The Impacts of Trypanosoma cruzi Strain Variability on Metabolism and Genetic Manipulation

Abstract

This research aimed to investigate how natural genetic variations in the eukaryotic unicellular parasite Trypanosoma cruzi, the causative agent of Chagas disease, impact its metabolism and ability to be genetically manipulated. To achieve this goal, the study focused on metabolic flexibility, antibiotic susceptibility, and the development of new tools for T. cruzi genetic manipulation. We examined ATP generation across three metabolic pathways in four T. cruzi strains: Esmeraldo, Sylvio-X10, CL Brener, and Tulahuen. Our findings revealed strain-specific variations, indicating two strains in particular preferentially utilize different metabolic pathways to maintain crucial ATP levels. This diversity of the T. cruzi species suggests a complex metabolic landscape with potential implications for parasite survival and pathogenicity within its host. Furthermore, we investigated antibiotic susceptibility among these four strains, and found significant variations in their susceptibility towards commonly used antibiotics for genetic manipulation. This demonstrates that empirically determining optimal selection markers for the strain being modified is very important. Notably, we found that genetic relatedness does not necessarily correlate with antibiotic susceptibility, as strains belonging to the same discrete typing units (DTUs) showed great variability in their responses to antibiotics. To facilitate future genetic studies in T. cruzi, we successfully developed a robust and flexible methodology for generating donor DNA constructs for CRISPR/Cas9-mediated gene editing. This approach, utilizing the NEBuilder HiFi DNA assembly method, enables the efficient assembly of complex donor DNA constructs from multiple fragments, streamlining the process of genetic manipulation in this parasite.Overall, this study provides a foundation for understanding the interplay between genetic variation, metabolism, and drug susceptibility in T. cruzi. Our long-term goal is to link strain-specific amino acid substitutions with different metabolic outcomes, ultimately leading to a more comprehensive understanding of T. cruzi biology and potentially revealing novel therapeutic targets for Chagas disease.