The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets.

Human trypanosomiasis and leishmaniasis are vector-borne neglected tropical diseases caused by infection with the protozoan parasites Trypanosoma spp. and Leishmania spp., respectively. Once restricted to endemic areas, these diseases are now distributed worldwide due to human migration, climate change, and anthropogenic disturbance, causing significant health and economic burden globally. The current chemotherapy used to treat these diseases has limited efficacy, and drug resistance is spreading. Hence, new drugs are urgently needed. Phenotypic compound screenings have prevailed as the leading method to discover new drug candidates against these diseases. However, the publication of the complete genome sequences of multiple strains, advances in the application of CRISPR/Cas9 technology, and in vivo bioluminescence-based imaging have set the stage for advancing target-based drug discovery. This review analyses the limitations of the narrow pool of available drugs presently used for treating these diseases. It describes the current drug-based clinical trials highlighting the most promising leads. Furthermore, the review presents a focused discussion on the most important biological and pharmacological challenges that target-based drug discovery programs must overcome to advance drug candidates. Finally, it examines the advantages and limitations of modern research tools designed to identify and validate essential genes as drug targets, including genomic editing applications and in vivo imaging.

Comparative Proteomic Analysis of Lysine Acetylation in Trypanosomes.

Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T. brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T. cruzi insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T. brucei stages and 18 in common between the two species. While K-ac proteins from T. cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T. brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival.

Transcriptional Regulation of Telomeric Expression Sites and Antigenic Variation in Trypanosomes.

INTRODUCTION: Trypanosoma brucei uses antigenic variation to evade the host antibody clearance by periodically changing its Variant Surface Glycoprotein (VSGs) coat. T. brucei encode over 2,500 VSG genes and pseudogenes, however they transcribe only one VSG gene at time from one of the 20 telomeric Expression Sites (ESs). VSGs are transcribed in a monoallelic fashion by RNA polymerase I from an extranucleolar site named ES body. VSG antigenic switching occurs by transcriptional switching between telomeric ESs or by recombination of the VSG gene expressed. VSG expression is developmentally regulated and its transcription is controlled by epigenetic mechanisms and influenced by a telomere position effect. CONCLUSION: Here, we discuss 1) the molecular basis underlying transcription of telomeric ESs and VSG antigenic switching; 2) the current knowledge of VSG monoallelic expression; 3) the role of inositol phosphate pathway in the regulation of VSG expression and switching; and 4) the developmental regulation of Pol I transcription of procyclin and VSG genes.

Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway.

The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5'-monophosphate (GMP) formation: conversion from xanthosine-5'-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 µM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche.

Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and ß) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.

Inhibition of isoleucyl-tRNA synthetase as a potential treatment for human African Trypanosomiasis.

Trypanosoma brucei sp. causes human African trypanosomiasis (HAT; African sleeping sickness). The parasites initially proliferate in the hemolymphatic system and then invade the central nervous system, which is lethal if not treated. New drugs are needed for HAT because the approved drugs are few, toxic, and difficult to administer, and drug resistance is spreading. We showed by RNAi knockdown that T. brucei isoleucyl-tRNA synthetase is essential for the parasites in vitro and in vivo in a mouse model of infection. By structure prediction and experimental analysis, we also identified small molecules that inhibit recombinant isoleucyl-tRNA synthetase and that are lethal to the parasites in vitro and highly selective compared with mammalian cells. One of these molecules acts as a competitive inhibitor of the enzyme and cures mice of the infection. Because members of this class of molecules are known to cross the blood-brain barrier in humans and to be tolerated, they may be attractive as leading candidates for drug development for HAT.

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles.

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-ß-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.

Erratum: Update Notice: Efficient Generation of Genome-wide Libraries for Protein-ligand Screens Using Gibson Assembly.

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Identifying Antigenic Switching by Clonal Cell Barcoding and Nanopore Sequencing in Trypanosoma brucei.

Many organisms alternate the expression of genes from large gene sets or gene families to adapt to environmental cues or immune pressure. The single-celled protozoan pathogen Trypanosoma brucei spp. periodically changes its homogeneous surface coat of variant surface glycoproteins (VSGs) to evade host antibodies during infection. This pathogen expresses one out of ~2,500 VSG genes at a time from telomeric expression sites (ESs) and periodically changes their expression by transcriptional switching or recombination. Attempts to track VSG switching have previously relied on genetic modifications of ES sequences with drug-selectable markers or genes encoding fluorescent proteins. However, genetic modifications of the ESs can interfere with the binding of proteins that control VSG transcription and/or recombination, thus affecting VSG expression and switching. Other approaches include Illumina sequencing of the VSG repertoire, which shows VSGs expressed in the population rather than cell switching; the Illumina short reads often limit the distinction of the large set of VSG genes. Here, we describe a methodology to study antigenic switching without modifications of the ES sequences. Our protocol enables the detection of VSG switching at nucleotide resolution using multiplexed clonal cell barcoding to track cells and nanopore sequencing to identify cell-specific VSG expression. We also developed a computational pipeline that takes DNA sequences and outputs VSGs expressed by cell clones. This protocol can be adapted to study clonal cell expression of large gene families in prokaryotes or eukaryotes. Key features • This protocol enables the analysis of variant surface glycoproteins (VSG) switching in T. brucei without modifying the expression site sequences. • It uses a streamlined computational pipeline that takes fastq DNA sequences and outputs expressed VSG genes by each parasite clone. • The protocol leverages the long reads sequencing capacity of the Oxford nanopore sequencing technology, which enables accurate identification of the expressed VSGs. • The protocol requires approximately eight to nine days to complete.

PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes.

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

Genome-Wide Libraries for Protozoan Pathogen Drug Target Screening Using Yeast Surface Display.

The lack of genetic tools to manipulate protozoan pathogens has limited the use of genome-wide approaches to identify drug or vaccine targets and understand these organisms' biology. We have developed an efficient method to construct genome-wide libraries for yeast surface display (YSD) and developed a YSD fitness screen (YSD-FS) to identify drug targets. We show the efficacy of our method by generating genome-wide libraries for Trypanosoma brucei, Trypanosoma cruzi, and Giardia lamblia parasites. Each library has a diversity of ∼105 to 106 clones, representing ∼6- to 30-fold of the parasite's genome. Nanopore sequencing confirmed the libraries' genome coverage with multiple clones for each parasite gene. Western blot and imaging analysis confirmed surface expression of the G. lamblia library proteins in yeast. Using the YSD-FS assay, we identified bonafide interactors of metronidazole, a drug used to treat protozoan and bacterial infections. We also found enrichment in nucleotide-binding domain sequences associated with yeast increased fitness to metronidazole, indicating that this drug might target multiple enzymes containing nucleotide-binding domains. The libraries are valuable biological resources for discovering drug or vaccine targets, ligand receptors, protein-protein interactions, and pathogen-host interactions. The library assembly approach can be applied to other organisms or expression systems, and the YSD-FS assay might help identify new drug targets in protozoan pathogens.

High-Efficiency Transformation and Expression of Genomic Libraries in Yeast.

Saccharomyces cerevisiae is a powerful system for the expression of genome-wide or combinatorial libraries for diverse types of screening. However, expressing large libraries in yeast requires high-efficiency transformation and controlled expression. Transformation of yeast using electroporation methods is more efficient than chemical methods; however, protocols described for electroporation require large amounts of linearized plasmid DNA and often yield approximately 106 cfu/µg of plasmid DNA. We optimized the electroporation of yeast cells for the expression of whole-genome libraries to yield up to 108 cfu/µg plasmid DNA. The protocol generates sufficient transformants for 10-100× coverage of diverse genome libraries with small amounts of genomic libraries (0.1 µg of DNA per reaction) and provides guidance on calculations to estimate library size coverage and transformation efficiency. It describes the preparation of electrocompetent yeast cells with lithium acetate and dithiothreitol conditioning step and the transformation of cells by electroporation with carrier DNA. We validated the protocol using three yeast surface display libraries and demonstrated using nanopore sequencing that libraries' size and diversity are preserved. Moreover, expression analysis confirmed library functionality and the method's efficacy. Hence, this protocol yields a sufficient representation of the genome of interest for downstream screening purposes while limiting the amount of the genomic library required.

Human plasma membrane-derived vesicles halt proliferation and induce differentiation of THP-1 acute monocytic leukemia cells.

Plasma membrane-derived vesicles (PMVs) are small intact vesicles released from the cell surface that play a role in intercellular communication. We have examined the role of PMVs in the terminal differentiation of monocytes. The myeloid-differentiating agents all-trans retinoic acid/PMA and histamine, the inflammatory mediator that inhibits promonocyte proliferation, induced an intracellular Ca(2+)-mediated PMV (as opposed to exosome) release from THP-1 promonocytes. These PMVs cause THP-1 cells to enter G(0)-G(1) cell cycle arrest and induce terminal monocyte-to-macrophage differentiation. Use of the TGF-ß receptor antagonist SB-431542 and anti-TGF-ß1 Ab showed that this was due to TGF-ß1 carried on PMVs. Although TGF-ß1 levels have been shown to increase in cell culture supernatants during macrophage differentiation and dendritic cell maturation, the presence of TGF-ß1 in PMVs is yet to be reported. In this study, to our knowledge we show for the first time that TGF-ß1 is carried on the surface of PMVs, and we confirm the presence within PMVs of certain leaderless proteins, with reported roles in myeloid cell differentiation. Our in vitro findings support a model in which TGF-ß1-bearing PMVs, released from promonocytic leukemia cells (THP-1) or primary peripheral blood monocytes on exposure to sublytic complement or after treatment with a differentiation therapy agent, such as all-trans retinoic acid, significantly reduce proliferation of THP-1 cells. Such PMVs also induce the terminal differentiation of primary peripheral blood monocytes as well as THP-1 monocytes.

