Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.     

DL-alpha-difluoromethylarginine inhibits intracellular Trypanosoma cruzi multiplication by affecting cell division but not trypomastigote-amastigote transformation.

DL-alpha-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), decreases the capacity of Trypanosoma cruzi to invade and multiply within different types of mammalian host cells in vitro. In this work we found that inhibition of intracellular growth results from selective impairment of amastigote division without appreciable alteration of the capacity of the invading trypomastigotes to transform into the replicative amastigote form. Addition of agmatine, the product of arginine decarboxylation, reversed the inhibitory effect of DFMA. Inhibition of ornithine decarboxylase activity by DL-alpha-difluoromethylornithine present in the medium prior to and during infection did not affect trypomastigote transformation or amastigote replication and did not change the magnitude of the inhibitory effect of DFMA on parasite multiplication. Hence, neither polyamine synthesis via the ornithine decarboxylase pathway nor salvage of host cell polyamines by T. cruzi appeared to be a likely explanation for the normal rate of parasite transformation that was seen in the presence of DFMA. Two clones of T. cruzi, TMSU-1 and TMSU-2, were tested for their degrees of sensitivity to the inhibitory effects of DFMA. Both trypomastigote association with (i.e., binding to and penetration of) myoblasts, and intracellular amastigote multiplication by either clone were found to be significantly (P less than 0.05) but not completely inhibited by DFMA. Therefore, the partial inhibition of T. cruzi infectivity and replication caused by DFMA is unlikely to represent a composite of effects of the drug on DFMA-sensitive and insensitive clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic Heterogeneity in Trypanosoma cruzi Strains From Naturally Infected Triatomine Vectors in Northeastern Brazil: Epidemiological Implications

Eighteen Trypanosoma cruzi strains isolated from naturally infected triatomines were studied genetically. The majority of the strains were from Triatoma brasiliensis, the principal vector of Chagas disease in the northeast of Brazil. Multilocus enzyme electrophoresis (MLEE) and randomly amplified polymorphic DNA (RAPD) analyses were used to investigate the genotypic diversity and the spread of the T. cruzi genotypes in different environments. MLEE clearly distinguished two distinct isoenzyme profiles, and RAPD analysis revealed 10 different genotypes circulating in rural areas. The strains could be typed as isoenzyme variants of the T. cruzi principal zymodeme Z1 (T. cruzi I). An effective program of epidemiological vigilance is required to prevent the spread of T. cruzi I strains into human dwellings.     

In vitro culture and biochemical characterization of six trypanosome isolates from Peru and Brazil

Six trypanosomatids isolated from different geographical areas from South America (Peru and Brazil) and different vectors and reservoir hosts (the triatomine Panstrongylus chinai [TP1], Triatoma infestans [TP2], Rhodnius ecuadorensis [TP3], R. prolixus [TB1], Didelphys marsupialis [TB2]), and one from a human asymptomatic patient [TB3], were characterized using lectin agglutination, isoenzyme profile, in vitro culture final metabolite patterns, and compared with a reference strain (Trypanosoma cruzi, Maracay strain [TC]). The different isolates were cultured in vitro in Grace's medium supplemented with 10% inactivated bovine foetal serum. According to our results and the statistical study, the isolate obtained from R. ecuadorensis should be designed as a Trypanosoma rangeli sp., showing all other isolates strong similarities to T. cruzi. Between them, two clusters could be identified, strongly correlating with the geographical origin. Cluster I grouped isolates from Peru and T. cruzi reference strain, and cluster II grouped the three Brazilian isolates.     

