Genetic variation and exchange in Trypanosoma cruzi isolates from the United States

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, is a multiclonal parasite with high levels of genetic diversity and broad host and geographic ranges. Molecular characterization of South American isolates of T. cruzi has demonstrated homologous recombination and nuclear hybridization, as well as the presence of 6 main genetic clusters or "discrete typing units" (DTUs). Few studies have extensively investigated such exchange events and genetic diversity in North American isolates. In the current study, we genetically characterized over 50 US isolates from wildlife reservoirs (e.g., raccoons, opossums, armadillos, skunks), domestic dogs, humans, nonhuman primates, and reduviid vectors from nine states (TX, CA, OK, SC, FL, GA, MD, LA, TN) using a multilocus sequencing method. Single nucleotide polymorphisms were identified in sequences of the mismatch-repair class 2 (MSH2) and Tc52 genes. Typing based on the two genes often paralleled genotyping by classic methodologies using mini-exon and 18S and 24Sα rRNA genes. Evidence for genetic exchange was obtained by comparing sequence phylogenies of nuclear and mitochondrial gene targets, dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the cytochrome oxidase subunit II- NADH dehydrogenase subunit I region (COII-ND1), respectively. We observed genetic exchange in several US isolates as demonstrated by incongruent mitochondrial and nuclear genes phylogenies, which confirms a previous finding of a single genetic exchange event in a Florida isolate. The presence of SNPs and evidence of genetic exchange illustrates that strains from the US are genetically diverse, even though only two phylogenetic lineages have been identified in this region.

Figure 1. Evolutionary relationships among mismatch-repair class 2 gene (MSH2) and the thiol-disulfide oxido-reductase Tc52 gene (Tc52) from 50 and 51 Trypanosoma cruzi isolates, respectively.

Three phylogenetic trees were created by neighbor-joining (NJ), minimum evolution (ME), and maximum parsimony (MP) methods from the alignment of each gene target and a consensus tree was interpreted. Numbers at the branches are bootstrap values >50% (500 replicates) for the same nodes of the NJ, ME, MP trees. Evolutionary distances were computed using the Kimura 2-parameter method . ▴ = the 17 US TcI isolates that were identical; • = the 24 or 27 US TcIIa isolates that were identical. * = reference strains: SilvioX10 cl1, Colombiana, P209 (TcI); X110/8 (TcIII); CANIII cl1, Dog Theis, Ecua6 (TcIV); CL Brener (TcVI).

Figure 2. Evolutionary relationships among dihydrofolate reductase-thymidylate synthase (DHFR-TS) from 43 Trypanosoma cruzi isolates.

Three phylogenetic trees were created by neighbor-joining (NJ), minimum evolution (ME), and maximum parsimony (MP) methods from the alignment of each gene target and a consensus tree was interpreted. Numbers at the branches are bootstrap values >50% (500 replicates) for the same nodes of the NJ, ME, MP trees. Evolutionary distances were computed using the Kimura 2-parameter method . The nine isolates with positions incongruent to the mitochondrial phylogenies (Fig. 3) are highlighted. * = reference strains. Sequences clustered in 3 clades: Clade 1 includes TcIV T. cruzi isolates from the US and reference TcIV and TcV S. America strains. T. cruzi isolates of TcI lineage from the US and reference strains clustered in Clade 2, while Clade 3 consists on TcII and TcVI S. American reference strains.

Figure 3. Evolutionary relationships among cytochrome oxidase subunit II- NADH dehydrogenase subunit I region (COII-ND1) from 43 Trypanosoma cruzi isolates.

Three phylogenetic trees were created by neighbor-joining (NJ), minimum evolution (ME), and maximum parsimony (MP) methods from the alignment of each gene target and a consensus tree was interpreted. Numbers at the branches are bootstrap values >50% (500 replicates) for the same nodes of the NJ, ME, MP trees. Evolutionary distances were computed using the Kimura 2-parameter method . The nine isolates with positions incongruent to the nuclear phylogenies (Fig. 2) are highlighted. * = reference strains. Sequences clustered in 4 distinct clades. Clade 1 contains exclusively US origin TcIV strains of T. cruzi. Reference TcIII-TcIV strains of T. cruzi clustered in Clade 2. TcI T. cruzi from the US and S. American reference strains clustered in Clade 3, while a separation of a TcII reference strain results in the fourth clade.