Phosphoproteomic analysis of the response to DNA damage in Trypanosoma brucei.

Damage to the genetic material of the cell poses a universal threat to all forms of life. The DNA damage response is a coordinated cellular response to a DNA break, key to which is the phosphorylation signalling cascade. Identifying which proteins are phosphorylated is therefore crucial to understanding the mechanisms that underly it. We have used SILAC-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following double stranded DNA breaks, at two distinct loci in the genome of the single cell eukaryote Trypanosoma brucei. Here, we report on the Trypanosoma brucei phosphoproteome following a single double strand break at either a chromosome internal or subtelomeric locus, specifically the Bloodstream form expression site. We detected >6500 phosphorylation sites, of which 211 form a core set of double strand break responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we have identified two novel phosphorylation events on Histone H2A and find that in response to a chromosome internal break, proteins are predominantly phosphorylated, while a greater proportion of proteins dephosphorylated following a DNA break at a subtelomeric bloodstream form expression site. Our data represents the first DNA damage phosphoproteome and provides novel insights into repair at distinct chromosomal contexts in Trypanosoma brucei.

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) procaviensis Isolate 253, Strain LV425.

Leishmania (Mundinia) procaviensis is a parasitic kinetoplastid that was first isolated from a rock hyrax in Namibia in 1975. We present the complete genome sequence of Leishmania (Mundinia) procaviensis isolate 253, strain LV425, sequenced using combined short- and long-read technologies. This genome will contribute to our understanding of hyraxes as a Leishmania reservoir.

Synchronization of Trypanosoma brucei by Counter-Flow Centrifugal Elutriation.

Centrifugal counter-flow elutriation is a non-invasive technique that separates cells based on their hydrodynamic volume in a specialized centrifugation chamber that allows the application of a counter-flow of buffer to oppose sedimentation. Here, we report a centrifugal counter-flow elutriation protocol for Trypanosoma brucei cells that is able to rapidly isolate highly enriched G1 subpopulations (>95%) of synchronized cells. The cells obtained are viable and proliferate without lag, allowing subsequent cell cycle phases to be obtained by continued culture. The synchronized cell cultures obtained by this process have uniform DNA content, a narrow size distribution, undergo synchronous division, and maintain synchrony into subsequent cell cycles.

Chromosome-Scale Assembly of the Complete Genome Sequence of Porcisia hertigi, Isolate C119, Strain LV43.

Porcisia hertigi is a parasitic kinetoplastid first isolated from porcupines (Coendou rothschildi) in central Panama in 1965. We present the complete genome sequence of P. hertigi, isolate C119, strain LV43, sequenced using combined short- and long-read technologies. This complete genome sequence will contribute to our knowledge of the parasitic genus Porcisia.

Chromosome-scale genome sequencing, assembly and annotation of six genomes from subfamily Leishmaniinae.

We provide the raw and processed data produced during the genome sequencing of isolates from six species of parasites from the sub-family Leishmaniinae: Leishmania martiniquensis (Thailand), Leishmania orientalis (Thailand), Leishmania enriettii (Brazil), Leishmania sp. Ghana, Leishmania sp. Namibia and Porcisia hertigi (Panama). De novo assembly was performed using Nanopore long reads to construct chromosome backbone scaffolds. We then corrected erroneous base calling by mapping short Illumina paired-end reads onto the initial assembly. Data has been deposited at NCBI as follows: raw sequencing output in the Sequence Read Archive, finished genomes in GenBank, and ancillary data in BioSample and BioProject. Derived data such as quality scoring, SAM files, genome annotations and repeat sequence lists have been deposited in Lancaster University's electronic data archive with DOIs provided for each item. Our coding workflow has been deposited in GitHub and Zenodo repositories. This data constitutes a resource for the comparative genomics of parasites and for further applications in general and clinical parasitology.

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) sp. Ghana, Isolate GH5, Strain LV757.

