Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state.

Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. In kinetoplastid protozoa, including T. brucei, posttranscriptional control mechanisms are the primary means of gene regulation, and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that reportedly interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We validate the DRBD18 interaction with translating ribosomes and the translation initiation factor, eIF3a. We further show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, that is, changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. In DRBD18-depleted cells, a set of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. RNA immunoprecipitation/qRT-PCR indicates that DRBD18 associates with members of both repressed and enhanced cohorts. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.

RNA-Seq reveals that overexpression of TcUBP1 switches the gene expression pattern toward that of the infective form of Trypanosoma cruzi.

Trypanosomes regulate gene expression mainly by using posttranscriptional mechanisms. Key factors responsible for carrying out this regulation are RNA-binding proteins, affecting subcellular localization, translation, and/or transcript stability. Trypanosoma cruzi U-rich RNA-binding protein 1 (TcUBP1) is a small protein that modulates the expression of several surface glycoproteins of the trypomastigote infective stage of the parasite. Its mRNA targets are known, but the impact of its overexpression at the transcriptome level in the insect-dwelling epimastigote cells has not yet been investigated. Thus, in the present study, by using a tetracycline-inducible system, we generated a population of TcUBP1-overexpressing parasites and analyzed its effect by RNA-Seq methodology. This allowed us to identify 793 up- and 371 downregulated genes with respect to the wildtype control sample. Among the upregulated genes, it was possible to identify members coding for the TcS superfamily, MASP, MUCI/II, and protein kinases, whereas among the downregulated transcripts, we found mainly genes coding for ribosomal, mitochondrial, and synthetic pathway proteins. RNA-Seq comparison with two previously published datasets revealed that the expression profile of this TcUBP1-overexpressing replicative epimastigote form resembles the transition to the infective metacyclic trypomastigote stage. We identified novel cis-regulatory elements in the 3'-untranslated region of the affected transcripts and confirmed that UBP1m, a signature TcUBP1 binding element previously characterized in our laboratory, is enriched in the list of stabilized genes. We can conclude that the overall effect of TcUBP1 overexpression on the epimastigote transcriptome is mainly the stabilization of mRNAs coding for proteins that are important for parasite infection.

Uncovering a multitude of stage-specific splice variants and putative protein isoforms generated along mouse spermatogenesis.

BACKGROUND: Mammalian testis is a highly complex and heterogeneous tissue. This complexity, which mostly derives from spermatogenic cells, is reflected at the transcriptional level, with the largest number of tissue-specific genes and long noncoding RNAs (lncRNAs) compared to other tissues, and one of the highest rates of alternative splicing. Although it is known that adequate alternative-splicing patterns and stage-specific isoforms are critical for successful spermatogenesis, so far only a very limited number of reports have addressed a detailed study of alternative splicing and isoforms along the different spermatogenic stages. RESULTS: In the present work, using highly purified stage-specific testicular cell populations, we detected 33,002 transcripts expressed throughout mouse spermatogenesis not annotated so far. These include both splice variants of already annotated genes, and of hitherto unannotated genes. Using conservative criteria, we uncovered 13,471 spermatogenic lncRNAs, which reflects the still incomplete annotation of lncRNAs. A distinctive feature of lncRNAs was their lower number of splice variants compared to protein-coding ones, adding to the conclusion that lncRNAs are, in general, less complex than mRNAs. Besides, we identified 2,794 unannotated transcripts with high coding potential (including some arising from yet unannotated genes), many of which encode unnoticed putative testis-specific proteins. Some of the most interesting coding splice variants were chosen, and validated through RT-PCR. Remarkably, the largest number of stage-specific unannotated transcripts are expressed during early meiotic prophase stages, whose study has been scarcely addressed in former transcriptomic analyses. CONCLUSIONS: We detected a high number of yet unannotated genes and alternatively spliced transcripts along mouse spermatogenesis, hence showing that the transcriptomic diversity of the testis is considerably higher than previously reported. This is especially prominent for specific, underrepresented stages such as those of early meiotic prophase, and its unveiling may constitute a step towards the understanding of their key events.

Transcriptome analysis of the allotetraploids of the Dilatata group of Paspalum (Poaceae): effects of diploidization on the expression of defensin and Snakin/GASA genes.

