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S J Turco

Publications and source records attributed to S J Turco.

At least 73 records · Page 4Linked to original sources
PubMed1992

Inhibition of macrophage protein kinase C-mediated protein phosphorylation by Leishmania donovani lipophosphoglycan.

The cell surface lipophosphoglycan (LPG) from Leishmania donovani promastigotes is a potent inhibitor of purified protein kinase C (PKC) activity in vitro. In this study, we have investigated the effect of LPG on the activation process of PKC in murine bone marrow-derived macrophages. The extent and kinetics of calcium ionophore A23187-induced [3H] phorbol dibutyrate binding to macrophages were not affected by LPG pretreatment or infection with either wild-type or LPG-deficient promastigotes, indicating no effect on the association of calcium-dependent PKC with the plasma membrane. In contrast, LPG inhibited the phosphorylation of both the PKC-specific VRKRTRLLR substrate peptide and MARCKS, and endogenous PKC substrate, in 1-oleoyl-2-acetyl-glycerol-stimulated macrophages. These observations provide direct evidence that LPG effectively inhibits PKC activity in intact macrophages. Finally, depletion of PKC rendered macrophages more permissive for the proliferation of L. donovani, suggesting that inhibition of PKC-dependent events contributes to the survival of this parasite within its host cell.

Amino Acid Sequence↗
PubMed1992

Stage-specific adhesion of Leishmania promastigotes to the sandfly midgut.

Although leishmaniasis is transmitted to humans almost exclusively by the bite of infected phlebotomine sandflies, little is known about the molecules controlling the survival and development of Leishmania parasites in their insect vectors. Adhesion of Leishmania promastigotes to the midgut epithelial cells of the sandfly was found to be an inherent property of noninfective-stage promastigotes, which was lost during their transformation to metacyclic forms, thus permitting the selective release of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release was found to be controlled by specific developmental modifications in terminally exposed saccharides on lipophosphoglycan, the major surface molecule on Leishmania promastigotes.

Animals↗
PubMed1992

Refined structure of the lipophosphoglycan of Leishmania donovani.

The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.

Animals↗
PubMed1992

Purification and characterization of an extracellular phosphoglycan from Leishmania donovani.

An extracellular phosphoglycan (exPG), present in the culture medium of the promastigote form of Leishmania donovani, was purified and structurally characterized. The purification scheme included ethanol precipitation of the culture medium, anion exchange chromatography, hydrophobic chromatography on phenyl-Sepharose, and preparative polyacrylamide gel electrophoresis. Structural analysis by 1H-1H NMR, methylation linkage analysis, and glycosidase digestion revealed that the exPG consisted of the following structure: (CAP)----[PO4-6Galp beta 1-4Manp alpha 1]10-11-PO4-6Galp beta 1-4Man. The cap was found to be one of several small, neutral oligosaccharides, the most abundant of which was the trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The results indicated structural analogy to the cellular-derived lipophosphoglycan (LPG) from L. donovani. The important exceptions are a lack of the lipid anchor, the entire phosphosaccharide core, and several of the repeating disaccharide units. Although the function of exPG is presently unknown, it may play a protective role for the promastigote in the insect vector or during infection of a mammalian host.

Animals↗
PubMed1992

Solution structure of the lipophosphoglycan of Leishmania donovani.

The three-dimensional solution structure of the repeating -PO4-6Gal beta 1-4Man alpha 1- disaccharide fragment of the lipophosphoglycan (LPG) derived from Leishmania donovani has been determined by use of a combination of homo- and heteronuclear NMR spin coupling constant measurements together with restrained molecular mechanical minimization and molecular dynamics simulations. The fragment exists with limited mobility in solution about the Gal beta 1-4Man linkages, whereas in contrast a variety of stable rotamers exist about the Man alpha 1-PO4-6Gal linkages. These rotamers define several major stable conformers in solution, which are discussed in terms of the proposed biological role of LPG.

Animals↗
PubMed1992

Stimulation of human T lymphocytes by Leishmania lipophosphoglycan-associated proteins.

Lipophosphoglycan (LPG) is a glycoconjugate present on the surface of Leishmania promastigotes that has been reported to promote intracellular survival of these parasites, to protect mice against leishmaniasis, and to elicit T cell responses in infected mice and humans. We investigated whether LPG and its components could elicit proliferative responses and cytokine secretion from leishmaniasis patient PBMC. LPG prepared by standard methods (LPG-1) stimulated patients T cells to proliferate and secrete IFN-gamma. LPG was fractionated into several components. An LPG-1-specific T cell line was shown to respond to the core region but not to the repeating saccharide units. LPG-1 was fractionated to yield an LPG-free- associated protein complex and an LPG-2 fraction that was more than 95% depleted of associated protein. The ability of LPG-2 to stimulate T cells was significantly decreased over that of LPG-1. In contrast, LPG-AP stimulated T cell proliferation and IFN-gamma production. Therefore, proteins associated with LPG were effective in eliciting patient T cell responses, whereas the glycolipid enriched moiety was weakly effective or ineffective at stimulating these responses.

Animals↗
PubMed1992

Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage.

Protozoan parasites of the genus Leishmania produce the novel surface glycoconjugate, lipophosphoglycan (LPG), which is required for parasite infectivity. In this study we show that LPG structure is modified during the differentiation of L. major promastigotes from a less infectious form in logarithmic growth phase to a highly infectious 'metacyclic' form during stationary growth phase. In both stages, the LPGs comprise linear chains of phosphorylated oligosaccharide repeat units which are anchored to the membrane via a glycosyl-phosphatidylinositol glycolipid anchor. During metacyclogenesis there is (i) an approximate doubling in the average number of repeat units per molecule from 14 to 30, (ii) a pronounced decrease in the relative abundance of repeat units with side chains of beta Gal or Gal beta 1-3Gal beta 1-, and a corresponding increase in repeat units with either no side chains or with side chains of Arap alpha 1-2 Gal beta 1- and (iii) a decrease in the frequency with which the glycolipid anchor is substituted with a single glucose alpha 1-phosphate residue. While the majority of the LPG phosphoglycan chains are capped with the neutral disaccharide, Man alpha 1-2Man, a significant minority of the chains appeared to terminate in non-phosphorylated repeat units and may represent incompletely capped species. We suggest that the developmental modification of LPG may be important in modulating the binding of promastigotes to receptors in the sandfly midgut and on human macrophages and in increasing the resistance of metacyclic promastigotes to complement-mediated lysis.

Animals↗
PubMed1992

Biosynthesis of lipophosphoglycan from Leishmania donovani: characterization of mannosylphosphate transfer in vitro.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes and is composed of a capped polymer of repeating PO4-6Gal(beta 1,4)Man alpha 1 disaccharide units linked via a phosphosaccharide core to a lyso-1-O-alkylphosphatidylinositol anchor. An exogenous acceptor composed of the glycolipid anchor portion of LPG was shown to stimulate the enzymatic synthesis of the repeating phosphorylated disaccharide units of LPG in a cell-free system. Using the exogenous acceptor, GDP-[3H]Man, [beta-32P]GDP-Man, and unlabeled UDP-Gal as substrates, membrane preparations from an LPG-defective mutant of L. donovani that lacks endogenous acceptors catalyzed the incorporation of the doubly labeled mannosylphosphate unit into a product that exhibited the chemical and chromatographic characteristics of LPG. Analysis of fragments generated by mild acid hydrolysis of the radiolabeled product indicated that [3H]mannose-1-[32P]PO4 had been transferred from the dual-labeled sugar nucleotide. These results are consistent with the proposal that the repeating units of the L. donovani LPG are synthesized by the alternating transfer of mannose 1-phosphate and galactose from their respective nucleotide donors.

Animals↗
PubMed1992

The lipophosphoglycan of Leishmania parasites.

Protozoan parasites of the genus Leishmania have the remarkable ability to avoid destruction in the hostile environments they encounter throughout their life cycle. The molecular details of how these pathogens persevere with impunity under harsh conditions are beginning to be understood. The fact that Leishmania parasites have adapted to not only survive, but to proliferate probably is due to the protection conferred by specialized molecules on the parasite's cell surface. One such macromolecule is a novel glycoconjugate called lipophosphoglycan. This heterogeneous, lipid-containing polysaccharide is the major surface molecule of the parasite and has been implicated in a surprisingly large number of functions that may contribute the the parasite's pathogenesis. This review emphasizes the structural aspects of lipophosphoglycan and its possible functions and biosynthesis.

