Perspectives on the Trypanosoma cruzi–host cell receptor interactions

Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets.     

Trypanosoma cruzi: experimental parasitism of bone and cartilage

Trypanosoma cruzi causes Chagas’ disease, a systemic infection that affects cells of meso-, endo-, and ectodermic origin. However, as far as we know, the presence of T. cruzi stages in bone has not been reported previously, and it has scarcely been investigated in cartilage. We inoculated 7- and 20-day-old (8 and 15 g) NMRI albino mice i.p. with metacyclic trypomastigotes from Rhodnius prolixus used for xenodiagnosis of mice previously infected with mammalian, human, and triatomines isolates, characterized by randomly amplified polymorphic DNA as zymodeme 1 (equivalent to T. cruzi I). Tissular parasitism (quantified according to the number of pseudocysts/50 fields 400×) showed amastigotes, intermediate forms, or trypomastigotes in sternum chondroblasts, osteoblasts, macrophages, and fibroblasts; chondrocyte and osteocyte invasion was rare. All isolates parasitized bone marrow macrophages, with few amastigotes. We observed marked associated myotropism, with or without inflammatory infiltration; there were small numbers of intensely parasitized mononuclear cells in perichondrium and periosteum. We discuss the results in relation to the marked differences of the T. cruzi tropism toward the different types of sternum cells, and, additionally, we outline the possibility of transmitting parasitized bone marrow through transplants. The fact of finding parasite stages in sternum bone and cartilage may be considered important due to the studies on Chagas’ disease paleoparasitology that are based on histological and molecular analysis.Declaration: The experiments conducted in this study comply with the current laws of Bioethics of the Venezuelan Ministry of Science and Technology (No. G-2005000406).     

Reversible acquisition of a host cell surface membrane antigen by Trypanosoma cruzi.

The ability of Trypanosoma cruzi, the etiological agent of Chagas' disease to acquire host cell surface antigen was tested. Parasites emerging after intracellular replication in WOS sarcoma monolayers expressed a sarcoma-associated surface antigen. This antigen was deleted from these parasites after replication in the MNS control monolayer, which does not express WOS sarcoma-associated surface antigen, or by replication in cell-free medium. This type of reversible acquisition of host surface antigen has not been previously described in T. cruzi or other intracellular protozoan parasites.     

A DTU-dependent blood parasitism and a DTU-independent tissue parasitism during mixed infection of Trypanosoma cruzi in immunosuppressed mice

Trypanosoma cruzi (Tc) diversity is determined by different biological, genetic, and biochemical markers and has been grouped into six discrete typing units (DTUs) or taxonomic groups (TcI–TcVI). This variability, coupled with natural reinfection or the hosts' immunosuppression, may play an important role in the pathogenesis of Chagas disease. Therefore, we evaluated the blood and tissue parasitism and genetic profile of mice coinfected with the TcII (JG) strain and TcI AQ1-7 (AQ) or MUTUM (MT) strains during the acute and chronic phases of the disease and during immunosuppression. T. cruzi blood populations in mixed infections were clearly associated with the TcII strain during acute and chronic phases or during immunosuppression. However, in tissues, the parasite populations were distributed according to the strain and the stage of infection. TcII populations overlapped TcI strains during the acute phase; in contrast, during chronic phase, both TcI strains were more prevalent than the TcII strain. The immunosuppression induced selective exacerbation of parasite populations, leading to reactivation of the TcII strain when associated with the AQ, but not with MT strain. Thus, a differential distribution of T. cruzi populations in blood and tissues with overlapping according to the stage of infection and strain used was observed. Blood parasitism was associated with the DTU TcII and tissue parasitism with a specific parasite strain and not with DTUs. Finally, to our knowledge, this is the first study to analyze subpatent blood parasitism and to simultaneously identify different T. cruzi populations in tissues and blood.     

