Bryan C. Jensen

Widespread variation in transcript abundance within and across developmental stages of Trypanosoma brucei.

BackgroundTrypanosoma brucei, the causative agent of African sleeping sickness, undergoes a complex developmental cycle that takes place in mammalian and insect hosts and is accompanied by changes in metabolism and cellular morphology. While differences in mRNA expression have been described for many genes, genome-wide expression analyses have been largely lacking. Trypanosomatids represent a unique case in eukaryotes in that they transcribe protein-coding genes as large polycistronic units, and rarely regulate gene expression at the level of transcription initiation.ResultsHere we present a comprehensive analysis of mRNA expression in several stages of parasite development. Utilizing microarrays that have multiple copies of multiple probes for each gene, we were able to demonstrate with a high degree of statistical confidence that approximately one-fourth of genes show differences in mRNA expression levels in the stages examined. These include complex patterns of gene expression within gene families, including the large family of variant surface glycoproteins (VSGs) and their relatives, where we have identified a number of constitutively expressed family members. Furthermore, we were able to assess the relative abundance of all transcripts in each stage, identifying the genes that are either weakly or highly expressed. Very few genes show no evidence of expression.ConclusionDespite the lack of gene regulation at the level of transcription initiation, our results reveal extensive regulation of mRNA abundance associated with different life cycle and growth stages. In addition, analysis of variant surface glycoprotein gene expression reveals a more complex picture than previously thought. These data provide a valuable resource to the community of researchers studying this lethal agent.

Extensive stage-regulation of translation revealed by ribosome profiling of Trypanosoma brucei

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.

A Novel Protein Kinase Localized to Lipid Droplets Is Required for Droplet Biogenesis in Trypanosomes

ABSTRACT Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.

Species Specificity in Ribosome Biogenesis: a Nonconserved Phosphoprotein Is Required for Formation of the Large Ribosomal Subunit in Trypanosoma brucei

ABSTRACT In the protozoan parasite Trypanosoma brucei, the large rRNA, which is a single 3.4- to 5-kb species in most organisms, is further processed to form six distinct RNAs, two larger than 1 kb (LSU1 and LSU2) and four smaller than 220 bp. The small rRNA SR1 separates the two large RNAs, while the remaining small RNAs are clustered at the 3′ end of the precursor rRNA. One would predict that T. brucei possesses specific components to carry out these added processing events. We show here that the trypanosomatid-specific nucleolar phosphoprotein NOPP44/46 is involved in this further processing. Cells depleted of NOPP44/46 by RNA interference had a severe growth defect and demonstrated a defect in large-ribosomal-subunit biogenesis. Concurrent with this defect, a significant decrease in processing intermediates, particularly for SR1, was seen. In addition, we saw an accumulation of aberrant processing intermediates caused by cleavage within either LSU1 or LSU2. Though it is required for large-subunit biogenesis, we show that NOPP44/46 is not incorporated into the nascent particle. Thus, NOPP44/46 is an unusual protein in that it is both nonconserved and required for ribosome biogenesis.

Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei.

Background The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage. Methodology/Principal Findings We therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D2, quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds. Conclusions/Significance Parasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent proliferation may identify new means to activate death pathways.

The Trypanosoma brucei Life Cycle Switch TbPTP1 Is Structurally Conserved and Dephosphorylates the Nucleolar Protein NOPP44/46

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.

Integrative analysis of the Trypanosoma brucei gene expression cascade predicts differential regulation of mRNA processing and unusual control of ribosomal protein expression

BackgroundTrypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model.ResultsWe refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated.ConclusionsLevels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism.

Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping.

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.

Trypanosoma brucei: Two mitogen activated protein kinase kinases are dispensable for growth and virulence of the bloodstream form

Mitogen activated protein kinase cascades function in eukaryotic responses to the environment and stress. Trypanosomatid parasites possess protein kinases with sequences characteristic of kinases in such cascades. In this report we use gene knockouts to demonstrate that two mitogen activated kinase kinase genes, MKK1 (Tb927.3.4860) and MKK5 (Tb927.10.5270), are not essential in the pathogenic bloodstream stage of Trypanosoma brucei, either in vitro or in vivo. Bloodstream forms lacking MKK1 showed decreased growth at 39°C as compared to the parental line. However, unlike its Leishmania orthologue, T. brucei MKK1 does not appear to play a significant role in flagellar biogenesis.

NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms

Highlights • Various genetic mutants of NDH2 were created in bloodstream form Trypanosoma brucei.• NDH2 null mutants showed a substantial reduction in growth.• NDH2 ablation in a complex I deficient background led to severe growth restriction.• Upon prolonged culture, parasites partially compensated for NDH2 deficiency.• Loss of NDH2 led to reduced acetate, potentially contributing to the growth defect.