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The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei the proteins that make up the kinetochore are highly divergent, with the inner kinetochore comprising at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2 and KKT3. We performed a phenotypic screen of T. brucei bloodstream forms with a Novartis kinase-focused inhibitor library, which identified a number of selective inhibitors with potent pan-kinetoplastid activity. Deconvolution of an amidobenzimidazole series using a selection of 37 T. brucei mutants that over-express known essential protein kinases identified CLK1 as the primary target. Biochemical studies show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with C215 in the ATP binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. We show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 to be essential for KKT2 function and for kinetochore assembly. We propose that CLK1 is part of a novel signalling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. This work highlights a novel drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

The leishmaniases, caused by Leishmania species of protozoan parasites, are neglected tropical diseases with millions of cases worldwide. Current therapeutic approaches are limited by toxicity, resistance, and cost. N-Myristoyltransferase (NMT), an enzyme ubiquitous and essential in all eukaryotes, has been validated via genetic and pharmacological methods as a promising anti-leishmanial target. Here we describe a comprehensive structure-activity relationship (SAR) study of a thienopyrimidine series previously identified in a high-throughput screen against Leishmania NMT, across 68 compounds in enzyme- and cell-based assay formats. Using a chemical tagging target engagement biomarker assay, we identify the first inhibitor in this series with on-target NMT activity in leishmania parasites. Furthermore, crystal structure analyses of 12 derivatives in complex with Leishmania major NMT revealed key factors important for future structure-guided optimization delivering IMP-105 (43), a compound with modest activity against Leishmania donovani intracellular amastigotes and excellent selectivity (>660-fold) for Leishmania NMT over human NMTs.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2’s unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant but exclude most other diamidine drugs. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family. African sleeping sickness is a potentially deadly illness caused by the parasite Trypanosoma brucei. The disease is treatable, but many of the current treatments are old and are becoming increasingly ineffective. For instance, resistance is growing against pentamidine, a drug used in the early stages in the disease, as well as against melarsoprol, which is deployed when the infection has progressed to the brain. Usually, cases resistant to pentamidine are also resistant to melarsoprol, but it is still unclear why, as the drugs are chemically unrelated. Studies have shown that changes in a water channel called aquaglyceroporin 2 (TbAQP2) contribute to drug resistance in African sleeping sickness; this suggests that it plays a role in allowing drugs to kill the parasite. This molecular 'drain pipe' extends through the surface of T. brucei, and should allow only water and a molecule called glycerol in and out of the cell. In particular, the channel should be too narrow to allow pentamidine or melarsoprol to pass through. One possibility is that, in T. brucei, the TbAQP2 channel is abnormally wide compared to other members of its family. Alternatively, pentamidine and melarsoprol may only bind to TbAQP2, and then 'hitch a ride' when the protein is taken into the parasite as part of the natural cycle of surface protein replacement. Alghamdi et al. aimed to tease out these hypotheses. Computer models of the structure of the protein were paired with engineered changes in the key areas of the channel to show that, in T. brucei, TbAQP2 provides a much broader gateway into the cell than observed for similar proteins. In addition, genetic analysis showed that this version of TbAQP2 has been actively selected for during the evolution process of T. brucei. This suggests that the parasite somehow benefits from this wider aquaglyceroporin variant. This is a new resistance mechanism, and it is possible that aquaglyceroporins are also larger than expected in other infectious microbes. The work by Alghamdi et al. therefore provides insight into how other germs may become resistant to drugs., This work was supported by the UK Medical Research Council (MRC) [grant G0701258 to HPdK] andby the US National Institutes of Health (NIH) [Grant No. GM111749 to DWB]. DC was supported byan MRC iCASE award [MR/R015791/1]. UZ acknowledges funding from the Scottish UniversitiesPhysics Alliance. AHA is funded through a PhD studentship from Albaha University, Saudi Arabia.GDC was funded by a PhD Studentship from Science Without Borders [206385/2014–5, CNPq,Brazil]. TS was funded via a Doctoral Training Programme of the MRC and the Cambridge Trust andSW was funded by a Sir Henry Dale fellowship of the Wellcome Trust and Royal Society. The authorsthank Dr Tansy Hammarton for the use of the CRK2 RNAi cell line and Prof David Horn for the use ofthe aqp1-3 null cell line. This work was supported by a grant from the Wellcome Trust (204697/Z/16/Z to MCF. The authors are grateful to Professor George Diallinas, University of Athens, Greece, forhis exceptionally insightful reviewer comments and have adopted several of his arguments inrevision.

Initiation of DNA replication depends upon recog- nition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei , an ancient unicellular eukary- ote, only one ORC-related initiator, TbORC1 / CDC6, has been identified by sequence homology. Here we show that three TbORC1 / CDC6-interacting fac- tors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1 / CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1 / CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, sug- gesting it positively regulates replication. This work shows that ORC architecture and regulation are di- verged features of DNA replication initiation in T. bru- cei , providing new insight into this key stage of eu- karyotic genome copying.

Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses.

