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Background: Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing. Methodology/Principal findings: Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed. Conclusions/Significance: We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples. Author summary: Chagas disease, caused by the parasite Trypanosoma cruzi, is a significant public health concern in Latin America. This parasite is genetically diverse and classified into different lineages. Proper strain typing techniques are necessary to study T. cruzi, because their lineages have significant biological differences. Several typing methods have been proposed, each of which has its own strengths and limitations. However, most of these methods lack sensitivity or fail for discriminating some lineages. Genetic markers with high copy numbers are required to gain sensitivity. Here, we deep sequenced DNA regions present in the large mitochondrion of the parasite (mHVRs) from strains belonging to the six main lineages to obtain reference mHVR sequences and develop a typing workflow. Amplicon sequencing of mHVR was conducted on 62 T. cruzi strains. Despite high sequence diversity, strains of the same lineage shared more sequences than strains of different lineages. Two reference sets of mHVR sequences were generated and evaluated for their ability to typify distinct types of T. cruzi samples. The workflow presented in this study could serve as a valuable resource for T. cruzi typing in future studies. [ABSTRACT FROM AUTHOR]

Background: Genetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. Methodology/Principal findings: In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them. Conclusions/significance: The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and explaining the relationships between these three clones are discussed. Author summary: Chagas disease, an important public health problem in Latin America, is caused by the parasite Trypanosoma cruzi. Despite being a widely studied parasite, several questions on the biology of genetic exchange remain unanswered. Population genomic studies have inferred meiosis in T. cruzi, but this cellular division mechanism has not been observed in laboratory yet. In addition, previous results suggest that mitochondrial DNA (called kDNA) may be inherited from both parents in hybrids. Here, we analyzed a hybrid strain and its potential parents to address the mechanisms of genetic exchange at nuclear and mitochondrial levels. We observed that the hybrid strain had heterozygous patterns and DNA content compatible with a meiosis event. Also, we observed that the evolutionary histories of nuclear DNA and kDNA maxicircles were discordant and that the three strains shared identical DNA sequences. Mitochondrial introgression of maxicircle DNA from one genotype to another may explain this observation. In addition, we demonstrated that the hybrid strain shared kDNA minicircles with both parental strains. Our results suggest that hybridization implied meiosis and biparental inheritance of the kDNA. Further research is required to address such phenomena in detail. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi, the protozoan causative of Chagas disease, is classified into six main Discrete Typing Units (DTUs): TcI-TcVI. This parasite has around 105 copies of the minicircle hypervariable region (mHVR) in their kinetoplastic DNA (kDNA). The genetic diversity of the mHVR is virtually unknown. However, cross-hybridization assays using mHVRs showed hybridization only between isolates belonging to the same genetic group. Nowadays there is no methodologic approach with a good sensibility, specificity and reproducibility for direct typing on biological samples. Due to its high copy number and apparently high diversity, mHVR becomes a good target for typing. Methodology/Principal findings: Around 22 million reads, obtained by amplicon sequencing of the mHVR, were analyzed for nine strains belonging to six T. cruzi DTUs. The number and diversity of mHVR clusters was variable among DTUs and even within a DTU. However, strains of the same DTU shared more mHVR clusters than strains of different DTUs and clustered together. In addition, hybrid DTUs (TcV and TcVI) shared similar percentages (1.9–3.4%) of mHVR clusters with their parentals (TcII and TcIII). Conversely, just 0.2% of clusters were shared between TcII and TcIII suggesting biparental inheritance of the kDNA in hybrids. Sequencing at low depth (20,000–40,000 reads) also revealed 95% of the mHVR clusters for each of the analyzed strains. Finally, the method revealed good correlation in cluster identity and abundance between different replications of the experiment (r = 0.999). Conclusions/Significance: Our work sheds light on the sequence diversity of mHVRs at intra and inter-DTU level. The mHVR amplicon sequencing workflow described here is a reproducible technique, that allows multiplexed analysis of hundreds of strains and results promissory for direct typing on biological samples in a future. In addition, such approach may help to gain knowledge on the mechanisms of the minicircle evolution and phylogenetic relationships among strains. [ABSTRACT FROM AUTHOR]

