Tryparedoxins (TXNs) are oxidoreductases unique to trypanosomatids (including Leishmania and Trypanosoma parasites) that transfer reducing equivalents from trypanothione, the major thiol in these organisms, to sulfur-dependent peroxidases and other dithiol proteins. The existence of a TXN within the mitochondrion of trypanosomatids, capable of driving crucial redox pathways, is considered a requisite for normal parasite metabolism. Here this concept is shown not to apply to Leishmania. First, removal of the Leishmania infantum mitochondrial TXN (LiTXN2) by gene-targeting, had no significant effect on parasite survival, even in the context of an animal infection. Second, evidence is presented that no other TXN is capable of replacing LiTXN2. In fact, although a candidate substitute for LiTXN2 (LiTXN3) was found in the genome of L. infantum, this was shown in biochemical assays to be poorly reduced by trypanothione and to be unable to reduce sulfur-containing peroxidases. Definitive conclusion that LiTXN3 cannot directly reduce proteins located within inner mitochondrial compartments was provided by analysis of its subcellular localization and membrane topology, which revealed that LiTXN3 is a tail-anchored (TA) mitochondrial outer membrane protein presenting, as characteristic of TA proteins, its N-terminal end (containing the redox-active domain) exposed to the cytosol. This manuscript further proposes the separation of trypanosomatid TXN sequences into two classes and this is supported by phylogenetic analysis: i) class I, encoding active TXNs, and ii) class II, coding for TA proteins unlikely to function as TXNs. Trypanosoma possess only two TXNs, one belonging to class I (which is cytosolic) and the other to class II. Thus, as demonstrated for Leishmania, the mitochondrial redox metabolism in Trypanosoma may also be independent of TXN activity. The major implication of these findings is that mitochondrial functions previously thought to depend on the provision of electrons by a TXN enzyme must proceed differently.

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNA(Sec) however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNA(Sec) transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNA(Sec) is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNA(Sec) genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNA(Sec) outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNA(Sec), in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNA(Sec) gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatids.

A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals, and plants. Recently Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has been suggested as candidate for heterologous genes expression. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein; the oligosaccharide structures are similar to those of mammals with N-linked galactose, and fucose residues. To date several heterologous proteins have been expressed in L. tarentolae including both cytoplasmic enzymes and membrane receptors. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of those tools coupled with a better understanding of the biology of Leishmania species will lead to value and power in commercial and research labs alike.

The micronutrient selenium is found in proteins as selenocysteine (Sec), the 21st amino acid cotranslationally inserted in response to a UGA codon. In vitro studies in archaea and mouse showed that Sec-tRNA(Sec) formation is a 3-step process starting with serylation of tRNA(Sec) by seryl-tRNA synthetase (SerRS), phosphorylation of serine to form phosphoserine (Sep)-tRNA(Sec) by phosphoseryl-tRNA(Sec) kinase (PSTK), and conversion to Sec-tRNA(Sec) by Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic selenoprotein synthesis has been lacking. Here, we present an analysis of Sec-tRNA(Sec) formation in the parasitic protozoon Trypanosoma brucei in vivo. Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis, demonstrating the essentiality of both enzymes for Sec-tRNA(Sec) formation. Growth of the 2 knockout strains was not impaired; thus, unlike mammals, trypanosomes do not require selenoproteins for viability. Analysis of conditional RNAi strains showed that SerRS, selenophosphate synthase, and the Sec-specific elongation factor, EFSec, are also essential for selenoprotein synthesis. These results with T. brucei imply that eukaryotes have a single pathway of Sec-tRNA(Sec) synthesis that requires Sep-tRNA(Sec) as an intermediate.

