The Effects of the Methylating Agent 1,2-Bis(methylsulfonyl)-1-methylhydrazine on Morphology, DNA Content and Mitochondrial Function of Trypanosoma brucei Subspecies

Repeated exposure of trypanosomes in vitro or in vivo to low concentrations of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine induces a series of moderately synchronous morphological and biochemical changes. Cell division halts and the long-slender bloodstream forms transform to short-stumpy forms via larger intermediate-stage cells which contain approximately double the normal G₂ content of DNA. In common with naturally occurring short-stumpy trypanosomes, drug-induced short-stumpy forms do not infect rodents and when transferred to Cunningham's medium, transform to and replicate as procylics. Furthermore, these short-stumpy forms exhibit α-ketoglutarate supported motility and oxygen consumption, acquire the ability to reduce nitroblue tetrazolium (NADH diaphorase positivity) and appear to be in the G₁ or G₀ stage of the cell cycle based upon DNA content.     

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml⁻¹ bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10⁻¹² mg ml⁻¹) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10⁻⁵-10⁻⁶ mg ml⁻¹. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.     

TbDSS-1, an essential Trypanosoma brucei exoribonuclease homolog that has pleiotropic effects on mitochondrial RNA metabolism

Mitochondrial gene expression in trypanosomes is controlled primarily at the levels of RNA processing and RNA stability. This regulation undoubtedly involves numerous ribonucleases. Here we characterize the Trypanosoma brucei homolog of the yeast DSS-1 mitochondrial exoribonuclease, which we term TbDSS-1. Biochemical fractionation indicates that TbDSS-1 is mitochondrially localized, as predicted by its N-terminal sequence. In contrast to its yeast homolog, TbDSS-1 does not appear to be associated with mitochondrial ribosomes. Targeted downregulation of TbDSS-1 by RNA interference in procyclic-form T. brucei results in a severe growth defect. In addition, TbDSS-1 depletion leads to a decrease in the levels of never edited cytochrome oxidase subunit I (COI) mRNA and both unedited and edited COIII mRNAs, indicating this enzyme functions in the control of mitochondrial RNA abundance. We also observe a considerable reduction in the level of edited apocytochrome b (CYb) mRNA and a corresponding increase in unedited CYb mRNA, suggesting that TbDSS-1 functions, either directly or indirectly, in the control of RNA editing. The abundance of both gCYb[560] and gA6[149] guide RNAs is reduced upon TbDSS-1 depletion, although the reduction in gCYb[560] is much more dramatic. The significant reduction in gCYb levels could potentially account for the observed decrease in CYb RNA editing. Western blot analyses of mitochondrial RNA editing and stability factors indicate that the perturbations of RNA levels observed in TbDSS-1 knock-downs do not result from secondary effects on other mitochondrial proteins. In all, these data demonstrate that TbDSS-1 is an essential protein that plays a role in mitochondrial RNA stability and RNA editing.     

Cloning, functional analysis, and mitochondrial localization of Trypanosoma brucei monothiol glutaredoxin-1

African trypanosomes encode three monothiol glutaredoxins (1-C-Grx1 to 3). 1-C-Grx1 has a putative CAYS active site and Cys181 as single additional cysteine. The recombinant protein forms non-covalent homodimers. As observed for other monothiol glutaredoxins, Trypanosoma brucei 1-C-Grx1 was not active in the glutaredoxin assay with hydroxyethyl disulfide and glutathione nor catalyzed the reduction of insulin disulfide. In addition, it lacked peroxidase activity and did not catalyze protein (de)glutathionylation. Upon oxidation, 1-C-Grx1 forms an intramolecular disulfide bridge and, to a minor degree, covalent dimers. Both disulfide forms are reduced by the parasite trypanothione/tryparedoxin system. 1-C-Grx1 shows mitochondrial localization. The total cellular concentration is at least 5 microm. Thus, 1-C-Grx1 is an abundant protein especially in the rudimentary organelle of the mammalian form of the parasite. Expression of 1-C-Grx1 in Grx5-deficient yeast cells with its authentic presequence targeted the protein to the mitochondria and partially restored the growth phenotype and aconitase activity of the mutant, and conferred resistance against hydroperoxides and diamide. The parasite Grx2 and 3 failed to substitute for Grx5. This is surprising because even bacterial and plant 1-Cys-glutaredoxins efficiently revert the defects, and may be due to the lack of two basic residues conserved in all but the trypanosomatid proteins.     