Leishmania (Mundinia) sp. Ghana is a kinetoplastid parasite isolated in 2015 in Ghana. We report the complete genome sequence of L. (M.) sp. Ghana, sequenced using combined short-read and long-read technologies. This will facilitate greater understanding of this novel pathogen and its relationships within the subgenus Mundinia.

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) enriettii, Isolate CUR178, Strain LV763.

Leishmania (Mundinia) enriettii is a parasitic kinetoplastid first isolated from a guinea pig in Brazil in 1946. We present the complete genome sequence of L. (M.) enriettii, isolate CUR178, strain LV763, sequenced using combined short-read and long-read technologies. This will facilitate a greater understanding of the genome diversity within L. (M.) enriettii.

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) orientalis, Isolate LSCM4, Strain LV768.

Leishmania (Mundinia) orientalis is a kinetoplastid parasite first isolated in 2014 in Thailand. We report the complete genome sequence of L. (M.) orientalis, sequenced using combined short-read and long-read technologies. This will facilitate greater understanding of this novel pathogen and its relationship to other members of the subgenus Mundinia.

Extensive Translational Regulation through the Proliferative Transition of Trypanosoma cruzi Revealed by Multi-Omics.

Trypanosoma cruzi is the etiological agent for Chagas disease, a neglected parasitic disease in Latin America. Gene transcription control governs the eukaryotic cell replication but is absent in trypanosomatids; thus, it must be replaced by posttranscriptional regulatory events. We investigated the entrance into the T. cruzi replicative cycle using ribosome profiling and proteomics on G1/S epimastigote cultures synchronized with hydroxyurea. We identified 1,784 translationally regulated genes (change > 2, false-discovery rate [FDR] < 0.05) and 653 differentially expressed proteins (change > 1.5, FDR < 0.05), respectively. A major translational remodeling accompanied by an extensive proteome change is found, while the transcriptome remains largely unperturbed at the replicative entrance of the cell cycle. The differentially expressed genes comprise specific cell cycle processes, confirming previous findings while revealing candidate cell cycle regulators that undergo previously unnoticed translational regulation. Clusters of genes showing a coordinated regulation at translation and protein abundance share related biological functions such as cytoskeleton organization and mitochondrial metabolism; thus, they may represent posttranscriptional regulons. The translatome and proteome of the coregulated clusters change in both coupled and uncoupled directions, suggesting that complex cross talk between the two processes is required to achieve adequate protein levels of different regulons. This is the first simultaneous assessment of the transcriptome, translatome, and proteome of trypanosomatids, which represent a paradigm for the absence of transcriptional control. The findings suggest that gene expression chronology along the T. cruzi cell cycle is controlled mainly by translatome and proteome changes coordinated using different mechanisms for specific gene groups. IMPORTANCE Trypanosoma cruzi is an ancient eukaryotic unicellular parasite causing Chagas disease, a potentially life-threatening illness that affects 6 to 7 million people, mostly in Latin America. The antiparasitic treatments for the disease have incomplete efficacy and adverse reactions; thus, improved drugs are needed. We study the mechanisms governing the replication of the parasite, aiming to find differences with the human host, valuable for the development of parasite-specific antiproliferative drugs. Transcriptional regulation is essential for replication in most eukaryotes, but in trypanosomatids, it must be replaced by subsequent gene regulation steps since they lack transcription initiation control. We identified the genome-wide remodeling of mRNA translation and protein abundance during the entrance to the replicative phase of the cell cycle. We found that translation is strongly regulated, causing variation in protein levels of specific cell cycle processes, representing the first simultaneous study of the translatome and proteome in trypanosomatids.

LGAAP: Leishmaniinae Genome Assembly and Annotation Pipeline.

We present the LGAAP computational pipeline, which was successfully used to assemble six genomes of the parasite subfamily Leishmaniinae to chromosome-scale completeness from a combination of long- and short-read sequencing data. LGAAP is open source, and we suggest that it may easily be ported for assembly of any genome of comparable size (∼35 Mb).