Leishmania infantum cytosolic tryparedoxin (LiTXN1) can be regarded as a potential candidate for drug targeting. This redox active molecule, which belongs to the thioredoxin superfamily, is one constituent of the hydroperoxide elimination cascade in L. infantum and may also be involved in other cellular processes such as DNA synthesis or host-parasite interaction. In order to validate LiTXN1 as a drug target we have employed a gene replacement strategy. We observed that substitution of both chromosomal LiTXN1 alleles was only possible upon parasite complementation with an episomal copy of the gene. Furthermore, contrary to control parasites carrying the empty vector, both the insect and the mammalian stages of L. infantum retained the episomal copy of LiTXN1 in the absence of drug pressure. These results confirm the essentiality of LiTXN1 throughout the life cycle of the parasite, namely in the disease-causing amastigote stage. In addition, the data obtained showed that disruption of one allele of this gene leads only to a 25% reduction in the expression of LiTXN1. Even though this does not affect promastigote growth and susceptibility to hydrogen peroxide, ex vivo infection assays suggest that wild-type levels of LiTXN1 are required for optimal L. infantum virulence.

All mitochondria have integral outer membrane proteins with beta-barrel structures including the conserved metabolite transporter VDAC and the conserved protein import channel Tom40. Bioinformatic searches of the Trypanosoma brucei genome for either VDAC or Tom40 identified a single open reading frame, with sequence analysis suggesting that VDACs and Tom40s are ancestrally related and should be grouped into the same protein family: the mitochondrial porins. The single T. brucei mitochondrial porin is essential only under growth conditions that depend on oxidative phosphorylation. Mitochondria isolated from homozygous knock-out cells did not produce ATP in response to added substrates, but ATP production was restored by physical disruption of the outer membrane. These results demonstrate that the mitochondrial porin identified in T. brucei is the main metabolite channel in the outer membrane and therefore the functional orthologue of VDAC. No distinct Tom40 was identified in T. brucei. In addition to mitochondrial proteins, T. brucei imports all mitochondrial tRNAs from the cytosol. Isolated mitochondria from the VDAC knock-out cells import tRNA as efficiently as wild-type. Thus, unlike the scenario in plants, VDAC is not required for mitochondrial tRNA import in T. brucei.

Like yeast, Trypanosoma brucei is a model organism and has a published genome sequence. Although T. b. brucei strain 427 is used for studies of trypanosome molecular biology, particularly antigenic variation, in many labs worldwide, this strain was not selected for the genome sequencing project as it is monomorphic and unable to complete development in the insect vector. Instead, the fly transmissible, mating competent strain TREU 927 was used for the genome project, but is not as easily grown or genetically manipulable as strain 427; furthermore, recent findings have spread concern on the potential human infectivity of TREU 927. Here we show that a 40-year-old cryopreserved line of strain 427, Variant 3, is fly transmissible and also able to undergo genetic exchange with another strain of T. b. brucei. Comparison of Variant 3 with lab isolates of 427 shows that all have variant surface glycoprotein genes 117, 121 and 221, and identical alleles for 3 microsatellite loci. Therefore, despite some differences in molecular karyotype, there is no doubt that Variant 3 is an ancestral line of present day 427 lab isolates. Since Variant 3 grows fast both as bloodstream forms and procyclics and is readily genetically manipulable, it may prove useful where a fly transmissible version of 427 is required.

The loci Tb927.3.4070, 927.3.4080, Tb927.3.4090, 927.3.4100 and 927.3.4110 of Trypanosoma brucei encode five similar proteins with 13-14 transmembrane domains. Corresponding mRNAs are more abundant in bloodstream-form trypanosomes than in procyclics. The 4070, 4090 and 4110 genes have almost identical 3'-intergenic regions and the predicted proteins share a short C-terminal extension; a reporter mRNA with the 4110 3'-untranslated region was more abundant in bloodstream forms than procyclic forms. The 3'-untranslated regions for 4080 and 4100 are different, and that of 4080 gave procyclic-specific reporter expression. TbUBP1 and 2 are proteins with low-specificity RNA-binding activity. Over-expression of TbUBP2 in bloodstream forms increased the overall abundance of mRNA encoding the transmembrane proteins, whereas TbUBP1 and 2 RNA interference decreased it. RNAi targeting TbUBP1 and 2 in bloodstream forms decreased mRNA with a 4110 3'-untranslated region, but increased it for 4080. Thus TbUBP and TbUBP2 may accentuate developmental regulation of the Tb927.3.4070-927.3.4110 mRNAs.