Structure and function of the native and recombinant mitochondrial MRP1/MRP2 complex from Trypanosoma brucei

The mitochondrial RNA-binding proteins (MRP) 1 and 2 play a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Here, we report the purification of a high molecular weight protein complex consisting solely of the MRP1 and MRP2 proteins from the mitochondrion of T. brucei. The MRP1/MRP2 complex natively purified from T. brucei and the one reconstituted in Escherichia coli in vivo bind guide (g) RNAs and pre-mRNAs with dissociation constants in the nanomolar range, and efficiently promote annealing of pre-mRNAs with their cognate gRNAs. In addition, the MRP1/MRP2 complex stimulates annealing between two non-cognate RNA molecules suggesting that along with the cognate duplexes, spuriously mismatched RNA hybrids may be formed at some rate in vivo. A mechanism of catalysed annealing of gRNA/pre-mRNA by the MRP1/MRP2 complex is proposed.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

The 14-3-3 proteins of Trypanosoma brucei function in motility, cytokinesis, and cell cycle

The cDNAs for two isoforms (I and II) of the 14-3-3 proteins have been cloned and functionally characterized in Trypanosoma brucei. The amino acid sequences of isoforms I and II have 47 and 50% identity to the human tau isoform, respectively, with important conserved features including a potential amphipathic groove for the binding of phosphoserine/phosphothreonine-containing motifs and a nuclear export signal-like domain. Both isoforms are abundantly expressed at approximately equal levels (1-2 x 10(6) molecules/cell) and localized mainly in the cytoplasm. Knockdown by induction of double-stranded RNA of isoform I and/or II in both bloodstream and procyclic forms resulted first in a reduction of cell motility and then significant reduction in cell growth rates and morphological changes; the changes include aberrant numbers of organelles and abnormal shapes and sizes that mimic phenotypes produced by various cytokinesis inhibitors. Morphological and fluorescence-activated cell sorting analysis of the cell cycle suggested that isoforms I and II might play important roles in nuclear (G2-M transition) and cell (M-G1 transition) division. These findings indicate that the 14-3-3 proteins play important roles in cell motility, cytokinesis, and the cell cycle.     

The in vitro and in vivo effects of mefloquine on Trypanosoma brucei brucei.

Blood stream forms of Trypanosoma brucei brucei were grown over mouse kidney (MK) cells in minimum essential medium with various concentrations of mefloquine. The drug was observed to inhibit multiplication of the parasites in vitro. Groups of male albino mice were treated with mefloquine at 24, 48 and hours after T. b. brucei infection. Mefloquine at 0.03 mg/kg body weight administered for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice for 90 days and over after the clearance of parasite from the blood. The doses for both the in vitro and in vivo therapy, were well below those prescribed for humans.     

Mitochondria morphology and DNA content upon sublethal exposure to beta-amyloid(1-42) peptide

Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between amyloid beta peptides (AP) and mitochondrial dysfunction has been established in cellular models of AD using Abeta concentrations capable of triggering massive neuronal death. However, mitochondrial changes related to sublethal exposure to Abeta are less known. Here we show that subtoxic, 1 microM Abeta(1-42) exposure does not change the mitochondrial shape of living cells, as visualized upon the uptake of the non-potentiometric fluorescent probe Mitotracker Green and enhanced yellow fluorescent protein (EYFP)-tagged cytochrome c oxidase expression. Immunolocalization of oxidative adducts 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanine and 8-hydroxyguanosine demonstrates that one-micromolar concentration of Abeta(1-42) is also not sufficient to elicit dramatic qualitative changes in the RNA/DNA oxidative products. However, in comparison with controls, semi-quantitative analysis of the overall mitochondrial mass by integrated fluorescence intensity reveals an ongoing down-regulation in mitochondrial biosynthesis or, conversely, an enhanced autophagic demise of Abeta treated cells. Furthermore, a significant increase of the full-length mitochondrial DNA (mtDNA) from Abeta-treated versus control cells is found, as measured by long range polymerase chain reaction (PCR). Such up-regulation is accompanied by extensive fragmentation of the unamplified mtDNA, probably due to the detrimental effect of Abeta. We interpret these results as a sequence of compensatory responses induced by mtDNA damage, which are devoted to repression of oxidative burst. In conclusion, our findings suggest that early therapeutic interventions aimed at prevention of mitochondrial oxidative damage may delay AD progression and help in treating AD patients.     

