Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon.

The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

Heritable integration of kDNA minicircle sequences from Trypanosoma cruzi into the avian genome: insights into human Chagas disease.

We demonstrate the genetic transfer of DNA between eukaryotes from different kingdoms. The mitochondrial kinetoplast DNA (kDNA) of the intracellular parasite Trypanosoma cruzi is transferred to human patients with Chagas disease. This transfer was reproduced experimentally in rabbits and chickens. The kDNA is integrated into the host genome. In the human chromosomes, five loci were identified as integration sites, and the beta-globin locus and LINE-1 retrotransposons were frequently targeted. Short repeated sequences in the parasite and the target host DNAs favor kDNA integration by homologous recombination. Introduced kDNA was present in offspring of chronically infected rabbits and in chickens hatched from T. cruzi-inoculated eggs. kDNA incorporated into the chicken germline was inherited through the F2 generation in the absence of persistent infection. kDNA integration represents a potential cause for the autoimmune response that develops in a percentage of chronic Chagas patients, which can now be approached experimentally.

Triatoma infestans apyrases belong to the 5'-nucleotidase family.

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.