The trypanosomatid protozoan Leishmania tarentolae has been extensively used as a model system for studying causative agents of several tropical diseases and more recently as a host for recombinant protein production. Here we analyze the rates of partial or complete deletions of expression cassettes integrated into small ribosomal RNA and tubulin gene clusters as well as into ornithine decarboxylase gene of L. tarentolae. In approximately 60% of cases gene conversion was responsible for the deletion while in the rest of the cases deletion occurred within the expression cassette. We used this observation to design constitutive and inducible expression vectors that could be stably integrated into the genome and subsequently depleted of the antibiotic resistance genes using thymidine kinase or bleomycin resistance genes as negative selection markers. This enabled us to obtain L. tarentolae strains containing constitutive or inducible markerless expression cassettes. Analysis of the markerless strains demonstrated that although stability varied among clones some were stable for as many as 200 generations. We expect that this approach will be useful for the construction of strains carrying multiple expression cassettes for analysis of trypanosomatid pathogenicity mechanisms and overexpression of multi-subunit protein complexes for biochemical and structural studies.

In trypanosomatids, uridylate residues are post-transcriptionally added to or deleted from pre-mRNAs during the complex process of RNA editing. Editing is carried out exclusively in the mitochondrion of these parasites and involves numerous proteins assembled into protein and ribonucleoprotein complexes. Previously we identified RNA-editing-associated protein -1 (REAP-1), an RNA binding protein found in the mitochondrion of Trypanosoma brucei. REAP-1 was shown to specifically recognize and bind to pre-mRNAs that require editing and was proposed to act as a recruitment factor to deliver pre-mRNAs to editing complexes. To help define the role of REAP-1, we have now constructed REAP-1 null mutants. We show that the null mutants, although viable, have a significant growth defect. RNA levels within the mitochondrion were evaluated using reverse transcriptase real-time PCR. Surprisingly, the results show that mitochondrial RNA levels are increased, regardless of the editing status of the RNA. All RNA tested, whether unedited, edited, or never edited were increased in the mutant cell line relative to wild-type levels. This study provides the first evidence for a role of REAP-1 in RNA metabolism.

Laser wakefield acceleration of electrons holds great promise for producing ultracompact stages of GeV scale, high-quality electron beams for applications such as x-ray free electron lasers and high-energy colliders. Ultrahigh intensity laser pulses can be self-guided by relativistic plasma waves (the wake) over tens of vacuum diffraction lengths, to give >1  GeV energy in centimeter-scale low density plasmas using ionization-induced injection to inject charge into the wake even at low densities. By restricting electron injection to a distinct short region, the injector stage, energetic electron beams (of the order of 100 MeV) with a relatively large energy spread are generated. Some of these electrons are then further accelerated by a second, longer accelerator stage, which increases their energy to ∼0.5  GeV while reducing the relative energy spread to <5% FWHM.

A laser wakefield acceleration study has been performed in the matched, self-guided, blowout regime producing 720+/-50 MeV quasimonoenergetic electrons with a divergence Deltatheta_{FWHM} of 2.85+/-0.15 mrad using a 10 J, 60 fs 0.8 mum laser. While maintaining a nearly constant plasma density (3x10;{18} cm;{-3}), the energy gain increased from 75 to 720 MeV when the plasma length was increased from 3 to 8 mm. Absolute charge measurements indicate that self-injection of electrons occurs when the laser power P exceeds 3 times the critical power P_{cr} for relativistic self-focusing and saturates around 100 pC for P/P_{cr}>5. The results are compared with both analytical scalings and full 3D particle-in-cell simulations.

The nucleotide sugar UDP-galactose is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-galactose could arise de novo by epimerization of UDP-glucose or by a salvage pathway involving phosphorylation of galactose and the action of UDP-glucose:alpha-D-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glucose, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-glucose was expected to deprive parasites of UDP-galactose required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the galactose rich phosphoglycans. Moreover, no alteration in the abundant galactose-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-glucose independent salvage pathway for UDP-galactose biosynthesis.