The in vitro and in vivo effects of metronidazole and chloroquine on Trypanosoma brucei brucei

Bloodstream forms of Trypanosoma brucei brucei were grown over baby hamster kidney cells in minimum essential medium with various concentrations of metronidazole (Flagyl) and chloroquine. Both drugs inhibited the multiplication of the parasite in vitro. The least effective concentrations for metronidazole and chloroquine were 0.003 mg/ml and 0.0024 mg/ml, respectively. Groups of 12-day-old female CDI mice were treated with 1 of the 2 drugs at 24, 48, and 72 hr after T. brucei infection. The drugs administered stat or daily reduced the number of parasites in the mice but did not effect a cure; they prolonged the survival period of the animals. However, metronidazole (0.1 mg/kg body weight) and chloroquine (0.08 mg/kg body weight) combined and given daily for 4 consecutive days cleared the infection. No trypanosomes were detected in the blood of these mice 3 mo after treatment. The dosages for both the in vitro (metronidazole 0.003 mg/ml; chloroquine 0.0024 mg/ml) and in vivo (metronidazole 0.1 mg/kg body weight; chloroquine 0.08 mg/kg body weight) were well below those prescribed for humans.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.     

TbSmee1 regulates hook complex morphology and the rate of flagellar pocket uptake in Trypanosoma brucei

Trypanosoma brucei uses multiple mechanisms to evade detection by its insect and mammalian hosts. The flagellar pocket (FP) is the exclusive site of uptake from the environment in trypanosomes and shields receptors from exposure to the host. The FP neck is tightly associated with the flagellum via a series of cytoskeletal structures that include the hook complex (HC) and the centrin arm. These structures are implicated in facilitating macromolecule entry into the FP and nucleating the flagellum attachment zone (FAZ), which adheres the flagellum to the cell surface. TbSmee1 (Tb927.10.8820) is a component of the HC and a putative substrate of polo‐like kinase (TbPLK), which is essential for centrin arm and FAZ duplication. We show that depletion of TbSmee1 in the insect‐resident (procyclic) form of the parasite causes a 40% growth decrease and the appearance of multinucleated cells that result from defective cytokinesis. Cells lacking TbSmee1 contain HCs with aberrant morphology and show delayed uptake of both fluid‐phase and membrane markers. TbPLK localization to the tip of the new FAZ is also blocked. These results argue that TbSmee1 is necessary for maintaining HC morphology, which is important for the parasite's ability to take up molecules from its environment.     

UTP-dependent turnover of Trypanosoma brucei mitochondrial mRNA requires UTP polymerization and involves the RET1 TUTase

Trypanosoma brucei mitochondria possess a unique RNA decay pathway in which rapid degradation of polyadenylated mRNAs is dependent on the addition of UTP, as measured by in organello pulse chase assays. To determine the mechanism by which UTP stimulates the degradation of polyadenylated RNAs, we performed in organello pulse chase assays under different conditions. Treatment of mitochondria with proteinase K revealed that UTP does not act through a receptor on the surface of the mitochondria. To determine if the UTP-stimulated RNA decay pathway is triggered by the mitochondrial energy state or ATP:UTP ratio, increasing ATP was added to a constant amount of UTP during the chase period of the assay. Results indicate that rapid turnover is responsive to UTP and not the ATP:UTP ratio. Experiments using UTP analogs demonstrate that UTP polymerization into RNAs is necessary for UTP-dependent degradation. Furthermore, experiments performed with RNAi cells indicate that the RET1 terminal uridylyl transferase (TUTase) is required for UTP-dependent decay of polyadenylated RNAs. Overall, these results show that degradation of polyadenylated RNAs in T. brucei mitochondria can occur through a unique mechanism that requires the polymerization of UTP into RNAs, presumably by the RET1 TUTase.     

Synthesis,cyclooxygenase inhibitory effects,and molecular modeling study of 4-aryl-5-(4-(methylsulfonyl)phenyl)-2-alkylthio and -2-alkylsulfonyl-1H-imidazole derivatives

A series of 4-aryl-5-(4-(methylsulfonyl)phenyl)-2-alkylthio and 2-alkylsulfonyl-1H-imidazole derivatives were synthesized. All compounds were tested in human blood assay to determine COX-1 and COX-2 inhibitory potency and selectivity. Among the synthesized compounds, 2-alkylthio series were more potent and selective than 2-sulfonylalkyl derivatives. In molecular modeling, interaction of 2-sulfonylalkyl moiety with Arg120 in COX-1 and an extra hydrogen bond with Tyr341 in COX-2 increased the residence time of ligands in the active site in 2-sulfonylalkyl and 2-alkylthio analogs, respectively.     

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1) from Trypanosoma brucei

We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.     

The multiple roles of cyclin E1 in controlling cell cycle progression and cellular morphology of Trypanosoma brucei

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro glutathione-S-transferase pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was impaired significantly only in cyclin E1+CRK1-depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if only CRK3 was depleted. Taken together, these data suggest multiple functions of cyclin E1: it forms a complex with CRK1 in promoting G1/S phase transition; it forms a complex with CRK2 in controlling the posterior morphogenesis during G1/S transition; and it forms a complex with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome.