Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen

<b>Background</b><br /> The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.<p></p> <b>Results</b><br /> Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.<p></p> <b>Conclusion</b><br /> Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.<p></p&gt

Tracking autophagy during proliferation and differentiation of trypanosoma brucei

Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. The Trypanosoma brucei genome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatible T. brucei were modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form of T. brucei

Studies on the energy metabolism of Leishmania mexicana mexicana

The metabolism of the mammalian form of Leishmania mexicana mexicana has been investigated by comparing the amastigote activities of a number of enzymes involved in the glycolytic sequence, tricarboxylic acid cycle and associated pathways with those of the promastigote. Initial studies suggested that there are only quantitive differences between the two forms with the greatest of the differences found in the area of CO2-fixation. Phosphoenolpyruvate carboxykinase (PEP carboxykinase) was detected at much higher activity in amastigotes than promastigotes. This enzyme was found to be ADP specific and to have an absolute requirement for Mn2+. There appeared to be no regulation of the amastigote enzyme in crude homogenates by ATP, GTP, ITP or the end products malate and succinate. Pyruvate carboxylase was undetectable in either parasite form and malic enzyme (carboxylating) was detected only at low activity in promastigotes. Amastigotes had low pyruvate kinase but high malate dehydrogenase activities in comparison to promastigotes. This suggests that in amastigotes the main catabolic route for PEP is to succinate, a major end-product of metabolism, by way of carboxylation and reduction involving malate dehydrogenase and fumarate reductase. In contrast, the high activity of pyruvate kinase in promastigotes suggests that, in this parasite form, PEP is more likely to be converted to pyruvate which either enters the TCA cycle or is transaminated to alanine. Promastigotes had a high NADH-linked glutamate dehydrogenase activity in comparison to amastigotes and may reflect the higher glutamate utilization by the insect form of the parasite. A low NADPH-linked glutamate dehydrogenase activity was found in amastigotes, whereas none was detected in promastigotes. Isocitrate lyase could not be detected in either form, suggesting that the glyoxylate cycle plays no part in L. m. mexicana metabolism. The theory that the glycolytic kinases, and in particular phosphofructokinase, are primary targets in the action of pentavalent antimonial drugs against Leishmania was investigated with L. m. mexicana. Pentostam (sodium stibogluconate), active in vivo against Leishmania, had no effect on promastigote growth in vitro even at concentrations of 100μg/ml. In contrast, Triostam, the trivalent analogue of Pentostam, inhibited the growth in vitro of L. m. mexicana promastigotes with an LD50 of 20μg/ml and an MLC of 400μg/ml. It was found to be almost as effective on a weight/weight basis as the trivalent arsenical melarsen oxide (LD50, 20μg/ml; MLC, 100μg/ml). The leishmanicidal effect of Triostam, however, could not be correlated with any enzyme inhibitory activity. Neither Pentostam (110μM) nor Triostam (600μM)inhibited hexokinase, phosphofructokinase, pyruvate kinase, malate dehydrogenase or PEP carboxykinase from either amastigote or promastigote. In contrast, melarsen oxide was a potent inhibitor of all leishmanial enzymes tested except hexokinase. The action of Triostam was antagonised by cysteine indicating that the drug's action may involve thiol groups in some way. Triostam, but not Pentostam, inhibited phosphofructokinase of adult Schistosoma mansoni with an I50 of 200μM, suggesting that antimonials may act in different ways against schistosomes and Leishmania. The subcellular organisation of L. m. mexicana promastigotes was investigated using differential and isopycnic centrifugation. Glycosomes and mitochondrial vesicles from culture promastigotes were separated on linear sucrose gradients. Hexokinase, glucose phosphate isomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and PEP carboxykinase were recovered largely in association with glycosomes (density; 1.215 g/ml). Phosphoglycerate kinase and glucose-6-phosphate dehydrogenase had some glycosomal activity but were mostly recovered in the soluble fractions. Pyruvate kinase was totally cytosolic. Malate dehydrogenase showed a broad peak corresponding to that of the mitochondrial marker oligomycinsensitive ATPase (density; 1.190g/ml). Glutamate dehydrogenase and alanine aminotransferase both showed small mitochondrial peaks, but most of the activities were recovered elsewhere on the gradient and in the soluble fractions. Amastigotes of L. m. mexicana were not successfully fractionated using the technique developed for promastigotes, therefore the subcellular location of enzymes in amastigotes was investigated by following the release of soluble enzymes from digitonin-treated amastigotes. This revealed distinct cytosolic, mitochondrial and glycosomal compartments. The findings give an insight into the organisation of L. m. mexicana promastigote and amastigote energy metabolism. The importance of PEP carboxykinase and malate dehydrogenase to the metabolism of the amastigote prompted a more detailed investigation of these two enzymes. Isoelectric focusing studies showed that amastigotes possessed particulate malate dehydrogenase isoenzymes apparently absent from promastigotes. The particulate activities of amastigote malate dehydrogenase and PEP carboxykinase were purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5' AMP-Sepharose 4B and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. PEP carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SOS gel electrophoresis were 39,8OO and 33,300 for malate dehydrogenase and 63,100 and 65,100 for PEP carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km NADH of 41μM and a Km oxaloacetic acid of 39μM. For malate oxidation there was a Km malate of 3.6mM and a Km NAD+ of 0.79mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than O.83mM and malate was found to be a product inhibitor at high concentrations, however there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts, nevertheless their apparent importance and unique subcellular organisation in the parasite make them potential targets for chemotherapeutic attack

