نبذة مختصرة : Background Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20–50 maxicircles (∼20 kb) and thousands of minicircles (0.5–10 kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30–35% develop potentially fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the diversity present within the various DTUs. Methodology/Principal Findings To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme based on ten gene fragments. A panel of 32 TcI isolates was genotyped using the mtMLST scheme, GPI, mini-exon and 25 microsatellite loci. Comparison of nuclear and mitochondrial data revealed clearly incongruent phylogenetic histories among different geographical populations as well as major DTUs. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain Sylvio X10/1, to provide the first evidence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi. Conclusions/Significance mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi.
Author Summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an important public health problem in Latin America. While molecular techniques can differentiate the major T. cruzi genetic lineages, few have sufficient resolution to describe diversity among closely related strains. The online availability of three mitochondrial genomes allowed us to design a multilocus sequence typing (mtMLST) scheme to exploit these rapidly evolving markers. We compared mtMLST with current nuclear typing tools using isolates belonging to the oldest and most widely occurring lineage TcI. T. cruzi is generally believed to reproduce clonally. However, in this study, distinct branching patterns between mitochondrial and nuclear phylogenetic trees revealed multiple incidences of genetic exchange within different geographical populations and major lineages. We also examined Illumina sequencing data from the TcI genome strain which revealed multiple different mitochondrial genomes within an individual parasite (heteroplasmy) that were, however, not sufficiently divergent to represent a major source of typing error. We strongly recommend this combined nuclear and mitochondrial genotyping methodology to reveal cryptic diversity and genetic exchange in T. cruzi. The level of resolution that this mtMLST provides should greatly assist attempts to elucidate the complex interactions between parasite genotype, clinical outcome and disease distribution.
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