Use of proteoliposome as a vaccine against Trypanosoma cruzi in mice

We have generated proteoliposomes carrying proteins of Trypanosoma cruzi for use as immunogens in BALB/c mice. T. cruzi trypomastigote and amastigote forms were sonicated and mixed with SDS, with 94% recovery of soluble proteins. To prepare proteoliposomes, we have used a protocol in which dipalmitoylphosphatidylcholine, dipalmitoyl-phosphatidylserine and cholesterol were incubated with the parasite proteins. BALB/c mice immunized with 20microg were able to generate antibodies which, in Western blotting, reacted with the proteins of T. cruzi. We further investigated the ability of peritoneal cells from immunized mice to arrest the intracellular replication of trypomastigotes, in vitro. After 72h of culture, the number of intracellular parasites in immunized macrophages decreased significantly, as compared to controls. Despite the fact that exposure of mice to T. cruzi proteins incorporated into proteoliposomes generate antibodies and activate macrophages, the immunized mice were not protected against T. cruzi intraperitoneal challenge.     

Trypanosoma cruzi: subtractive hybridization as a molecular strategy to generate new targets to distinguish groups and hybrids

RAPD analysis and sequences of the mini-exon and ribosomal genes show that Trypanosoma cruzi can be clustered into two phylogenetic groups-T. cruzi I and II. Herein, the Representational Difference Analysis (RDA) method was used, providing new targets specific for each group. After three rounds of RDA hybridizing F strain (tester) with Y strain (driver) and vice-versa, an F-specific (F#30) and Y-specific (Y#22) clone were obtained specifically recognizing isolates from Amazonas (T. cruzi I) and Piauí (T. cruzi II). These segments corresponded to an unspecified protein (F#30) and a trans-sialidase (Y#22). Analysis of the F#30 sequence in T. cruzi I, T. cruzi II and zymodeme 3 samples displayed negligible specific differences that distinguished each group. In addition this F#30 gene has great potential as a hybrid marker.     

Production and characterization of clones of the Brazil strain of Trypanosoma cruzi

Six clones and 4 subclones were isolated from the Brazil strain of Trypanosoma cruzi and were passaged in C3H(He) mice. Parasitemia levels and survival times of mice infected with 8 of the isolates were equivalent to the Brazil strain in virulence. Two clones, designated WFTc-5.1 and WFTc-6.1 (WFTc = Wake Forest Trypanosoma cruzi) were of lower virulence in C3H mice than the other isolates and the Brazil strain. C57BL/6 mice infected with WFTc-5.1 had significantly lower parasitemias and higher survival rates than C57BL/6 mice infected with the Brazil strain or a clone designated WFTc-3.2. Levels of anti-T. cruzi IgM and IgG antibodies were the same in mice infected with higher virulence or lower virulence isolates. Based on these results the Brazil strain of T. cruzi is composed of distinct subpopulations which are heterogeneous with respect to virulence.     

The anticancer drug tamoxifen is active against Trypanosoma cruzi in vitro but ineffective in the treatment of the acute phase of Chagas disease in mice

The activity of the antineoplastic drug tamoxifen was evaluated against Trypanosoma cruzi. In vitro activity was determined against epimastigote, trypomastigote and amastigote forms of CL14, Y and Y benznidazole resistant T. cruzi strains. Regardless of the strain used, the drug was active against all life-cycle stages of the parasite with a half maximal effective concentration ranging from 0.7-17.9 μM. Two experimental models of acute Chagas disease were used to evaluate the in vivo efficacy of treatment with tamoxifen. No differences in parasitemia and mortality were observed between control mock-treated and tamoxifen-treated mice.     