The tumoricidal effect of Trypanosoma cruzi: its intracellular cycle and the immune response of the host

Many experimental evidences indicate that infection with Trypanosoma cruzi delays or inhibits the growth of malignant tumors in different strains of mice and in rats. These facts were verified by different workers. Although earlier workers proposed that this effect would be due to a toxin of T. cruzi, most of the accumulated evidences do not agree with such proposal. This present hypothesis agrees with the experimental data and proposes that the liberation of many endocellular antigens by destruction of some cancer cells, infected with T. cruzi, gives rise to an autoimmune response against antigens of analogous cancer cells, which limits or inhibits tumor growth. This point of view is supported by experimental studies on Chagas' disease which showed the role of T. cruzi, to induce autoimmune reactions against target organs of the disease. On the basis of this hypothesis I postulate a new way to stimulate the immune system of the host against cancer.     

Selective binding of Trypanosoma cruzi to host cell membrane polypeptides.

An adaptation of the immunoblotting technique was used to investigate binding interactions between Trypanosoma cruzi and mammalian host cells at the molecular level. A specific binding interaction was observed between T. cruzi and two host cell membrane polypeptides with molecular masses of approximately 32 and 34 kilodaltons. This molecular interaction was observed with antigen extracts of T. cruzi and with live, infective trypomastigote stages of the parasite, suggesting that the observed phenomenon may have relevance to the initial attachment of the parasite to the host cell membrane before invasion.     

Trypanosoma cruzi heparin-binding proteins and the nature of the host cell heparan sulfate-binding domain

Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)-binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S(35)]methionine labeled proteins eluted from heparin-sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi-cardiomyocyte interaction.     

Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi.

We have used antibodies to the Trypanosoma cruzi trans-sialidase and to its product, the host cell invasion-related Ssp-3 epitope, to study the expression of the corresponding antigens during the intracellular development of the parasite and in the extracellular trypomastigotes. As soon as 2 h after host cell invasion, trans-sialidase was no longer detected, whereas the Ssp-3 epitope was still present on intracellular parasites. The amastigotes which subsequently developed remained nonreactive with the antibodies. Expression of enzymatically active T. cruzi trans-sialidase started again only after transformation of the amastigotes into trypomastigotes 72 h after host cell invasion. trans-Sialidase was shed from the trypanosomes into the host cell cytoplasm, where the enzyme accumulated until release of the parasites. All released trypomastigotes expressed trans-sialidase on their surfaces and in the flagellar pockets, but stumpy trypomastigotes were stained more intensely than slender trypomastigotes. Ssp-3, the sialylated reaction product of trans-sialidase, was assembled only after rupture of the host cell membrane and was detected on the plasma membranes and in the flagellar pockets of all trypomastigotes.     

Trypanosoma cruzi infection induces a global host cell response in cardiomyocytes

Chagas' disease, caused by the hemoflagellate protozoan Trypanosoma cruzi, affects millions of people in South and Central America. Chronic chagasic cardiomyopathy, the most devastating manifestation of this disease, occurs in approximately one-third of infected individuals. Events associated with the parasite's tropism for and invasion of cardiomyocytes have been the focus of intense investigation in recent years. In the present study, we use murine microarrays to investigate the cellular response caused by invasion of primary murine cardiomyocytes by T. cruzi trypomastigotes. These studies identified 353 murine genes that were differentially expressed during the early stages of invasion and infection of these cells. Genes associated with the immune response, inflammation, cytoskeleton organization, cell-cell and cell-matrix interactions, apoptosis, cell cycle, and oxidative stress are among those affected during the infection. Our data indicate that T. cruzi induces broad modulations of the host cell machinery in ways that provide insight into how the parasite survives, replicates, and persists in the infected host and ultimately defines the clinical outcome of the infection.     

Reversible intracellular displacement of the cytoskeletal protein and organelles by ultracentrifugation of the sympathetic ganglion.