The self-guiding of relativistically intense but ultrashort laser pulses has been experimentally investigated as a function of laser power, plasma density, and plasma length in the blowout regime. The extent of self-guiding, observed by imaging the plasma exit, is shown to be limited by nonlinear pump depletion with observed self-guiding of over tens of Rayleigh lengths. Spectrally resolved images of the plasma exit show evidence consistent with self-guiding in the plasma wake. Minimal losses of the self-guided pulse resulted when the initial spot size was matched to the blowout radius.

An ultrarelativistic 28.5 GeV, 700-microm-long positron bunch is focused near the entrance of a 1.4-m-long plasma with a density n(e) between approximately equal to 10(13) and approximately equal to 5 x 10(14) cm(-3). Partial neutralization of the bunch space charge by the mobile plasma electrons results in a reduction in transverse size by a factor of approximately equal to 3 in the high emittance plane of the beam approximately equal to 1 m downstream from the plasma exit. As n(e) increases, the formation of a beam halo containing approximately 40% of the total charge is observed, indicating that the plasma focusing force is nonlinear. Numerical simulations confirm these observations. The bunch with an incoming transverse size ratio of approximately 3 and emittance ratio of approximately 5 suffers emittance growth and exits the plasma with approximately equal sizes and emittances.

The electron hosing instability in the blow-out regime of plasma-wakefield acceleration is investigated using a linear perturbation theory about the electron blow-out trajectory in Lu et al.[in Phys. Rev. Lett. 96, 165002 (2006)10.1103/PhysRevLett.96.165002]. The growth of the instability is found to be affected by the beam parameters unlike in the standard theory Whittum et al.[Phys. Rev. Lett. 67, 991 (1991)10.1103/PhysRevLett.67.991] which is strictly valid for preformed channels. Particle-in-cell simulations agree with this new theory, which predicts less hosing growth than found by the hosing theory of Whittum et al.

A Thomson scattering opticals system is described with the following characteristics:(1) it allows scattering angles down to 1 mrad before collection optics interfere with beam dumping;(2) it gives excellent k resolution for angles of > or approximately 1.5 mrad;(3) it collects light from a scattering volume which can be variably positioned without optical realignment; and (4) it is compact in size. The design, test data, and an application to ruby-laser scattering from 100-microm wavelength plasma waves are presented.

We have developed a three-stage CO(2) master-oscillator-amplifier system that produces 1.1 TW of peak power. The system generates 170 J of energy in a diffraction-limited 160+/-10ps pulse on the 10P(20) line. We also report the realization of a two-wavelength terawatt-peak-power CO(2) laser that can be tuned to an arbitrary pair of lines. A two-stage semiconductor switching system driven by a picosecond-pulse Nd:YAG laser was used to slice a short, low-power 10.6-mum pulse for amplification. A simple plasma shutter helped to compensate for gain narrowing in a final three-pass amplifier and to shorten the pulse.

We report a factor-of-6 shortening of the 240-ps (FWHM) pulses in a triple-pass, 2.5-atm CO(2) amplifier. This technique is based on the self-phase modulation of a 10-mum pulse in a plasma after the first pass of amplification, followed by narrowing of this chirped pulse during further amplification. Subsequently, strong power broadening provides the necessary bandwidth to amplify 40-ps pulses to terawatt power levels.

Purine nucleoside and nucleobase transporters play a vital role in the metabolism and survival of Trypanosoma brucei because this parasitic protozoan is unable to synthesize purines de novo and thus must acquire preformed purines from its hosts. These parasites express a variety of nucleoside and nucleobase permeases with diverse substrate specificities and distinct patterns of expression during the trypanosome life cycle. We report here that expression of the newly characterized T. brucei nucleoside transporter 10 gene (TbNT10) is up-regulated in the short stumpy form of the life cycle, the bloodstream form of the parasite that is pre-adapted for infection of the tsetse fly vector. Functional expression of TbNT10 in Saccharomyces cerevisiae reveals that the TbNT10 gene encodes an adenosine/guanosine/inosine transporter with apparent Km values of approximately 1 microM and hence is a high affinity purine nucleoside transporter. The restricted expression of TbNT10 during the life cycle suggests that the functional properties of this permease may be specialized to support development and growth of the differentiated short stumpy form or to promote the transformation of short stumpy to procyclic forms within the insect vector.