The SNARE protein family of Leishmania major

BACKGROUND: Leishmania major is a protozoan parasite with a highly polarised cell shape that depends upon endocytosis and exocytosis from a single area of the plasma membrane, the flagellar pocket. SNAREs (soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors) are key components of the intracellular vesicle-mediated transports that take place in all eukaryotic cells. They are membrane-bound proteins that facilitate the docking and fusion of vesicles with organelles. The recent availability of the genome sequence of L. major has allowed us to assess the complement of SNAREs in the parasite and to investigate their location in comparison with metazoans. RESULTS: Bioinformatic searches of the L. major genome revealed a total of 27 SNARE domain-containing proteins that could be classified in structural groups by phylogenetic analysis. 25 of these possessed the expected features of functional SNAREs, whereas the other two could represent kinetoplastid-specific proteins that might act as regulators of the SNARE complexes. Other differences of Leishmania SNAREs were the absence of double SNARE domain-containing and of the brevin classes of these proteins. Members of the Qa group of Leishmania SNAREs showed differential expressions profiles in the two main parasite forms whereas their GFP-tagging and in vivo expression revealed localisations in the Golgi, late endosome/lysosome and near the flagellar pocket. CONCLUSION: The early-branching eukaryote L. major apparently possess a SNARE repertoire that equals in number the one of metazoans such as Drosophila, showing that the machinery for vesicle fusion is well conserved throughout the eukaryotes. However, the analysis revealed the absence of certain types of SNAREs found in metazoans and yeast, while suggesting the presence of original SNAREs as well as others with unusual localisation. This study also presented the intracellular localisation of the L. major SNAREs from the Qa group and reveals that these proteins could be useful as organelle markers in this parasitic protozoon

Bloodstream form trypanosoma brucei depend upon multiple metacaspases associated with RAB11-positive endosomes

Trypanosoma brucei possesses five metacaspase genes. Of these, MCA2 and MCA3 are expressed only in the mammalian bloodstream form of the parasite, whereas MCA5 is expressed also in the insect procyclic form. Triple RNAi analysis showed MCA2, MCA3 and MCA5 to be essential in the bloodstream form, with parasites accumulating pre-cytokinesis. Nevertheless, triple null mutants (Δmca2/3Δmca5) could be isolated after sequential gene deletion. Thereafter, Δmca2/3Δmca5 mutants were found to grow well both in vitro in culture and in vivo in mice. We hypothesise that metacaspases are essential for bloodstream form parasites, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of Δmca2/3Δmca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin D2. The metacaspases were found to colocalise with RAB11, a marker for recycling endosomes. However, variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, Δmca2/3Δmca5 and triple RNAi induced parasites. Thus, the data provide no support for the direct involvement of T. brucei metacaspases in programmed cell death and suggest that the proteins have a function associated with RAB11 vesicles that is independent of known recycling processes of RAB11-positive endosomes

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana

AbstractImcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucel and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. Imcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity

Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi

BACKGROUND: The trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids. RESULTS: Bioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs. CONCLUSION: Trypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function based solely on sequence similarity. Hence the connection of stimuli to protein phosphorylation networks remains enigmatic. The presence of numerous PKs with significant sequence similarity to known drug targets, as well as a large number of unusual kinases that might represent novel targets, strongly argue for functional analysis of these molecules

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/ UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function

High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs

Ubiquitin and ubiquitin-like conjugation systems in trypanosomatids

In eukaryotic cells, reversible attachment of ubiquitin and ubiquitin-like modifiers (Ubls) to specific target proteins is conducted by multicomponent systems whose collective actions control protein fate and cell behaviour in precise but complex ways. In trypanosomatids, attachment of ubiquitin and Ubls to target proteins regulates the cell cycle, endocytosis, protein sorting and degradation, autophagy and various aspects of infection and stress responses. The extent of these systems in trypanosomatids has been surveyed in recent reports, while in Leishmania mexicana, essential roles have been defined for many ubiquitin-system genes in deletion mutagenesis and life-cycle phenotyping campaigns. The first steps to elucidate the pathways of ubiquitin transfer among the ubiquitination components and to define the acceptor substrates and the downstream deubiquitinases are now being taken