Superior cervical ganglia of rats were centrifuged at 40,000 rpm (160,500 g) for 1 h at 4 degrees C. Most neuronal somata exhibit a minor centripetal domain free of organelles and a major centrifugal domain rich in organelles. The former is occupied by numerous fine granules having low electron density unlike ribosomes in epoxy sections stained with uranium and lead, and is occupied by a meshwork of microtrabecular or filamentous elements similar to that of the centrifugal domain as well as that of the normal cells in PEG (polyethylene glycol)-processed embedment-free sections without staining. The latter centrifugal domain contains regular cell organelles except for neurofilaments without stratification. All the organelles are suspended in the meshwork of microtrabecular or filamentous elements. In immunolight microscopy, NFPs (neurofilament proteins) are confined to the centripetal domain. In immunoelectron microscopy using ultrathin cryosections and the protein A-gold labeling, numerous gold-particles for NFPs were deposited randomly in the centripetal cytoplasmic domain without long linear alignment. In the PEG sections the gold-labelings for NFPs are randomly deposited on portions of the microtrabecular strands in the centripetal domain. After incubation of the centrifuged ganglia in the anterior eye chamber overnight, NFPs-immunoreactivity appears again diffusely throughout the entire cytoplasm of all neuronal somata in immunolight microscopy. The organelle-free domain of the cytoplasm is no longer visible in electron microscopy. The present findings are discussed in relation to the state of the cytoplasmic soluble proteins and the reality of the microtrabecular or filamentous elements in the cytoplasm.     

Actin cytoskeleton-dependent and -independent host cell invasion by Trypanosoma cruzi is mediated by distinct parasite surface molecules

The disassembly of host cell actin cytoskeleton as a facilitator of Trypanosoma cruzi invasion has been reported by some authors, while other workers claim that it instead inhibits internalization of the parasite. In this study we aimed at elucidating the basis of this discrepancy. We performed experiments with metacyclic trypomastigotes of T. cruzi strains G and CL, which differ markedly in infectivity and enter target cells by engaging the surface molecules gp35/50 and gp82, respectively, which have signaling activity. Treatment of HeLa cells with the F-actin-disrupting drug cytochalasin D or latrunculin B inhibited the invasion by strain G but not the invasion by strain CL. In contrast to cells penetrated by strain CL, which were previously shown to have a disrupted actin cytoskeleton architecture, no such alteration was observed in HeLa cells invaded by strain G, and parasites were found to be closely associated with target cell actin. Coinfection with enteroinvasive Escherichia coli (EIEC), which recruits host cell actin for internalization, drastically reduced entry of strain CL into HeLa cells but not entry of strain G. In contrast to gp82 in its recombinant form, which induces disruption of F-actin and inhibits EIEC invasion, purified mucin-like gp35/50 molecules promoted an increase in EIEC uptake by HeLa cells. These data, plus the finding that drugs that interfere with mammalian cell signaling differentially affect the internalization of metacyclic forms of strains G and CL, indicate that the host cell invasion mediated by gp35/50 is associated with signaling events that favor actin recruitment, in contrast to gp82-dependent invasion, which triggers the signaling pathways leading to disassembly of F-actin.     

Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR

A real-time PCR technique that allows for accurate and sensitive quantitation of tissue parasite burden in animals infected with the protozoan parasite Trypanosoma cruzi was developed. The utility of this method was demonstrated by confirmation of higher parasite load in mice with acute infections in comparison to chronically infected mice, detection of tissue-restricted parasite persistence in different parasite:host strain combinations, and the observation of increased tissue parasite burden with higher infective doses. This method should be a useful tool for monitoring parasite burden in hosts under various treatment regimens.     

Antibody-induced capping of the intracellular stage of Trypanosoma cruzi.

Intracellular-stage Trypanosoma cruzi were isolated from infected mice. The effect of specific human antibodies on the membrane surface antigens was studied in vitro, using the immunofluorescent technique. The immune serum induced the aggregation of surface antigens, forming polar caps on the amastigotes.     

