Post-transcriptional regulation of the trypanosome heat shock response by a zinc finger protein.

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3'-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures.

Multiple genetic mechanisms lead to loss of functional TbAT1 expression in drug-resistant trypanosomes.

The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [(3)H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a DeltaTbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.

The role of deadenylation in the degradation of unstable mRNAs in trypanosomes.

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5'-3' exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5' and 3' ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA.

Predictive computational models of substrate binding by a nucleoside transporter.

Transporters play a vital role in both the resistance mechanisms of existing drugs and effective targeting of their replacements. Melarsoprol and diamidine compounds similar to pentamidine and furamidine are primarily taken up by trypanosomes of the genus Trypanosoma brucei through the P2 aminopurine transporter. In standardized competition experiments with [(3)H]adenosine, P2 transporter inhibition constants (K(i)) have been determined for a diverse dataset of adenosine analogs, diamidines, Food and Drug Administration-approved compounds and analogs thereof, and custom-designed trypanocidal compounds. Computational biology has been employed to investigate compound structure diversity in relation to P2 transporter interaction. These explorations have led to models for inhibition predictions of known and novel compounds to obtain information about the molecular basis for P2 transporter inhibition. A common pharmacophore for P2 transporter inhibition has been identified along with other key structural characteristics. Our model provides insight into P2 transporter interactions with known compounds and contributes to strategies for the design of novel antiparasitic compounds. This approach offers a quantitative and predictive tool for molecular recognition by specific transporters without the need for structural or even primary sequence information of the transport protein.

An RNAi screen of the RRM-domain proteins of Trypanosoma brucei.

In eukaryotes, proteins containing RNA Recognition Motifs (RRMs) are involved in many different RNA processing reactions, RNA transport, and mRNA decay. Kinetoplastids rely extensively on post-transcriptional mechanisms to control gene expression, so RRM domain proteins are expected to play a prominent role. We here describe the results of an RNA interference screen targeting 37 of the 72 RRM-domain proteins of Trypanosoma brucei. RNAi targeting 8 of the genes caused clear growth inhibition in bloodstream trypanosomes, and milder effects were seen for 9 more genes. The small, single-RRM protein TbRBP3 specifically associated with 10 mRNAs in trypanosome lysates, but RBP3 depletion did not affect the transcriptome.

Detection of arsenical drug resistance in Trypanosoma brucei with a simple fluorescence test.

The resurgence of human African trypanosomiasis (HAT), coupled with an increased incidence of drug resistance, is of concern. We report a quick, simple, and sensitive test for identification of parasites resistant to melarsoprol, the main drug used to treat late stage HAT. Resistant parasites are defective in a plasma membrane transporter responsible for drug uptake. The same transporter carries the fluorescent diamidine DB99 (2,5-bis-(4-amidinophenyl)-3,4-dimethylfuran) into trypanosomes. The two DNA-containing structures in the trypanosome--the nucleus and the kinetoplast--begin to fluoresce within 1 min of introduction of DB99, unless drug resistant.

Design and synthesis of a series of melamine-based nitroheterocycles with activity against Trypanosomatid parasites.

The parasites that give rise to human African trypanosomiasis (HAT) are auxotrophs for various nutrients from the human host, including purines. They have specialist nucleoside transporters to import these metabolites. In addition to uptake of purine nucleobases and purine nucleosides, one of these transporters, the P2 transporter, can carry melamine derivatives; these derivatives are not substrates for the corresponding mammalian transporters. In this paper, we report the coupling of the melamine moiety to selected nitro heterocycles with the aim of selectively delivering these compounds to the parasites. Some compounds prepared have similar in vitro trypanocidal activities as melarsoprol, the principal drug used against late-stage HAT, with 50% growth inhibitory concentrations in the submicromolar range. Selected compounds were also evaluated in vivo in rodent models infected with Trypanosoma brucei brucei and T. brucei rhodesiense and showed pronounced activity and in two cases were curative without overt signs of toxicity. Compounds were also tested against other trypanosomatid pathogens, Leishmania donovani and Trypanosoma cruzi, and significant activity in vitro was noted for T. cruzi against which various nitro heterocycles are already registered for use.

The Trypanosoma brucei CCCH zinc finger proteins ZC3H12 and ZC3H13.

