TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei.

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more 'metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.

Decoding the network of Trypanosoma brucei proteins that determines sensitivity to apolipoprotein-L1.

In contrast to Trypanosoma brucei gambiense and T. b. rhodesiense (the causative agents of human African trypanosomiasis), T. b. brucei is lysed by apolipoprotein-L1 (apoL1)-containing human serum trypanolytic factors (TLF), rendering it non-infectious to humans. While the mechanisms of TLF1 uptake, apoL1 membrane integration, and T. b. gambiense and T. b. rhodesiense apoL1-resistance have been extensively characterised, our understanding of the range of factors that drive apoL1 action in T. b. brucei is limited. Selecting our bloodstream-form T. b. brucei RNAi library with recombinant apoL1 identified an array of factors that supports the trypanocidal action of apoL1, including six putative ubiquitin modifiers and several proteins putatively involved in membrane trafficking; we also identified the known apoL1 sensitivity determinants, TbKIFC1 and the V-ATPase. Most prominent amongst the novel apoL1 sensitivity determinants was a putative ubiquitin ligase. Intriguingly, while loss of this ubiquitin ligase reduces parasite sensitivity to apoL1, its loss enhances parasite sensitivity to TLF1-dominated normal human serum, indicating that free and TLF1-bound apoL1 have contrasting modes-of-action. Indeed, loss of the known human serum sensitivity determinants, p67 (lysosomal associated membrane protein) and the cathepsin-L regulator, 'inhibitor of cysteine peptidase', had no effect on sensitivity to free apoL1. Our findings highlight a complex network of proteins that influences apoL1 action, with implications for our understanding of the anti-trypanosomal action of human serum.

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei.

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

Genome-wide and protein kinase-focused RNAi screens reveal conserved and novel damage response pathways in Trypanosoma brucei.

All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.

VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes.

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion.

Distinct activation mechanisms trigger the trypanocidal activity of DNA damaging prodrugs.

Quinone-based compounds have been exploited to treat infectious diseases and cancer, with such chemicals often functioning as inhibitors of key metabolic pathways or as prodrugs. Here, we screened an aziridinyl 1,4-benzoquinone (ABQ) library against the causative agents of trypanosomiasis, and cutaneous leishmaniasis, identifying several potent structures that exhibited EC50 values of <100 nM. However, these compounds also displayed significant toxicity towards mammalian cells indicating that they are not suitable therapies for systemic infections. Using anti-T. brucei ABQs as chemical probes, we demonstrated that these exhibit different trypanocidal modes of action. Many functioned as type I nitroreductase (TbNTR) or cytochrome P450 reductase (TbCPR) dependent prodrugs that, following activation, generate metabolites which promote DNA damage, specifically interstrand crosslinks (ICLs). Trypanosomes lacking TbSNM1, a nuclease that specifically repairs ICLs, are hypersensitive to most ABQ prodrugs, a phenotype exacerbated in cells also engineered to express elevated levels of TbNTR or TbCPR. In contrast, ABQs that contain substituent groups on the biologically active aziridine do not function as TbNTR or TbCPR-activated prodrugs and do not promote DNA damage. By unravelling how ABQs mediate their activities, features that facilitate the desired anti-parasitic growth inhibitory effects could be incorporated into new, safer compounds targeting these neglected tropical diseases.

Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.

Trypanosoma brucei, protozoan parasites that cause human African trypanosomiasis (HAT), depend on ornithine uptake and metabolism by ornithine decarboxylase (ODC) for survival. Indeed, ODC is the target of the WHO "essential medicine" eflornithine, which is antagonistic to another anti-HAT drug, suramin. Thus, ornithine uptake has important consequences in T. brucei, but the transporters have not been identified. We describe these amino acid transporters (AATs). In a heterologous expression system, TbAAT10-1 is selective for ornithine, whereas TbAAT2-4 transports both ornithine and histidine. These AATs are also necessary to maintain intracellular ornithine and polyamine levels in T. brucei, thereby decreasing sensitivity to eflornithine and increasing sensitivity to suramin. Consistent with competition for histidine, high extracellular concentrations of this amino acid phenocopied a TbAAT2-4 genetic defect. Our findings established TbAAT10-1 and TbAAT2-4 as the parasite ornithine transporters, one of which can be modulated by histidine, but both of which affect sensitivity to important anti-HAT drugs.-Macedo, J. P., Currier, R. B., Wirdnam, C., Horn, D., Alsford, S., Rentsch, D. Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei.

High-throughput decoding of antitrypanosomal drug efficacy and resistance.

The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT). The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown. Here we use all five current HAT drugs for genome-scale RNA interference target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. RIT-seq profiling identifies both known drug importers and the only known pro-drug activator, and links more than fifty additional genes to drug action. A bloodstream stage-specific invariant surface glycoprotein (ISG75) family mediates suramin uptake, and the AP1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis, all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H(+)-ATPases to pentamidine action, and trypanothione and several putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of antitrypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented molecular insight into the mode of action of antitrypanosomal drugs.

Co-dependence between trypanosome nuclear lamina components in nuclear stability and control of gene expression.

The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component.

Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes.

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes.

Cathepsin-L can resist lysis by human serum in Trypanosoma brucei brucei.

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence 'hits' were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.

DNA break site at fragile subtelomeres determines probability and mechanism of antigenic variation in African trypanosomes.