Efficient Generation of Genome-wide Libraries for Protein-ligand Screens Using Gibson Assembly.

Genome-wide screens using yeast or phage displays are powerful tools for identifying protein-ligand interactions, including drug or vaccine targets, ligand receptors, or protein-protein interactions. However, assembling libraries for genome-wide screens can be challenging and often requires unbiased cloning of 10 5 -10 7 DNA fragments for a complete representation of a eukaryote genome. A sub-optimal genomic library can miss key genomic sequences and thus result in biased screens. Here, we describe an efficient method to generate genome-wide libraries for yeast surface display using Gibson assembly. The protocol entails genome fragmentation, ligation of adapters, library cloning using Gibson assembly, library transformation, library DNA recovery, and a streamlined Oxford nanopore library sequencing procedure that covers the length of the cloned DNA fragments. We also describe a computational pipeline to analyze the library coverage of the genome and predict the proportion of expressed proteins. The method allows seamless library transfer among multiple vectors and can be easily adapted to any expression system.

Protein acetylation in the critical biological processes in protozoan parasites.

Protein lysine acetylation has emerged as a major regulatory post-translational modification in different organisms, present not only on histone proteins affecting chromatin structure and gene expression but also on nonhistone proteins involved in several cellular processes. The same scenario was observed in protozoan parasites after the description of their acetylomes, indicating that acetylation might regulate crucial biological processes in these parasites. The demonstration that glycolytic enzymes are regulated by acetylation in protozoans shows that this modification might regulate several other processes implicated in parasite survival and adaptation during the life cycle, opening the chance to explore the regulatory acetylation machinery of these parasites as drug targets for new treatment development.

The phosphoinositide regulatory network in Trypanosoma brucei: Implications for cell-wide regulation in eukaryotes.

The unicellular eukaryote Trypanosoma brucei undergoes extensive cellular and developmental changes during its life cycle. These include regulation of mammalian stage surface antigen variation and surface composition changes between life stages; switching between glycolysis and oxidative phosphorylation; differential mRNA editing; and changes in posttranscriptional gene expression, protein trafficking, organellar function, and cell morphology. These diverse events are coordinated and controlled throughout parasite development, maintained in homeostasis at each life stage, and are essential for parasite survival in both the host and insect vector. Described herein are the enzymes and metabolites of the phosphatidylinositol (PI) cellular regulatory network, its integration with other cellular regulatory systems that collectively control and coordinate these numerous cellular processes, including cell development and differentiation and the many associated complex processes in multiple subcellular compartments. We conclude that this regulation is the product of the organization of these enzymes within the cellular architecture, their activities, metabolite fluxes, and responses to environmental changes via signal transduction and other processes. We describe a paradigm for how these enzymes and metabolites could function to control and coordinate multiple cellular functions. The significance of the PI system's regulatory functions in single-celled eukaryotes to metazoans and their potential as chemotherapeutic targets are indicated.

ATR Kinase Is a Crucial Player Mediating the DNA Damage Response in Trypanosoma brucei.

DNA double-strand breaks (DSBs) are among the most deleterious lesions that threaten genome integrity. To address DSBs, eukaryotic cells of model organisms have evolved a complex network of cellular pathways that are able to detect DNA damage, activate a checkpoint response to delay cell cycle progression, recruit the proper repair machinery, and resume the cell cycle once the DNA damage is repaired. Cell cycle checkpoints are primarily regulated by the apical kinases ATR and ATM, which are conserved throughout the eukaryotic kingdom. Trypanosoma brucei is a divergent pathogenic protozoan parasite that causes human African trypanosomiasis (HAT), a neglected disease that can be fatal when left untreated. The proper signaling and accuracy of DNA repair is fundamental to T. brucei not only to ensure parasite survival after genotoxic stress but also because DSBs are involved in the process of generating antigenic variations used by this parasite to evade the host immune system. DSBs trigger a strong DNA damage response and efficient repair process in T. brucei, but it is unclear how these processes are coordinated. Here, by knocking down ATR in T. brucei using two different approaches (conditional RNAi and an ATR inhibitor), we show that ATR is required to mediate intra-S and partial G1/S checkpoint responses. ATR is also involved in replication fork stalling, is critical for H2A histone phosphorylation in a small group of cells and is necessary for the recruitment and upregulation of the HR-mediated DNA repair protein RAD51 after ionizing radiation (IR) induces DSBs. In summary, this work shows that apical ATR kinase plays a central role in signal transduction and is critical for orchestrating the DNA damage response in T. brucei.

Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes.

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.