Wide reference databases for typing Trypanosoma cruzi based on amplicon sequencing of the minicircle hypervariable region.

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.

Hydrophobicity-Driven Increases in Editing in Mitochondrial mRNAs during the Evolution of Kinetoplastids.

Kinetoplastids are a diverse group of flagellates which exhibit editing by insertion/deletion of Us in the mitochondrial mRNAs. Some mRNAs require editing to build most of their coding sequences, a process known as pan-editing. Evidence suggests that pan-editing is an ancestral feature in kinetoplastids. Here, we investigate how the transition from nonedited to pan-edited states occurred. The mitochondrial mRNAs and protein sequences from nine kinetoplastids and related groups (diplonemids, euglenids, and jakobids) were analyzed. RNA editing increased protein hydrophobicity to extreme values by introducing Us in the second codon position, despite the absence of editing preferences related to codon position. In addition, hydrophobicity was maintained by purifying selection in species that lost editing by retroposition of the fully edited mRNA. Only a few hydrophobic to hydrophilic amino acid changes were inferred for such species. In the protein secondary structure, these changes occurred spatially close to other hydrophilic residues. The analysis of coevolving sites showed that multiple changes are required together for hydrophobicity to be lost, which suggest the proteins are locked into extended hydrophobicity. Finally, an analysis of the NAD7 protein-protein interactions showed they can also influence hydrophobicity increase in the protein and where editing can occur in the mRNA. In conclusion, our results suggest that protein hydrophobicity has influenced editing site selection and how editing expanded in mRNAs. In effect, the hydrophobicity increase was entrenched by a neutral ratchet moved by a mutational pressure to introduce Us, thus helping to explain both RNA editing increase and, possibly, persistence.

Genome data vs MLST for exploring intraspecific evolutionary history in bacteria: Much is not always better.

Genome-based phylogeny has been proposed to be more accurate than phylogeny based in a few genes as MLST-based phylogeny. However, much is not always better. Here we analyzed 368 complete genomes corresponding to 9 bacterial species in order to address intraspecific phylogeny. The studied species were: Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia trachomatis, Helicobacter pylori, Klebsiella pneumoniae, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus and Streptococcus pyogenes. The intra-specific phylogenies were inferred using the complete genome sequences of different strains of these species and their MLST schemes. A supermatrix approach was used to infer maximum likelihood phylogenies in both cases. The phylogenetic incongruence between the supermatrix-based genome or MLST tree and individual trees (constructed from genome fragments or MLST genes, respectively) was analyzed. In supermatrix-based trees for genomes, most branches showed a high branch support; however, a high number of branches also showed high percentage of topologically incongruent individual trees. Interestingly, genome and MLST trees showed similar levels of incongruence in the phylogeny for each bacteria specie. Both genome and MLST approaches showed that C. trachomatis and S. aureus have a tree-like evolutionary history (low levels of internal incongruence). Instead, B. pseudomallei and S. pyogenes show high levels of incongruence (network-like evolutionary story) probably caused by HGT (horizontal gene transfer). Concluding, our analysis showed that: high branch supports obtained in genome phylogenies could be an artifact probably caused by data size; MLST is valid to address intraspecific phylogenetic structure; and, each species has its own evolutionary history, which could be affected by HGT to different extents.

Guide RNA Repertoires in the Main Lineages of Trypanosoma cruzi: High Diversity and Variable Redundancy Among Strains.

Trypanosoma cruzi, as other kinetoplastids, has a complex mechanism of editing of mitochondrial mRNAs that requires guide RNAs (gRNAs) coded in DNA minicircles in the kinetoplast. There are many variations on this mechanism among species. mRNA editing and gRNA repertoires are almost unknown in T. cruzi. Here, gRNAs were inferred based on deep-sequenced minicircle hypervariable regions (mHVRs) and editing cascades were rebuilt in strains belonging to the six main T. cruzi lineages. Inferred gRNAs were clustered according to their sequence similarity to constitute gRNA classes. Extreme diversity of gRNA classes was observed, which implied highly divergent gRNA repertoires among different lineages, even within some lineages. In addition, a variable gRNA class redundancy (i.e., different gRNA classes editing the same mRNA region) was detected among strains. Some strains had upon four times more gRNA classes than others. Such variations in redundancy affected gRNA classes of all mRNAs in a concerted way, i.e., there are correlated variations in the number of gRNAs classes editing each mRNA. Interestingly, cascades were incomplete for components of the respiratory complex I in several strains. Finally, gRNA classes of different strains may potentially edit mitochondrial mRNAs from other lineages in the same way as they edit their own mitochondrial mRNAs, which is a prerequisite for biparental inheritance of minicircle in hybrids. We propose that genetic exchange and biparental inheritance of minicircles combined with minicircle drift due to (partial) random segregation of minicircles during kDNA replication is a suitable hypothesis to explain the divergences among strains and the high levels of gRNA redundancy in some strains. In addition, our results support that the complex I may not be required in some stages in the life cycle as previously shown and that linkage (in the same minicircle) of gRNAs that edit different mRNAs may prevent gRNA class lost in such stage.

A Novel Genotype and First Record of Trypanosoma lainsoni in Argentina.

Trypanosomes are a group of parasitic flagellates with medical and veterinary importance. Despite many species having been described in this genus, little is known about many of them. Here, we report a genetic and morphological characterization of trypanosomatids isolated from wild mammals from the Argentine Chaco region. Parasites were morphologically and ultrastructurally characterized by light microscopy and transmission electron microscopy. Additionally, 18s rRNA and gGAPDH genes were sequenced and analyzed using maximum likelihood and Bayesian inference. Morphological characterization showed clear characteristics associated with the Trypanosoma genus. The genetic characterization demonstrates that the studied isolates have identical sequences and a pairwise identity of 99% with Trypanosoma lainsoni, which belongs to the clade of lizards and snakes/rodents and marsupials. To date, this species had only been found in the Amazon region. Our finding represents the second report of T. lainsoni and the first record for the Chaco region. Furthermore, we ultrastructurally described for the first time the species. Finally, the host range of T. lainsoni was expanded (Leopardus geoffroyi, Carenivora, Felidae; and Calomys sp., Rodentia, Cricetidae), showing a wide host range for this species.

Evidence of hybridization, mitochondrial introgression and biparental inheritance of the kDNA minicircles in Trypanosoma cruzi I.

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.

Elucidating diversity in the class composition of the minicircle hypervariable region of Trypanosoma cruzi: New perspectives on typing and kDNA inheritance.

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.