi. Evidences indicate that T. cruzi entry may occur by endocytosis/phagocytosis or by an active manner. Although macropinocytosis is largely considered an endocytic process where cells internalize only large amounts of solutes, several pathogens use this pathway to enter into host cells. To investigate whether T. cruzi entry into peritoneal macrophages and LLC-MK2 epithelial cells can be also mediated through a macropinocytosis-like process, we used several experimental strategies presently available to characterize macropinocytosis such as the use of different inhibitors. These macropinocytosis' inhibitors blocked internalization of T. cruzi by host cells. To further support this, immunofluorescence microscopy and scanning electron microscopy techniques were used. Field emission scanning electron microscopy revealed that after treatment, parasites remained attached to the external side of host cell plasma membrane. Proteins such as Rabankyrin 5, tyrosine kinases, Pak1 and actin microfilaments, which participate in macropinosome formation, were localized at T. cruzi entry sites. We also observed co-localization between the parasite and an endocytic fluid phase marker. All together, these results indicate that T. cruzi is able to use multiple mechanisms of penetration into host cell, including macropinocytosis.     

Trypanosoma cruzi: Activity of heterocyclic cationic molecules in vitro

Chagas disease remains a serious public health problem in several Latin American countries. New chemotherapy is urgently needed since current drugs are limited in efficacy and exhibit undesirable side effects. Aromatic diamidines and analogs are well known anti-parasitic agents and in this study, we have evaluated the in vitro trypanocidal effect of several different heterocyclic cationic compounds, including diamidines (DB1195, DB1196 and DB1345), a monoamidine (DB824), an arylimidamide (DB613A) and a guanylhydrazone (DB1080) against amastigotes and bloodstream trypomastigotes of Trypanosoma cruzi, the etiological agent of Chagas disease. Our present findings showed that all compounds exerted, at low-micromolar doses, a trypanocidal effect upon both intracellular parasites and bloodstream trypomastigotes of T. cruzi. The activity of DB1195, DB1345, DB824 and DB1080 against bloodstream forms was reduced when these compounds were assayed in the presence of mouse blood possibly due to their association with plasma constituents and/or due to metabolic instability of the compounds. However, trypanocidal effects of DB613A and DB1196 were not affected by plasma constituents, suggesting their potential application in the prophylaxis of banked blood. In addition, potency and selectivity of DB613A, towards intracellular parasites, corroborate previous results that demonstrated the highly promising activity of arylimidamides against this parasite, which justify further studies in experimental models of T. cruzi infection.     

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.     

Invitro evaluation of granulocyte differentiation of diffusion chamber inocula

The semi-soft agar colony assay permits an $\text{in}$$\text{vitro}$ analysis of committed myeloid stem cell (CFU-c) proliferation capacities. In this paper this procedure has been used in combination with prior diffusion chamber culturing to determine the effect of host influences upon this committed stem cell population. This “double-seeding” procedure of first culturing bone marrow cells in diffusion chambers and then re-seeding them in agar furnishes data suggesting a relationship between $\text{in}$$\text{vivo}$ diffusion chamber transitional lymphocytes and $\text{in}$$\text{vitro}$ CFU-c seeding capacities. Diffusion chamber culturing offers a means of monitoring granulopoiesis and selects for enrichment of stem cell numbers. Detection and quantification of diffusion chamber stem cell enrichment is easily assessed by seeding chamber contents into the agar colony assay.     

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites’ antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson’s coefficient: 0.617, 0.771, 0.499; respectively, P < 0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.     

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.     

Catabolic metabolism in Trypanosoma cruzi

Culture, blood and intracellular forms of Trypanosoma cruzi have a high rate of endogenous oxygen uptake and probably utilize amino acids and carbohydrates as their exogenous energy sources. It is likely that triglyceride is the main energy reserve. Oxidation of carbohydrate by all forms is probably via a glycolytic sequence and a complete tricarboxylic acid cycle. These data suggest that the substrates utilized and catabolic pathways present in mammalian forms of T. cruzi are similar to those of culture forms of the organism and are quite distinct from those of the bloodstream forms of African trypanosomes.     

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.