Trypanosoma cruzi: Inhibition of host cell uptake of infective bloodstream forms by alpha-2-macroglobulin

The infection of murine macrophages and fibroblasts by recently isolated infective bloodstream trypomastigotes ofTrypanosoma cruzi is inhibited by the addition of human plasma protease inhibitor alpha-2-macroglobulin (₂M) or of soybean trypsin inhibitor. The ingestion of the non-infective epimastigotes by macrophages is not affected by the physiological protease inhibitor. Incubation of bloodstream trypomastigotes for 20 h in a serum-free axenic medium enhances their ability to infect macrophages in a process influenced by the temperature and sensitive to ₂M. After this period the infectivity of the parasites to cells was not sensitive to ₂M. These observations suggest that proteases located on the surface and/or secreted by the bloodstream trypomastigote form ofT. cruzi may modulate its ability to infect host cells.     

Role of iron in intracellular growth of Trypanosoma cruzi

Host hypoferremic responses occur during infection with Trypanosoma cruzi, presumably through the transfer of iron to the sites of intracellular parasite replication. Depletion of host intracellular iron stores reduces parasite pathogenicity. It has therefore been hypothesized that T. cruzi requires iron for optimal growth in host cells and that, unlike extracellular pathogens, T. cruzi may benefit from host hypoferremic responses. These hypotheses were examined by the in vitro infection of peritoneal macrophages with T. cruzi. Various doses of desferrioxamine or referrated desferrioxamine were added to the culture medium, and parasite growth was monitored. The influence of treatment on uninfected macrophage morphology, function, and iron content was also verified. Desferrioxamine reduced the rate of amastigote replication in a dose-dependent fashion, whereas referrated desferrioxamine did not. The iron content of desferrioxamine-treated macrophages was decreased by 55% without provoking significant morphological or functional changes. Thus, amastigotes used host cell iron stores for optimal growth, and desferrioxamine reduced growth by depleting host cell iron. Hence, it was suggested that depletion of host intracellular iron stores may protect against T. cruzi and, furthermore, that host responses which transfer iron to the intracellular sites of T. cruzi replication may enhance parasite pathogenicity.     

Trypanosoma cruzi: experimental parasitism in the central nervous system of albino mice

Trypanosoma cruzi causes a pan-infection, Chagas disease, in American mammals through fecal transmission by triatomine insects, resulting in an acute phase parasitemia with intracellularity mainly in the myocells and cells of the central nervous system (CNS).The parasites, due to the immune response, then decrease in number, characteristic of the life-long chronicity of the disease. We infected a mouse model with isolates obtained from reservoirs and vectors from rural and urban endemic areas in Venezuela. Intracellular proliferation and differentiation of the parasite in astrocytes, microglia, neurons, endothelial cells of the piarachnoid, cells of the Purkinje layer, and spinal ganglion cells, as well as extracellularly in the neuropil, were evaluated during the acute phase. Damages were identified as meningoencephalitis, astrocytosis, reactive microglia, acute neuronal degeneration by central chromatolysis, endothelial cell hyperplasia, edema of the neuropil, and satellitosis. This is the first time that satellitosis has been reported from a mammal infected with T. cruzi. Intracellular T. cruzi and inflammatory infiltrates were found in cardiac and skeletal myocytes and liver cells. No parasitism or alterations to the CNS were observed in the chronic mice, although they did show myocarditis and myocitis with extensive infiltrates. Our results are discussed in relation to hypotheses that deny the importance of the presence of tissue parasites versus the direct relationship between these and the damages produced during the chronic phase of Chagas disease. We also review the mechanisms proposed as responsible for the nervous phase of this parasitosis.     

