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

<div><p><em>Trypanosoma cruzi</em>, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the <em>T. cruzi</em> 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 <em>T. cruzi</em> parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single <em>T. cruzi</em> 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 <em>T. cruzi</em> 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 <em>T. cruzi</em> 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 <em>T. cruzi</em>. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality.</p> </div

Allele sizes of TcTAT20 microsatellite and 24Sα rDNA gene amplified from A316A R7 single cells.

<p>Allele sizes are presented in base pairs.</p><p>(-), Amplification failure probably due to allele drop-out effect. <i>T. cruzi</i> DTUs according to Zingales <i>et al.</i>, 2009.</p><p>In this table are shown only those wells that presented positive amplification for both markers.</p

Allele sizes for each microsatellite locus amplified from A316A R7 single sorted cells.

<p>Allele sizes are presented in base pairs.</p><p>(-), Amplification failure probably due to allele drop-out effect; n.a, not amplified.</p

Electrofluorogram of TcAAT8 (A) and TcAAAT6 (B) loci for A316A R7 before and after sorting.

<p>Numbers at the peaks refer to the size of the amplicons in base pairs. The peaks presenting values of 75, 210, and 320 bp refer to internal markers used for alignment in the Allelelocator software. The wells containing single cells that showed positive amplification are also indicated. Control wells presenting DNA of the original strain pool of parasites were also included.</p

24Sα rDNA profiles of single parasites from the SilvioX10 cl1 and Esmeraldo cl3 artificial mixture.

<p>DNA from Esmeraldo cl3 (125 bp), Silvio X10 cl1 (110 bp), and SO3 cl5 (110/125) were used as controls for rDNA type 1 (<i>T. cruzi</i> II), rDNA type 2 (<i>T. cruzi</i> I), and rDNA type 1/2 (<i>T. cruzi</i> V), respectively. Corresponding microplate wells of the positive amplifications are also indicated. Control wells were programmed to contain 0, 2, 5, or 10 parasites. MW: Molecular weight of 25 bp (Invitrogen, USA).</p

Allele sizes for each microsatellite amplified from Be-78 1B and Be-78 25B single sorted cells.

<p>Allele sizes are presented in base pairs.</p><p>(-), Amplification failure probably due to allele drop-out effect; n.a, not amplified or not analyzed.</p><p>In this table are shown the alleles from wells derived from different experiments that presented positive amplification for at least three microsatellite loci.</p

PCR-RFLP profiles of the COII gene from the A316A R7 single sorted parasites.

<p>After digestion with <i>Alu</i>I the COII amplicons generate three RFLP patterns for <i>T. cruzi</i>: the presence of a fragment of 264 bp characterizes a mitochondria haplotype A, associated with <i>T. cruzi</i> I (e.g., Silvio X10 cl1); the presence of a fragment of 212 bp characterizes a mitochondria haplotype C, associated with <i>T. cruzi</i> II (e.g., Esmeraldo cl3), and the presence of a fragment of 294 bp characterizes a mitochondria haplotype B associated with <i>T. cruzi</i> III-VI DTUs (e.g., S03 cl5). Only wells that resulted in positive amplification are shown. Control wells were programmed to contain 0, 2, 5, or 10 parasites. MW: Molecular weight of 25 bp (Invitrogen, USA); NC – RFLP mix: negative control containing only the reaction mix.</p

Primers used for amplification of the <i>T. cruzi</i> 24Sα rDNA, COII, and microsatellite markers.

<p>Primers used for amplification of the <i>T. cruzi</i> 24Sα rDNA, COII, and microsatellite markers.</p

Electrofluorogram presenting the profiles obtained for 24Sα rDNA on A316A R7 single sorted parasites.

<p>Numbers at the peaks refer to the size of the amplicons in base pairs. The peaks presenting values of 75 and 155 bp refer to internal markers used for alignment in the Allelelocator software. The wells containing single cells that showed positive amplification are also indicated. Control wells presenting DNA of the original strain pool of parasites or 10 sorted parasites were also included.</p