The status of Leishmania tarentolae/Trypanosoma platydactyli

Taxonomic studies and classification of Leishmania species have developed rapidly in recent years, but controversy still surrounds the relationships between those species infecting lizards and those infecting mammals. Some authorities maintain that the leishmanias o f lizards form a sufficiently distinct group to be ranked as a separate genus - Sauroleishmania(1,2) - while Wallbanks et al. have gone further to suggest that such species might be classified within the genus Trypanosoma(3). This suggestion followed from work showing that promostigote forms of Trypanosoma platydactyli from a gecko, had similar isoenzyme profiles to Leishmania tarentolae, a well-known species from lizards. In this article however, Larry Simpson and George Holz Jr discuss conflicting evidence, concluding from recent studies of DNA and lipid composition that the lizard leishmanias are more closely related to mammalian leishmanias than to trypanosomes.0.     

Pteridine Requirement of the Hemoflagellate Leishmania tarentolae

SYNOPSIS. Leishmania tarentolae grown in a defined medium required both folic acid and an unconjugated pteridine. Continuous growth was obtained with folic acid at a minimal level of 0.34 ng/ml and biopterin at 1.7 ng/ml. Neopterin was about 1/100 as active as biopterin.     

Leishmania tarentolae: streptomycin and chloramphenicol resistance of promastigotes.

Stable mutant strains of Leishmania tarentolae promastigotes resistant to chloramphenicol (CAP) were isolated by replica-plating techniques. In addition, cell lines stress-adapted to streptomycin and to high culture temperature (33 C) were obtained. Drug resistance was influenced by temperature, culture media, and plating technique. Inhibition of amino acid incorporation into protein occurs in CAP-resistant cells when exposed to 600 μg CAP/ml but this inhibition was 50–80% lower than that found in wild type sensitive cells. The primary site of CAP action appears to be inhibition of protein synthesis. CAP also adversely affected proline oxidation.     

Isozymes in the Culture Forms of Leishmania tarentolae*

SYNOPSIS. Leishmania tarentolae grown in Trager's defined medium C, blood brain heart infusion broth and blood agar contained 2 forms of malic dehydrogenase (MDH) after zone electrophoresis in potato starch: one at the point of origin and the other migrating towards the anode. The pH optimum with oxaloacetate as substrate was ? 8.35 for the anodal form and 7.50 for the point of origin enzyme. The Michaelis constant (Km) with oxaloacetate was 1.8–2.8 × 10^?5 M for the anodal form and 4.0 × 10^?5 M for the nonmigratory form. At pH 7.4, both MDHs were inhibited by oxaloacetate concentrations greater than 3.75 × 10^?4 M. Ratios of activity with different NAD analogs were dissimilar. A few of the non-migratory enzyme ratios corresponded with those reported for mitochondrial MDH. There was no correspondence between the ratios shown by anodal MDH and ratios reported either for mitochondrial MDH or for cytoplasmic MDH. The thionicotinamide analog was not utilized by point of origin MDH; however, the anodal form did show greater activity with this analog which is a characteristic of cytoplasmic MDH. Anodal MDH was more stable than non-migratory enzyme. Heat inactivation studies indicated 80% inactivation at 68°C for the anodal form and 100% inactivation at 37°C for the other form. The point of origin enzyme had a half life of about 48 hours at 4°C whereas anodal MDH was stable for at least one week at 4°C. Addition of enzyme stabilizing agents (Cleland's reagent, mercaptoethanol and gelatin) did not prevent breakdown of the non-migrating enzyme. Phosphate buffer increased the activity of the point of origin enzyme but had no effect on anodal MDH. On the basis of the above results, non-migratory enzyme is thought to be a variant of mitochondrial MDH. The characteristics of the anodal MDH do not readily indentify it as a typical mitochondrial or cytoplasmic type and it may be a modified type similar to those found in parasitic protozoa by other workers.     

Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Kinetoplast DNA (? = 1.703 g/ml.) was isolated by preparative cesium chloride ultracentrifugation in a fixed-angle rotor from total cell DNA of Leishmania tarentolae and examined in terms of sedimentation properties, melting characteristics, and appearance in the electron microscope. It consisted of several molecular types, either free or bound together in associations of variable size: minicircles (molecular weight = 0.56 ± 0.03 × 10⁶), catenated minicircles, “figure 8” molecules, and long molecules. The associations seem to be held together by the long molecules threading through the smaller circles and catenanes. The large associations could be broken down by sonication, DNase II-treatment, or shear forces. Minicircles, catenated dimers, trimers, and small linear fragments were separated on preparative sucrose gradients of sonicated DNA, and S_20,w values were assigned to each molecular type by band sedimentation in the analytical ultracentrifuge.     

Exploiting Leishmania tarentolae cell-free extracts for the synthesis of human solute carriers

Abstract

Cell-free protein production offers a versatile alternative to complement in vivo expression systems. However, usage of prokaryotic cell-free systems often leads to non-functional proteins. We modified a previously designed cell-free system based on the protozoan Leishmania tarentolae, a parasite of the Moorish gecko Tarentola mauritanica, together with a species-independent translational sequences-based plasmid to produce human membrane proteins in 2 hours reaction time. We successfully established all four commonly used expression modes for cell-free synthesis of membrane proteins with a human organic anion transporter, SLC17A3, as a model membrane protein: (i) As precipitates without the addition of any hydrophobic environment, (ii) in the presence of detergents, (iii) with the addition of liposomes, and (iv) supplemented with nanodiscs. We utilized this adapted system to synthesize 22 human solute carriers from 20 different families. Our results demonstrate the capability of the Leishmania tarentolae cell-free system for the production of a huge variety of human solute carriers in the precipitate mode. Furthermore, purified SLC17A3 shows the formation of an oligomeric state.     

Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.     

Behavior of the Kinetoplast of Leishmania tarentolae upon Cell Rupture*

SYNOPSIS. The kinetoplast of L. tarentolae remains attached to the basal body upon cell rupture by detergent lysis, sonication, or hypotonic lysis in 0.02 M Tris buffer (pH 7.9) at 0–4 C. Hypotonic lysis in 0.02 M Tris-HCl-2 mM EDTA at 0–4 C and application of mild shearing forces bring about release of most of the swollen kinetoplasts. The kinetoplast DNA can be seen in phase contrast microscopy as a dark mass contiguous to the kinetoplast membrane directly opposite the basal body. Upon return to isotonic media, the kinetoplast shrinks; the membranes of such kinetoplasts are impermeable to added DNAase.     

Potential application of Leishmania tarentolae as an alternative platform for antibody expression

Through the discovery of monoclonal antibody (mAb) technology, profound successes in medical treatment against a wide range of diseases have been achieved. This has led antibodies to emerge as a new class of biodrugs. As the “rising star” in the pharmaceutical market, extensive research and development in antibody production has been carried out in various expression systems including bacteria, insects, plants, yeasts, and mammalian cell lines. The major benefit of eukaryotic expression systems is the ability to carry out posttranslational modifications of the antibody. Glycosylation of therapeutic antibodies is one of these important modifications, due to its influence on antibody structure, stability, serum half-life, and complement recruitment. In recent years, the protozoan parasite Leishmania tarentolae has been introduced as a new eukaryotic expression system. L. tarentolae is rich in glycoproteins with oligosaccharide structures that are very similar to humans. Therefore, it is touted as a potential alternative to mammalian expression systems for therapeutic antibody production. Here, we present a comparative review on the features of the L. tarentolae expression system with other expression platforms such as bacteria, insect cells, yeasts, transgenic plants, and mammalian cells with a focus on mAb production.     

Crystal structure of adenine phosphoribosyltransferase from Leishmania tarentolae: potential implications for APRT catalytic mechanism

The three-dimensional structure of Leishmania tarentolae adenine phosphoribosyltransferase (APRT) in complex with adenosine-5-monophosphate (AMP) and a phosphate ion has been solved. Refinement against X-ray diffraction data extending to 2.2-A resolution led to a final crystallographic R factor of 18.3%. Structural comparisons amongst this APRT enzyme and other 'type I' PRTases whose structures have been determined reveal several important features of the PRTases catalytic mechanism. Based on structural superpositions and molecular interaction potential calculations, it was possible to suggest that the PRPP is the first substrate to bind, while the AMP is the last product to leave the active site, in accordance to recent kinetic studies performed with the Leishmania donovani APRT.     

Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae

The coding sequence of several mitochondrial mRNAs of the trypanosomatid family of protozoa is created by the guide RNA-directed insertion and deletion of uridylates (Us). Selection-amplification was used to explore the sequence and structure of the guide RNA and mRNA required for efficient insertional editing within a mitochondrial extract prepared from Leishmania tarentolae. This study identifies several novel features of the editing reaction in addition to several that are consistent with the previous mutagenesis and phylogenetic analysis of the reaction in Trypanosoma brucei, a distantly related trypanosomatid. Specifically, there is a strong bias against cytidines 5' of the editing sites and guanosines immediately 3' of guiding nucleotides. U insertions are directed both 5' and 3' of a genomically encoded U, which was previously assumed not to occur. Base pairing immediately flanking an editing site can significantly stimulate the editing reaction and affect the reaction fidelity but is not essential. Likewise, single-stranded RNA in the region upstream of the editing site, not necessarily immediately adjacent, can facilitate editing but is also not essential. The editing of an RNA containing many of the optimal features is linear with increasing quantities of extract permitting specific activity measurements to be made that are not possible with previously described T. brucei and L. tarentolae assays. The reaction catalyzed by the L. tarentolae extract can be highly accurate, which does not support a proposed model for editing that was based largely on the inaccuracy of an earlier in vitro reaction.     

Differential localization of nuclear-encoded tRNAs between the cytosol and mitochondrion in Leishmania tarentolae

All mitochondrial tRNAs of the kinetoplastid protozoan Leishmania tarentolae are encoded in the nucleus and are imported from the cytosol into the mitochondrion. We previously reported the partitioning of five tRNAs and found that all were shared between the two compartments to different extents. To increase our knowledge of the tRNAs of this organism, and to attempt to understand the signals involved in their subcellular localization, a method to RT-PCR amplify new tRNAs was developed. Various tRNAs were 3' polyadenylated and reverse transcribed with a sequence-tagged primer. The cDNA was tagged by ligation to an anchor oligonucleotide, and the resulting double-tagged cDNA was amplified by PCR. Four new tRNAs were obtained, bringing to 20 the total number of L. tarentolae tRNAs identified to date. The subcellular localization of 17 tRNAs was quantitatively analyzed by two-dimensional gel electrophoresis and northern hybridization. In general, the previously suggested operational classification of tRNAs into three groups (mainly cytosolic, mainly mitochondrial, and shared between the two compartments) is still valid, but the relative abundance of each tRNA in the cytosol or mitochondrion varied greatly as did the level of expression.     

Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa Leishmania tarentolae could be used for production of amino acid specific (15)N-labeled recombinant proteins. Using synthetic growth medium we were able to express in L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the final yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identification of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the L. tarentolae expression system for production of isotopically labeled proteins.