Plant Snakin/GASA and defensin peptides are cysteine-rich molecules with a wide range of biological functions. They are included within the large family of plant antimicrobial peptides (AMPs), characterized by their structural stability, broad spectrum of activity, and diverse mechanisms of action. The Dilatata group of Paspalum includes five allotetraploids that share an equivalent genomic formula IIJJ. From RNA-seq data of seedling tissues, we performed an in silico characterization of the defensin and Snakin/GASA genes in these species and diploids with a II and JJ genome formula and studied the evolutionary consequences of polyploidy on the expression of the two AMPs families. A total of 107 defensins (distributed in eight groups) and 145 Snakin/GASA (grouped in three subfamilies) genes were identified. Deletions, duplications and/or gene silencing seem to have mediated the evolution of these genes in the allotetraploid species. In defensin genes, the IIJJ allopolyploids retained the I subgenome defensin copies in some of the identified groups supporting the closeness of their nuclear genome with the I subgenome species. In both AMPs families, orthologous genes in tetraploids exhibit higher similarity to each other than with diploids. This data supports the theory of a single origin for the allotetraploids. Several copies of both defensin and Snakin/GASA genes were detected in the five polyploids which could have arisen due to duplication events occurring independently during the diploidization processes in the allotetraploid taxa.

Genomic characterization of Moraxella bovis and Moraxella bovoculi Uruguayan strains isolated from calves with infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.

Crystal structure of Trypanosoma cruzi heme peroxidase and characterization of its substrate specificity and compound I intermediate.

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

RENANO: a REference-based compressor for NANOpore FASTQ files.

MOTIVATION: Nanopore sequencing technologies are rapidly gaining popularity, in part, due to the massive amounts of genomic data they produce in short periods of time (up to 8.5 TB of data in <72 h). To reduce the costs of transmission and storage, efficient compression methods for this type of data are needed. RESULTS: We introduce RENANO, a reference-based lossless data compressor specifically tailored to FASTQ files generated with nanopore sequencing technologies. RENANO improves on its predecessor ENANO, currently the state of the art, by providing a more efficient base call sequence compression component. Two compression algorithms are introduced, corresponding to the following scenarios: (1) a reference genome is available without cost to both the compressor and the decompressor and (2) the reference genome is available only on the compressor side, and a compacted version of the reference is included in the compressed file. We compare the compression performance of RENANO against ENANO on several publicly available nanopore datasets. RENANO improves the base call sequences compression of ENANO by 39.8% in scenario (1), and by 33.5% in scenario (2), on average, over all the datasets. As for total file compression, the average improvements are 12.7% and 10.6%, respectively. We also show that RENANO consistently outperforms the recent general-purpose genomic compressor Genozip. AVAILABILITY AND IMPLEMENTATION: RENANO is freely available for download at: https://github.com/guilledufort/RENANO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Real-Time Genomic Surveillance for SARS-CoV-2 Variants of Concern, Uruguay.

We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.

ENANO: Encoder for NANOpore FASTQ files.

MOTIVATION: The amount of genomic data generated globally is seeing explosive growth, leading to increasing needs for processing, storage and transmission resources, which motivates the development of efficient compression tools for these data. Work so far has focused mainly on the compression of data generated by short-read technologies. However, nanopore sequencing technologies are rapidly gaining popularity due to the advantages offered by the large increase in the average size of the produced reads, the reduction in their cost and the portability of the sequencing technology. We present ENANO (Encoder for NANOpore), a novel lossless compression algorithm especially designed for nanopore sequencing FASTQ files. RESULTS: The main focus of ENANO is on the compression of the quality scores, as they dominate the size of the compressed file. ENANO offers two modes, Maximum Compression and Fast (default), which trade-off compression efficiency and speed. We tested ENANO, the current state-of-the-art compressor SPRING and the general compressor pigz on several publicly available nanopore datasets. The results show that the proposed algorithm consistently achieves the best compression performance (in both modes) on every considered nanopore dataset, with an average improvement over pigz and SPRING of >24.7% and 6.3%, respectively. In addition, in terms of encoding and decoding speeds, ENANO is 2.9× and 1.7× times faster than SPRING, respectively, with memory consumption up to 0.2 GB. AVAILABILITY AND IMPLEMENTATION: ENANO is freely available for download at: https://github.com/guilledufort/EnanoFASTQ. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of µORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers.

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis' gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

Conservation and diversification of small RNA pathways within flatworms.