Differential expression of Trypanosoma cruzi I associated with clinical forms of Chagas disease: overexpression of oxidative stress proteins in acute patient isolate

Chagas disease has a variable clinical course with different manifestations and heterogenous geographical distribution. Some studies suggest that this clinical variability could be influenced by the genetic variability of T. cruzi. Here we present the differential protein expression among trypomastigotes and amastigotes of T. cruzi group I isolates from patients with acute and chronic form of Chagas disease from Santander, Colombia. A total of 29 proteins were identified by MALDI-TOF and LC-MS/MS; twenty in trypomastigote and nine in amastigote stage. The 29 proteins identified were grouped in 7 functional categories: 1) metabolism 31%, 2) assembly of cytoskeleton 13.7%, 3) protein destination 13.7%, 4) defenses antioxidants 20.6%, 5) protein synthesis and cellular cycle 13.7%, 6) catabolism 6.8%, and 7) adhesion 3.4%. Tryparedoxin peroxidase, lipoamide dehydrogenase, tyrosine amino transferase and HSP70 were overexpressed in the acute Chagas isolate. Tryparedoxin peroxidase overexpression in the acute isolate was confirmed by Western blot analysis. Most of these proteins are associated with resistance to oxidative stress facilitating their survival within host cells. Therefore, these proteins may represent virulence factors associated with the development of the acute form of the disease and could be used as biomarkers of the clinical course of disease and as drug targets.     

Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.     

New enzyme immunoassays for specific assay and general detection of Trypanosoma cruzi, epimastigotes.

A highly specific competitive enzyme-linked immunosorbent assay for the epimastigote of Tulahuen strain was developed by using the usual 3 immunological reagents, a rabbit antiserum specific for T. cruzi, epimastigote of Tulahuen strain, beta-D-galactosidase-labeled goat anti-rabbit immunoglobulin G and the solid-phase cell fragments of the epimastigote of Tulahuen strain. A new method, the selected antibody enzyme immunoassay (SAEIA) which generally detected all strains of the epimastigote tested with the same working range, was developed by changing only the solid-phase antigen to the epimastigote of Y strain among the 3 immunological reagents. Both assays permitted us to measure accurately as little as 1,000 parasites per assay tube. Scope of the SAEIA was limited to the epimastigote. Both life-cycle forms of T. cruzi which appear in mammals, amastigote and trypomastigote, and other kinetoplastids showed low cross-reaction values by the assay. The assay principle of the new method and a preliminary study to apply the SAEIA for finding the field T. cruzi-infected insect vectors were also reported.     

Randomly Amplified Polymorphic DNA (RAPD) and Isoenzyme Analysis of Trypanosoma rangeli Strains

ABSTRACT. Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co-infections with Trypanosoma cruzi , demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of T. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.     

Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages

In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T. cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes. No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T. cruzi II) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T. cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T. cruzi I extracellular amastigotes, especially of the G strain, were more infective than T. cruzi II parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. The fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process.     

Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts

Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.     

Trypanosoma cruzi in wild raccoons and opossums in North Carolina.

Trypanosoma cruzi was isolated from 1 of 12 (8.3%) opossums and 3 of 20 (15%) raccoons from the piedmont area of North Carolina. Although T. cruzi has been isolated previously from wild mammals in the southern United States, the present study is the first published report of naturally occurring T. cruzi infection of wild mammals in North Carolina. All 4 isolates were maintained successfully in axenic culture and in murine fibroblasts. In addition, intraperitoneal injection of 1 x 10(6) culture forms of 1 of the opossum isolates into C3H mice resulted in low but detectable parasitemias as early as day 6 of infection. These mice resolved parasitemia and survived infection. Intraperitoneal injection of 1 x 10(6) culture forms of a raccoon isolate resulted in the death of 3 out of 4 mice. Surprisingly, parasitemias were never detected in the peripheral blood of these mice. Infection of murine fibroblasts in vitro resulted in the presence of intracellular amastigote stages characteristic of T. cruzi.     

Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagle dogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.     

Pre-Columbian Chagas disease in Brazil: Trypanosoma cruzi I in the archaeological remains of a human in Peruaçu Valley, Minas Gerais, Brazil

We evaluated the presence and distribution of Trypanosoma cruzi DNA in a mummy presenting with megacolon that was dated as approximately 560 +/- 40 years old. The mummy was from the Perua?u Valley in the state of Minas Gerais, Brazil. All samples were positive for T. cruzi minicircle DNA, demonstrating the presence and broad dissemination of the parasite in this body. From one sample, a mini-exon gene fragment was recovered and characterized by sequencing and was found to belong to the T. cruzi I genotype. This finding suggests that T. cruzi I infected humans during the pre-Columbian times and that, in addition to T. cruzi infection, Chagas disease in Brazil most likely preceded European colonization.     