Chromosome-Scale Assembly of the Complete Genome Sequence of Leishmania (Mundinia) martiniquensis, Isolate LSCM1, Strain LV760.

Leishmania (Mundinia) martiniquensis is a kinetoplastid parasite that was first isolated in 1995 on Martinique. We report the first complete genome for Leishmania martiniquensis from Asia, isolate LSCM1, strain LV760, which was sequenced using combined short-read and long-read technologies. This will facilitate greater understanding of the evolution of the geographically dispersed subgenus Mundinia.

Re-evaluation of Diadenosine Tetraphosphate (Ap4A) From a Stress Metabolite to Bona Fide Secondary Messenger.

Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (NpnNs), the best studied of which is diadenosine tetraphosphate (Ap4A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap4A and Ap4N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the "friend" and "foe" scenarios. The "friend (or alarmone) hypothesis" suggests that ApnN act as bona fide secondary messengers mediating responses to stress. In contrast, the "foe" hypothesis proposes that ApnN and other NpnN are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for ApnN in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to ApnN. Finally, areas for future research are discussed with possible but unproven roles for intracellular ApnN to encourage further research into the signaling networks that are regulated by these nucleotides.

Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggests atypical mechanisms for thermotolerance.

The symptoms of African sleeping sickness, caused by the parasite Trypanosoma brucei, can include periods of fever as high as 41 °C which triggers a heat shock response in the parasite. To capture events involved in sensing and responding to heat shock in the mammalian infective form we have conducted a SILAC-based quantitative proteomic and phosphoproteomic analysis of T. brucei cells treated at 41 °C for 1h. Our analysis identified 193 heat shock responsive phosphorylation sites with an average of 5-fold change in abundance, but only 20 heat shock responsive proteins with average of 1.5-fold change. These data indicate that protein abundance does not rapidly respond (≤1 h) to heat shock, and that the changes observed in phosphorylation site abundance are larger and more widespread. The heat shock responsive phosphorylation sites showed enrichment of RNA binding proteins with putative roles in heat shock response included P-body / stress granules and the eukaryotic translation initiation 4F complex. The ZC3H11-MKT1 complex, which stabilises mRNAs of thermotolerance proteins, appears to represent a key signal integration node in the heat shock response. SIGNIFICANCE: We report the first quantitative study of changes in protein and phosphorylation site abundance in response to heat shock in the clinically relevant form of the human parasite Trypanosoma brucei. The identification of heat shock responsive phosphorylation sites on proteins with putative roles in thermotolerance including the ZC3H11-MKT1 complex provides evidence of the role dynamic phosphorylation of RNA binding proteins in co-ordinating heat shock. Temperature changes in the host are a major physiological challenge to parasites and factors conferring tolerance to heat shock constitute overlooked virulence factors. A better understanding of these virulence factors will pave the way for the development of novel drug therapies which selectively target T. brucei.

Proteome-Wide Quantitative Phosphoproteomic Analysis of Trypanosoma brucei Insect and Mammalian Life Cycle Stages.

Mass spectrometry based proteomics allows for the identification and quantification of protein and phosphorylation site abundance on a proteome wide scale. Here we describe the metabolic labeling of cultured Trypanosoma brucei cells in either the bloodstream or procyclic life cycle stage using stable isotope labeling of amino acids in cell culture (SILAC), and the production of samples suitable for analysis by liquid chromatography tandem mass spectrometry. The protocols require little specialist equipment, and they typically enable quantification of over 4500 proteins and 9000 phosphorylation sites.

A mechanism-inspired UDP-N-acetylglucosamine pyrophosphorylase inhibitor.

