Trypanosoma cruzi: Impact of dual-clone infections on parasite biological properties in BALB/c mice

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 different dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our findings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. Significant changes in the expected values were observed in 4.2-79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of significant differences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas' disease.     

Evidence for multiple hybrid groups in Trypanosoma cruzi

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.     

Trypanosoma cruzi (Kinetoplastida Trypanosomatidae): ecology of the transmission cycle in the wild environment of the Andean valley of Cochabamba, Bolivia

An active Trypanosoma cruzi transmission cycle maintained by wild rodents in the Andean valleys of Cochabamba Bolivia is described. Wild and domestic Triatoma infestans with 60% infection with T. cruzi were found and was evidenced in 47.5% (rodents) and 26.7% (marsupial) by parasitological and/or serologycal methods. Phyllotis ocilae and the marsupial species Thylamys elegans, are the most important reservoirs followed by Bolomys lactens and Akodon boliviensis. In spite of both genotypes (TCI and TCII) being prevalent in Bolivia, in our study area only T. cruzi I is being transmitted. Our data suggest that wild T. infestans and wild small mammals play an important role in the maintenance of the transmission cycle of T. cruzi. Furthermore, the finding of high prevalence of T. cruzi infection in wild T. infestans point to the risk of the dispersion of Chagas' disease.     

Trypanosoma cruzi: Mutations in the 3' untranslated region of calmodulin gene are specific for lineages T. cruzi I, T. cruzi II, and the Zymodeme III isolates

Populations of Trypanosoma cruzi can be clustered in two main phylogenetic lineages, T. cruzi I and T. cruzi II and a third group denominated Zymodeme III (ZIII) has been described. Using 23 isolates representing the two major T. cruzi groups and the Zymodeme III, the 3' untranslated region (3'UTR) of the calmodulin gene was analyzed. Several mutations located on a 330 bp segment of this 3'UTR were observed, among which three important insertion/deletion events, namely (1) a dinucleotide AG present only in ZIII isolates; (2) a 13 bases purine block missing only in ZIII; and (3) a five base GT block in T. cruzi II. Minimum free energy dot plots show that T. cruzi I and T. cruzi ZIII exhibit similar patterns of optimal and sub-optimal folding of this segment. These mutations in 3'UTR of calmodulin raise the possibility that T. cruzi I and ZIII group are sharing common functional routes.     

Fibrillogenesis of apomyoglobin facilitated by aggregation sequence of yeast Sup35 in various regions

To examine the effect of aggregation sequence QGGYQQQYNP from yeast Sup35 on fibril formation of sperm whale apomyoglobin (apoMb), we constructed several mutants via substitution. Urea-induced unfolding of apoMb confirms that the substitution of the aggregation sequence does not significantly affect the stability of the mutants compared to wild type (WT) at pH 4.2. Under this condition, however, despite the difference in rate most apoMb mutants form fibrils more readily than WT with distinct morphology. These results suggest that the aggregation sequence facilitates fibril assembly of apoMb at acidic pH in vitro and this facilitation depends on the regions replaced.     

Real time PCR strategy for the identification of major lineages of Trypanosoma cruzi directly in chronically infected human tissues

Two evolutionary lineages, called Trypanosoma cruzi I and II, have been identified in T. cruzi, the etiologic agent of human Chagas disease. Here, we describe a molecular strategy for direct genetic typing of these major groups of T. cruzi directly in human tissues. The protocol is based on heminested PCR amplification of the D7 region of the 24Salpha ribosomal DNA (rDNA), followed by identification of the products using denaturation curves in real time PCR. The repetitive nature of the gene, and the heminested PCR format insured the high sensitivity necessary to detect the presence of the very scarce T. cruzi DNA present in the chronically infected human tissues. There is 80% DNA sequence homology between the two 24Salpha rDNA alleles that define the T. cruzi I and II groups, sufficient to produce different thermal denaturation curves with melting temperature (TM) values of 81.7+/-0.43 and 78.2+/-0.33 degrees C (mean+/-SEM). Using this technical approach, we analysed tissue samples (esophagi, hearts and colon) from 25 different patients with the gastrointestinal or cardiac forms of Chagas disease; in all of them we found only the presence of T cruzi II. Previous epidemiological and immunological findings had already led to the idea that chronic human infections occurring in Brazil and Argentina might be primarily due to T. cruzi II strains, but all the evidence available had been indirect. Our findings provide definitive proof of this hypothesis and will also allow the establishment of which group of T. cruzi is responsible for Chagas disease in other countries.     

