What are the consequences of mitogenic stimulation for Trypanosoma cruzi, the etiological agent of Chagas disease?

• Grant number: 24/07821-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2024
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Marcelo Santos da Silva
• Grantee: Fernanda Merchiori Gerolamo
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Parasitic infections remain a major cause of morbidity and mortality worldwide. Most parasitic infections in humans and domestic animals can be attributed to protozoan pathogens, including T. cruzi, the causative agent of Chagas disease (CD). This illness affects ~ 10 million people on the American continent and is the main cause of congestive heart failure in Latin America. There are no effective vaccines against CD, and treatments rely on drugs that, in addition to having side effects, allow the emergence of dormant (persistent) parasites. Here, we intend to investigate the effect of mitogenic stimulation on T. cruzi, focusing on its DNA damage response (DDR). We assume (hypothesize) that cells with a high proliferative rate would be more likely to suffer DNA damage due to replication stress. If this hypothesis supports our experimental tests, we will be faced with a potential route of intervention, as in addition to eliminating the parasites due to excessive DNA damage, mitogenic stimulation would possibly activate the dormant parasites, leading them to enter the cell cycle, become susceptible to a possible combined treatment using other drugs. We intend to use epimastigote forms of T. cruzi to carry out a proof of concept of our hypothesis. Initially, we will perform preliminary kinetic assays to figure out the optimal concentration of the mitogen (TGF-± or LB-100) and the treatment period to be used in this approach. Next, we will verify the viability of the T. cruzi cells, as well as their DDR and the presence of replication stress by immunofluorescence assays (using ±-³H2A and native BrdU detection). We also intend to analyze the cell cycle profile by flow cytometry and CeCyD software. Finally, we intend to carry out infection assays to analyze the behavior of amastigote forms of T. cruzi within human cardiomyocytes during mitogenic stimulation. In case epimastigote forms of T. cruzi are not stimulated by any of the proposed mitogens, we intend to report this peculiarity and work only with the amastigote forms. If the amastigotes forms are also not stimulated by the mitogens, we will report this curious finding and develop a new hypothesis to try to understand this unusual behavior of T. cruzi. We strongly believe that the findings obtained from this proposal will help us better understand de DDR from T. cruzi and pave the way for a new approach to combating CD.