Summary Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility. DOI: http://dx.doi.org/10.7554/eLife.12765.001, eLife digest The African trypanosome, Trypanosoma brucei, is a parasite that is transmitted between mammals by the tsetse fly, and causes a disease known as sleeping sickness in humans. Like many other parasites, trypanosomes have evolved ways to avoid being killed by their hosts. One such survival strategy involves the parasites constantly changing the molecules that coat their surface, which are the main targets recognized by their hosts’ immune systems. Switching one coat protein for another similar protein, a process called antigenic variation, allows a parasite to evade an attack and establish a persistent infection. Antigenic variation also makes it almost impossible to develop a vaccine that will offer lasting protection against the parasite. Previous research suggested that a trypanosome might deliberately break its own DNA and then exploit a repair process to switch its current coat protein-encoding gene for another one located elsewhere within its genetic material. Devlin, Marques et al. now reveal that it is unlikely that trypanosomes use a specific enzyme to break DNA deliberately during coat switching. Instead, experiments using whole-genome sequencing suggest that coat-gene-switching might arise from the strategies trypanosomes use to copy their genetic material during cell division. These findings bring researchers closer to understanding how trypanosomes start antigenic variation in order to evade their hosts’ immune responses. In addition, the findings suggest a new model that could help researchers answer an important question: how does the timing of genome copying vary from cell to cell? Nevertheless, the hypothesis proposed by Devlin, Marques et al. will now require rigorous testing. Future studies could also ask if other parasites use similar strategies to survive being attacked by their host’s immune systems. DOI: http://dx.doi.org/10.7554/eLife.12765.002

The prime function of classically activated macrophages (activated by Th1-type signals, such as IFN-γ) is microbial destruction. Alternatively activated macrophages (activated by Th2 cytokines, such as IL-4 and IL-13) play important roles in allergy and responses to helminth infection. We utilize a murine model of filarial infection, in which adult nematodes are surgically implanted into the peritoneal cavity of mice, as an in vivo source of alternatively activated macrophages. At 3 wk postinfection, the peritoneal exudate cell population is dominated by macrophages, termed nematode-elicited macrophages (NeMφ), that display IL-4-dependent features such as the expression of arginase 1, RELM-α (resistin-like molecule α), and Ym1. Since increasing evidence suggests that macrophages show functional adaptivity, the response of NeMφ to proinflammatory Th1-activating signals was investigated to determine whether a switch between alternative and classical activation could occur in macrophages differentiated in an in vivo infection setting. Despite the long-term exposure to Th2 cytokines and antiinflammatory signals in vivo, we found that NeMφ were not terminally differentiated but could develop a more classically activated phenotype in response to LPS and IFN-γ. This was reflected by a switch in the enzymatic pathway for arginine metabolism from arginase to inducible NO synthase and the reduced expression of RELM-α and Ym1. Furthermore, this enabled NeMφ to become antimicrobial, as LPS/IFN-γ-treated NeMφ produced NO that mediated killing of Leishmania mexicana. However, the adaptation to antimicrobial function did not extend to key regulatory pathways, such as IL-12 production, which remained unaltered.

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.

N-Myristoyltransferase (NMT) is a potential drug target in Leishmania parasites. Scaffold-hopping from published inhibitors yielded the serendipitous discovery of a chemotype selective for Leishmania donovani NMT; development led to high affinity inhibitors with excellent ligand efficiency. The binding mode was characterised by crystallography and provides a structural rationale for selectivity.

Truncation converted a Plasmodium N-myristoyltransferase inhibitor into a Leishmania-selective series, leading to a potent L. donovani NMT inhibitor through structure-guided design., N-Myristoyltransferase (NMT) is a potential drug target in Leishmania parasites. Scaffold-hopping from published inhibitors yielded the serendipitous discovery of a chemotype selective for Leishmania donovani NMT; development led to high affinity inhibitors with excellent ligand efficiency. The binding mode was characterised by crystallography and provides a structural rationale for selectivity.

Inhibitors of Leishmania N-myristoyltransferase (NMT), a potential target for the treatment of leishmaniasis, obtained from a high-throughput screen, were resynthesized to validate activity. Crystal structures bound to Leishmania major NMT were obtained, and the active diastereoisomer of one of the inhibitors was identified. On the basis of structural insights, enzyme inhibition was increased 40-fold through hybridization of two distinct binding modes, resulting in novel, highly potent Leishmania donovani NMT inhibitors with good selectivity over the human enzyme.

Dendritic cells (DCs), as professional antigen-presenting cells, are members of the innate immune system and function as key players during the induction phase of adaptive immune responses. Uptake, processing, and presentation of antigens direct the outcome toward either tolerance or immunity. The cells of the immune system are among the most highly radiosensitive cells in the body. For high doses of ionizing radiation (HD-IR) both immune-suppressive effects after whole body irradiation and possible immune activation during tumor therapy were observed. On the other hand, the effects of low doses of ionizing radiation (LD-IR) on the immune system are controversial and seem to show high variability among different individuals and species. There are reports revealing that protracted LD-IR can result in radioresistance. But immune-suppressive effects of chronic LD-IR are also reported, including the killing or sensitizing of certain cell types. This article shall review the current knowledge of radiation-induced effects on the immune system, paying special attention to the interaction of DCs and T cells.

Investigating the proteome of intracellular Leishmania amastigotes has recently become possible due to the exploitation of fluorescence activated intracellular parasite sorting. Here, we employed this technology in combination with gel free analysis to greatly improve proteome coverage and suggest proteins putatively secreted by the parasites. In total, 1764 proteins were identified of which 741 had not been reported before. Protein abundance indices were calculated to rank individual proteins according to their abundance in vivo. Using the LeishCyc resource, an overview of metabolically relevant proteins was produced that integrated protein abundance data. Bioinformatic analysis identified 143 proteins possibly secreted by L. mexicana amastigotes, half of which have no known function. The data provide a useful resource, e.g. for modelling metabolic flux or selecting novel vaccine antigens.

The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5(+) stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp-rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite-containing phagosomes in primary host cells. Using real-time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite-infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5(+) compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life-cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5(+) phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication. - Lippuner, C., Paape, D., Paterou, A., Brand, J., Richardson, M., Smith, A. J., Hoffmann, K., Brinkmann, V., Blackburn, C., Aebischer, T. Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice. FASEB J. 23, 483-491 (2009)

SummaryN-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen.