Leishmania donovani: promastigote--macrophage surface interactions in vitro.

The mechanism by which promastigotes of Leismania donovani enter hamster peritoneal macrophages was studied in vitro by light and electron microscopy. Quantitative light microscope studies showed a time-dependent increase of intracellular parasites, which had no preferable orientation during entry. Scanning and transmission electron microscopy revealed striking host-parasite surface interactions marked by the formation of whorled pseudopodia around the promastigotes in the early stage and engulfment of the parasites akin to normal phagocytosis in the later phase. Early host-parasite interactions were categorized quantitatively by scanning electron microscopy into several types, of which “head-first entry” and “tail-first entry” were approximately equal in frequency of occurrence, confirming light microscope observations. Cytochalasin B at 10 μg/ml prevented the intracellular entry of the parasites and the formation of macrophage-originated pseudopodia normally seen to seize the promastigotes. Killed, but morphologically intact, promastigotes were poorly taken up by macrophages and lacked certain types of interactions normally encountered with macrophage pseudopodia. Motility of promastigotes and their affinity to the surface of macrophages are suggested as elements of importance which parasites contribute to aid the process of their entry. The above results indicate that promastigotes of L. donovani depend on phagocytic activity of macrophages to gain intracellular entrance, but parasite-specific activities and/or properties may also play a role. It is suggested that “facilitated phagocytosis” may be used to describe this unique type of endocytosis associated with leishmania-macrophage interactions.     

Evidence for phosphorylation of the extracellular acid phosphatase of Leishmania donovani

The presence of two phosphorylated molecular species in the culture supernatants of axenically cultivated Leishmania donovani promastigotes was demonstrated by biosynthetically labeling cultures with [32P]phosphate. One of these species was resolved into two bands with Mr's of 149,000 and 97,000 by dissociating polyacrylamide gel electrophoresis and copurified with the extracellular acid phosphatase activity produced by the promastigotes. The site of phosphorylation of the extracellular acid phosphatase is not yet known.     

Evidence for Phosphorylation of the Extracellular Acid Phosphatase of Leishmania donovani1

The presence of two phosphorylated molecular species in the culture supernatants of axenically cultivated Leishmania donovani promastigotes was demonstrated by biosynthetically labeling cultures with [³²P]phosphate. One of these species was resolved into two bands with M_r's of 149,000 and 97,000 by dissociating polyacrylamide gel electrophoresis and copurified with the extracellular acid phosphatase activity produced by the promastigotes. The site of phosphorylation of the extracellular acid phosphatase is not yet known.     

Function and assembly of the Leishmania surface coat

Like many trypanosomatids, the cell surface coat of Leishmania spp. is responsible for mediating various host-parasite interactions as well as acting as a dense physical barrier. This confers protection to the parasites in the hostile environments of the sandfly midgut and the macrophage phagolysosome. The major components of the surface coat are tethered to the cell surface via glycosylphosphatidylinositol glycolipids, and the composition of this surface coat is exquisitely regulated during the course of the parasite life-cycle. In this paper, we review what is known about the composition, biosynthesis and function of these glycosylphosphatidylinositol-containing molecules found within the parasite surface coat.     

Evidence for the extracellular reduction of alpha-lipoic acid by Leishmania donovani promastigotes: a transplasma membrane redox system

Leishmania donovani cells, capable of reducing certain electron acceptors with redox potentials at pH 7.0 down to -290 mV, outside the plasma membrane, can reduce the oxidised form of alpha-lipoic acid. alpha-Lipoic acid has been used as natural electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane alpha-lipoic acid reduction by Leishmania was not inhibited by mitochondrial inhibitors as azide, cyanide, rotenone or antimycin A, but responded to hemin, modifiers of sulphhydryl groups and inhibitor of glycolysis. The protonophores carbonyl cyanide chlorophenylhydrazone and 2,4-dinitrophenol showed inhibition of alpha-lipoic acid reduction. This transmembrane redox system differs from that of mammalian cells in respect to its sensitivity of UV irradiation and stimulation by diphenylamine. Thus a naphthoquinone coenzyme appears to be involved in alpha-lipoic acid reduction by Leishmania cells.     

The comparative fine structure and surface glycoconjugate expression of three life stages of Leishmania major

The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electron-density. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.     

