Development of a defined medium for heterologous expression in Leishmania tarentolae

A defined medium is essential for studying nutritional requirements of microorganisms and for selective supplementation of necessary substances. Hemin is an essential ingredient for growth of Leishmania tarentolae, but tends to precipitate in aqueous solutions without further stabilization. These aggregates disturb the measurement of the optical density or the cell density and the following downstream processing. Therefore, we were looking for stabilizing substances and established a PEG-hemin-solution, which avoided flocculation and allowed the cultivation of L. tarentolae in a medium, which we termed SFP(II) medium. With addition of RNA from Saccharomyces cerevisiae to SFP(II) medium the SFP(III) medium was established. In this medium, the specific cell division rate was increased (0.103 h(-1)) and stable for longer periods of time. The evaluation of the SFP(III) medium was done in shaker flasks by successful expression and segregation of the SAG2 protein, one of the main surface antigens of Toxoplasma gondii. With establishment and evaluation of this defined medium, the status of the Leishmania tarentolae expression system as an alternative to commonly used cell cultures is supported.     

Characterization of the growth behavior of Leishmania tarentolae: a new expression system for recombinant proteins

Biotechnological production of recombinant proteins for human therapy requires a cultivation of the host organism in a nutrient medium free of animal substances. Therefore, various nutrient media for the new expression system Leishmania tarentolae were developed and examined according to their cultivation conditions as static suspension culture and agitated culture. Investigations resulted in the development of a serum-free but hemin containing medium, based on yeast extract and buffer salts. Here we report that a high and stable specific growth rate of 0.103 h(-1) and a maximal cell density of 1 x 10(9) cells ml(-1) is obtained in an alternative medium, the YE-medium. For the newly developed medium, the successful expression of enhanced green fluorescent protein and the adaptation of the cultivation from the agitated culture to the bioreactor could be shown. Furthermore, an analytical method for detection of the essential, organic iron source hemin was established. The consumption of hemin was monitored because hemin is a potentially important process parameter for bioprocess control. With knowledge of these results, an improved expression system is available as an alternative to commonly used cell cultures for the production of recombinant proteins.     

Isolation and characterization of mitochondrial ribosomes and ribosomal subunits from Leishmania tarentolae

We have analyzed Leishmania tarentolae mitochondrial ribonucleoprotein (RNP) complexes using the 9S small subunit (SSU) rRNA and the 12S large subunit (LSU) rRNA as markers, and have identified a 50S RNP particle as the putative mitochondrial monosome, a 40S particle as the putative LSU and a 30S particle as the putative SSU. These assignments are supported by morphological analysis by cryo-electron microscopy and proteomics analyses by mass spectrometry. The presence of additional rRNA-containing particles complicated the analysis and most likely was the basis for previous difficulties in identification of these ribosomes; thus, in addition to the monosomes and their subunits, there are abundant stable 45S particles (SSU(*)) containing only 9S rRNA, which may represent homodimers of the SSU or SSU associated with additional proteins, and variable minor amounts of 65S and 70S particles, which represent homodimers of the LSU and SSU(*), respectively. These additional rRNA particles might be due to the lengthy mitochondrial isolation and ribosome isolation procedures or may be present in vivo and play yet undetermined roles.     

Recurrent polymorphisms in small chromosomes of Leishmania tarentolae after nutrient stress or subcloning.

Molecular karyotypes of the UC, LEM87 and LEM115 Leishmania tarentolae strains were obtained. All strains had 24-28 chromosomal bands which varied in size between 300 kb and 2.9 Mb. Several recurrent chromosomal polymorphisms occurred in LEM115 after nutrient shock or subcloning. One type of polymorphism involves the truncation of a 365-kb chromosome which contains the miniexon genes. This specific chromosome breakage appears to be induced by the nutrient shock or subcloning process and also occurs spontaneously during routine passage. Another polymorphism is the appearance of a 90-kb minichromosome (115-SNA1) after severe nutrient shock. This appears to be selection of a pre-existing cell type from a mosaic population. The 115-SNA1 minichromosome has sequence homology with a minichromosome in LEM87 cells but shows no homology with any chromosomes in 115wt or other strains. The copy number of 115-SNA1 varies with culture conditions, suggesting a relaxed centromeric control. The nature and origin of this minichromosome is not known.     

A new expression vector for Crithidia fasciculata and Leishmania

Crithidia fasciculata is a monogenetic parasite of insects. It grows in fully defined media without requiring serum, which facilitates biochemical analysis. We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania. These cells can be readily transfected with plasmid DNA by electroporation and transformants selected with various antibiotic resistance markers. 5'-Trans-splicing signals and poorly defined regions within the 3'-untranslated regions of genes are required for optimal expression of genes in trypanosomatids. We, therefore, inserted the intergenic region of the C. fasciculata phosphoglycerate kinase (PGK) genes A and B, which allows polyadenylation of the target gene and spliced leader addition to the selectable marker gene. Part of the intergenic region of the PGK locus was added upstream of the target gene to permit its trans-splicing. A 3'-untranslated sequence from the Crithidia glutathionylspermidine synthetase (GSPS) was also added to allow the polyadenylation of the selectable marker gene. Genes can be readily inserted using a multiple cloning site and can be expressed as a fusion protein with a poly-histidine sequence at either the N or C-terminus or fused with green fluorescent protein. Biologically active proteins can be expressed in C. fasciculata or L. amazonensis promastigotes and purified by affinity chromatography using a metal chelating column.     

Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein

The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.     

Protein methylation and protein methylases in Leishmania donovani and Leishmania tropica promastigotes

We studied the content of acid-stable methylated amino acids of soluble proteins in promastigotes of Leishmania donovani and L. tropica. epsilon-N-Trimethyllysine and NG,NG-dimethylarginine were found in both Leishmania species after culture in the presence of [methyl-14C]methionine. In addition, 3-N-methylhistidine was found only in L. tropica and epsilon-N-dimethyllysine only in proteins of L. donovani. As sinefungin, an antileishmanial nucleoside antibiotic, is a known transmethylase inhibitor, its effect on protein methylation was studied, in whole cells and in vitro. In the first case the drug had no effect on the content of methylated amino acid residues of soluble proteins. In vitro, histone methylation by crude extracts was studied at pH 7.2 and 9.0, known in other organisms as optimum pH values for arginine and lysine methylation, respectively. Surprisingly, arginine methylation by extracts of L. donovani was the same at both pH values while lysine residues were more efficiently methylated at pH 7.2 than at pH 9 by the extracts of the two species. These results indicate that the properties of protein methylases I and III of these parasites are different from those of other organisms hitherto studied. The inhibition constants of sinefungin for the leishmanial protein methylases were weak in comparison with those for enzymes from other sources, with the exception of the constant of L. donovani enzyme at pH 9.     

Intracellular protein degradation in Leishmania tropica promastigotes

A labile class of proteins in the range of M_r = 30 000–60 000 which turn over rapidly have been demonstrated in Leishmania tropica promastigotes. The rate of protein degradation is increased by exhaustion of nutrients or inhibition of energy metabolism. Proteolysis is reduced when the parasites are provided with a readily utilizable carbon and energy source such as glucose, glutamate or proline. The breakdown of proteins in L. tropica is dependent on continuous protein synthesis probably for protease synthesis. It is suggested that the relatively high rate of intracellular protein degradation is an auxiliary means for generating carbon and energy sources during nutritional stress.     

Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.     

Cloning,heterologous expression,and substrate specificities of protein farnesyltransferases from Trypanosoma cruzi and Leishmania major

Chagas disease and leishmaniasis are tropical diseases caused by the protozoan parasites, Trypanosoma cruzi and Leishmania species, respectively. Protein farnesyltransferase (PFT) is being investigated as a target for anti-trypanosomatid agents because inhibitors of this enzyme are highly toxic to these parasites compared to mammalian cells. Here, we report the cloning of the alpha- and beta-subunit genes of PFT from T. cruzi and Leishmania major. The proteins encoded by these genes are considerably larger than those of mammalian PFTs due to the presence of a number of inserts of >25 amino acids that map to junctions between helical structural elements. These inserts are not part of the active site or the interface between the two subunits. Northern blots demonstrate expression of messenger RNA for the PFT subunits in both mammalian and insect life-cycle stages of these parasites. The T. cruzi, Trypanosoma brucei, and L. major PFTs were overexpressed in the Sf9 cell/baculovirus system as active enzyme forms. Kinetic studies with a panel of CALX-containing peptides with all 20 amino acids in the X-position show that trypanosomatid PFTs have similar substrate specificities and these are different from the mammalian PFT substrate specificity patterns.     

The heat shock protein 90 of Leishmania donovani

The 90-kDa heat shock protein (Hsp90) of Leishmania donovani is a highly abundant cytoplasmic protein and is involved in a variety of cellular processes. Pharmacological deactivation of Hsp90 leads to growth arrest and induces the synthesis of heat shock proteins. Moreover, treatment of promastigote parasites with Hsp90 inhibitors induces the synthesis of amastigote-specific marker proteins and a morphological alteration similar to axenic amastigote differentiation. We propose a role for Hsp90 in the feedback control of the cellular stress response and in the control of the parasite's life cycle.     

Putrescine transport system in Leishmania infantum promastigotes

A transport system for putrescine in Leishmania infantum (= L. donovani infantum) promastigotes has been identified and characterized by measuring the uptake of radioactively labelled putrescine. Putrescine uptake was time- and temperature-dependent without any accumulation taking place at 0 degrees C. Uptake of putrescine was maximal at pH values near neutrality. Putrescine uptake showed an apparent Km = 1.08 +/- 0.12 microM and a Vmax = 1.74 + 0.62 pmol min-1 (10(6) cells)-1. The effect of metabolic poisons and uncoupling agents suggests that the putrescine uptake was energy-dependent. The transport system seemed to be highly specific for putrescine as neither aminoacids nor related compounds tested showed any competition with putrescine uptake. The trypanocide Berenil inhibited almost completely the putrescine uptake with non-competitive kinetics and a value of Ki = 43 microM.     

Comparison of the effects of Leishmania major or Leishmania donovani infection on macrophage gene expression

The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45β, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.