Insect Trypanosomatid Research Articles

Analysis of ribosomal RNA genes suggests that trypanosomes are monophyletic.

To further investigate the phylogeny of protozoa from the order Kinetoplastida we have sequenced the small subunit (SSU) and a portion of the large subunit (LSU) nuclear rRNA genes. The SSU and LSU sequences were determined from a lizard trypanosome, Trypanosoma scelopori and a bodonid, Rhynchobodo sp., and the LSU sequences were determined from an insect trypanosomatid, Crithidia oncopelti, and a bodonid, Dimastigella trypaniformis. Contrary to previous results, in which trypanosomes were found to be paraphyletic, with Trypanosoma brucei representing the earliest-diverging lineage, we have now found evidence for the monophyly of trypanosomes. Addition of new taxa which subdivide long branches (such as that of T. brucei) have helped to identify homoplasies responsible for the paraphyletic trees in previous studies. Although the monophyly of the trypanosome clade is supported in the bootstrap analyses for maximum likelihood at 97% and maximum parsimony at 92%, there is only a small difference in ln-likelihood value or tree length between the most optimal monophyletic tree and the best suboptimal paraphyletic tree. Within the trypanosomatid subtree, the clade of trypanosomes is a sister group to the monophyletic clade of the nontrypanosome genera. Different groups of trypanosomes group on the tree according to their mode of transmission. This suggests that the adaptation to invertebrate vectors plays a more important role in the trypanosome evolution than the adaptation to vertebrate hosts.

Further Evidence for the Presence of Mitochondrially Encoded Subunits in Cytochrome c Oxidase of the Trypanosomatid Crithidia fasciculata

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.

Purification and characterization of cytochrome c oxidase from the insect trypanosomatid Crithidia fasciculata

Cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid Crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. The purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of Cu A in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but not heterologous, cytochrome c. Two-dimensional PAGE showed that C. fasciculata cytochrome c oxidase consists of at least 10 different subunits. N-terminal sequences were obtained from the six smallest subunits of the complex, one of them showing significant similarity to Neurospora crassa cytochrome c oxidase subunit V. The N-terminus of each of the four largest subunits was found to be blocked.

Putrescine active uptake system in the Trypanosomatid Crithidia fasciculata.

Using the insect Trypanosomatid Crithidia fasciculata as a model parasite of mammalian pathogenic flagellates, i.e. Leishmania and Trypanosoma spp., we have studied the kinetic and regulatory characteristics of the polyamine uptake system. Putrescine transport was age-dependent with maximum expression values at the proliferative logarithmic phase. Putrescine transport in Crithidia fasciculata was energy-dependent and against a putrescine concentration gradient. The integrity of the membrane sulfhydryl groups was absolutely required for optimum transport rates. The specificity of this mechanism was studied in the presence of a series of different chain length aliphatic diamines, showing the high specificity for putrescine and the poor effect of this series at the highest concentration analyzed as well as the higher polyamines spermidine and spermine. Finally, the well-known inhibitor of polyamine biosynthesis, DFMO, led to an upward regulation of putrescine uptake correlating with the depletion of intracellular polyamine pool. In addition, the presence of high concentrations of putrescine in the culture medium produced a downward regulation of this system.

Structural determinants of putrescine uptake inhibition produced by cationic diamidines in the model of trypanosomatid Crithidia fasciculata.

The effect of a heterologous series of cationic diamidines has been tested on cell growth and polyamine uptake on the model of insect Trypanosomatid Crithidia fasciculata. The maximum inhibitory effect on both parameters was found for pentamidine and dibromopropamidine, which exhibit a longer distance between amino and imino substituents. A minimum inhibitory effect was found with amicarbalide. A good relationship was obtained when the distance between amino moieties was plotted versus the inhibitory effect on putrescine uptake, suggesting a role of this structural property on polyamine transport in Crithidia fasciculata. In addition, a similar correlation was obtained for another Trypanosomatid parasite, Leishmania infantum.

Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidia fasciculata, a member of the most primitive eukaryotic group to contain mitochondria. Separation of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55, 46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, E1α and E1β, respectively. The purified complex did not exhibit endogenous PDH a kinase activity. p70 was much less abundant than p60. Polyclonal antisera raised against p70 did not cross-react with p60, and antisera raised against p60 did not cross-react with p70, suggesting that p60 did not arise from p70 by proteolysis. Both p70 and p60 contained similar amino terminal sequences. Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources. Incubation of the purified PDC with [2- 14C]pyruvate in the absence of CoA resulted in the acetylation of both p70 and p60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for p60. Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2 l and E2 i , and one major acetylated fragment of 58 kDa derived from p70. Therefore, these results suggest that p70 is an E3BP and given its apparent M r and degree of acetylation, it contains multiple lipoyl domains.

A possible role for the guide RNA U-tail as a specificity determinant in formation of guide RNA-messenger RNA chimeras in mitochondrial extracts of Crithidia fasciculata

Chimeric g(uide) RNA:pre-mRNA molecules are potential intermediates of the RNA editing process in kinetoplastid mitochondria. We have studied the characteristics of chimeric molecules formed in mitochondrial extracts of the insect trypanosomatid Crithidia fasciculata which had been supplied with synthetic NADH dehydrogenase (ND) subunit-7 gRNA and pre-mRNA variants. The ability of a gRNA to participate in chimera formation in this system depends on the possibility of base pairing with the pre-mRNA via the anchor sequence, but not on the presence of a U-tail or a full-length informational part. Chimeras formed with a specific gRNA:pre-mRNA pair displayed a large variation in length, due to variably sized 3′ end truncations of the gRNA moieties and variation in the sites in the pre-mRNA to which the gRNAs were attached. Surprisingly, the presence of a U-tail in the gRNA for a large part determined the specificity of the linkage. In 60% of the cases gRNAs possessing a U-tail of at least one residue were attached to an editing site, whereas 75% of the gRNAs without Us were attached to non-editing sites. Furthermore, the chimera forming activity was greatly stimulated by the addition of ATP but not by AMP-CPP, and ATP-analogue with a non-hydrolyzable α-β phosphate bond. This suggests the involvement in the chimera formation of an RNA ligase.

Reverse transcriptase encoded by a retrotransposon from the trypanosomatid Crithidia fasciculata.

The long interspersed nuclear element (LINE)-like elements are a distinct family of eukaryotic transposons that contain a long open reading frame with limited sequence homology to retroviral reverse transcriptases. Unlike many retrotransposons, they lack long terminal repeats. The mechanism by which LINE-like elements move within the genomes of their hosts remains speculative. We have used an unusual approach to express and detect enzymatic activities associated with Crithidia retrotransposable element 1 (CRE1), a site-specific LINE-like element found in the insect trypanosomatid Crithidia fasciculata. A chimeric gene fusing the yeast retrotransposon Ty1 and the CRE1 open reading frame is constructed and then overexpressed in yeast. Fusion proteins are packaged into virus-like particles, which can be partially purified and directly analyzed for enzymatic activity. Here we demonstrate that CRE1 encodes an RNA-directed DNA polymerase. These data provide direct biochemical evidence that this widely distributed class of retrotransposons encodes reverse transcriptase and sets the stage for a detailed understanding of the mechanisms involved in LINE-like element transposition.

Stable transformation of Leptomonas seymouri by circular extrachromosomal elements.

To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri. The vector is relatively small (6 kilobase pairs) and contains a portion of the L. seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418. This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid. In L. seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon. Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells.

Identification of C3 acceptors responsible for complement activation in Crithidia fasciculata.

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg+2-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [131I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 x 10(5) C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.

Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata.

Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.

Trypanothione dependent peroxide metabolism in Crithidia fasciculata and Trypanosoma brucei

A trypanothione-dependent peroxidase activity has been identified in the insect trypanosomatid Crithidia fasciculata and in the mammalian trypanosome Trypanosoma brucei. Using organic hydroperoxides as oxidant, specific peroxidase activities in these organisms are 5.0 and 1.0 nmol min −1 (10 8 cells) −1 respectively. The T. brucei peroxidase had an activity of 0.4 nmol min −1 (10 8 cells) −1 using hydrogen peroxide as oxidant. The enzyme is specific for the N 1, N 8-bis(glutathionyl)spermidine conjugate (dihydrotrypanothione); N 1-mono-glutathionylspermidine is not a substrate. Experiments to demonstrate that this parasite peroxidase may contain selenium were inconclusive. However, bloodstream T. brucei can incorporate radiolabelled selenite into proteins.

DNA sequence of Crithidia fasciculata kinetoplast minicircles

Kinetoplast DNA (kDNA) networks of the insect trypanosomatid Crithidia fasciculata strain CF-C1 contain a nearly homogeneous population of kDNA minicircles as judged by restriction enzyme cleavage analysis. We have determined the entire nucleotide sequence of the major class of minicircles by analyzing M13 phage clones carrying half-length segments of the kDNA minicircle molecules. The 12 nucleotide sequence d(G-G-G-G-T-T-G-G-T-G-T-A) is the longest sequence common to kDNA minicircles from several trypanosome species examined to date. Two copies of this universal minicircle sequence were identified 180° apart as direct repeats within the C. fasciculata kDNA minicircles. In addition, these universal minicircle sequences are contained within direct repeats with nearly identical sequences of 173 and 177 base pairs (bp) in length. These sequences are also conserved in the same arrangement in minor sequence classes of minicircles from this strain. Site-specific discontinuities on both strands of the minicircle, identified previously in minicircle replication intermediates, were localized within the 173 and 177 bp conserved sequences. These sequences were also found to have extensive homology with similar conserved sequences in kDNA minicircles from Leishmania tarentolae. We suggest that the two conserved sequences, each containing a single copy of the universal minicircle sequence, represent replication origins in the Crithidia minicircles.

The biosynthesis of trypanothione and N1-glutathionylspermidine in Crithidia fasciculata

Studies on the biosynthesis of trypanothione [ N 1, N 8-bis(glutathionyl)-spermidine] in the insect trypanosomatid Crithidia fasciculata have led to the discovery of an additional sulfur-containing peptide conjugated to spermidine. Labelling studies with [ 3H]spermidine show that 50% of the total intracellular spermidine is incorporated into peptide conjugates, the major component being N 1-glutathionylspermidine. This compound has previously been identified in Escherichia coli, as the principal low molecular weight thiol in stationary phase, but not the logarithmic phase of growth. In contrast, in C. fasciculata, this compound is present in all phases of growth. In the presence of glutathione and ATP, extracts of C. fasciculata can catalyse conversion of spermidine to N 1-glutathionylspermidine and trypanothione. Both N 1- and N 8-regioisomers of glutathionylspermidine will replace spermidine in the reaction, suggesting they may be intermediates in the biosynthetic pathway to trypanothione. The antiprotozoal drugs berenil, pentamidine, ethidium bromide, imidocarb, methylglyoxal-bis(guanylhydrazone) and 1,3-diacetylbenzene-bis(guanylhydrazone) had no effect on the synthesis of N 1-glutathionylspermidine or trypanothione in vitro.

Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids.

Glutathione reductase from trypanosomes and leishmanias, unlike glutathione reductase from other organisms, requires an unusual low molecular weight cofactor for activity. The cofactor was purified from the insect trypanosomatid Crithidia fasciculata and identified as a novel glutathione-spermidine conjugate, N1,N8-bis(L-gamma-glutamyl-L-hemicystinyl-glycyl)spermidine, for which the trivial name trypanothione is proposed. This discovery may open a new chemotherapeutic approach to trypanosomiasis and leishmaniasis.

Pyruvate decarboxylase and alcohol dehydrogenase in the insect trypanosomatid, Crithidia fasciculata

1. 1. Cell-free extracts of the insect trypanosomatid Crithidia fasciculata, obtained by grinding with glass powder in a mortar, contained activity of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1). Both enzymes were found in the soluble fraction (105,000 g supernatant) and were partially purified by ammonium sulphate fractionation and gel filtration on Sephadex G-200. 2. 2. Pyruvate decarboxylase, free of lactate dehydrogenase and with little endogenous alcohol dehydrogenase, was assayed spectrophotometrically with commercial yeast alcohol dehydrogenase as coupled enzyme. The activity was increased nearly 2-fold by the addition of thiamine pyrophosphate (0.1 mM) and MgCl 2 (1 mM). The apparent K m for pyruvate was 2 mM, and the optimal pH was 6.0. 3. 3. Alcohol dehydrogenase was nearly 14-fold more active in the direction of acetaldehyde reduction than in the direction of ethanol oxidation. The optimal pH was about 7.0, and the apparent K m values were 35 μM for acetaldehyde (at 0.15 mM NADH) and 14 μM for NADH (at 60 μM acetaldehyde). 4. 4. Both enzymes were strongly inhibited by thiol reagents such as p-chloromercuribenzoate and phenolphthalein mercuric acetate; pyruvate decarboxylase was the more sensitive. 5. 5. The properties of both enzymes suggest that the enzyme system for ethanol production by C. fasciculata is very similar to that present in baker's yeast.

One strand of ars189 from the maxicircle of Crithidia fasciculata, transforms Saccharomyces cerevisiae more efficiently than its complementary strand as a single stranded DNA

An autonomously replicating element ( ars 189) has been isolated from the maxicircle DNA of an insect trypanosomatid Crithidia fasciculata. This 189-bp fragment contains two copies of the yeast consensus ARS sequence of (A/T)TTTATPuTTT(T/A), has an A + T composition of 79.4%, and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. The complementary strands of ars 189 have been cloned into an M 13 vector containing the URA3 gene of Saccharomyces cerevisiae. When these circular single-stranded (ss) DNAs were used to transform yeast spheroplasts, the M 13 chimeric DNA carrying the strand of ars189 rich in adenine generated approximately four times more yeast Ura + transformants than the construct containing the thymine-rich strand. In contrast, both strands of yeast ARS1 cloned into an M 13 vector transformed yeast at an equivalent level. The conversion of ARS -containing ss DNAs to duplex forms in vivo and their subsequent autonomous replication have been verified by Southern hybridization analysis of extracts from yeast transformants.

A 189-bp fragment of Crithidia fasciculata maxicircle DNA confers autonomous replication in Saccharomyces cerevisiae

A 189-bp fragment capable of promoting high-frequency transformation in Saccharomyces cerevisiae has been isolated from the maxicircle of the insect trypanosomatid Crithidia fasciculata. Chimeric plasmids containing this autonomously replicating sequence ( ars) are maintained as extrachromosomal elements in S. cerevisiae. The nucleotide sequence of the maxicircle fragment, termed ars 189, has been determined, and its position has been mapped in the maxicircle. The ars 189 fragment has an A + T content of 79.4% and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. In one strand (the T strand) thymine accounts for 118 out of 189 nucleotides while adenine accounts for only 32 nucleotides. The arc 189 DNA does not hybridize with minicircles, and its sequence appears to be unique in the C. fasciculata maxicircle genome. This sequence also shows extensive homology to a sequence within a 2.6-kb arc fragment of the Leishmania tarentolae maxicircle. In addition, ars 189 contains two copies of a yeast consensus ars sequence (A/T) TTTATPuTTT (T/A).

Identification of autonomous replication sequences in genomic and mitochondrial DNA of Crithidia fasciculata

High frequency transformation of Saccharomyces cerevisiae was used as a functional assay to isolate autonomous replication sequences ( ars) from the genomic and kinetoplast DNA of the insect trypanosomatid Crithidia fasciculata. Three independent cloned genomic sequences and one kinetoplast DNA sequence promoted high frequency transformation and extrachromosomal maintenance of the YIp5 plasmid DNA in yeast. The kinetoplast DNA clone was sub-cloned to further localize the DNA sequence essential for ars activity. This element was shown to be contained in a 2 kb HindIII- EcoRI fragment derived from a 8 kb HindIII fragment of the maxicircle component of the kinetoplast DNA. This 2 kb fragment is within a DNA sequence that has been shown to strongly hybridize to Trypanosoma brucei maxicircle DNA.

On the DNA content and ploidy of trypanosomes

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture form were not significantly different. In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1, 221–246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.