A High-Throughput Method for the Analysis of Larval Developmental Phenotypes in Caenorhabditis elegans

Caenorhabditis elegans postembryonic development consists of four discrete larval stages separated by molts. Typically, the speed of progression through these larval stages is investigated by visual inspection of the molting process. Here, we describe an automated method to monitor the timing of these discrete phases of C. elegans maturation, from the first larval stage through adulthood, using bioluminescence. The method was validated with a lin-42 mutant strain that shows delayed development relative to wild-type animals and with a daf-2 mutant that shows an extended second larval stage. This new method is inherently high-throughput and will finally allow dissecting the molecular machinery governing the speed of the developmental clock, which has so far been hampered by the lack of a method suitable for genetic screens.     

Developmental variation in Rab11-dependent trafficking in Trypanosoma brucei

In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.     

Mining Chemical Activity Status from High-Throughput Screening Assays

High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare.     

Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.     

Biosynthesis of trypanothione in Trypanosoma brucei brucei

Trypanothione [T(SH)2], the major redox mediator in pathogenic trypanosomatids, is synthetized stepwise by two distinct enzymes in Crithidia fasciculata, while in Trypanosoma cruzi a single enzyme catalyzes both steps. A full-length reading frame presumed to encode trypanothione synthetase (TryS) was obtained by PCR using DNA of T. brucei as template and primers based on fragments of putative TryS genes. The recombinant protein produced by E. coli Origami (DE3) was purified to homogeneity by chelate and ion exchange chromatography. The enzyme catalyzed both reactions of T(SH)2 biosynthesis. Thus, T(SH)2 synthesis appears to be similar in African (T. brucei) and New World (T. cruzi) trypanosomes but distinct from that of Crithidia.     

Cell-Based Assays for High-Throughput Screening

Cell-based assays represent approximately half of all high-throughput screens currently performed. Here, we review in brief the history and status of high-throughput screening (HTS), and summarize some of the challenges and benefits associated with the use of cell-based assays in HTS. Approaches for successful experimental design and execution of cell-based screens are introduced, including strategies for assay development, implementation of primary and secondary screens, and target identification. In doing so, we hope to provide a comprehensive review of the cell-based HTS process and an introduction to the methodologies and techniques used.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

A Search for Trypanosoma brucei rhodesiense Diagnostic Antigens by Proteomic Screening and Targeted Cloning

Background

The only available diagnostic method for East African trypanosomiasis is light microscopy of blood samples. A simple immunodiagnostic would greatly aid trypanosomiasis control.

Methodology and Principal Findings

To find trypanosome proteins that are specifically recognised by sera from human sleeping sickness patients, we have screened the Trypanosoma brucei brucei proteome by Western blotting. Using cytosolic, cytoskeletal and glycosomal fractions, we found that the vast majority of abundant trypanosome proteins is not specifically recognised by patient sera. We identified phosphoglycerate kinase (PGKC), heat shock protein (HSP70), and histones H2B and H3 as possible candidate diagnostic antigens. These proteins, plus paraflagellar rod protein 1, rhodesain (a cysteine protease), and an extracellular fragment of the Trypanosoma brucei nucleoside transporter TbNT10, were expressed in E. coli and tested for reactivity with patient and control sera. Only TbHSP70 was preferentially recognized by patient sera, but the sensitivity and specificity were insufficient for use of TbHSP70 alone as a diagnostic. Immunoprecipitation using a native protein extract revealed no specifically reacting proteins.

Conclusions

No abundant T. brucei soluble, glycosomal or cytoskeletal protein is likely to be useful in diagnosis. To find useful diagnostic antigens it will therefore be necessary to use more sophisticated proteomic methods, or to test a very large panel of candidate proteins.     