UDP-N-acetylglucosamine pyrophosphorylase (UAP1) catalyses the last step in eukaryotic biosynthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), converting UTP and GlcNAc-1P to the sugar nucleotide. Gene disruption studies have shown that this gene is essential in eukaryotes and a possible antifungal target, yet no inhibitors of fungal UAP1 have so far been reported. Here we describe the crystal structures of substrate/product complexes of UAP1 from Aspergillus fumigatus that together provide snapshots of catalysis. A structure with UDP-GlcNAc, pyrophosphate and Mg2+ provides the first Michaelis complex trapped for this class of enzyme, revealing the structural basis of the previously reported Mg2+ dependence and direct observation of pyrophosphorolysis. We also show that a highly conserved lysine mimics the role of a second metal observed in structures of bacterial orthologues. A mechanism-inspired UTP α,ß-methylenebisphosphonate analogue (meUTP) was designed and synthesized and was shown to be a micromolar inhibitor of the enzyme. The mechanistic insights and inhibitor described here will facilitate future studies towards the discovery of small molecule inhibitors of this currently unexploited potential antifungal drug target.

Organising the cell cycle in the absence of transcriptional control: Dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally.

The cell division cycle of the unicellular eukaryote Trypanosome brucei is tightly regulated despite the paucity of transcriptional control that results from the arrangement of genes in polycistronic units and lack of dynamically regulated transcription factors. To identify the contribution of dynamic phosphorylation to T. brucei cell cycle control we have combined cell cycle synchronisation by centrifugal elutriation with quantitative phosphoproteomic analysis. Cell cycle regulated changes in phosphorylation site abundance (917 sites, average 5-fold change) were more widespread and of a larger magnitude than changes in protein abundance (443 proteins, average 2-fold change) and were mostly independent of each other. Hierarchical clustering of co-regulated phosphorylation sites according to their cell cycle profile revealed that a bulk increase in phosphorylation occurs across the cell cycle, with a significant enrichment of known cell cycle regulators and RNA binding proteins (RBPs) within the largest clusters. Cell cycle regulated changes in essential cell cycle kinases are temporally co-ordinated with differential phosphorylation of components of the kinetochore and eukaryotic initiation factors, along with many RBPs not previously linked to the cell cycle such as eight PSP1-C terminal domain containing proteins. The temporal profiles demonstrate the importance of dynamic phosphorylation in co-ordinating progression through the cell cycle, and provide evidence that RBPs play a central role in post-transcriptional regulation of the T. brucei cell cycle. Data are available via ProteomeXchange with identifier PXD013488.

Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis.

Visceral leishmaniasis causes considerable mortality and morbidity in many parts of the world. There is an urgent need for the development of new, effective treatments for this disease. Here we describe the development of an anti-leishmanial drug-like chemical series based on a pyrazolopyrimidine scaffold. The leading compound from this series (7, DDD853651/GSK3186899) is efficacious in a mouse model of visceral leishmaniasis, has suitable physicochemical, pharmacokinetic and toxicological properties for further development, and has been declared a preclinical candidate. Detailed mode-of-action studies indicate that compounds from this series act principally by inhibiting the parasite cdc-2-related kinase 12 (CRK12), thus defining a druggable target for visceral leishmaniasis.

Dynamic regulation of the Trypanosoma brucei transferrin receptor in response to iron starvation is mediated via the 3'UTR.

The bloodstream form of the parasite Trypanosoma brucei obtains iron from its mammalian host by receptor-mediated endocytosis of host transferrin through its own unique transferrin receptor (TbTfR). Expression of TbTfR rapidly increases upon iron starvation by post-transcriptional regulation through a currently undefined mechanism that is distinct from the mammalian iron response system. We have created reporter cell lines by fusing the TbTfR 3'UTR or a control Aldolase 3'UTR to reporter genes encoding GFP or firefly Luciferase, and inserted the fusions into a bloodstream form cell line at a tagged ribosomal RNA locus. Fusion of the TbTfR 3'UTR is sufficient to significantly repress the expression of the reporter proteins under normal growth conditions. Under iron starvation conditions we observed upregulation of the mRNA and protein level of the TbTfR 3'UTR fusions only, with a magnitude and timing consistent with that reported for upregulation of the TbTfR. We conclude that the dynamic regulation of the T. brucei transferrin receptor in response to iron starvation is mediated via its 3'UTR, and that the effect is independent of genomic location.