Trypanosoma cruzi: acute and long-term infection in the vertebrate host can modify the response to benznidazole

We analyzed the influence of Trypanosoma cruzi maintenance in different hosts (dog and mouse) on its susceptibility to benznidazole treatment. Five T. cruzi stocks were isolated from dogs inoculated with Be-62 or Be-78 strain (both sensitive to benznidazole) 2-10 years ago, and the benznidazole sensitivity was then determined using the mouse as experimental model. The different T. cruzi stocks obtained from long-term infected dogs showed 50-90% drug resistance right after isolation. However, maintenance of these T. cruzi stocks in mice, by successive blood passages (2.5 years), led to either a decrease or stability of the drug resistance pattern and an increase in parasite virulence. We also demonstrated the effectiveness of the induction of parasitemia reactivation by cyclophosphamide immunosuppression in the evaluation of the response to the specific drug treatment.     

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization.     

Ancestral genomes, sex, and the population structure of Trypanosoma cruzi

Acquisition of detailed knowledge of the structure and evolution of Trypanosoma cruzi populations is essential for control of Chagas disease. We profiled 75 strains of the parasite with five nuclear microsatellite loci, 24Salpha RNA genes, and sequence polymorphisms in the mitochondrial cytochrome oxidase subunit II gene. We also used sequences available in GenBank for the mitochondrial genes cytochrome B and NADH dehydrogenase subunit 1. A multidimensional scaling plot (MDS) based in microsatellite data divided the parasites into four clusters corresponding to T. cruzi I (MDS-cluster A), T. cruzi II (MDS-cluster C), a third group of T. cruzi strains (MDS-cluster B), and hybrid strains (MDS-cluster BH). The first two clusters matched respectively mitochondrial clades A and C, while the other two belonged to mitochondrial clade B. The 24Salpha rDNA and microsatellite profiling data were combined into multilocus genotypes that were analyzed by the haplotype reconstruction program PHASE. We identified 141 haplotypes that were clearly distributed into three haplogroups (X, Y, and Z). All strains belonging to T. cruzi I (MDS-cluster A) were Z/Z, the T. cruzi II strains (MDS-cluster C) were Y/Y, and those belonging to MDS-cluster B (unclassified T. cruzi) had X/X haplogroup genotypes. The strains grouped in the MDS-cluster BH were X/Y, confirming their hybrid character. Based on these results we propose the following minimal scenario for T. cruzi evolution. In a distant past there were at a minimum three ancestral lineages that we may call, respectively, T. cruzi I, T. cruzi II, and T. cruzi III. At least two hybridization events involving T. cruzi II and T. cruzi III produced evolutionarily viable progeny. In both events, the mitochondrial recipient (as identified by the mitochondrial clade of the hybrid strains) was T. cruzi II and the mitochondrial donor was T. cruzi III.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

Protection of steroid hormone receptors by protease inhibitors

Steroid hormone receptors are proteolyzed by different types of enzymes present in target tissues. Effective protease inhibitors protecting steroid hormone receptors in various target tissues were investigated. Progesterone receptor (PR) in hen oviduct and estrogen receptor (ER) in cow uterus were specifically protected by relatively low concentrations (0.5 mM) of leupeptin or antipain (inhibitors of serine and thiol proteases). It was indicated that two different types of enzymes which modify native glucocorticoid receptor (GR) are present in rat liver. One was inhibited by 1 mM leupeptin or 1 mM antipain, while the other was inhibited by 1 mM phosphoramidon (inhibitor of thermolysin like proteases) or 10 mM sodium molybdate. Native PR, ER, and GR were shown to have similar Stokes radii (44 Å).     

