Dados do Trabalho

Título

Trypanosoma cruzi Exploits Extracellular Vesicles to Transfer Kinetoplast DNA to Host Cells, Modulating LINE-1 Expression

Introdução

Horizontal DNA Transfer (HDT) acts as a principal catalyst in the grand scheme of evolutionary progression, as documented across diverse species, encompassing both parasites and host organisms. HDT can be favored by the release of extracellular vesicles (EVs) and the activity of retroelements. The parasite Trypanosoma cruzi (Tc), notoriously attributed to Chagas disease, has been proven capable of translocating its kinetoplast DNA (kDNA) into the host genome. Nonetheless, the intricate mechanisms permitting kDNA to traverse from the protist's cytoplasm to host cells, and the consequential effects on host cells, remains poorly understood.

Objetivo (s)

In this study, we endeavored to detect and verify the association of Tc-secreted extracellular vesicles with kDNA and scrutinize the influence of the parasite upon the host’s Long Interspersed Nuclear Element-1 (LINE-1) activity. 

Material e Métodos

To achieve these ends, ultracentrifugation and a transwell system were implemented for isolation of extracellular vesicles, followed by subsequent analysis of vesicle content via restriction enzymes and classic PCR (cPCR). With the aid of the CRISPR/dCas9 system, it was ascertained whether the kDNA minicircles were effectively integrated into the host cell genome. Furthermore, the consequent alteration of the host's LINE-1 expression due to the parasite was evaluated using quantitative PCR (qPCR). 

Resultados e Conclusão

Throughout our analysis, we were able to discern double-stranded kDNA minicircles encapsulated within Tc-secreted vesicles, in absence of kDNA maxicircles or nuclear DNA. Confirmed through high-intensity signals in the nucleus via CRISPR/dCas9 experiments, transferred kDNA undeniably integrated within the host genome. Subsequent to parasite exposure or their secretome, host cells were found to exhibit up to 15- and 3-fold increase in LINE-1 expression, respectively. The utilization of the retroelement inhibitor, Ziduvudine, efficaciously blocked kDNA assimilation into the host genome. These findings expand our understanding of how kDNA minicircles are exchanged between Tc and its hosts and highlight the potential role of the retrotransposition machinery in kDNA integration and spread throughout the genome. Further studies are needed to elucidate the step-by-step route taken by kDNA minicircles from their release by the parasite through the host cell cytoplasm and into the nucleus to their final sites of integration in the chromosomes. 

Palavras Chave

Trypanosoma cruzi; kDNA; horizontal DNA transfer; extracellular vesicles; LINE-1