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S J Turco

Publications and source records attributed to S J Turco.

At least 19 recordsLinked to original sources
PubMed2000

Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major.

Protozoan parasites of the genus Leishmania undergo a complex life cycle involving transmission by biting sand flies and replication within mammalian macrophage phagolysosomes. A major component of the Leishmania surface coat is the glycosylphosphatidylinositol (GPI)-anchored polysaccharide called lipophosphoglycan (LPG). LPG has been proposed to play many roles in the infectious cycle, including protection against complement and oxidants, serving as the major ligand for macrophage adhesion, and as a key factor mitigating host responses by deactivation of macrophage signaling pathways. However, all structural domains of LPG are shared by other major surface or secretory products, providing a biochemical redundancy that compromises the ability of in vitro tests to establish whether LPG itself is a virulence factor. To study truly lpg(-) parasites, we generated Leishmania major lacking the gene LPG1 [encoding a putative galactofuranosyl (Gal(f)) transferase] by targeted gene disruption. The lpg1(-) parasites lacked LPG but contained normal levels of related glycoconjugates and GPI-anchored proteins. Infections of susceptible mice and macrophages in vitro showed that these lpg(-) Leishmania were highly attenuated. Significantly and in contrast to previous LPG mutants, reintroduction of LPG1 into the lpg(-) parasites restored virulence. Thus, genetic approaches allow dissection of the roles of this complex family of interrelated parasite virulence factors, and definitively establish the role of LPG itself as a parasite virulence factor. Because the lpg1(-) mutant continue to synthesize bulk GPI-anchored Gal(f)-containing glycolipids other than LPG, a second pathway distinct from the Golgi-associated LPG synthetic compartment must exist.

Animals↗
PubMed2000

The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits.

LPG2 (a gene involved in lipophosphoglycan assembly) encodes the Golgi GDP-Man transporter of the protozoan parasite Leishmania and is a defining member of a new family of eukaryotic nucleotide-sugar transporters (NSTs). Although NST activities are widespread, mammalian cells lack a GDP-Man NST, thereby providing an ideal heterologous system for probing the LPG2 structure and activity. LPG2 expression constructs introduced into either mammalian cells or a Leishmania lpg2(-) mutant conferred GDP-Man, GDP-Ara, and GDP-Fuc (in Leishmania only) uptake in isolated microsomes. LPG2 is the first NST to be associated with multiple substrate specificities. Uptake activity showed latency, exhibited an antiport mechanism of transport with GMP, and was susceptible to the anion transport inhibitor DIDS. The apparent K(m) for GDP-Man uptake was similar in transfected mammalian cells (12.2 microM) or Leishmania (6.9 microM). Given the evolutionary distance between protozoans and vertebrates, these data suggest that LPG2 functions autonomously to provide transporter activity. Using epitope-tagged LPG2 proteins, we showed the existence of hexameric LPG2 complexes by immunoprecipitation experiments, glycerol gradient centrifugation, pore-limited native gel electrophoresis, and cross-linking experiments. This provides strong biochemical evidence for a multimeric complex of NSTs, a finding with important implications to the structure and specificity of NSTs in both Leishmania and other organisms. Inhibition of essential GDP-Man uptake in fungal and protozoan systems offers an attractive target for potential chemotherapy.

Animals↗
PubMed2000

The role of phosphoglycans in Leishmania-sand fly interactions.

Leishmania promastigotes synthesize an abundance of phosphoglycans, either attached to the cell surface through phosphatidylinositol anchors (lipophosphoglycan, LPG) or secreted as protein-containing glycoconjugates. These phosphoglycans are thought to promote the survival of the parasite within both its vertebrate and invertebrate hosts. The relative contributions of different phosphoglycan-containing molecules in Leishmania-sand fly interactions were tested by using mutants specifically deficient in either total phosphoglycans or LPG alone. Leishmania donovani promastigotes deficient in both LPG and protein-linked phosphoglycans because of loss of LPG2 (encoding the Golgi GDP-Man transporter) failed to survive the hydrolytic environment within the early blood-fed midgut. In contrast, L. donovani and Leishmania major mutants deficient solely in LPG expression because of loss of LPG1 (involved in biosynthesis of the core oligosaccharide LPG domain) had only a slight reduction in the survival and growth of promastigotes within the early blood-fed midgut. The ability of the LPG1-deficient promastigotes to persist in the midgut after blood meal excretion was completely lost, and this defect was correlated with their inability to bind to midgut epithelial cells in vitro. For both mutants, when phosphoglycan expression was restored to wild-type levels by reintroduction of LPG1 or LPG2 (as appropriate), then the wild-type phenotype was also restored. We conclude, first, that LPG is not essential for survival in the early blood-fed midgut but, along with other secreted phosphoglycan-containing glycoconjugates, can protect promastigotes from the digestive enzymes in the gut and, second, that LPG is required to mediate midgut attachment and to maintain infection in the fly during excretion of the digested blood meal.

Agglutinins↗
PubMed1999

Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan.

The life cycle of Leishmania parasites within its sand fly vector involves the development of extracellular promastigotes from a noninfective, "procyclic" stage into an infective, "metacyclic" stage that is adapted for transmission in the fly and survival in the mammalian host. Lipophosphoglycan (LPG), the predominant surface glycoconjugate in both procyclic and metacyclic stages, is a critical virulence determinant. LPG is a multidomain molecule; the structural polymorphisms among species lie in branching from the backbone 6Galbeta1,4Man(alpha1)-PO(4) repeat units and in the composition of the small oligosaccharide caps. We have recently demonstrated that the LPG from an Indian isolate of Leishmania donovani differs from a Sudanese strain by possessing one or two side chain beta(1,3)-linked glucose residues. We now have characterized the glucosyltransferase activities responsible for glucosylating the LPG. When incubated with UDP-[(3)H]glucose and Mn(2+), microsomal membranes from the Indian isolate transferred [(3)H]glucose to the repeat units of the exogenous acceptor Sudanese L. donovani LPG, which does not contain any side chain branching. Glucose addition was maximal at 28 degrees C, the optimal growth temperature of procyclic L. donovani. Consistent with the lack of side chain branching in its LPG, Sudanese L. donovani showed minimal glucosyltransferase activity. Indian metacyclic promastigotes, in contrast to procyclic promastigotes, express no glucose side chains off the repeat units. Therefore, we compared the relative activity of the glucosyltransferases in microsomes from procyclic and metacyclic promastigotes and observed approximately 80% less activity in the latter. These results provide evidence that the glucose side chain addition to LPG is developmentally regulated during the parasite's life cycle and that the glucosyltransferases of L. donovani are strain specific.

Animals↗
PubMed1999

Glycoconjugates in Leishmania infectivity.

Leishmaniasis is a major health problem to humans and is caused by one of the world's major pathogens, the Leishmania parasite. These protozoa have the remarkable ability to avoid destruction in hostile environments they encounter throughout their life cycle. That Leishmania parasites have adapted to not only survive, but to proliferate largely is due to the protection conferred by unique glycoconjugates that are either on the parasites' cell surface or secreted. Most of these specialized molecules are members of a family of phosphoglycans while others are a family of glycosylinositol phospholipids. Together they have been implicated in a surprisingly large number of functions for the parasites throughout their life cycle and, therefore, are key players in their pathogenesis. This review summarizes the biological roles of these glycoconjugates and how they are believed to contribute to Leishmania survival in destructive surroundings.

Animals↗
PubMed1999

Intra-species and stage-specific polymorphisms in lipophosphoglycan structure control Leishmania donovani-sand fly interactions.

The Leishmania lipophosphoglycan conveys the ability for the parasites to avoid destruction in diverse host environments. During its life cycle within the sand fly vector, the parasite differentiates from a dividing procyclic promastigote stage that avoids expulsion from the midgut by attaching to the gut wall, to a nondividing metacyclic promastigote stage that is unable to attach to the midgut and migrates to the mouth parts for reinfection of a mammalian host. Lipophosphoglycan plays an integral role during this transition. Structurally, lipophosphoglycan is a multidomain glycoconjugate whose polymorphisms among species lie in the backbone Gal(beta 1,4)Man(alpha 1)-PO(4) repeating units and the oligosaccharide cap. We have characterized the lipophosphoglycan from an Indian L. donovani isolate. Unlike East African isolates, which express unsubstituted repeats and a galactose- and mannose-terminating cap, procyclic lipophosphoglycan from the Indian isolate consists of beta1,3-linked glucose residues that branch off the backbone repeats (n approximately 17) and also terminate the cap. Of biological significance, metacyclic lipophosphoglycan lacks the glucose residues while doubling the number of repeats. The importance of these developmental modifications in lipophosphoglycan structure was determined using binding experiments to Phlebotomus argentipes midguts. Procyclic promastigotes and procyclic LPG were able to bind to sand fly midguts in vitro whereas metacyclic parasites and LPG lost this capacity. These results demonstrate that the Leishmania adapts the synthesis of terminally exposed sugars of its LPG to manipulate parasite-sand fly interactions.

Animals↗
PubMed1999

A high-yield, enzymatic synthesis of GDP-D-[3H]arabinose and GDP-L-[3H]fucose.

For assays involving glycosyltransferases or transporters, several GDP-sugars are either commercially unavailable or expensive. We describe an enzymatic synthesis of GDP-d-[3H]arabinosep and GDP-l-[3H]fucose that yields 66-95% nucleotide-sugar from the appropriate radiolabeled sugar in less than 30 min. The coupled reaction requires Mg2+, ATP, and GTP along with the appropriate radioactive monosaccharide, sugar-1-kinase, and pyrophosphorylase. The latter two activities are present in a cytosolic fraction of Crithidia fasciculata, which is easily grown at room temperature in simple culture medium without serum or added CO2. Addition of commercial yeast inorganic pyrophosphatase shifts the equilibrium of the pyrophosphorylase reaction toward nucleotide-sugar formation. To verify that these nucleotide-sugars are biologically active, we tested their ability to serve as substrates for glycosyltransferases. GDP-l-[3H]fucose functions as the donor substrate for recombinant human fucosyltransferase V, and GDP-d-[3H]arabinosep serves as the donor substrate for the arabinosyltransferase activities present in Leishmania major microsomes.

Animals↗
PubMed1999

Adversarial relationship between the leishmania lipophosphoglycan and protein kinase C of host macrophages.

The dominant glycoconjugate on the cell surface of all Leishmania promastigotes is an unusual glycoconjugate named lipophosphoglycan (LPG). Its relative abundance, unique structure, and cellular location have implicated LPG as an essential virulence determinant. One feature of LPG resides in its strong inhibitory effect on the activity of protein kinase C (PKC) of host macrophages. This article summarizes the evidence that LPG is inhibitory toward PKC activation in macrophages and discusses the implication of such inhibition on intramacrophage survival of the parasite.

Animals↗
PubMed1999

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.

3T3 Cells↗
PubMed1999

Employee and independent contractor relationships.

Most practitioners find themselves at a disadvantage in dealing with business issues and relationships. As health care continues to change, knowledge of contracts and business relationships will help CRNA practitioners navigate new as well as traditional practice settings. This article discusses the advantages and disadvantages of two business relationships: employee and independent contractor.

Contract Services↗
PubMed1998

Lipophosphoglycan of Leishmania donovani inhibits lipid vesicle fusion induced by the N-terminal extremity of viral fusogenic simian immunodeficiency virus protein.

Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania parasites, was recently shown to be a potent inhibitor of viral infection. The mechanism by which this natural membrane amphiphile compound inhibits membrane fusion was investigated in this study using a simple model membrane system and a synthetic peptide corresponding to the fusion peptide of simian immunodeficiency virus (SIV). At low concentration (< 10 microM), LPG inhibits SIV-induced lipid mixing of large unilamellar vesicles composed of an equimolar mixture of egg phosphatidylcholine and egg phosphatidylethanolamine. Importantly, this inhibition was observed regardless of which LPG was inserted in the inner monolayer, the outer monolayer or both sides of the membrane, suggesting that the inner monolayer plays a determining role in membrane fusion. Fourier transform infrared spectroscopy revealed that LPG induced a conformational change of SIV fusion peptide without affecting its capacity to interact with the lipid membrane. This structural change was shown not to depend on the LPG localization and was observed even when LPG was exclusively associated to the inner lipid membrane.

Animals↗
PubMed1998

Fusion of Sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy.

Fusion between Sendai virus (SV) and individual host cells was investigated with confocal laser scanning microscopy (CLSM) and image correlation spectroscopy (ICS). SV was labeled with the fluorescent probe 7-octadecylamino-4-nitrobenz-2-oxa-1,3-diazole (NBD-NH-C18) and was allowed to bind to host cells (HEp-2, BALB-3T3) at 4 degrees C. The effect of lipophosphoglycan (LPG), isolated from Leishmania donovani, on virus fusion was investigated by incorporation of LPG (0, 5, 10 or 20 microM) into the host cell membrane (HEp-2) before addition of SV. LPG did not affect the number of SV bound per cell. After incubation at 37 degrees C for 15 min without LPG, CLSM revealed a redistribution of NBD-NH-C18 from the SV envelope to the host cell membrane and an increase in average fluorescence intensity, indicating dequenching. ICS analysis of images obtained after incubation at 37 degrees C showed an increased mean cluster density to 260% of the value at 4 degrees C, reflecting the disappearance of labeled SV from the cell surface and diffusion of NBD-NH-C18 into the host cell membrane. Preincubation of the cells with LPG inhibited the temperature-induced redistribution and dequenching of NBD-NH-C18 in a concentration-dependent manner, with a total inhibition of fusion at 20 microM LPG. Together, the results demonstrate that CLSM combined with ICS is a powerful tool for studies of fusion of enveloped viruses with individual host cells and that LPG inhibits the fusion process at or before the hemifusion (lipid mixing) stage of SV interaction with cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

Characterization of developmentally-regulated activities in axenic amastigotes of Leishmania donovani.

Leishmania donovani is an obligatory intracellular parasite which cycles between the midgut of sand flies (extracellular promastigote) and the phagolysosomes of mammalian macrophages (intracellular amastigote). Promastigotes have been readily cultured, whereas axenic cultures of amastigotes have only recently been developed. A new method for in vitro differentiation of L. donovani promastigotes into amastigotes is presented, in which promastigotes are exposed to environmental changes that mimic the in vivo process. First, promastigotes are subjected to 37 degrees C + 5% CO2 for 24 h, and then are shifted to pH 5.5. Under these conditions, differentiation is completed within 120 h. In the reverse process, amastigotes are induced to differentiate back to promastigotes by transferring them to promastigote growth conditions (medium 199 at pH 7.4 and 26 degrees C). Axenic amastigotes closely resemble animal-derived amastigotes. They manifest all seven proteins of the amastigote-specific A2 gene family. They down-regulate lipophosphoglycan (LPG) synthesis and do not express it on their surface. LPG is up-regulated 2 h after inducing amastigotes to differentiate to promastigotes. Within 6 h, parasites resume the promastigote level of this molecule, although differentiation is completed only after 48 h. Axenic amastigotes also express amastigote-like metabolic activities of proline uptake, as well as thymidine and proline incorporation. In conclusion, the results indicate that the method developed for in vitro differentiation of L. donovani promastigotes to amastigotes is efficient and yields organisms resembling animal-derived amastigotes. Being able to induce in vitro differentiation of L. donovani provides us with an excellent tool to study Leishmania development and differentiation.

Animals↗
PubMed1998

Leishmania donovani has distinct mannosylphosphoryltransferases for the initiation and elongation phases of lipophosphoglycan repeating unit biosynthesis.

