No abstract

DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.

Edited by Michael IbbaReplication protein A (RPA), a heterotrimeric complex, is the major singlestranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.

Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cellpenetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi. The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK 2 monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulinubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi. Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

Bacteroides genus are commonly found on mucous membranes, including the female genital tract, acting as agents for several site infections. Anaerobic infections are usually polymicrobial and endogenous. Trichomonas vaginalis, the trichomoniasis etiologic agent, is a facultative anaerobic flagellated parasite spread worldwide. The purpose of this study was to explore the association between vaginal bacteria and T. vaginalis, as well as to understand factors that may favour the infection of T. vaginalis. We have, therefore, used T. vaginalis trophozoites and the species Bacteroides fragilis, which is considered the most important in its genus, onceit is the most commonly isolated bacteria from endogenous infections. The parasite-bacteria interaction was performed in different proportions in periods varying from 1 to 12 hours applying viability tests. The data were analyzed to compare the parasite viability in vitro in the presence and absence of B. fragilis. The results indicate that in the 1:100 proportion postinteraction analysis, ultrastructural alterations were noticeable after 6 hours. After 8 hours, T. vaginalis viability decreased, and after 12 hours of interaction no viable trophozoites were found. These data suggest that the parasite can deal with B. fragilis in short interaction periods. However, in longer interaction periods the trophozoites collapse, indicating that B. fragilis may produce toxic metabolites against T. vaginalis activity. KEY WORDS: Bacteroides fragilis; Trichomonas vaginalis; parasite-bacteria interaction; parasiteenvironment; electron microscopy; gynecologic infections.