Type IB topoisomerases are enzymes essential for the orderly synthesis of nucleic acids and are the molecular target for antitumor camptothecins. In dozens of organisms, including eukaryotes, bacteria, and viruses, this enzyme is monomeric. However, we previously found that topoisomerase IB in trypanosomes is a heteromultimer, comprised of two distinct subunits encoded by separate genes. A large 90 kDa subunit contains the DNA binding domain and a small 36 kDa subunit contains the catalytic domain. In this study we use RNA interference to silence each of the subunits separately. For each subunit, tetracycline-induced expression of double-stranded RNA results in drastic reduction of cognate mRNA and protein. For the large subunit, nucleic acid biosynthesis (as monitored by the incorporation of radiolabeled precursors into DNA and RNA) is halved by 39 h, and cell growth halts by 72 h, after induction. The steady state level of both nuclear and mitochondrial mRNAs is reduced. Virtually identical results are obtained by silencing the small subunit. Interestingly, although interference is specific at the level of mRNA, silencing of one subunit leads to a profound reduction in the level of protein for both subunits, suggesting that survival, or perhaps synthesis, of each subunit depends upon the presence of the other. These findings underscore the essential nature of type IB topoisomerase activity in Trypanosoma brucei and its suitability as a target for rational drug design.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. The method of choice today to examine gene function in these organisms is RNA interference (RNAi). Messenger RNA (mRNA) degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. This chapter describes some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA, and analysis of dsRNA and target mRNA.

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites of mammals that were first described over 100 hundred years ago. They have long been the subjects of biological investigation, which has yielded insights into a number of fundamental, as well as novel, cellular processes in all organisms. In the last decade or so, genetic manipulation of trypanosomes has become possible through DNA transformation, allowing yet more detailed analysis of the biology of the parasite. One facet of this is that DNA transformation has itself been used as an assay for recombination and will undoubtedly lead to further genetic approaches to examine this process. Here we describe protocols for DNA transformation of Trypanosoma brucei, including two different life cycle stages and two different strain types that are distinguished by morphological and developmental criteria. We consider the application of transformation to recombination, as well as the uses of transforming the different life cycle stages and strain types.

Genomic DNA fragments encoding nine, novel, P-type ATPases in trypanosomatid organisms were amplified in PCR, using degenerate oligonucleotide primers that recognize the ATP-binding and -phosphorylation sites present in all P-type ATPases. Subsequent phylogenetic analysis, based on the presence of conserved motifs in predicted peptide sequences for six Trypanosoma brucei, T. cruzi or Leishmania donovani PCR fragments, identified calcium-, proton- and phospholipid-translocating ATPases. DNA fragments that predict proteins homologous to the fungal, type-IID, P-type, ATPase pumps that transport Na(+) or K(+) ions were also present in T. brucei (TBCA1; 1022 nucleotides representing 340 amino acids), T. cruzi (TCNA1; 1022 nucleotides representing 340 amino acids) and L. donovani (LDCA1; 1031 nucleotides representing 343 amino acids). Southern blots showed that the Na(+)-ATPases were each present as a single-copy gene. The LDCA1 fragment was used to clone the complete LDCA1 gene from an L. donovani genomic-DNA library. The LDCA1 gene encodes a protein, of 1047 amino acids, with a predicted molecular mass of 115,501 Da. The results of analyses based on northern blots and the rapid amplification of cDNA ends (RACE) indicated that LDCA1 was expressed in promastigotes and amastigotes from axenic cultures and in animal-derived amastigotes. TBCA1 was expressed, as a 5.0-kb transcript, in procyclic culture stages and bloodstream trypomastigotes, with the 5.0-kb message up-regulated six-fold in the trypomastigote stage. Western blots probed with an antibody to the partial TBCA1 peptide identified a 150-kDa protein that was detected, by immunofluorescence, on the surface membrane of procyclic T. brucei.

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.