Trypanosoma cruzi infection by oral route: how the interplay between parasite and host components modulates infectivity

Trypanosoma cruzi infection by oral route constitutes the most important mode of transmission in some geographical regions, as illustrated by reports on microepidemics and outbreaks of acute Chagas' disease acquired by ingestion of food contaminated with parasites from triatomine insects. In the mouse model, T. cruzi metacyclic trypomastigotes invade the gastric mucosal epithelium, a unique portal of entry for systemic infection. High efficiency of metacyclic forms in establishing infection by oral route is associated with expression of gp82, a stage-specific surface molecule that binds to gastric mucin and to epithelial cells. Gp82 promotes parasite entry by triggering the signaling cascades leading to intracellular Ca²⁺ mobilization. T. cruzi strains deficient in gp82 can effectively invade cells in vitro, by engaging the Ca²⁺ signal-inducing surface glycoprotein gp30. However, they are poorly infective in mice by oral route because gp30 has low affinity for gastric mucin. Metacyclic forms also express gp90, a stage-specific surface glycoprotein that binds to host cells and acts as a negative regulator of invasion. T. cruzi strains expressing gp90 at high levels, in addition to gp82 and gp30, are all poor cell invaders in vitro. Notwithstanding, their infectivity by oral route may vary because, unlike gp82 and gp30, which resist degradation by pepsin in the gastric milieu, the gp90 isoforms of different strains have varying susceptibility to peptic digestion. For instance, in a T. cruzi isolate, derived from an acute case of Chagas' disease acquired by oral route, gp90 is extensively degraded by gastric juice in the mouse stomach and this renders the parasite highly invasive towards target cells. If such an exacerbation of infectivity occurs in humans, it may be responsible for the severity of the disease reported in outbreaks of oral infection.     

Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Trypanosoma cruzi surface molecule gp90 downregulates invasion of gastric mucosal epithelium in orally infected mice

Experiments were performed to elucidate why Trypanosoma cruzi isolates 573 and 587 differ widely in their efficiency to infect gastric mucosal epithelium when administered orally to mice. These isolates have the same surface profile and a similar capacity to enter host cells in vitro. Metacyclic forms of isolates 573 and 587 and the control CL isolate expressed similar levels of gp82, which is a cell invasion-promoting molecule. Expression of gp90, a molecule that downregulates cell invasion, was lower in the CL isolate. Consistent with this profile, approximately threefold fewer parasites of isolates 573 and 587 entered epithelial HeLa cells, as compared to the CL isolate. No difference in the rate of intracellular parasite replication was observed between isolates. When given orally to mice, metacyclic forms of isolate 573, like the CL isolate, produced high parasitemia (>10(6) parasites per ml at the peak), killing approximately 40% of animals, whereas infection with isolate 587 resulted in low parasitemia (<10(5) parasites per ml), with zero mortality. On the fourth day post-inoculation, tissue sections of the mouse stomach stained with hematoxylin and eosin showed a four to sixfold higher number of epithelial cells infected with isolate 573 or CL than with isolate 587. The rate of intracellular parasite development was similar in all isolates. Mimicking in vivo infection, parasites were treated with pepsin at acidic pH and then assayed for their ability to enter HeLa cells or explanted gastric epithelial cells. Pepsin extensively digested gp90 from isolate 573 and significantly increased invasion of both cells, but had minor effect on gp90 or infectivity of isolates 587 and CL. The profile of g82 digestion was similar in isolates 573 and 587, with partial degradation to a approximately 70 kDa fragment, which preserved the target cell binding domain as well as the region involved in gastric mucin adhesion. Gp82 from CL isolate was resistant to pepsin. Assays with parasites recovered from the mouse stomach 2 h after oral infection showed an extensive digestion of gp90 and increased infectivity of isolate 573, but not of isolate 587 or CL. Our data indicate that T. cruzi infection in vitro does not always correlate with in vivo infection because host factors may act on parasites, modulating their infectivity, as is the case of pepsin digestion of isolate 573 gp90.     

Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages

, , and 1986. Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages. International Journal for Parasitology 16: 629–632. Prelabelling of lysosomes with acridine orange has been performed in order to verify whether metacyclic forms of Trypanosoma cruzi are capable of inhibiting lysosomal fusion during the first moments of interiorization in non-sensitized mouse peritoneal macrophages. Thus, the degree of degranulation (lysosomal fusion) in metacyclic forms is low while epimastigote forms present higher levels. When epimastigote forms are made to interact with the macrophages in the presence of various concentrations of the medium used for transformations of epimastigotes to metacyclic forms or when interaction was performed in the presence of NH₄Cl, the degree of degranulation was similar to that obtained when interaction was carried out with metacyclic forms.

