Genotypic and phenotypic variability of Trypanosoma cruzi isolated from Cynomolgus macaques and tools for multi-gene expression
Yao, Phil Yu
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The protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, is a promiscuous pathogen infecting not only humans, but also many mammalian species. A community of 64 cynomolgus macaques housed in southern United States provided opportunity for investigation into natural T. cruzi infection in an entire population from a geographically restricted locale. Genotyping of hemoculture-isolated T. cruzi revealed the presence of two lineages: TcI and TcIV, which are the most common lineages identified by previous studies in infections originating in North America. All tested isolates were infective and virulent in an experimental mouse model, establishing chronic infection and inducing immune responses against an epitope from a highly variable trans-sialidase gene family, consistent with those observed in infections by long-maintained laboratory-adapted strains of T. cruzi. Although infection with the majority of fresh isolates was susceptible to clearance by treatment with benznidazole, isolates that appear to be naturally resistant to benznidazole treatment were identified. A subset of isolates was found to be amenable to genetic manipulation by heterologous gene expression using pTREX and gene knockout with CRISPR-Cas9, which are powerful tools for understanding Trypanosoma cruzi biology and host-parasite interactions. Among the limitations in the stable expression of multiple genes of interest by T. cruzi is the restricted set of antibiotics available to select for the desired transgenic parasites and the extended time (often 4-6 weeks) required for this selection. These limitations make generating parasite lines that co-express multiple genes both time-consuming and inefficient. In this study 3 approaches were evaluated for co-expressing transgenes in T. cruzi by separating transgenes using: a) previously defined minimal trypanosome 3’ and 5’ untranslated regions; b) full length intergenic regions from T. cruzi genes; and c) 2A intraribosomal self-cleaving peptide skip sequences. These tools can be applied to future work in assessing the ability of multiple immune-modifying proteins, expressed by T. cruzi, to alter the mammalian host immune response to infection.