Systematic identification of genes encoding cell surface and secreted proteins that are essential for in vitro growth and infection in Leishmania donovani.

Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease.

The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei.

The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1-3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1-3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1-3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1-3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1-3 loss-of-function is a mechanism of antifolate drug resistance.

Homoserine and quorum-sensing acyl homoserine lactones as alternative sources of threonine: a potential role for homoserine kinase in insect-stage Trypanosoma brucei.

De novo synthesis of threonine from aspartate occurs via the ß-aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L-homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the ß-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.

Dissecting the metabolic roles of pteridine reductase 1 in Trypanosoma brucei and Leishmania major.

Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H(4)B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (k(cat)/K(m)) for LmPTR1 are similar with dihydrobiopterin (H(2)B) and quinonoid dihydrobiopterin (qH(2)B) as substrates and about 20-fold lower than LmQDPR with qH(2)B. In contrast, TbPTR1 shows a 10-fold higher k(cat)/K(m) for H(2)B over qH(2)B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H(4)B (430 nM, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H(4)B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H(4)B as a medium supplement. These cells grew normally with H(4)B, which spontaneously oxidizes to qH(2)B, but were unable to survive in the absence of pterin or with either biopterin or H(2)B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target.

Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in mice.

Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines ((oe)RNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the (oe)RNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase.

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.

Characterisation of a putative glutamate 5-kinase from Leishmania donovani.

Previous metabolic studies have demonstrated that leishmania parasites are able to synthesise proline from glutamic acid and threonine from aspartic acid. The first committed step in both biosynthetic pathways involves an amino acid kinase, either a glutamate 5-kinase (G5K; EC2.7.2.11) or an aspartokinase (EC2.7.2.4). Bioinformatic analysis of multiple leishmania genomes identifies a single amino acid-kinase gene (LdBPK 262740.1) variously annotated as either a putative glutamate or aspartate kinase. To establish the catalytic function of this Leishmania donovani gene product, we have determined the physical and kinetic properties of the recombinant enzyme purified from Escherichia coli. The findings indicate that the enzyme is a bona fide G5K with no activity as an aspartokinase. Tetrameric G5K displays kinetic behaviour similar to its bacterial orthologues and is allosterically regulated by proline, the end product of the pathway. The structure-activity relationships of proline analogues as inhibitors are broadly similar to the bacterial enzyme. However, unlike G5K from E. coli, leishmania G5K lacks a C-terminal PUA (pseudouridine synthase and archaeosine transglycosylase) domain and does not undergo higher oligomerisation in the presence of proline. Gene replacement studies are suggestive, but not conclusive that G5K is essential. ENZYMES: Glutamate 5-kinase (EC2.7.2.11); aspartokinase (EC2.7.2.4).

Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase.

Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome.

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug class.

Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1.

Genetic studies indicate that the enzyme pteridine reductase 1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T. brucei PTR1 (K(i)(app)=7 nM), which had high selectivity over both human and T. brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low K(m) to K(i) ratio (K(m)=25 nM and K(i)=2.3 nM, respectively).