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.
Chagas disease, an American protozoonosis caused by Trypanosoma cruzi, is characterized by various clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive injuries. This complex ailment still afflicts 10 million people across Latin America and another 90 million are at risk of acquiring the disease [1]. A vaccine or specific treatment for large-scale public health interventions is still not available, so the main control strategy relies on prevention of transmission by controlling insect vectors and contamination by food or blood transfusion [2]. T. cruzi is a very polymorphic species, as extensively demonstrated by biological, biochemical, and molecular studies [3], and this certainly contributes to the pleomorphism of the symptoms and to the difficulty in controlling the disease [4]. T. cruzi was recently subdivided into six discrete taxonomic units (DTUs) named T. cruzi I to T. cruzi VI [5], of which at least four are involved with human pathology [6]. Indirect evidence suggests that part of T. cruzi populations is multiclonal. This is in accordance with the theoretical expectation, since patients in endemic areas are infected by multiple contacts with different triatomines and these, in turn, may feed on different infected individuals. Most of the putative multiclonal populations were suggested based on the identification of more than two alleles for different markers [7]–[9]. Whether these reported populations are really multiclonal remains controversial because we cannot exclude the possibility of aneuploidy [7], [8], [10], [11]. Thus, direct evidence for the multiclonality of sylvatic and domestic populations is lacking. Indeed, it is not possible to identify all the clones that constitute a given multiclonal population because current conventional cloning methods (micromanipulation, limiting dilution or cloning in blood-agar plates) may favor individuals over-represented in the original population and/or presenting higher growth rates [12]–[15]. To answer these questions we devised a new strategy for sorting single T. cruzi parasites, adapted from methods originally described for analyzing single spermatozoids [16]. Using this innovative approach the results presented herein may contribute to settling the debate regarding the multiclonality of three T. cruzi representative strains isolated from human and vector hosts. The methodology described here is able to completely dissect the complexity of strains and estimate the relative contribution of each subpopulation, and will therefore have important implications for the study of T. cruzi biology and disease.
In this study we used four multiclonal T. cruzi populations: one artificially composed of Esmeraldo cl3 (a standard T. cruzi II strain) and Silvio X10 cl1 (a standard T. cruzi I strain), a naturally occurring putatively multiclonal vector strain called A316A R7, and two populations derived from a naturally occurring putatively multiclonal human strain named Be-78 1B and Be-78 25B. The A316A R7 strain was isolated from a Triatoma sordida circulating in northwestern Paraná state, Brazil [17], [18]. The Be-78 1B and Be-78 25B are two populations derived from a chronic chagasic outbred dog infected with Be-78, originally isolated from Berenice, the first patient of Carlos Chagas [19], harvested after 1 or 25 successive blood passages in mice [20]. About 1. 5 mL (106 cells) of T. cruzi epimastigotes cultured in Liver Infusion Tryptose (LIT) medium were transferred to a siliconed Vacutainer tube (Itupeva, Brazil). The culture was centrifuged at 30×g for 10 minutes at 4°C to separate intact cells from cellular debris. After this centrifugation, the culture was incubated for 10 minutes at room temperature to allow mobile epimastigotes to reach the surface. The collected cells were submitted to three washes with 1× PBS pH 7. 4 at 4°C sterilized using 0. 22 µM Millex GV filter (Millipore, MA, USA) and centrifuged at 1000×g for 10 minutes at 4°C. After the last wash the cells were suspended in 1 mL of 1× PBS pH 7. 4 and fixed for 30 minutes using absolute ethanol maintained at −20°C. Finally, the fixed cells were stored at 4°C overnight, after which they were submitted to sorting on the FACS Vantage apparatus. To calibrate the powerful single-cell sorter FACS Vantage to achieve single T. cruzi specimens, we used fluorescent beads as an indirect control because bead size is similar to the epimastigote form of the parasite [21]. Fluorescent beads (Spherotech Inc. , IL, USA) measuring 10. 0 to 14. 0 µm in diameter were sorted in microscope slides according to the manufacturer' s protocol (Becton Dickinson, CA, USA). We found that the counting of the sorted beads under fluorescence microscope always showed good correspondence with the number of beads programmed for each well. In wells programmed to contain one bead we found an occasional absence of beads, but never more than one bead. To sort T. cruzi strains, small aliquots of the fixed cells were transferred to a Falcon tube (Becton Dickinson, CA, USA) for FACS counting. The solution containing the parasites was diluted in 1× PBS pH 7. 4 until the FACS counting indicated approximately 100 events per second. Samples were gated forward scatter (FSC) versus side scatter (SSC) to select cell populations of interest (Figure S1). The autofluorescence pattern presented by T. cruzi allows cell sorting without staining with fluorescent dyes or reagents. Then, the Clone Cyt apparatus (Becton Dickinson, CA, USA) was used to sort single cells directly into 96-wells plate (Sorensen BioScience, UT, USA) containing 5 µL of 10% Triton X-100. Control wells were programmed to contain 0,2, 5, or 10 sorted parasites from each T. cruzi population. After sorting, a drop of mineral oil was added to each well and the microplates were stored at −20°C until needed. Before the PCR assays, the single sorted T. cruzi was lysed by exposing the microplates to 80°C for 1 hour in a thermocycler. Cells were genotyped by using polymorphic markers to mitochondrial (Cytochrome Oxidase subunit II- COII) and nuclear (24Sα rDNA) genes and four microsatellite loci.
To evaluate whether the previously used bead-calibrated method could be successfully used to separate single T. cruzi parasites, we initially sorted an artificial mixture of two monoclonal populations: Silvio X10 cl1 (T. cruzi I) and Esmeraldo cl3 (T. cruzi II). Next, we confirmed separation by genotyping the 24Sα rRNA gene of the single sorted parasites and the controls present in each well (Figure 1). In the control wells A1, A2, A3, and A4 programmed to contain 10 or 5 cells, we detected the expected amplicons of 110 and 125 bp, demonstrating that the two types of cells used in the artificial mixture were present. From the 83 wells programmed to contain the sorted single parasites, 40% (33 wells) showed the amplification of only the 125 bp or the 110 bp amplicon, therefore confirming single cell sorting. This efficiency is approximately the double that obtained with a previously published protocol [25]. Moreover, none of the wells programmed to contain a single cell showed the amplification of both fragments. These results indicate that the FACS Vantage sorting method we modified could be used efficiently to separate single T. cruzi parasites. The A316A R7 strain was isolated from a triatomine bug in Paraná, southern Brazil. Because this strain presented more than two alleles for some of the DNA markers used during its genotyping, it was suggested that it was constituted of a mixed population [17]. However, considerable controversy persisted on whether the multiclonal status of this and other T. cruzi strains is real [7], [8], [26]–[28] or just reflects aneuploidy for the analyzed loci [10], [11]. We therefore used the single T. cruzi sorting and genotyping approach proposed here to address this question. To this end, we sorted single parasites from the A316A R7 strain and genotyped them by using three sets of markers: COII, 24Sα rDNA, and microsatellites (Figures 2,3, 4).
To bring further light to the debate regarding the multiclonal status of T. cruzi strains, an innovative approach was successfully adapted from a method originally described to genotype single sorted spermatozoids, to separate single parasites. The evidence described herein reveals that multiclonality holds true for two representative T. cruzi human and vector strains. The approach we developed employed the FACS Vantage sorting method, which allowed us to estimate the relative contribution of each parasite subpopulation within the strains analyzed. The efficiency of the approach was initially established by sorting and genotyping single parasites from an artificially composed multiclonal population and not only all the individuals present in the original mixture could be identified, but also the real proportion of each population could be assigned. The devised method was then used to scrutinize three naturally occurring putative multiclonal populations originally isolated from a vector (A316A R7) and two derived from a patient (Be-78 1B and Be-78 25B). A long-held debate was whether T. cruzi strains presenting more than two alleles for some polymorphic markers were really multiclonal or aneuploid populations [7], [9], [10]. Previous work suggested that the A316A R7 strain was a mixture of distinct populations [18], [29]. Our new results involving the microsatellite analysis of single sorted parasites confirmed the presence of different subpopulations within this strain, ruling out aneuploidy as a possible explanation for the complex genetic patterns presented by this population. Interesting results were observed with the human T. cruzi strain. Great controversy remains on whether or not Berenice, the first patient of Carlos Chagas, has been infected with a multiclonal T. cruzi. This was due to the fact that two different but apparently monoclonal populations (Be-62 and Be-78) were isolated from her [34]. The observation that Be-78 has changed its molecular and biochemical characteristics after passages in dogs and mice strengthened this possibility, suggesting that the passage in different hosts could have grown previously undetectable subpopulations within the original strain. Here we showed that Be-78 1B and Be-78 25B (two of the T. cruzi populations derived from a dog infected with Be-78 after 1 or 25 successive passages in mice) were constituted by three subpopulations: one compatible with Be62, another equivalent to the Be78, and a third one not identified before. Therefore, the complexity of this T. cruzi strain is even greater than originally expected. We also detected alleles not previously reported for the analyzed strains. The probable reason they were not found before is that when alleles of less