Characterization and functional analysis of the proteins Prohibitin 1 and 2 in Trypanosoma cruzi

Background Chagas disease is the third most important neglected tropical disease. There is no vaccine available, and only two drugs are generally prescribed for the treatment, both of which with a wide range of side effects. Our study of T. cruzi PHBs revealed a pleiotropic function in different stages of the parasite, participating actively in the transformation of the non-infective replicative epimastigote form into metacyclic trypomastigotes and also in the multiplication of intracellular amastigotes. Methodology/principal findings To obtain and confirm our results, we applied several tools and techniques such as electron microscopy, immuno-electron microscopy, bioinformatics analysis and molecular biology. We transfected T. cruzi clones with the PHB genes, in order to overexpress the proteins and performed a CRISPR/Cas9 disruption to obtain partially silenced PHB1 parasites or completely silenced PHB2 parasites. The function of these proteins was also studied in the biology of the parasite, specifically in the transformation rate from non-infective forms to the metacyclic infective forms, and in their capacity of intracellular multiplication. Conclusion/significance This research expands our understanding of the functions of PHBs in the life cycle of the parasite. It also highlights the protective role of prohibitins against ROS and reveals that the absence of PHB2 has a lethal effect on the parasite, a fact that could support the consideration of this protein as a possible target for therapeutic action. Author summary Trypanosoma cruzi is the etiological agent of American trypanosomiasis or Chagas disease. Among the trypanosomes known to affect humans, this is the only one with an obligatory intracellular multiplication stage in the mammalian host called amastigote. In this work, we describe for the first time the function and location of Trypanosoma cruzi proteins Prohibitin 1 and 2. Prohibitins are highly conserved proteins that belong to the Stomatin-Prohibitin-Flotillin-HflC/K (SPFH) protein superfamily and share a SPFH domain. PHBs are ubiquitous and play important roles in many cellular processes. Although they are mainly located in yeast mitochondria, nematodes and humans, PHBs could also be present in the nucleus and in cytosolic membranes. The localization of T. cruzi PHBs is stage-specific, showing a cytoplasmic membrane distribution in the human infective trypomastigote stages and in the amastigote forms. In the epimastigote form, PHBs bind Fe3+ ions, exerting a protective effect against oxidative stress. While the overexpression of PHB1 and PHB2 showed no significant differences in the parasite's infectivity, it caused an increase in the intracellular multiplication of the amastigote form. The deletion of the PHB2 gene had a lethal effect on the parasite, whereas PHB1 partial silencing caused a significant decrease in ROS detoxification. Together, these results indicate that PHBs could participate in metacyclogenesis in ROS detoxification and could play a role in the replication of the intracellular stage of T. cruzi. Besides, our results suggest the potential role of PHB2 as a target for drug therapy.

Automated summary

Chagas disease is a neglected tropical disease with no available vaccine and limited treatment options. The study on T. cruzi PHBs reveals their pleiotropic function in different parasite stages and highlights their potential as therapeutic targets. PHB2 deletion has a lethal effect on the parasite, while PHBs play roles in metacyclogenesis and ROS detoxification.