Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.

The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis.     

Dynamics of polymorphism of acidocalcisomes in<Emphasis Type="Italic"> Leishmania</Emphasis> parasites

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 m diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warrens medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Characterisation of a new Leishmania META gene and genomic analysis of the META cluster

The META1 gene of Leishmania is upregulated in metacyclic promastigotes and encodes a 12 kDa virulence-related protein, conserved in all Leishmania species analysed. In this study, the genomic region adjacent to the Leishmania amazonensis META1 gene was characterised and compared to the Leishmania major META1 locus as well as to syntenic loci identified in Trypanosoma brucei and Trypanosoma cruzi. Three new genes expressed with increased abundance of steady state mRNA in L. amazonensis promastigotes were identified, two of which are upregulated in stationary phase promastigotes, sharing the pattern of expression previously described for the META1 mRNA. One of these new genes, named META2, encodes a polypeptide of 444 amino acid residues with a repetitive structure showing three repeats of the META domain (defined as a small domain family found in the Leishmania META1 protein and in bacterial proteins hypothetically secreted and/or implicated in motility) and a carboxyl-terminal region similar to several putative calpain-like proteins of Trypanosoma and Leishmania.     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.     

Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani

Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.     

Studies on Stibanate unresponsive isolates of<Emphasis Type="Italic">Leishmania donovani</Emphasis>

Visceral leishmaniasis, also known as kala-azar (KA) is generally caused byLeishmania donovani. Organic pentavalent antimonials (SbV) is the first line of treatment for KA. However, the number of KA patients unresponsive to treatment with Sb(V) is steadily increasing in India and elsewhere. The primary objective of this work is to determine the factor(s) associated with the rise of unresponsiveness. Analysis of the clonal population of parasites clearly indicated that wild type parasites isolated from KA patients who were clinically cured after treatment with Sb(V), were a mixture of resistant and sensitive cells. The resistant promastigotes were also resistant as amastigotesin vivo. It was further observed that Stibanate sensitive parasites can be made resistant to the drug by repeated passages in experimental animals followed by incomplete treatment with suboptimal doses of the drug. These results suggest that the steady rise in Sb(V) unresponsiveness of KA patients in India is due to infection with resistant parasites, generated as a result of irregular and often incomplete treatment of the patients.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Klaivanolide, an antiprotozoal lactone from Uvaria klaineana

Bioguided-fractionation of a CH(2)Cl(2) extract of the stems of Uvaria klaineana (Annonaceae) led to isolation of klaivanolide, a novel bisunsaturated 7-membered lactone (5-acetoxy-7-benzoyloxymethyl-7H-oxepin-2-one), together with benzyl benzoate. Klaivanolide showed potent in vitro antileishmanial activity against both sensitive and amphotericin B-resistant promastigote forms of Leishmania donovani with IC(50) values of 1.75 and 3.12 microM, respectively. The compound also showed in vitro trypanocidal activity against trypomastigote forms of Trypanosoma brucei brucei GVR 35. Its structure was established by 1D and 2D NMR and other spectroscopic techniques.