BackgroundTrypanosoma brucei subspecies infect humans and animals in sub-Saharan Africa. This early diverging eukaryote shows many novel features in basic biological processes, including the use of polycistronic transcription to generate all protein-coding mRNAs. Therefore we hypothesized that translational control provides a means to tune gene expression during parasite development in mammalian and fly hosts.ResultsWe used ribosome profiling to examine genome-wide protein synthesis in animal-derived slender bloodstream forms and cultured procyclic (insect midgut) forms. About one-third of all CDSs showed statistically significant regulation of protein production between the two stages. Of these, more than two-thirds showed a change in translation efficiency, but few appeared to be controlled by this alone. Ribosomal proteins were translated poorly, especially in animal-derived parasites. A disproportionate number of metabolic enzymes were up-regulated at the mRNA level in procyclic forms, as were variant surface glycoproteins in bloodstream forms. Comparison with cultured bloodstream forms from another strain revealed stage-specific changes in gene expression that transcend strain and growth conditions. Genes with upstream ORFs had lower mean translation efficiency, but no evidence was found for involvement of uORFs in stage-regulation.ConclusionsRibosome profiling revealed that differences in the production of specific proteins in T. brucei bloodstream and procyclic forms are more extensive than predicted by analysis of mRNA abundance. While in vivo and in vitro derived bloodstream forms from different strains are more similar to one another than to procyclic forms, they showed many differences at both the mRNA and protein production level.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-911) contains supplementary material, which is available to authorized users.

BackgroundLeishmania use exosomes to communicate with their mammalian hosts and these secreted vesicles appear to contribute to pathogenesis by delivering protein virulence factors to macrophages. In other eukaryotes, exosomes were found to carry RNA cargo, such as mRNAs and small non-coding RNAs, capable of altering recipient cell phenotype. Whether leishmania exosomes also contain RNAs which they are able to deliver to bystander cells is not known. Here, we show that leishmania exosomes indeed contain RNAs and compare and contrast the RNA content of exosomes released by Leishmania donovani and Leishmania braziliensis.ResultsWe purified RNA from exosomes collected from axenic amastigote culture supernatant and found that when compared with total leishmania RNA, exosomes mainly contained short RNA sequences. Exosomes with intact membranes were capable of protecting their RNA cargo from degradation by RNase. Moreover, exosome RNA cargo was delivered to host cell cytoplasm in vitro. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA species such as rRNA and tRNA. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. In addition, we observed the presence of sequences mapping to siRNA-coding regions in L. braziliensis , but not in L. donovani exosomes.ConclusionsThese results show that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs. These exosomes are competent to deliver their cargo of novel, potential small regulatory RNAs to macrophages where they may influence parasite-host cell interactions. The remarkably high degree of congruence in exosomal RNA content between L. donovani and L. braziliensis, argues for the presence of a conserved mechanism for exosomal RNA packaging in leishmania. These findings open up a new avenue of research on non-canonical, small RNA pathways in this trypanosomatid, which may elucidate pathogenesis and identify novel therapeutic approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1260-7) contains supplementary material, which is available to authorized users.

Next Generation Sequencing (NGS) strategies, like RNA-Seq, have revealed the transcription of a wide variety of long non-coding RNAs (lncRNAs) in the genomes of several organisms. In the present work we assessed the lncRNAs complement of Schistosoma mansoni, the blood fluke that causes schistosomiasis, ranked among the most prevalent parasitic diseases worldwide. We focused on the long intergenic/intervening ncRNAs (lincRNAs), hidden within the large amount of information obtained through RNA-Seq in S. mansoni (88 libraries). Our computational pipeline identified 7029 canonically-spliced putative lincRNA genes on 2596 genomic loci (at an average 2.7 isoforms per lincRNA locus), as well as 402 spliced lncRNAs that are antisense to protein-coding (PC) genes. Hundreds of lincRNAs showed traits for being functional, such as the presence of epigenetic marks at their transcription start sites, evolutionary conservation among other schistosome species and differential expression across five different life-cycle stages of the parasite. Real-time qPCR has confirmed the differential life-cycle stage expression of a set of selected lincRNAs. We have built PC gene and lincRNA co-expression networks, unraveling key biological processes where lincRNAs might be involved during parasite development. This is the first report of a large-scale identification and structural annotation of lncRNAs in the S. mansoni genome.

