Microevolution of Trypanosoma cruzi reveals hybridization and clonal mechanisms driving rapid genome diversification.

Protozoa and fungi are known to have extraordinarily diverse mechanisms of genetic exchange. However, the presence and epidemiological relevance of genetic exchange in Trypanosoma cruzi, the agent of Chagas disease, has been controversial and debated for many years. Field studies have identified both predominantly clonal and sexually recombining natural populations. Two of six natural T. cruzi lineages (TcV and TcVI) show hybrid mosaicism, using analysis of single-gene locus markers. The formation of hybrid strains in vitro has been achieved and this provides a framework to study the mechanisms and adaptive significance of genetic exchange. Using whole genome sequencing of a set of experimental hybrids strains, we have confirmed that hybrid formation initially results in tetraploid parasites. The hybrid progeny showed novel mutations that were not attributable to either (diploid) parent showing an increase in amino acid changes. In long-term culture, up to 800 generations, there was a variable but gradual erosion of progeny genomes towards triploidy, yet retention of elevated copy number was observed at several core housekeeping loci. Our findings indicate hybrid formation by fusion of diploid T. cruzi, followed by sporadic genome erosion, but with substantial potential for adaptive evolution, as has been described as a genetic feature of other organisms, such as some fungi.

Prevalence of Genetically Complex Leishmania Strains With Hybrid and Mito-Nuclear Discordance.

Approximately 20 Leishmania species are known to cause cutaneous, mucocutaneous, and visceral disorders in humans. Identification of the causative species in infected individuals is important for appropriate treatment and a favorable prognosis because infecting species are known to be the major determinant of clinical manifestations and may affect treatments for leishmaniasis. Although Leishmania species have been conventionally identified by multilocus enzyme electrophoresis, genetic analysis targeting kinetoplast and nuclear DNA (kDNA and nDNA, respectively) is now widely used for this purpose. Recently, we conducted countrywide epidemiological studies of leishmaniasis in Ecuador and Peru to reveal prevalent species using PCR-RFLP targeting nDNA, and identified unknown hybrid parasites in these countries together with species reported previously. Furthermore, comparative analyses of kDNA and nDNA revealed the distribution of parasites with mismatches between these genes, representing the first report of mito-nuclear discordance in protozoa. The prevalence of an unexpectedly high rate (~10%) of genetically complex strains including hybrid strains, in conjunction with the observation of mito-nuclear discordance, suggests that genetic exchange may occur more frequently than previously thought in natural Leishmania populations. Hybrid Leishmania strains resulting from genetic exchanges are suggested to cause more severe clinical symptoms when compared with parental strains, and to have increased transmissibility by vectors of the parental parasite species. Therefore, it is important to clarify how such genetic exchange influences disease progression and transmissibility by sand flies in nature. In addition, our aim was to identify where and how the genetic exchange resulting in the formation of hybrid and mito-nuclear discordance occurs.

Study on the Occurrence of Genetic Exchange Among Parasites of the Leishmania mexicana Complex.

In Leishmania, genetic exchange has been experimentally demonstrated to occur in the sand fly vector and in promastigote axenic cultures through a meiotic-like process. No evidence of genetic exchange in mammalian hosts have been reported so far, possibly due to the fact that the Leishmania species used in previous studies replicate within individual parasitophorous vacuoles. In the present work, we explored the possibility that residing in communal vacuoles may provide conditions favorable for genetic exchange for L. mexicana and L. amazonensis. Using promastigote lines of both species harboring integrated or episomal drug-resistance markers, we assessed whether genetic exchange can occur in axenic cultures, in infected macrophages as well as in infected mice. We obtained evidence of genetic exchange for L. amazonensis in both axenic promastigote cultures and infected macrophages. However, the resulting products of those putative genetic events were unstable as they did not sustain growth in subsequent sub-cultures, precluding further characterization.

Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi.

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

Analysis of DNA exchange using Thymidine analogs (ADExTA) in Trypanosoma cruzi

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

Evidence and importance of genetic exchange among field populations of Trypanosoma cruzi.

Many eukaryotic pathogenic microorganisms that were previously assumed to propagate clonally have retained cryptic sexual cycles. The principal reproductive mode of Trypanosoma cruzi, the aetiological agent of Chagas disease, remains a controversial topic. Despite the existence of two recent natural hybrid lineages, a pervasive view is that recombination has been restrained at an evolutionary scale and is of little epidemiological relevance to contemporary parasite populations. This article reviews the growing number of field studies which indicate that natural hybridization in T. cruzi may be frequent, non-obligatory and idiosyncratic; potentially involving independent exchange of kinetoplast and nuclear genetic material as well as canonical meiotic mechanisms. Together these observations now challenge the traditional paradigm of preponderate clonal evolution in T. cruzi and highlight the need for additional, intensive and appropriately sampled field surveys, complemented by high resolution, combined nuclear and mitochondrial population genetics analyses.

Preponderant clonal evolution of Trypanosoma cruzi I from Argentinean Chaco revealed by Multilocus Sequence Typing (MLST).

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales, the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study, we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area. Finally, considering our findings we discuss the scenario for the genetic structure of TcI.

Reproductive clonality in protozoan pathogens--truth or artefact?

The debate around the frequency and importance of genetic exchange in parasitic protozoa is now several decades old. Recently, fresh assertions have been made that predominant clonal evolution explains the population structures of several key protozoan pathogens. Here, we present an alternative perspective. On the assumption that much apparent clonality may be an artefact of inadequate sampling and study design, we review current research to define why sex might be so difficult to detect in protozoan parasite populations. In doing so, we contrast laboratory models of genetic exchange in parasitic protozoa with natural patterns of genetic diversity and consider the fitness advantage of sex at different evolutionary scales. We discuss approaches to improve the accuracy of efforts to characterize genetic exchange in the field. We also examine the implications of the first population genomic studies for the debate around sex and clonality in parasitic protozoa and discuss caveats for the future.