Lipophosphoglycan (LPG) is the predominant surface glycoconjugate of Leishmania promastigotes and plays several roles in the infectious cycle of this protozoan parasite. The salient feature of LPG is the presence of 15-30 copies of a disaccharide-phosphate repeating unit Gal(beta1,4)Man(alpha1-PO4), which is also found on many other secreted molecules (secretory acid phosphatase, phosphoglycan, proteophosphoglycan). This structural diversity suggests that a multiplicity of enzymes mediating repeating unit addition may exist, especially for the mannosylphosphoryltransferases (MPTs), which initiate repeating unit synthesis. This work has taken a combined biochemical-genetic approach to resolve this issue. An lpg- mutant of Leishmania donovani, JEDI, was obtained by antibody selection against cells expressing a repeating unit epitope of LPG. Metabolic and surface labeling experiments revealed that JEDI cells accumulated a truncated form of LPG bearing only a single repeating unit: [Gal(beta 1,4)Man(alpha1-PO4)][Gal(alpha1,6)Gal(alpha1,3)Gal(f)(beta1,3)[Glc(alpha 1-PO4)]Man(alpha1,3)Man(alpha1,4)GlcN(alpha1,6)]-PI. Enzymatic assays of microsomal preparations showed that JEDI lacked MPT activity when tested with a repeating unit acceptor but retained wild-type levels of the MPT activity with an LPG glycan core acceptor. These data indicate that at least two distinct MPT activities are required for LPG repeating unit synthesis: one involved in the 'initiation' of repeating unit synthesis on the LPG core (iMPT), and a second (lacking in JEDI) participating in the 'elongation' phase of repeating unit addition (eMPT), leading to the mature full-length LPG.

Animals↗
PubMed1998

Leishmania lipophosphoglycan reduces monocyte transendothelial migration: modulation of cell adhesion molecules, intercellular junctional proteins, and chemoattractants.

We previously identified the structural requirement for the inhibitory activity of Leishmania lipophosphoglycan (LPG) to block endothelial adhesion to monocytes. Here we showed that LPG reduces transendothelial migration of monocytes. LPG pretreatment of endothelial cells (2 microM, 1 h) reduced monocyte migration across endothelial cells activated by bacterial endotoxin (LPS) or IL-1beta (60 and 46%, respectively). A fragment of LPG (i.e., repeating phosphodisaccharide (consisting of galactosyl-mannose)) and LPG coincubated with LPG-neutralizing mAb lacks inhibitory activity on monocyte migration. Pretreatment of monocytes with LPG (2 microM, 1 h) also did not affect monocyte migration through control or LPS-activated endothelial cells. FACS analysis reveals that LPG treatment blocked the LPS-mediated expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells and monocyte adhesion without altering the integrity of the endothelial monolayer. LPG (2 microM, 1 h) alone was capable of altering the expression and distribution of two junctional adhesion molecules, CD31 and vascular endothelium cadherin, as well as reversing the effects of LPS on these proteins. The induction of endothelial cells by LPS to transcribe and release monocyte chemoattractant protein-1 (MCP-1) was significantly reduced by LPG (40-65%). LPG treatment of nonactivated endothelial cells also suppressed by 55 to 75% the monocyte migration triggered by a MCP-1 chemoattractant gradient, and coincubation of LPG with neutralizing mAb abrogated the inhibitory activity. Together, these data point to a novel anti-inflammatory function of LPG in reducing monocyte migration across endothelial cells via a mechanism of inhibition of endothelial expression of cell adhesion molecules, modulation of intercellular junctional proteins, and synthesis of MCP-1.

Animals↗
PubMed1998

Microbial glycoconjugates.

The surfaces of all microbes are 'sugar coated' with molecules such as lipopolysaccharides in Gram-negative bacteria, capsular polysaccharides in bacteria, lipoarabinomannans in mycobacteria and lipophosphoglycan in Leishmania. The basic structures of these glycoconjugates are known and, in the case of pathogens, they can function as virulence determinants. Recent publications have refined some of these structures and have elucidated interesting genes and proteins responsible for their biosynthesis.

Animals↗
PubMed1998

Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania.

Leishmania exploits several strategies to survive within the phagolysosome of vertebrate macrophages and be transmitted by sand fly vectors. Recent advances in functional genetic analysis provide a new avenue for identifying genes implicated in the infectious cycle of the parasite, such as those necessary for the synthesis and expression of the key surface glycoconjugate, lipophosphoglycan (LPG).

Animals↗