Antigenic variation in African trypanosomes requires monoallelic transcription and switching of variant surface glycoprotein (VSG) genes. The transcribed VSG, always flanked by '70 bp'-repeats and telomeric-repeats, is either replaced through DNA double-strand break (DSB) repair or transcriptionally inactivated. However, little is known about the subtelomeric DSBs that naturally trigger antigenic variation in Trypanosoma brucei, the subsequent DNA damage responses, or how these responses determine the mechanism of VSG switching. We found that DSBs naturally accumulate close to both transcribed and non-transcribed telomeres. We then induced high-efficiency meganuclease-mediated DSBs and monitored DSB-responses and DSB-survivors. By inducing breaks at distinct sites within both transcribed and silent VSG transcription units and assessing local DNA resection, histone modification, G2/M-checkpoint activation, and both RAD51-dependent and independent repair, we reveal how breaks at different sites trigger distinct responses and, in 'active-site' survivors, different switching mechanisms. At the active site, we find that promoter-adjacent breaks typically failed to trigger switching, 70 bp-repeat-adjacent breaks almost always triggered switching through 70 bp-repeat recombination (∼60% RAD51-dependent), and telomere-repeat-adjacent breaks triggered switching through loss of the VSG expression site (25% of survivors). Expression site loss was associated with G2/M-checkpoint bypass, while 70 bp-repeat-recombination was associated with DNA-resection, γH2A-focus assembly and a G2/M-checkpoint. Thus, the probability and mechanism of antigenic switching are highly dependent upon the location of the break. We conclude that 70 bp-repeat-adjacent and telomere-repeat-adjacent breaks trigger distinct checkpoint responses and VSG switching pathways. Our results show how subtelomere fragility can generate the triggers for the major antigenic variation mechanisms in the African trypanosome.

NUP-1 Is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions.

A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.

High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.

African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000 integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and "hypothetical" genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides much needed genetic validation to identify new drug targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.

Antigenic variation in African trypanosomes: the importance of chromosomal and nuclear context in VSG expression control.

African trypanosomes are lethal human and animal parasites that use antigenic variation for evasion of host adaptive immunity. To facilitate antigenic variation, trypanosomes dedicate approximately one third of their nuclear genome, including many minichromosomes, and possibly all sub-telomeres, to variant surface glycoprotein (VSG) genes and associated sequences. Antigenic variation requires transcription of a single VSG by RNA polymerase I (Pol-I), with silencing of other VSGs, and periodic switching of the expressed gene, typically via DNA recombination with duplicative translocation of a new VSG to the active site. Thus, telomeric location, epigenetic controls and monoallelic transcription by Pol-I at an extranucleolar site are prominent features of VSGs and their expression, with telomeres, chromatin structure and nuclear organization all making vitally important contributions to monoallelic VSG expression control and switching. We discuss VSG transcription, recombination and replication control within this chromosomal and sub-nuclear context.

Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei.

Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G2/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G2/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.

Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA.

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

Genetic dissection of drug resistance in trypanosomes.

The trypanosomes cause two neglected tropical diseases, Chagas disease in the Americas and African trypanosomiasis in sub-Saharan Africa. Over recent years a raft of molecular tools have been developed enabling the genetic dissection of many aspects of trypanosome biology, including the mechanisms underlying resistance to some of the current clinical and veterinary drugs. This has led to the identification and characterization of key resistance determinants, including transporters for the anti-Trypanosoma brucei drugs, melarsoprol, pentamidine and eflornithine, and the activator of nifurtimox-benznidazole, the anti-Trypanosoma cruzi drugs. More recently, advances in sequencing technology, combined with the development of RNA interference libraries in the clinically relevant bloodstream form of T. brucei have led to an exponential increase in the number of proteins known to interact either directly or indirectly with the anti-trypanosomal drugs. In this review, we discuss these findings and the technological developments that are set to further revolutionise our understanding of drug-trypanosome interactions. The new knowledge gained should inform the development of novel interventions against the devastating diseases caused by these parasites.

Bis-6-amidino-benzothiazole Derivative that Cures Experimental Stage 1 African Trypanosomiasis with a Single Dose.

We designed and synthesized a series of symmetric bis-6-amidino-benzothiazole derivatives with aliphatic central units and evaluated their efficacy against bloodstream forms of the African trypanosome Trypanosoma brucei. Of these, a dicationic benzothiazole compound (9a) exhibited sub-nanomolar in vitro potency with remarkable selectivity over mammalian cells (>26,000-fold). Unsubstituted 5-amidine groups and a cyclohexyl spacer were the crucial determinants of trypanocidal activity. In all cases, mice treated with a single dose of 20 mg kg-1 were cured of stage 1 trypanosomiasis. The compound displayed a favorable in vitro ADME profile, with the exception of low membrane permeability. However, we found evidence that uptake by T. brucei is mediated by endocytosis, a process that results in lysosomal sequestration. The compound was also active in low nanomolar concentrations against cultured asexual forms of the malaria parasite Plasmodium falciparum. Therefore, 9a has exquisite cross-species efficacy and represents a lead compound with considerable therapeutic potential.

Epigenetic mechanisms, nuclear architecture and the control of gene expression in trypanosomes.

The control of gene expression, and more significantly gene cohorts, requires tight transcriptional coordination and is an essential feature of probably all cells. In higher eukaryotes, the mechanisms used involve controlled modifications to both local and global DNA environments, principally through changes in chromatin structure as well as cis-element-driven mechanisms. Although the mechanisms regulating chromatin in terms of transcriptional permissiveness and the relation to developmental programmes and responses to the environment are becoming better understood for animal and fungal cells, it is only just beginning to become clear how these processes operate in other taxa, including the trypanosomatids. Recent advances are now illuminating how African trypanosomes regulate higher-order chromatin structure, and, further, how these mechanisms impact on the expression of major surface antigens that are of fundamental importance to life-cycle progression. It is now apparent that several mechanisms are rather more similar between animal and fungal cells and trypanosomes than it originally appeared, but some aspects do involve gene products unique to trypanosomes. Therefore, both evolutionarily common and novel mechanisms cohabit in trypanosomes, offering both important biological insights and possible therapeutic opportunity.