Leishmaniasis is a worldwide public health problem caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the most important species responsible for tegumentary leishmaniases in Brazil. An understanding of the molecular mechanisms underlying the success of this parasite is urgently needed. An in-depth study on the modulation of gene expression across the life cycle stages of L. braziliensis covering coding and noncoding RNAs (ncRNAs) was missing and is presented herein. Analyses of differentially expressed (DE) genes revealed that most prominent differences were observed between the transcriptomes of insect and mammalian proliferative forms (6,576 genes). Gene ontology (GO) analysis indicated stage-specific enriched biological processes. A computational pipeline and 5 ncRNA predictors allowed the identification of 11,372 putative ncRNAs. Most of the DE ncRNAs were found between the transcriptomes of insect and mammalian proliferative stages (38%). Of the DE ncRNAs, 295 were DE in all three stages and displayed a wide range of lengths, chromosomal distributions and locations; many of them had a distinct expression profile compared to that of their proteincoding neighbors. Thirty-five putative ncRNAs were submitted to northern blotting analysis, and one or more hybridization-positive signals were observed in 22 of these ncRNAs. This work presents an overview of the L. braziliensis transcriptome and its adjustments throughout development. In addition to determining the general features of the transcriptome at each life stage and the profile of proteincoding transcripts, we identified and characterized a variety of noncoding transcripts. The novel putative ncRNAs uncovered in L. braziliensis might be regulatory elements to be further investigated.

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.

Next-generation DNA sequencing is rapidly becoming a powerful tool for food animal management. One valuable use of this technology is to re-examine long-standing observations of performance differences associated with animal husbandry practices to better understand how these differences may be modulated by the gastrointestinal (GI) microbiome. The influences of environmental parameters such as air temperature and relative humidity on broiler chicken performance have commonly been observed, but how the GI microbiome may respond to seasonal environmental changes remains largely unknown. The purposes of this study were therefore to: (1) characterize the cecal microflora of commercial broilers (N = 87) collected at harvest across all 4 seasons, and (2) identify any significant changes of the GI microbiome and specific taxa according to season and Campylobacter status. Finding taxa with significant positive or negative correlations with Campylobacter could be useful by identifying indicator or antagonistic taxa and could also inform inferences regarding the ecological niche of Campylobacter. Whole GI tracts were removed from commercial broilers representing 87 independent flocks between April 2013 and May 2014 in the U.S. state of Georgia. Intact ceca were separated, cultured for Campylobacter and cecal contents were frozen. The cecal microbiome was characterized using barcoded sequencing of 16S rRNA genes on the Illumina MiSeq platform. The composition of the microbiome measured at processing was generally not affected by Campylobacter status but was most significantly affected by season of grow-out. Significantly fewer bacterial genera were found in winter than spring or summer. Bacterial genera with prior evidence for both positive or negative influences on gut health outcomes were significantly less abundant in the fall. Identifying specific members of the GI microbiota that vary according to season may help develop novel interventions to improve husbandry practices and growth performance.

BackgroundThe possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells.Methodology/Principal findingsAdult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways.Conclusions/SignificanceThe results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents.

Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we make use of existing Trypanosoma brucei ribosome profiling data to provide evidence of ribosome occupancy (and likely translation) of mRNAs from 225 currently unannotated coding sequences (CDSs). A small number of these putative genes correspond to extra copies of previously annotated genes, but 85% are novel. The median size of these novels CDSs is small (81 aa), indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs confirmed here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as protein-coding genes. Nonetheless, approximately one-third of the new CDSs were found only in T. brucei subspecies. Using ribosome footprints, RNA-Seq and spliced leader mapping data, we updated previous work to definitively revise the start sites for 414 CDSs as compared to the current gene models. The data pointed to several regions of the genome that had sequence errors that altered coding region boundaries. Finally, we consolidated this data with our previous work to propose elimination of 683 putative genes as protein-coding and arrive at a view of the translatome of slender bloodstream and procyclic culture form T. brucei.