People living in areas with active vector-borne transmission of Chagas disease have multiple contacts with its causative agent, Trypanosoma cruzi. Reinfections by T. cruzi are possible at least in animal models leading to lower or even hardly detectable parasitaemia. In humans, although reinfections are thought to have major public health implications by increasing the risk of chronic manifestations of the disease, there is little quantitative knowledge about their frequency and the timing of parasite re-inoculation in the course of the disease. Here, we implemented stochastic agent-based models i) to estimate the rate of re-inoculation in humans and ii) to assess how frequent are reinfections during the acute and chronic stages of the disease according to alternative hypotheses on the adaptive immune response following a primary infection. By using a hybrid genetic algorithm, the models were fitted to epidemiological data of Argentinean rural villages where mixed infections by different genotypes of T. cruzi reach 56% in humans. To explain this percentage, the best model predicted 0.032 (0.008–0.042) annual reinfections per individual with 98.4% of them occurring in the chronic phase. In addition, the parasite escapes to the adaptive immune response mounted after the primary infection in at least 20% of the events of re-inoculation. With these low annual rates, the risks of reinfection during the typically long chronic stage of the disease stand around 14% (4%-18%) and 60% (21%-70%) after 5 and 30 years, with most individuals being re-infected 1–3 times overall. These low rates are better explained by the weak efficiency of the stercorarian mode of transmission than a highly efficient adaptive immune response. Those estimates are of particular interest for vaccine development and for our understanding of the higher risk of chronic disease manifestations suffered by infected people living in endemic areas. [ABSTRACT FROM AUTHOR]

Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared. Author Summary: The single-celled parasite Trypanosoma cruzi occurs in mammals and insect vectors in the Americas. When transmitted to humans it causes Chagas disease (American trypanosomiasis) a major public health problem. T. cruzi is genetically diverse and currently split into six groups, known as TcI to TcVI. Multilocus sequence typing (MLST) is a method used for studying the population structure and diversity of pathogens and involves sequencing DNA of several different genes and comparing the sequences between isolates. Here, we assess 13 T. cruzi genes and select the best combination for diversity studies. Outputs reveal that a combination of 7 genes can be used for both lineage assignment and high resolution studies of genetic diversity, and a reduced combination of four loci for lineage assignment. Application of MLST for assigning field isolates of T. cruzi to genetic groups and for detailed investigation of diversity provides a valuable approach to understanding the taxonomy, population structure, genetics, ecology and epidemiology of this important human pathogen. [ABSTRACT FROM AUTHOR]

The model of predominant clonal evolution (PCE) proposed for micropathogens does not state that genetic exchange is totally absent, but rather, that it is too rare to break the prevalent PCE pattern. However, the actual impact of this “residual” genetic exchange should be evaluated. Multilocus Sequence Typing (MLST) is an excellent tool to explore the problem. Here, we compared online available MLST datasets for seven eukaryotic microbial pathogens: Trypanosoma cruzi, the Fusarium solani complex, Aspergillus fumigatus, Blastocystis subtype 3, the Leishmania donovani complex, Candida albicans and Candida glabrata. We first analyzed phylogenetic relationships among genotypes within each dataset. Then, we examined different measures of branch support and incongruence among loci as signs of genetic structure and levels of past recombination. The analyses allow us to identify three types of genetic structure. The first was characterized by trees with well-supported branches and low levels of incongruence suggesting well-structured populations and PCE. This was the case for the T. cruzi and F. solani datasets. The second genetic structure, represented by Blastocystis spp., A. fumigatus and the L. donovani complex datasets, showed trees with weakly-supported branches but low levels of incongruence among loci, whereby genetic structuration was not clearly defined by MLST. Finally, trees showing weakly-supported branches and high levels of incongruence among loci were observed for Candida species, suggesting that genetic exchange has a higher evolutionary impact in these mainly clonal yeast species. Furthermore, simulations showed that MLST may fail to show right clustering in population datasets even in the absence of genetic exchange. In conclusion, these results make it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens. Moreover, our results reveal different problems of MLST to determine the genetic structure in these organisms that should be considered. [ABSTRACT FROM AUTHOR]

Background: We have previously shown that the transcription factor c-Fos is also capable of associating to endoplasmic reticulum membranes (ER) and activating phospholipid synthesis. Herein we examined phospholipid synthesis status in brain tumors from human patients and from NPcis mice, an animal model of the human disease Neurofibromatosis Type 1 (NF1). Principal Findings: In human samples, c-Fos expression was at the limit of detection in non-pathological specimens, but was abundantly expressed associated to ER membranes in tumor cells. This was also observed in CNS of adult tumor-bearing NPcis mice but not in NPcis fos(-/-) KO mice. A glioblastoma multiforme and a malignant PNS tumor from a NF1 patient (MPNST) showed a 2- and 4- fold c-Fos-dependent phospholipid synthesis activation, respectively. MPNST samples also showed increased cell proliferation rates and abundant c-Fos expression. Conclusions: Results highlight a role of cytoplasmic c-Fos as an activator of phospholipid synthesis in events demanding high rates of membrane biogenesis as occurs for the exacerbated growth of tumors cells. They also disclose this protein as a potential target for controlling tumor growth in the nervous system. [ABSTRACT FROM AUTHOR]