cAMP signalling in Trypanosoma brucei

Cyclic AMP was the first second messenger to be identified. After five decades of research, much is currently known about its biological functions and clinical implications. Several components of the cAMP signalling pathways, such as the G-protein coupled receptors and the phosphodiesterases, have become sensitive and specific drug targets for a host of clinical applications. Surprisingly, very little effort has been invested so far into the study of cAMP signalling in parasites, and its significance in host/parasite interaction. Our laboratory has embarked on a study of cAMP signalling in Trypanosoma brucei. A newly identified adenylyl cyclase, GRESAG4.4B, a member of a small family of closely related genes, is being used as a model molecule for investigating the mechanisms which control cyclase activity in the T. brucei cell. On the other hand, a number of genes for different families of cAMP-specific phosphodiesterases have been identified and characterised. One enzyme, TbPDE1, is coded for by a single-copy gene. Knock-outs of this gene display an almost normal phenotype in culture, indicating that TbPDE1 is not an essential enzyme under culture conditions. A second phosphodiesterase which is being studied in detail, TbPDE2A, is clearly different from TbPDE1, and it is coded for by a member of a small gene family containing about six similar, but non-identical genes. TbPDE2A, as TbPDE1, is specific for cAMP. In its N-terminal, it contains a GAF domain which may represent an allosteric cGMP-binding site. The other members of the TbPDE2 family all exhibit strongly conserved catalytic domains, but vary widely in their N-terminal regulatory domains. With regard to downstream signalling by the cAMP generated through the interplay of adenylyl cyclases and phosphodiesterases, we have recently identified a single-copy gene (TbRSU1) which codes for a putative regulatory subunit of the cAMP-regulated protein kinase A. This protein exhibits considerable similarity with its mammalian counterparts. Immunoprecipitation co-precipitates a protein kinase activity with the characteristics of protein kinase A.     

High-Throughput Screening Techniques for Biomass Conversion

High-throughput (HTP) screening of biomass or biomass-degrading enzymes, regardless of the desired outcome, is fraught with obstacles and challenges not typically faced in more traditional biotechnology. The enzyme systems are complex and synergistic and the substrate is highly heterogeneous, insoluble, and difficult to dispense. Digestions are often carried out for days at temperatures of 50°C or higher, leading to significant challenges regarding evaporation control in small well volumes. Furthermore, it is often desirable to condition or “pretreat” the biomass at extreme temperatures and/or pH to enhance enzyme digestibility. Once the substrate has been saccharified, evaluation of the extent and efficiency of conversion is made more difficult by time-consuming and tedious techniques used to measure the sugar products. Over the past decade or so, biomass researchers have creatively addressed these challenges by developing techniques to reduce biomass heterogeneity, uniformly distribute biomass samples at the small scale, pretreat the biomass at the small scale, quantitatively load these samples with enzymes, control evaporation of small reaction volumes for multiday incubations, and rapidly quantify the products. Other aspects of these measurements remain problematic and are being addressed. This review will address some of these challenges in detail, but more importantly, we will endeavor to educate the reader about the trials, tribulations, and pitfalls of carrying out HTP screening in biomass conversion research.     

Golgi duplication in Trypanosoma brucei

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Trypanosoma brucei brucei: endocytic recycling is important for mouse infectivity

Endocytosis in the African trypanosome, Trypanosoma brucei, is intimately involved in maintaining homeostasis of the cell surface proteome, morphology of the flagellar pocket and has recently been demonstrated as a bona fide drug target. RNAi-mediated knockdown of many factors required for endocytic transport, including several small GTPases, the major coat protein clathrin and a clathrin-associated receptor, epsinR, results in rapid cell death in vitro. Rapid loss of viability in vitro precludes meaningful investigation by RNAi of the roles of trypanosome endocytosis in vivo. Here we have sought to address this issue using strategies designed to produce milder effects on the endocytic system than complete functional ablation. We created a trypanosome clathrin heavy chain hemizygote and several lines expressing mutant forms of Rab5 and Rab11, described previously. All are viable in in vitro culture, with negligible impact to proliferative rates or cell cycle. Clathrin hemizygotes express clathrin heavy chain at ∼50% of wild type levels, but despite this demonstrate no defect to growth in mice, while none of the Rab5 mutants affected proliferation in vivo, despite clear evidence for effects on endocytosis. By contrast we find that expressing a dominantly active Rab11 mutant led to compromised growth in mice. These data indicate that trypanosomes likely tolerate the effects of partly decreased clathrin expression and alterations in early endocytosis, but are more sensitive to alterations in the recycling arm of the pathway.     

Artificial linear mini-chromosomes for Trypanosoma brucei.

We have constructed artificial linear mini- chromosomes for the parasitic protozoan Trypanosoma brucei. These chromosomes exist at approx. 2 copies per cell, are indefinitely stable under selection but are lost from 50% of the transformed population in approx. 7 generations when grown in the absence of selective pressure. Consistent with results obtained earlier with natural chromosomes in T.brucei, the telomeres on these artificial chromosomes grow, adding approx. 1- 1.5 telomeric repeats per generation. The activity of a procyclic acidic repetitive protein (parp) gene promoter on these elements is unaffected by its proximity to a telomere, implying the lack of a telomere-proximal position effect (TPE) in procyclic trypanosomes. Among other things, these autonomously replicating dispensable genetic elements will provide a defined system for the study of nuclear DNA replication, karyotypic plasticity and other aspects of chromosomal behavior in this ancient eukaryotic lineage.