CCCH-type zinc finger proteins have various roles in RNA metabolism. We here analysed the functional relevance of two such proteins from Trypanosoma brucei, TbZC3H12 and TbZC3H13. Each protein has a single CCCH motif very similar to those seen in metazoan proteins that regulate mRNA degradation. TbZC3H12 is expressed in bloodstream form parasites at low levels. It is phosphorylated, cytosolic and not required for normal growth of cultured bloodstream trypanosomes. RNA interference targeting TbZC3H13, on a TbZC3H12 null background, also had no effect on bloodstream trypanosome growth, but over-expression of tagged TbZC3H13 inhibited procyclic trypanosome growth. Tandem affinity purification of both proteins revealed various interesting potential interactions; specificity was assessed against a list of proteins that were found in 24 other pull-down experiments, which is provided. The conservation of TbZC3H12 in all kinetoplastids, and TbZC3H13 in Salivaria, suggests that the two proteins may be required for optimal growth at some stage of the parasite life-cycle.

Processing of a phosphoglycerate kinase reporter mRNA in Trypanosoma brucei is not coupled to transcription by RNA polymerase II.

Capping of mRNAs is strictly coupled to RNA polymerase II transcription and there is evidence, mainly from metazoans, that other steps in pre-mRNA processing show a similar linkage. In trypanosomes, however, the mRNA cap is supplied by a trans spliced leader sequence. Thus pre-mRNAs transcribed by RNA Polymerase I are capped by trans splicing, and translation-competent transgenic mRNAs can be produced by RNA Polymerase I and T7 RNA polymerase so long as the primary transcript has a splice acceptor signal. We quantified the efficiency of processing of trypanosome pre-mRNAs produced from a plasmid integrated either at the tubulin locus, or in an rRNA spacer, and transcribed by RNA polymerase II, RNA polymerase I or T7 RNA polymerase. The processing efficiencies were similar for primary transcripts from the tubulin locus, produced by RNA polymerase II, and for RNA from an rRNA spacer, transcribed by RNA polymerase I. Primary transcripts produced by T7 RNA polymerase from the tubulin locus were processed almost as well. There was therefore no evidence for recruitment of the 3'-splicing apparatus by the RNA polymerase. Abundant transcripts transcribed from the rRNA locus by T7 RNA polymerase were somewhat less efficiently processed.

Trypanosomes as a model to investigate mRNA decay pathways.

In trypanosomes, individual mRNAs arise by the processing of primary polycistronic transcripts. Consequently, mRNA degradation rates are critical determinants of mRNA abundance. In this chapter, we summarize the various options for genetic manipulation in trypanosomes with the goal of analyzing mRNA stability, including RNA interference. We describe a method for measuring the half-lives of trypanosome mRNAs, including those that are very unstable, and also the isolation of tagged protein-RNA complexes by IgG affinity chromatography. Last, we detail our current methods for RNA analysis with microarrays.

Control of mRNA degradation in trypanosomes.

Control of gene expression in trypanosomes relies almost exclusively on post-transcriptional mechanisms. Trypanosomes have the normal enzymes for mRNA decay: both the exosome and a 5'-3'-exoribonuclease are important in the degradation of very unstable transcripts, whereas the CAF1/NOT complex plays a major role in the degradation of all mRNAs tested. Targeted RNA interference screening was used to identify RNA-binding proteins that regulate mRNA degradation, and it revealed roles for proteins with RNA recognition motifs or pumilio domains.

Control and regulation of gene expression: quantitative analysis of the expression of phosphoglycerate kinase in bloodstream form Trypanosoma brucei.

Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e.g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation.

Small trypanosome RNA-binding proteins TbUBP1 and TbUBP2 influence expression of F-box protein mRNAs in bloodstream trypanosomes.

In the African trypanosome Trypanosoma brucei nearly all control of gene expression is posttranscriptional; sequences in the 3'-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins, TbUBP1 and TbUBP2, containing a single RNA recognition motif, in trypanosome gene expression. TbUBP1 and TbUBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of TbUBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the CFB1 mRNA, which encodes a protein with a cyclin F-box domain. CFB1 regulation was mediated by the 3'-untranslated region and involved stabilization of the mRNA. Depletion of TbUBP2 and TbUBP1 inhibited growth and downregulated expression of the cyclin F box protein gene CFB2; trans splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to TbUBP2 or TbUBP1, with some preference for CFB1. We suggest that TbUBP1 and TbUBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.

Ab initio prediction of metabolic networks using Fourier transform mass spectrometry data.

Fourier transform mass spectrometry has recently been introduced into the field of metabolomics as a technique that enables the mass separation of complex mixtures at very high resolution and with ultra high mass accuracy. Here we show that this enhanced mass accuracy can be exploited to predict large metabolic networks ab initio, based only on the observed metabolites without recourse to predictions based on the literature. The resulting networks are highly information-rich and clearly non-random. They can be used to infer the chemical identity of metabolites and to obtain a global picture of the structure of cellular metabolic networks. This represents the first reconstruction of metabolic networks based on unbiased metabolomic data and offers a breakthrough in the systems-wide analysis of cellular metabolism.

Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance.

A novel trypanocide, 2,5-bis(4-amidinophenyl)furan (DB75), in its prodrug amidoxime-derivative form, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), is in trials as the first orally administered drug for human African trypanosomiasis. DB75 is a diamidine. Resistance to some diamidines correlates to loss of uptake via the P2 aminopurine transporter. We show here that uptake of DB75 into Trypanosoma brucei also occurs principally via the P2 transporter. Uptake of tritiated DB75 occurred via a high-affinity (K(m app), 3.2 microM) carriermediated route that was inhibited by adenosine, adenine, and pentamidine, all known substrates of the P2 transporter. Trypanosomes lacking the TbAT1 gene that encodes the P2 transporter demonstrated an 11-fold reduction in sensitivity to DB75 when measured under controlled in vitro conditions. These knockout cells were also less sensitive to DB75 than wild-type cells in mice. Initial uptake rates of DB75 into the Deltatbat1 knockout cell line were greatly reduced compared with rates in wild-type cells. A trypanosome cell line selected in vitro for DB75 resistance was shown to have lost P2-mediated DB75 uptake. The TbAT1 gene was mapped to chromosome V of the T. brucei genome and the DB75-resistant parasites were shown to have deleted both alleles of this gene. Fluorescence microscopy of DB75-treated trypanosomes revealed that DB75 fluorescence localizes rapidly within the DNA-containing organelles of wild-type trypanosomes, whereas no fluorescence was observed in Deltatbat1-null parasites or in the parasites selected for resistance to DB75.

Interaction of monobenzamidine-linked trypanocides with the Trypanosoma brucei P2 aminopurine transporter.

Single benzamidine group-carrying compounds were shown to interact with the Trypanosoma brucei P2 aminopurine transporter. Replacement of the amidine with a guanidine group decreased affinity. Trypanocidal activity was evident, but compounds were equally toxic against trypanosomes lacking the P2 transporter, which indicates additional uptake routes for monobenzamidine-derived compounds.

Activity of megazol, a trypanocidal nitroimidazole, is associated with DNA damage.

DNA damage associated with the trypanocidal activity of megazol [2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole] was shown in experiments in which DNA repair-deficient RAD51(-/-) Trypanosoma brucei mutants were found to be hypersensitive to the drug. Parasites resistant to megazol were selected and showed modest cross-resistance to other trypanocides, although neither drug efflux nor changes to intracellular thiols correlated with resistance.

The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in african trypanosomes.

Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [(3)H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a K(m) of 0.45 +/- 0.11 micro M, which was dose dependently inhibited by pentamidine and adenosine. The K(i) values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [(3)H]diminazene and [(3)H]pentamidine. TbAT1-null mutants had lost almost all capacity for [(3)H]diminazene transport. However, this cell line still displayed a small but detectable rate of [(3)H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [(3)H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Trypanocidal activity of melamine-based nitroheterocycles.

A series of nitroheterocyclic compounds were designed with linkages to melamine or benzamidine groups that are known substrates of the P2 aminopurine and other transporters in African trypanosomes of the brucei group. Several compounds showed in vitro trypanotoxicity with 50% inhibitory concentrations in the submicromolar range. Although most compounds interacted with the P2 transporter, as judged by their ability to inhibit adenosine transport via this carrier, uptake through this route was not necessary for activity since TbAT1-null mutant parasites, deficient in this transporter, retained sensitivity to these drugs. One compound, a melamine-linked nitrofuran, also showed pronounced activity against parasites in mice. Studies into the mode of action of this compound indicated that neither reductive, nor oxidative, stress were related to its trypanocidal activity ruling out a genotoxic effect in T. brucei, distinguishing it from some other, mammalian cell toxic, trypanocidal nitroheterocycles.

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei.

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.