Growth characteristics in axenic and cell cultures, protein profiles, and zymodeme typing of three Trypanosoma cruzi isolates from Louisiana mammals

Rates of trypomastigote adherence, interiorization, amastigote division in, and trypomastigote release from Vero cells were measured for Trypanosoma cruzi isolates from a dog (Tc-D), opossum (Tc-O), and an armadillo (Tc-A) from Louisiana. Because the Tc-O and Tc-A (wild isolates) trypomastigotes became interiorized rapidly, the media were quickly depleted of trypomastigotes thus reducing the numbers available to adhere to cells. In contrast, the Tc-D trypomastigote interiorization rate was slower. Intracellular amastigote division rate was slower for the Tc-D than the wild isolates. The Tc-D trypomastigotes were released from cells approximately 2 days later than wild isolate trypomastigotes, but twice the number were released. Growth rate for Tc-D epimastigotes in liver infusion tryptose media was faster than that of wild isolates. The doubling times for Tc-D, Tc-O, and Tc-A were 48.0, 69.0, and 67.4 hr, respectively. Soluble parasite extract was produced from epimastigotes of each isolate by freeze/thawing, sonication, and high-speed centrifugation. Proteins were separated on an SDS-PAGE slab gel and stained with Coomassie blue. Although similar bands were present in each preparation, the general pattern of staining was similar only between the Tc-O and Tc-A preparations, which showed some differences from the Tc-D preparation. Each isolate was zymodeme typed using 5 enzymes in lysates produced from epimastigotes of each isolate. Enzymes were separated electrophoretically and stained. Wild isolates showed similar patterns as zymodeme 1 reference stock, whereas the Tc-D isolate produced a pattern that did not resemble any of the reference stocks examined.     

Characterization of Trypanosoma rangeli strains isolated in Central and South America: an overview

Trypanosoma rangeli is a hemoflagelate parasite that infects domestic and sylvatic animals, as well as man, in Central and South America. T. rangeli has an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, sharing several animal reservoirs and triatomine vectors. We have isolated T. rangeli strains in the State of Santa Catarina, in southern Brazil, which dramatically increased the distribution area of this parasite. This brief review summarizes several studies comparing T. rangeli strains isolated in Santa Catarina with others isolated in Colombia, Honduras and Venezuela. The different methods used include indirect immunofluorescence and western blot assays, lectin agglutination, isoenzyme electrophoresis and random amplified polymorphic DNA analysis, triatomine susceptibility, in vitro cell infection assays, and mini-exon gene analysis.     

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility. Two Trypanosoma cruzi isolates, TCR-4 from Costa Rica and UES-1 from El Salvador, were studied in vitro to compare their infectivity or resistance and intracellular replication in myocardial cells in three strains of mice and rats: NGP white mice, C3 H mice and Sprague Dowley rats. Myocardial cells were cultured on coverslips at 37 degrees C in a humid 10% CO2 atmosphere and then infected at a ratio of one tripomastigote per cell. Samples were studied after 24, 72, 96 and 120 h of infection to determine parasite infection capacity and intracellular multiplication. Both parasites had the highest infection capacity in C3 H mice, followed by NGP mice cells with a very low infection rate. Lastly, almost no Trypanosoma cruzi multiplication was observed in Sprague Dowley rats, suggesting a strong natural resistance in this animal to both strains of the parasite. The UES-1 isolate presented higher multiplication and greater invasion than the TCR-4 strain, showing greater virulence of UES-1 in heart cells, at least in vitro.