Evidence for a conserved microbiota across the different developmental stages of Plodia interpunctella

Diversity and composition of lepidopteran microbiotas are poorly investigated, especially across the different developmental stages. To improve this knowledge, we characterize the microbiota among different developmental stages of the Indian meal moth, Plodia interpunctella, which is considered one of the major pest of commodities world-wide. Using culture-independent approach based on Illumina 16S rRNA gene sequencing we characterized the microbiota of four developmental stages: eggs, first-, and last-instar larvae, and adult. A total of 1022 bacterial OTUs were obtained, showing a quite diversified microbiota associated to all the analyzed stages. The microbiotas associated with P. interpunctella resulted almost constant throughout the developmental stages, with approximately 77% of bacterial OTUs belonging to the phylum of Proteobacteria. The dominant bacterial genus is represented by Burkholderia (?64%), followed by Propionibacterium, Delftia, Pseudomonas, and Stenotrophomonas. A core bacterial community, composed of 139 OTUs, was detected in all the developmental stages, among which 112 OTUs were assigned to the genus Burkholderia. A phylogenetic reconstruction, based on the 16S rRNA, revealed that our Burkholderia OTUs clustered with Burkholderia cepacia complex, in the same group of those isolated from the hemipterans Gossyparia spuria and Acanthococcus aceris. The functional profiling, predicted on the base of the bacterial 16S rRNA, indicates differences in the metabolic pathways related to metabolism of amino acids between preimaginal and adult stages. We can hypothesize that bacteria may support the insect host during preimaginal stages.     

Deletion study of DNA topoisomerase IB from Leishmania donovani: searching for a minimal functional heterodimer

The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved "core" domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIDeltaS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids placed at the N-terminal end and a variable region comprised between Ala548 to end of the C-terminal extension of LdTopIL were enzymatically dispensable. Altogether this combinatorial approach provides important structural insights of the regions involved in relaxation activity and for understanding the atypical structure of this heterodimeric enzyme.     

Identification of a surface membrane proton-translocating ATPase in promastigotes of the parasitic protozoan Leishmania donovani.

ATPase activities were measured in surface membranes and mitochondria isolated from promastigotes of the parasitic protozoan Leishmania donovani. The two enzymes were differentiated on the basis of pH optima, inhibitor sensitivity and by immunochemical methods. The surface-membrane (SM-) ATPase had an activity of 100 nmol/min per mg of protein, which was optimal at pH 6.5. The enzyme was Mg2+-dependent, partially inhibited by Ca2+, and unaffected by Na+ or K+. The SM-ATPase was inhibited by orthovanadate, NN'-dicyclohexylcarbodi-imide, and N-ethylmaleimide [IC50 (concentration causing half-maximal inhibition) 7.5, 25 and 520 microM respectively]; however, it was unaffected by ouabain, azide or oligomycin. The SM-ATPase demonstrated a Km of 1.05 mM and a Vmax. of 225 nmol/min per mg of protein. Moreover, fine-structure cytochemical results demonstrated that the SM-ATPase was localized to the cytoplasmic lamina of the parasite SM. A method was devised for the isolation of SM-derived vesicles. These were used to demonstrate the proton-pumping capacity of the SM-ATPase. Cumulatively, these results constitute the first demonstration of a surface-membrane proton-translocating ATPase in a parasitic protozoan.     

Leishmania donovani: purification and partial characterization of a glycophosphosphingolipid antigen expressed on promastigote surface.

A ceramide-anchored glycophosphosphingolipid antigen was isolated from the lipid extract of Leishmania donovani promastigotes. The affinity-purified glycolipid antigen contained galactose, mannose, myo-inositol, phosphate, ceramide, and hexosamine but no sialic acid. The phosphate group was present internally at the core of the structure: inositol (1-O)-phosphorylceramide. The phosphate group became susceptible to alkaline phosphatase only after alkali-catalyzed hydrolysis of the glycolipid.     

Leishmania donovani: regulated changes in the level of expression of the surface 3'-nucleotidase/nuclease

Leishmania donovani promastigotes have previously been shown to possess a surface membrane bound 3'-nucleotidase/nuclease (3'-N'ase) capable of hydrolyzing both nucleic acids and 3'-ribonucleotides. The specific activity of the 3'-N'ase was increased following transfer of the parasites to fresh, nutrient-replete media or to media lacking purines and/or inorganic phosphate (Pi). In nutrient-replete media, the enzyme activity was transiently elevated during the lag and early logarithmic phases of the growth curve; enzyme activity fell as the cells continued into late log and stationary phases. Purine- and Pi-starved cells exhibited significantly greater levels of 3'-N'ase activity than nutrient-replete cells. These levels remained elevated as long as the organisms were maintained in the deficient media. Nutrient-replete and purine-starved 125I surface-labeled parasites displayed differences in electrophoretic patterns. Upon purine starvation, incorporation of radiolabel was increased in proteins which migrated with apparent molecular weights of 70, 43, and 40 kDa. Comigration, in both one- and two-dimensional systems, of 3'-N'ase activity with the radiolabeled 43-kDa band demonstrated that this band was the catalytically active protein. Peptide mapping of the 70-, 43-, and 40-kDa proteins failed to demonstrate similarities in peptide sequence consistent with either a degradation or a precursor/product relationship. Treatment of the 43- and 40-kDa peptides with N-Glycanase indicated that they were differentially glycosylated. The cumulative results of these studies indicated that L. donovani can respond to altered culture conditions by the differential expression of surface proteins. In particular, the differential expression of the protein responsible for 3'-N'ase activity is consistent with the role of this enzyme in purine acquisition.     