The present results suggest that during the first moments of the interaction of T. cruzi, only the infective forms may increase the cytoplasmic pH value of the host phagocytic cell, avoiding lysosomal fusion and the subsequent destruction of the parasite.     

Trypanosoma cruzi: skin-penetration kinetics of vector-derived metacyclic trypomastigotes

In order to investigate the natural route of infection of nude and normal BALB/c mice with Trypanosoma cruzi via the skin, a drop of vector faeces/urine containing metacyclic trypomastigotes was placed onto the puncture site of a bite from Triatoma infestans. The periods of exposure, i.e. until removal of flagellates from the skin, and the time elapsed until surgical removal of the skin around the puncture were varied. After 15 min of exposure, T. cruzi developed in all nude mice without surgery, and in four of 10 mice if the puncture region of the skin was removed directly after exposure. In a shaved puncture region, 5 min of exposure were sufficient to infect all normal BALB/c mice without surgery and one of four mice with direct removal of the puncture region. Longer periods of exposure or time until removal of the skin only sometimes resulted in higher infection rates. Prepatent periods and the development of parasitaemia varied irrespective of the period of exposure or the period until skin removal at the puncture site. The importance of these findings is that they clearly prove that T. cruzi can rapidly invade the host via the puncture site of the bite of the vector and that at least some parasites are immediately transported away from this site.     

Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas'' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

Correlation between conformation and antibody binding: NMR structure of cross-reactive peptides from T. cruzi, human and L. braziliensis

The structure of peptides corresponding to the C-terminal residues from Trypanosoma cruzi (R13), human (H13) and Leishmania braziliensis (A13) ribosomal proteins were determined using nuclear magnetic resonance. Although there is only one amino acid difference between them, the peptides present distinct structures in solution: R13 adopts a random coil conformation while H13 and A13 form a bend. Interaction of these peptides with polyclonal antibodies from chronic Chagas’ disease patients and a monoclonal antibody raised against T. cruzi ribosomal P2β protein was probed by transferred NOE. The results show that the flexibility of R13 is fundamental for the binding to the antibody.     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH(4)Cl, which releases Ca(2+) from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.     

Synthesis and anti-Trypanosoma cruzi activity of derivatives from nor-lapachones and lapachones

New naphthoquinone derivatives were synthesized and assayed against bloodstream trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas’ disease. The compounds were rationalized based on hybrid drugs and appear as important compounds against this parasite. From nor-lapachol were prepared five substituted ortho-naphthofuranquinones, a non-substituted para-naphthofuranquinone, a new oxyrane and an azide and from -lapachone a new non-substituted para-naphthofuranquinone. Other five substituted ortho-naphthofuranquinones recently designed as cytotoxic, were also evaluated. The most active compounds were the ortho naphthofuranquinones 3-(4-methoxyphenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione and 3-(3-nitrophenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione with trypanocidal activity higher than that of benznidazole, the standard drug. The compounds were rationalized based on hybrid drugs and appear as important compounds against T. cruzi. The trypanocidal activity of these substances endowed with redox properties representing a good starting point for a medicinal chemistry program aiming the chemotherapy of Chagas’ disease.     

Structure and expression of three gp82 gene subfamilies of Trypanosoma cruzi

The glycoprotein gp82 is a GPI-anchored cell surface protein of Trypanosoma cruzi and is involved in cell invasion. Gp82 is encoded by multiple genes. To investigate the genetic basis of its biological function, we analyzed structure and expression of gp82 multigene family members in the Peruvian and Guatemalan strains. Three major groups of gp82 genes (A, B and C) were categorized by analyzing multiple DNA clones from the genomic PCR products. Within each group, 95–97% homology was observed, whereas between the groups, homology was 67–79%. The copy numbers of groups A, B and C as determined by real-time PCR were 18, 8 and 7 copies, respectively, in the Peru-2 strain. Significant elevation of the mRNA expression levels (5–10 times more) of all the subfamily genes was observed in the metacyclic stage compared with the epimastigote stage. When we focused on the binding motif sequence reported previously, we found substantial difference between that of A and C. However, the peptide inhibition invasion assay showed no functional difference. Taken together, we demonstrated that three subfamilies of gp82 were in the genome of T. cruzi and maintained their functional structure, and that the mRNA expressions of those genes were equally controlled in a stage-specific manner.     

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.     

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiophen-4,9-quinones

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reductase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 μM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 °C against trypomastigotes in murine blood as well against the enzyme trypanothione reductase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.     