BACKGROUND: Small non-coding RNAs, including miRNAs, and gene silencing mediated by RNA interference have been described in free-living and parasitic lineages of flatworms, but only few key factors of the small RNA pathways have been exhaustively investigated in a limited number of species. The availability of flatworm draft genomes and predicted proteomes allowed us to perform an extended survey of the genes involved in small non-coding RNA pathways in this phylum. RESULTS: Overall, findings show that the small non-coding RNA pathways are conserved in all the analyzed flatworm linages; however notable peculiarities were identified. While Piwi genes are amplified in free-living worms they are completely absent in all parasitic species. Remarkably all flatworms share a specific Argonaute family (FL-Ago) that has been independently amplified in different lineages. Other key factors such as Dicer are also duplicated, with Dicer-2 showing structural differences between trematodes, cestodes and free-living flatworms. Similarly, a very divergent GW182 Argonaute interacting protein was identified in all flatworm linages. Contrasting to this, genes involved in the amplification of the RNAi interfering signal were detected only in the ancestral free living species Macrostomum lignano. We here described all the putative small RNA pathways present in both free living and parasitic flatworm lineages. CONCLUSION: These findings highlight innovations specifically evolved in platyhelminths presumably associated with novel mechanisms of gene expression regulation mediated by small RNA pathways that differ to what has been classically described in model organisms. Understanding these phylum-specific innovations and the differences between free living and parasitic species might provide clues to adaptations to parasitism, and would be relevant for gene-silencing technology development for parasitic flatworms that infect hundreds of million people worldwide.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi.

BACKGROUND: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms. RESULTS: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage. CONCLUSIONS: A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.

Transcriptomic analysis of N-terminal mutated Trypanosoma cruzi UBP1 knockdown underlines the importance of this RNA-binding protein in parasite development.

BACKGROUND: During its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms. METHODS: In an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells. RESULTS: We analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells. CONCLUSION: Together, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.

RNA-seq data exploration after trypanosome RNA-binding protein UBP1 expression is altered by CRISPR-Cas9 gene editing and overexpression.

Previous studies have shown that overexpression of the Trypanosoma cruzi U-rich RNA-binding protein 1 (TcUBP1) in insect-dwelling epimastigotes results in a gene expression pattern resembling that of the infective form of the pathogen. Here, we used CRISPR-Cas9-induced edition of TcUBP1 and full-length protein overexpression in epimastigote cells to monitor transcriptomic changes during the epimastigote-to-metacyclic trypomastigote stage transition of T. cruzi. This dataset includes the bioinformatics analysis of three different RNA-seq samples, each with three biological replicates, showing differential mRNA abundances. The current transcriptome report has the potential to shed light on the quantitative variances in the expression of significant up- or down-regulated mRNAs as a consequence of the levels of the UBP1 protein. Raw data files were deposited at the NCBI Sequence Read Archive - SRA at http://ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers PRJNA907231 and PRJNA949967.

Evidence for a negative feedback control mediated by the 3' untranslated region assuring the low expression level of the RNA binding protein TcRBP19 in T. cruzi epimastigotes.

Because of their relevant role in the post-transcriptional regulation of the expression of a multitude of genes, RNA-binding proteins (RBPs) need to be accurately regulated in response to environmental signals in terms of quantity, functionality and localization. Transcriptional, post-transcriptional and post-translational steps have all been involved in this tight control. We have previously identified a Trypanosoma cruzi RBP, named TcRBP19, which can barely be detected at the replicative intracellular amastigote stage of the mammalian host. Even though protein coding genes are typically transcribed constitutively in trypanosomes, TcRBP19 protein is undetectable at the epimastigote stage. Here, we show that this protein expression pattern follows the steady-state of its mRNA. Using a T. cruzi reporter gene approach, we could establish a role for the 3' UTR of the tcrbp19 mRNA in transcript down-regulation at the epimastigote stage. In addition, the binding of the TcRBP19 protein to its encoding mRNA was revealed by in vitro pull down followed by qRT-PCR and confirmed by CLIP assays. Furthermore, we found that forced over-expression of TcRBP19 in T. cruzi epimastigotes decreased the stability of the endogenous tcrbp19 mRNA. These results support a negative feedback control of TcRBP19 to help maintain its very low concentration of TcRBP19 in the epimastigote stage. To our knowledge, this is the first RBP reported in trypanosomatids capable of negatively regulating its own mRNA. The mechanism revealed here adds to our limited but growing number of examples of negative mRNA autoregulation in the control of gene expression.

Implication of CA repeated tracts on post-transcriptional regulation in Trypanosoma cruzi.

In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.

Transcriptomic analysis of the adaptation to prolonged starvation of the insect-dwelling Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.

Improving genome-wide mapping of nucleosomes in Trypanosome cruzi.

In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain.

Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state.

Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. Whereas most well-studied organisms rely on transcriptional control as the main regulator of gene expression, post-transcriptional control mechanisms are particularly important in T. brucei , and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, i.e . changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. Proteomic analysis validates these data. In DRBD18-depleted cells, a cohort of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.