Glyoxalase I from Leishmania donovani: a potential target for anti-parasite drug

Glyoxalases are involved in a ubiquitous detoxification pathway. In pursuit of a better understanding of the biological function of the enzyme, the recombinant glyoxalase I (LdGLOI) protein has been characterized from Leishmania donovani, the most important pathogenic Leishmania species that is responsible for visceral leishmaniasis. A 24kDa protein was heterologously expressed in Escherichia coli. LdGLOI showed a marked substrate specificity for trypanothione hemithioacetal over glutathione hemithioacetal. Antiserum against recombinant LdGLOI protein could detect a band of anticipated size approximately 16kDa in promastigote extracts. Several inhibitors of human GLOI showed that they are weak inhibitors of L. donovani growth. Overexpression of GLOI gene in L. donovani using Leishmania expression vector pspalpha hygroalpha, we detected elevated expression of GLOI RNA and protein. Comparative modelling of the 3-D structure of LDGLOI shows that substrate-binding region of the model involves important differences compared to the homologues, such as E. coli, specific to glutathione. Most notably a substrate-binding loop of LDGLOI is characterized by a deletion of five residues compared to the E. coli homologue. Further, a critical Arg in the E. coli variant at the substrate-binding site is replaced by Tyr in LDGLOI. These major differences result in entirely different shapes of the substrate-binding loop and presence of very different chemical groups in the substrate-binding site of LDGLOI compared to E. coli homologue suggesting an explanation for the difference in the substrate specificity. Difference in the substrate specificity of the human and LDGLOI enzyme could be exploited for structure-based drug designing of selective inhibitors against the parasite.     

Lipophosphoglycan masks recognition of the Leishmania donovani promastigote surface by human kala-azar serum

Markedly elevated titers of anti-leishmanial antibodies are a hallmark of kala-azar. We investigated the role played by the lipophosphoglycan (LPG) in determining the reactivity of kala-azar serum with the surface of Leishmania donovani promastigotes. In assays performed with liver parasites there was negligible agglutination or fluorescent staining of LPG-bearing promastigotes by kala-azar serum, but strong reactivity in both assays with the use of an L. donovani mutant strain (R2D2) that lacks surface expression of LPG. Immunoprecipitation of lysates of 125I surface-labeled promastigotes indicated that kala-azar serum has reactivity with several surface proteins common to both the wild-type and R2D2 strains, and no reactivity with surface proteins unique to R2D2. Although direct ELISA showed that kala-azar serum recognizes purified promastigote LPG, inhibition ELISA suggested that such recognition is based solely upon reactivity with the normally unexposed core-anchor region of the molecule. We conclude that the poor reactivity of kala-azar serum with the surface of L. donovani promastigotes is caused by its lack of recognition of the exposed phosphodisaccharide repeat units of LPG, which in turn effectively mask the surface molecules that are recognized by kala-azar serum antibodies.     

Cultivation of Leishmania donovani and Leishmania braziliensis in Defined Media: Nutritional Requirements

SYNOPSIS Nutritional requirements of promastigotes of Leishmania donovani and Leishmania braziliensis were studied in modifications of a simple defined culture medium. "Continuous growth," considered as propagation through 10 successive passages, was supported by inorganic salts, 14 l -amino acids (arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine), glucose, adenosine, and a mixture of 11 vitamins and related growth factors. Purified defatted bovine serum albumin proved beneficial. The nutritional needs of the above species of Leishmania differ from those of 2 other hemoflagellate species, Leishmania tarentolae and Crithidia fasciculata , for which glucose, proline and glutamine were found to be nonessential. It is suggested that lower hemoflagellates may be capable of synthesizing these substrates de novo. Leishmania donovani and L. braziliensis required higher levels of folic acid than L. tarentolae , probably due to the fact that folates are involved as cofactors in the biosyntheses of pyrimidines and serine. Although the mixtures reported here cannot be regarded as "minimal essential" media, they are considerably less complex than the ones employed so far for cultivating hemoflagellates, and are therefore well suited for studies related to nutrition and biosynthetic capabilities of Trypanosomatids.