Unequivocal Identification of Subpopulations in Putative Multiclonal Trypanosoma cruzi Strains by FACs Single Cell Sorting and Genotyping

Abstract

Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations – Silvio X10 cl1 (TcI) and Esmeraldo cl3 (TcII) – and three naturally occurring strains, one isolated from a vector (A316A R7) and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality.

Identification of Trypanosoma cruzi Discrete Typing Units (DTUs) through the implementation of a High-Resolution Melting (HRM) genotyping assay

“…A similar initiative was proposed to develop a Multilocus Sequence Typing (MLST) scheme based on the analysis of a panel of four molecular targets: Met-III, RB19, TcGPXII, and DHFR-TS; however, the development of this process requires both bioinformatic analysis and sequencing steps [9]. Recently, the analysis of the polymorphisms of the 24sα rDNA gene, the subunit II of the cytochrome oxidase, and four microsatellite loci has been shown to be an alternative method [10]. This technique is highly efficient but requires multiple amplification steps that can lead to an increased risk of contamination.…”

Molecular Epidemiology of Human Oral Chagas Disease Outbreaks in Colombia

“…This premise emerges in the light of the absence of triple peaks among the loci studies or heterozygosis status; only locus TcATT14 showed heterozygosis and the rest of loci employed were homozygous, this could be explained by a low resolution power displayed by the microsatellite markers employed since parasite DNA yield was high (Table 2). Additionally, the multi-copy arrangement that SL-IR displays and the presence of null alleles and/or aneuploidy could explain these premises but further testing is required [44].…”

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

“…Such coupled genotyping would enhance resolution of parasite-vector genetic co-structure and thus, for example, help quantify rates of parasite transmission from domiciliating vectors or determine whether parasite gene flow proxies for (or improves understanding of) dispersal patterns in more slowly evolving vectors or hosts. It would also be interesting to test in how far deep-sequenced GLST libraries could help in detecting (and reconstructing distinct MLGs from) multiclonal T. cruzi infections without the use of cloning tools 101 , e.g., using bioinformatic strategies developed for malaria research 102–105 . Multiclonality has important implications for public health 106,107 but its potential prevalence in T. cruzi vectors and hosts 108,101,109 is difficult to describe from cultured cells 108,110 .…”