representative subpopulations are lower than 15% in the DNA artificial mixture, they cannot be detected by conventional PCR (our unpublished data). Another remarkable result was observed regarding the TcAAAT6 profiles of the single sorted parasites. The analysis of the vector strain showed the presence of two alleles of 251 and 275 bp, initially interpreted as a typical heterozygote genotype. However, the analysis of the single parasites using this locus showed the presence of two different subpopulations both presenting homozygote profiles. This finding emphasizes the advantage of genotyping single cells instead of pools of parasites, because conventional PCR cannot distinguish between the presence of a homogeneous population presenting two different alleles, and a heterogeneous population constituted by two different and homozygous individuals. Interestingly, by using the methodology presented here we successfully identified the subpopulations presented in a multiclonal strain as well as estimate the proportion of each subpopulation. A drawback inherent to use of a single cell is failure of amplification of one of the two alleles of some loci, a phenomenon known as allele drop-out (ADO). Due to its characteristics, ADO can be misleading as false homozygosity, as one of the alleles in heterozygote loci is undetectable [35]. Although we observed ADO in some of our analyses, it did not interfere with the single-cell genotyping because we analyzed simultaneously 96 plated cells and a variety of loci. Contrasting to real homozygosis, ADO is a random phenomenon and it does not occur in the same way in all wells and loci. Thus, the more plates and therefore the more single cells analyzed, more reliable the results will be, a possibility easily achievable by the cell sorting method devised. In summary, by using the strategy proposed here for sorting and genotyping T. cruzi single cells we successfully identified the complexity of representative human and vector T. cruzi strains and estimated the relative contribution of each subpopulation. These results have important implications in settling the debate regarding multiclonality versus aneuploidy in these representative strains. Moreover, our results indicate that the complexity of T. cruzi strains may be even greater than initially expected. It is worth noting that the methodology described herein has the potential to close the discussion concerning multiclonality. Knowledge about complexity of T. cruzi strains is essential for determining the aspects involved in differential parasite tissue tropism, clinical manifestations of the disease, and drug resistance. The single-cell genotyping approach allowed the analysis of the intrapopulation diversity to a degree not achieved previously by conventional methods, and may represent a powerful and new tool for the understanding of the population structure and dynamics of T. cruzi.
Trypanosoma cruzi, the cause of Chagas disease, is responsible for high levels of mortality and morbidity in many countries of Latin America. This species is very polymorphic and was recently classified into six discrete typing units (TcI-VI) that are associated with different geographical distribution, transmission cycles, and varying disease symptoms. Natural parasite populations are thought to have a complex structure since in endemic areas the same hosts may be simultaneously infected by different T. cruzi strains due to frequent exposure to insects as well as the variety of reservoirs and vectors in the sylvatic environment and the heterogeneous composition of the parasite populations. Most of the putative multiclonal populations have been suggested based on indirect evidence, or current conventional cloning methods that may favor some individuals' growth. In an effort to elucidate the complexity of T. cruzi isolates we adapted a methodology originally described for analyzing single spermatozoids, to sort single parasites. This approach allowed us to completely dissect the complexity of four strains and estimate the relative contribution of each subpopulation. Knowledge about complexity of T. cruzi strains is essential for determining the aspects involved in differential parasite tissue tropism, clinical manifestations of the disease, and drug resistance.
population genetics
ploidy
microbiology
parastic protozoans
dna
genetic polymorphism
dna amplification
biology
molecular biology
trypanosoma
nucleic acids
genetics
protozoology
molecular cell biology
genetics and genomics
Unequivocal Identification of Subpopulations in Putative Multiclonal Trypanosoma cruzi Strains by FACs Single Cell Sorting and Genotyping