The Influence of Recombinational Processes to Induce Dormancy in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects around 8 million people worldwide. Chagas disease can be divided into two stages: an acute stage with high parasitemia followed by a low parasitemia chronic stage. Recently, the importance of dormancy concerning drug resistance in T. cruzi amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different T. cruzi DTUs during their replicative epimastigote and amastigote stages. For this study, cells of T. cruzi CL Brener (DTU TcVI); Bug (DTU TcV); Y (DTU TcII); and Dm28c (DTU TcI) were used. In order to determine the proliferation rate and percentage of dormancy in epimastigotes, fluorescent-labeled cells were collected every 24 h for flow cytometer analysis, and cells showing maximum fluorescence after 144 h of growth were considered dormant. For the quantification of dormant amastigotes, fluorescent-labeled trypomastigotes were used for infection of LLC-MK2 cells. The number of amastigotes per infected LLC-MK2 cell was determined, and those parasites that presented fluorescent staining after 96 h of infection were considered dormant. A higher number of dormant cells was observed in hybrid strains when compared to non-hybrid strains for both epimastigote and amastigote forms. In order to investigate, the involvement of homologous recombination in the determination of dormancy in T. cruzi, we treated CL Brener cells with gamma radiation, which generates DNA lesions repaired by this process. Interestingly, the dormancy percentage was increased in gamma-irradiated cells. Since, we have previously shown that naturally-occurring hybrid T. cruzi strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from T. cruzi clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this T. cruzi CL Brener RAD51 mutant, evidencing a role of homologous recombination in the process of dormancy in this parasite. Altogether, our data suggest the existence of an adaptive difference between T. cruzi strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.

Selective Antiprotozoal Activity of Nitric Oxide-releasing Chitosan Nanoparticles Against Trypanosoma cruzi: Toxicity and Mechanisms of Action.

BACKGROUND: Chagas' disease, caused by Trypanosoma cruzi, was described for the first time over a hundred years ago. Nonetheless, clinically available drugs still lack effective and selective properties. Nitric oxide (NO) produced by activated macrophages controls the progression of disease by killing the parasite. METHODS AND RESULTS: Here, chitosan nanoparticles (CS NPs) were synthesized and mercaptosuccinic acid (MSA), the NO donor precursor, was encapsulated into CS NPs, forming MSA-CS NPs, which had hydrodynamic size of 101.0±2.535 nm. Encapsulated MSA was nitrosated forming NO donor S-nitrosomercaptosuccinic acid-containing nanoparticles (S-nitroso-MSA-CS NPs). Kinetic data revealed a sustained release of NO from the nanoparticles. S-nitroso-MSA-CS NPs inhibited epimastigote proliferation and trypomastigote viability of T. cruzi, with IC50=75.0±6.5 µg·mL-1 and EC50=25.0±5.0 µg·mL-1, respectively. Treatment of peritoneal macrophages with nanoparticles decreased the number of T. cruzi-infected cells and the average number of intracellular replicative amastigotes per infected cells. Besides, the results have showed a selective behaviour of S-nitroso-MSA-CS NPs to parasites. Morphological and biochemical changes induced by these NO-releasing nanoparticles, such as cell shrinkage, cell cycle arrest, mitochondrial membrane depolarization and phosphatidylserine exposure on cell surface indicate that epimastigotes death is associated to the apoptotic pathway. CONCLUSION: S-nitroso-MSA-CS NPs are promising nanocarriers for the treatment of Chagas's disease.

Dissecting biochemical peculiarities of the ATPase activity of TcSub2, a component of the mRNA export pathway in Trypanosoma cruzi.

The RNA helicase DEAD-box protein Sub2 (yeast)/UAP56 (mammals) is conserved across eukaryotes and is essential for mRNA export in trypanosomes. Despite the high conservation of Sub2 in lower eukaryotes such as Trypanosoma cruzi, the low conservation of other mRNA export factors raises questions regarding whether the mode of action of TcSub2 is similar to that of orthologs from other eukaryotes. Mutation of the conserved K87 residue of TcSub2 abolishes ATPase activity, showing that its ATPase domain is functional. However, the Vmax of TcSub2 was much higher than the Vmax described for the human protein UAP56, which suggests that the TcSub2 enzyme hydrolyzes ATP faster than its human homolog. Furthermore, we demonstrate that RNA association is less important to the activity of TcSub2 compared to UAP56. Our results show differences in activity of this protein, even though the structure of TcSub2 is very similar to UAP56. Functional complementation assays indicate that these differences may be common to other trypanosomatids. Distinct features of RNA influence and ATPase efficiency between UAP56 and TcSub2 may reflect distinct structures for functional sites of TcSub2. For this reason, ligand-based or structure-based methodologies can be applied to investigate the potential of TcSub2 as a target for new drugs.