Mechanisms of invasion and replication of the intracellular stage in Trypanosoma cruzi

Amastigotes obtained from spleens of mice infected with different strains of Trypanosoma cruzi were examined for their ability to invade macrophages and L929 cells and to initiate infection in mice. Both types of cells were readily invaded by organisms of the strains Y, MR, and Tulahuen. Organisms of the CL strain were taken up by both types of cells at a rate that was significantly lower than that for organisms of the other strains. However, all strains multiplied intracellularly. Activated macrophages inhibited the replication of intracellular organisms. Treatment of normal macrophages with cytochalasin B, trypsin, chymotrypsin, or pronase significantly inhibited phagocytosis, but the inhibitory effect was reversible. Mice injected with spleen amastigotes developed parasitemia and died of the infection. These results demonstrate that spleen amastigotes are able to infect, survive, and replicate within professional and nonprofessional phagocytes and to initiate infection in vivo. Interiorization of spleen amastigotes is by phagocytosis and is dependent upon a protease-sensitive receptor(s) on the cell surfaces of host macrophages.     

Modulation of Trypanosoma cruzi adhesion to host muscle cell membranes by ligands of muscarinic cholinergic and beta adrenergic receptors

Plasma membrane vesicles (PMVs) of Trypanosoma cruzi adhered to L6 myoblast host cells as a function of time and concentration in saturation phenomena in a similar fashion to that reported in a previous publication. The initial adhesion rate (A0) of T. cruzi PMVs to L6 myoblasts in tissue culture was inhibited by acetylcholine (10(-5) M), isoproterenol (10(-5) M) and norepinephrine (10(-8) M) (range 29.1-50.3% of control). Atropine, the antagonist of muscarinic cholinergic receptors (10(-5) M), and propranolol or pindolol, the antagonists of beta-adrenergic receptors (10(-5) M), were also equal inhibitors of the T. cruzi PMV to L6 myoblast adhesion rate (range 26.1 to 55.5% of control). The alpha-adrenergic receptor ligands yohimbine and phentolamine (10(-5) M) showed no A0 inhibitory activity in similar assays. The interaction of T. cruzi PMVs with type I host muscle sarcolemma receptors was clearly defined in assays which used porcine heart atrial membranes (PAMs) immobilized on cationic polyacrylamide beads. In this parasite membrane-host cell membrane assay system, 10(-10) M atropine and 5 x 10(-9) M propranolol produced a shift of an S-shaped T. cruzi PMV to PAM saturation isotherm to the right, suggesting that a negative cooperative interaction was produced between the intramembrane ligand binding site and another, surface heterotropic T. cruzi PMV adhesion site. Atropine and propranolol were equieffective inhibitors of the T. cruzi striated muscle sarcolemma recognition process, raising the possibility that T. cruzi attachment molecules annexed pairs of muscarinic cholinergic and beta-adrenergic receptors to effect adhesion of the two membrane surfaces.     

Role of membrane N-linked oligosaccharides in host cell interaction with invasive forms of Trypanosoma cruzi

The removal of N-linked oligosaccharides by peptide-N4-[N-acetyl-beta-glucoseaminyl]asparagine amidase (previously known as aspartoglycosylamine amidohydrolase and abbreviated N-glycanase) from the surface of blood or insect-transmissible forms of Trypanosoma cruzi markedly increased the capacity of these organisms to associate with (i.e., bind and penetrate) either mouse peritoneal macrophages or rat heart myoblasts. This effect was evidenced by a significant elevation in both the percentage of infected host cells and the average number of parasites per 100 cells. Conversely, N-glycanase treatment of either host cell markedly reduced both parameters to levels significantly below those obtained with cells mock treated with medium alone. The N-glycanase effect on the parasites was inhibited by heat inactivation of the enzyme or by the presence of fetuin, an N-glycanase substrate. The enhanced capacity of N-glycanase-treated T. cruzi to engage the host cells started to subside 2 h after the treatment, indicating the reversibility of the effect. The decreased reactivity of N-glycanase-treated macrophages or myoblasts with T. cruzi suggests that N-linked oligosaccharides on these host cells are involved in the initial phase of the cell infection process. Instead, because T. cruzi interacted more effectively with host cells after treatment with N-glycanase, parasite surface N-linked oligosaccharides would seem to interfere with the association.     

Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (−)- cubebin (CUB) and (−)-hinokinin (HNK), was evaluated in the chronic phase of Chagas’ disease by quantifying the enzyme β-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas’ disease.