Laboratory maintenance of Trypanosoma (Herpetosoma) rangeli Tejera, 1920

Two laboratory maintenance systems of Trypanosoma rangeli were compared. The maintenance by weekly subinoculations in Tobie's culture medium and the intrafemoral inoculation of Rhodnius prolixus with cultured flagellates, resulted in loss of infectivity of the metacyclic salivarian trypomastigotes for mice, ten months after maintenance in culture. With the system of cyclical passes through culture-Rhodnius-mouse-culture-Rhodnius, the infectivity of the metacyclic trypomastigotes for mice, was maintained during the three years of the experiment. The number and percentage of metacyclic trypomastigotes formed in the salivary glands of R. prolixus, previously inoculated intrafemorally or intracoelomically with culture forms of T. rangeli, did not show correlation with the inoculated dose, however the inoculated quantity demonstrated a direct relation with the mortality rate of the insects. The results indicate that T. rangeli requires an adequate maintenance system, so that under experimental condition the biological characteristics, normally expressed under natural conditions, are conserved.     

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.     

Structural Basis of the Interaction of a Trypanosoma cruzi Surface Molecule Implicated in Oral Infection with Host Cells and Gastric Mucin

Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogenic microorganisms. As concerns Trypanosoma cruzi, which causes Chagas disease, the insect vector-derived metacyclic trypomastigotes (MT) initiate infection by invading host cells, and later blood trypomastigotes disseminate to diverse organs and tissues. Studies with MT generated in vitro and tissue culture-derived trypomastigotes (TCT), as counterparts of insect-borne and bloodstream parasites, have implicated members of the gp85/trans-sialidase superfamily, MT gp82 and TCT Tc85-11, in cell invasion and interaction with host factors. Here we analyzed the gp82 structure/function characteristics and compared them with those previously reported for Tc85-11. One of the gp82 sequences identified as a cell binding site consisted of an α-helix, which connects the N-terminal β-propeller domain to the C-terminal β-sandwich domain where the second binding site is nested. In the gp82 structure model, both sites were exposed at the surface. Unlike gp82, the Tc85-11 cell adhesion sites are located in the N-terminal β-propeller region. The gp82 sequence corresponding to the epitope for a monoclonal antibody that inhibits MT entry into target cells was exposed on the surface, upstream and contiguous to the α-helix. Located downstream and close to the α-helix was the gp82 gastric mucin binding site, which plays a central role in oral T. cruzi infection. The sequences equivalent to Tc85-11 laminin-binding sites, which have been associated with the parasite ability to overcome extracellular matrices and basal laminae, was poorly conserved in gp82, compatible with its reduced capacity to bind laminin. Our study indicates that gp82 is structurally suited for MT to initiate infection by the oral route, whereas Tc85-11, with its affinity for laminin, would facilitate the parasite dissemination through diverse organs and tissues.     

Infectivity and route of penetration in rats after oral and intraperitoneal inoculations of bloodstream and in vitro-cultured metacyclic forms of Trypanosoma lewisi

Morphological changes of Trypanosoma lewisi blood trypomastigotes cultured in Schneider's Drosophila medium (SDM), supplemented or not with uric acid (SDM + UA), were compared to those that occurred in a control medium (M-199). No difference in trypanosome morphology and numbers was observed between SDM + UA and SDM cultures; there was little transformation into metacyclic stages in M-199. No difference was observed between the capacity of SDM- or SDM + UA-cultured metacyclic stages to infect rats. The infectivity of bloodstream forms was always higher than that of the SDM- or SDM + UA-cultured forms, whether inoculated orally or intraperitoneally. The oral inoculation of rats with tritium-labeled culture and bloodstream forms showed that the metatrypanosomes from the cultures remained longer in the salivary glands and tongue of the animal than the blood trypanosomes.     

Comparison of the infectivity of Trypanosoma cruzi insect-derived metacyclic trypomastigotes after mucosal and cutaneous contaminative challenges

Trypanosoma cruzi infects humans when infected triatomine vector excreta contaminate breaks in skin or mucosal surfaces. T. cruzi insect-derived metacyclic trypomastigotes (IMT) invade through gastric mucosa after oral challenges without any visible inflammatory changes, while cutaneous and conjunctival infections result in obvious local physical signs. In this study we compared the infectivity of T. cruzi IMT in mice after cutaneous and oral contaminative challenges simulating natural infections. The 50% infective dose (ID50) for oral challenge was 100 fold lower than the ID50for cutaneous challenge, indicating that oral mucosal transmission is more efficient than cutaneous transmission.