Animals↗
PubMed1991

The Leishmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex.

Lymphocytes from mice immunized with Leishmania donovani (LPG) were specifically stimulated to proliferate in vitro by purified LPG or its delipidated congener, phosphoglycan. The response was dose dependent and required prior immunization with either LPG or phosphoglycan. Proliferation was eliminated by specific depletion of Thy-1+ cells with antisera and C and the proliferating T cell subset was shown to be CD4+CD8-. Tests of various LPG fragments indicated that the T cell stimulation was associated with the core structure of LPG rather than the lipid or phosphoglycan repeat structure. However, amino acid analysis of LPG and active LPG fragments, after acid hydrolysis, showed the presence of amino acids in peptide linkage. Specific hydrolysis of the glycosidic linkages in LPG with trifluoromethanesulfonic acid provided polypeptide material reactive with two mAb previously believed to be LPG carbohydrate core specific. The protein was separated from LPG by reverse phase chromatography and shown to be a complex of proteins with common epitopes recognized by the two mAb. The dominant species isolated from LPG was a set of small, approximately 11,000 Mr, molecules. Subsequent T cell proliferation studies showed that the lymphocyte stimulation was associated with the protein component of LPG and not the glycan.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities.

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.

Animals↗
PubMed1991

Leishmania donovani lipophosphoglycan selectively inhibits signal transduction in macrophages.

The effect of purified lipophosphoglycan (LPG) of Leishmania donovani on signal transduction and gene expression in murine bone marrow-derived macrophages was investigated. LPG stimulated the rapid expression of both c-fos and TNF genes within 30 min after exposure, suggesting that the interaction of LPG with its receptor stimulated a specific signal transduction pathway. Macrophages pretreated with LPG for 3 h became unresponsive to subsequent stimulation with LPS and the activators of protein kinase C, 1-oleoyl-2-acetyl-glycerol, and calcium ionophore A23187. Moreover, LPG induced a rapid down-modulation of TNF receptors. In contrast, the ability of macrophages to express the c-fos gene in response to the cAMP analogue, dibutyryl cAMP, was not impaired by LPG. Fragmentation of LPG revealed that the inhibitory activity of LPG required both the repeating phosphorylated disaccharides and the phosphosaccharide core. Collectively, these data demonstrate that LPG selectively impaired signal transduction in macrophages and suggest a role for this molecule in the survival of the parasite within the macrophage.

Animals↗
PubMed1991

Expression of a stage-specific lipophosphoglycan in Leishmania major amastigotes.

Amastigotes of Leishmania major were isolated from infected mice and radiolabeled for 2 h with [3H]galactose. An acidic [3H]glycoconjugate was extracted from a dilipidated residue fraction with the solvent water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactivity labeled glycoconjugate was found to possess the following characteristics that were similar to the lipophosphoglycan extractable from promastigotes: (i) migrated as a broad band upon electrophoresis on SDS polyacrylamide gels; (ii) deaminated with nitrous acid; and (iii) hydrolyzed with phosphatidylinositol-specific phospholipase C. Furthermore, analysis of the aqueous soluble material released by the latter enzyme revealed a negatively-charged [3H]polysaccharide intermediate in size compared to the analogous portions of LPG isolated from non-infective and metacyclic promastigotes. Most importantly, the [3H]polysaccharide was found to contain phosphate and was susceptible to mild acid hydrolysis, establishing that the intact molecule is a lipophosphoglycan. A structural difference, however, was found in the major, mild acid-generated fragment of the amastigote phosphoglycan, which was larger in size and not as anionic as the analogous fragment from the promastigote phosphoglycans. These results indicate that the amastigotes do express a lipophosphoglycan, but that it is structurally distinct from its promastigote counterparts.

Animals↗
PubMed1991

Stable DNA transfection of a wide range of trypanosomatids.