Trypanosoma cruzi: Correlation of Growth Kinetics to Zymodeme Type in Clones Derived from Various Sources*†

SYNOPSIS. Trypanosoma ( Schizotrypanum ) cruzi clones were derived from isolates of an acute human case of Chagas' disease (strain Esmereldo), a human case of T. cruzi infection (strain CAN-III) and from a naturally infected opossum (strain WA-250). The isoenzyme patterns and growth rates of the clones were stable during long-term cultivation, by serial passages, of the parasites in liquid medium. Both clones of strain Esmereldo were zymodeme II; the 2 clones of strain CAN-III, zymodeme III; and the 5 clones of strain WA-250, zymodeme I. The range in doubling times of the parasite populations in liquid medium were 36–49. 7 h (strain WA-250), 117.2–133.7 h (Esmereldo clones) and 169–208 h (CAN-III clones).     

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Delta7-24, 25-dihydrolanosterol

Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.     

Trypanosoma cruzi: exploring the nuclear genome of zymodeme 3 stocks by chromosome size polymorphism

Chagas disease is emerging in the Brazilian Amazon. We evaluated the position of eight zymodeme 3 isolates from Amazonian sylvatic vectors and one human case in relation to Trypanosoma cruzi I and II major groups and hybrid strains by chromosome size polymorphism. Nineteen isolates were analyzed by mapping nine coding sequences on chromosomal bands (0.6-3.3Mbp). Numerical analysis was based on the absolute chromosomal size difference index (aCSDI). A dendrogram was obtained applying the minimum evolution criterion and considering the aCSDI values to estimate the branch lengths. The isolates were distributed in four groups. Group A clustered hybrid isolates; Groups B and C, T. cruzi II and T. cruzi I isolates, respectively. Seven Z3 stocks were clustered in Group D, which showed low intra-group diversity and was the most divergent. The proportion of two different-sized homologous chromosomes was determined. Wild vectors harboring Z3 stocks constitute a potential reservoir of human infection in the Amazon.     

The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.     

Biological and molecular characterization of a raccoon isolate of Trypanosoma cruzi from South Carolina

Biological and molecular characteristics of a raccoon isolate of Trypanosoma cruzi (R36) were compared with those of a known virulent strain (Brazil). Included in the characterization were growth rate in liver infusion tryptose medium, infectivity for murine fibroblasts, intracellular amastigote replication and trypomastigote release rates, polymerase chain reaction (PCR) profiling of the mini-exon gene, isoenzyme and random amplified polymorphic DNA (RAPD) profiles, and in vivo virulence for C3H/HeJ mice. Similar growth curves were noted for both strains; however, infectivity and rates of intracellular amastigote replication and trypomastigote release were significantly lower for the R36 isolate than for the Brazil strain. To determine virulence, C3H/ HeJ mice were exposed intraperitoneally to the R36 isolate. No parasite was observed in blood by direct examination or in tissues by histology; however, T. cruzi was detected by PCR in tissues (quadriceps and spleen) at 21 days postinfection. Analyses of the mini-exon gene, isoenzyme, and RAPD profiles indicate that R36 is in the T. cruzi II group and the Brazil strain is in the T. cruzi I group. Although infectivity and virulence of the raccoon isolate were lower than those for the Brazil strain, autochthonous infections in the United States have been reported, which suggests the need for further study of local T. cruzi isolates.     

Trypanosoma cruzi (kinetoplastida Trypanosomatidae): biological heterogeneity in the isolates derived from wild hosts

The course of experimental infection of Swiss mice with 95 sylvatic Trypanosoma cruzi isolates included in TCI or TCII genotype was characterized. The purpose was to verify biological properties and its eventual correspondence with original host species, genotype or zymodeme. The isolates of T. cruzi were 100% infective, 55% resulted in patent parasitemia with 69% (36/52) of mortality. A meaningful biological heterogeneity was observed in both, TCI and TCII isolates. TCII isolates resulted in higher patent parasitemia 64% (38/59), in contrast to the 41% TCI infected Swiss mice (14/34). Parasitemia was not always associated to mortality. Higher biological heterogeneity was observed in T. cruzi II isolates derived from L. rosalia from the Atlantic Coastal Rain forest. TCII isolates derived from marsupials resulted in very similar infection profile in Swiss mice.     

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.