We have shown that the Leishmania major transfection vector pR-NEO (or derivatives thereof) can be introduced and stably maintained in four species complexes of pathogenic Leishmania (L. tropica, L. mexicana, L. donovani, L. braziliensis), and the genera Endotrypanum and Crithidia; transfection of Trypanosoma cruzi or Trypanosoma brucei was not successful. Quantitative plating assays showed that the transfection efficiencies were high in L. major and Leishmania amazonensis (5x10(-5)/cell) and about 10-fold less for Leishmania panamaensis and Crithidia. Leishmania donovani transfected with pR-NEO retained the ability to infect hamsters, and amastigotes recovered after 2 months yielded G418-resistant promastigotes which retained high levels of extrachromosomal pR-NEO DNA. In promastigotes, the transfected DNA existed as extrachromosomal circles, and expressed the predicted 2.4-kb hybrid NEO/DHFR-TS mRNA bearing the trans-spliced miniexon. Large quantitative differences were observed only in Crithidia: relative to transfected Leishmania species, the copy number of pR-NEO was elevated 20-fold, while the levels of the NEO/DHRFR-TS mRNA or Escherichia coli beta-galactosidase (synthesized from the expression vector pX-beta GAL) were reduced 80 and more than 1000-fold, respectively. Thus, genetic signals derived from L. major DNA that mediate RNA expression or stability are recognized by the heterologous Leishmania species but less efficiently by Crithidia. These studies suggest that pR-NEO derived vectors may be applied to the study of genes expressed throughout the life cycle in a wide range of pathogenic trypanosomatids.

Animals↗
PubMed1990

Effect of glycolipids of Leishmania parasites on human monocyte activity. Inhibition by lipophosphoglycan.

Lipophosphoglycan (LPG) and glycosyl phosphatidylinositol Ag (GPI), are glycolipids present on the membrane of Leishmania parasites. Both glycolipids have been chemically characterized. LPG is a polysaccharide of repeating phosphorylated units linked to a phosphocarbohydrate core that is anchored to the membrane by lysoalkyl phosphatidylinositol (PI). The GPI are smaller glycolipids with a structure resembling the phosphocarbohydrate core of the LPG. They are anchored to the membrane by alkyl acyl PI. Their relative abundance, uniqueness of structure, and cellular location suggest a role in interactions of the parasites with host cells. In the present study we examined the effect of LPG and GPI on the activation of human peripheral blood monocytes. Three parameters were studied: the production of IL-1, chemotactic locomotion, and oxidative burst. We found that whereas neither GPI nor LPG directly affected monocyte activity, preincubation of the monocytes with LPG strongly inhibited further activation: The production of IL-1, after stimulation with LPS, was decreased in a dose-dependent manner. Previous incubation with LPG also inhibited chemotactic locomotion of monocytes and neutrophils in response to diacylglycerol, zymosan-activated serum, FMLP and LTB4. Luminol-dependent chemiluminiscence caused by stimulation of the monocytes with streptococci and histone was also inhibited. After fragmentation of the LPG into phosphoglycan and 1-O-alkylglycerol by phosphatidylinositol-phospholipase C, only the phosphoglycan retained inhibitory activity. No difference in inhibitory activity was found between LPG prepared from Leishmania major or Leishmania donovani promastigotes. These results show that the phosphoglycan of LPG inhibits the immunologic response of normal human monocytes and neutrophils, and suggest that LPG may influence the nature of the inflammatory response surrounding infected cells.

Animals↗
PubMed1990

Requirement of lipophosphoglycan for intracellular survival of Leishmania donovani within human monocytes.

The function of the lipophosphoglycan of Leishmania donovani parasites was investigated in human peripheral monocytes. In contrast to wild-type L. donovani which grow in monocytes, incubation of monocytes with two variant lines of L. donovani, defective in lipophosphoglycan expression, resulted in the entry of the variant cells into the monocytes and their subsequent destruction. Passive transfer of lipophosphoglycan to the variant cells led to prolonged survival in monocytes. These results indicate that lipophosphoglycan is required by the parasite for intracellular survival. To investigate one possible protective role of the glycoconjugate, preincubation of monocytes with a suspension of lipophosphoglycan and subsequent treatment of the cells with PMA or opsonized zymosan resulted in an attenuation of the oxidative burst; the attenuation effect was concentration dependent on the glycoconjugate and independent of preincubation time. Moreover, hydrophobic beads, coated with lipophosphoglycan, were phagocytized by monocytes and found to inhibit oxygen consumption in monocytes activated with PMA. These results suggest a possible relationship between the absence of lipophosphoglycan in the variant parasites and their inability to survive within monocytes. Although the precise molecular basis remains to be elucidated, the ability of lipophosphoglycan to impair the microbial oxidative response may be a contributing factor in its requirement for intracellular survival.

Animals↗