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S J Turco

Publications and source records attributed to S J Turco.

At least 55 records · Page 3Linked to original sources
PubMed1996

Deficiency in beta1,3-galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences the Leishmania-sand fly interaction.

To study the function of side chain oligosaccharides of the cell-surface lipophosphoglycan (LPG), mutagenized Leishmania major defective in side chain biosynthesis were negatively selected by agglutination with the monoclonal antibody WIC79.3, which recognizes the galactose-containing side chains of L. major LPG. One such mutant, called Spock, lacked the ability to bind significantly to midguts of the natural L. major vector, Phlebotomus papatasi, and to maintain infection in the sand fly after excretion of the digested bloodmeal. Biochemical characterization of Spock LPG revealed its structural similarity to the LPG of Leishmania donovani, a species whose inability to bind to and maintain infections in P. papatasi midguts has been strongly correlated with the expression of a surface LPG lacking galactose-terminated oligosaccharide side chains. An in vitro galactosyltransferase assay using wild-type or Spock membranes was used to determine that the defect in Spock LPG biosynthesis is a result of defective beta1,3-galactosyltransferase activity as opposed to a modification of LPG, which would prevent it from serving as a competent substrate for galactose addition. The results of these experiments show that Spock lacks the beta1, 3-galactosyltransferase for side chain addition and that the LPG side chains are required for L. major to bind to and to produce transmissible infection in P. papatasi.

Animals↗
PubMed1996

Use of the green fluorescent protein as a marker in transfected Leishmania.

We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.

Amino Acid Sequence↗
PubMed1995

Inhibition of HIV-1-induced syncytia formation and infectivity by lipophosphoglycan from Leishmania.

In HIV-1 infection, the appearance of syncytia-inducing (SI) isolates is associated with a more rapid decline of CD4+ cells and progression to AIDS. Agents that inhibit either virus infection or syncytia formation have the potential to be therapeutically useful. Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania, was recently shown to be a potent nonspecific inhibitor of viral membrane fusion. In this study, LPG demonstrated a dose-dependent inhibition of HIV-1-induced syncytia formation in CD4+ MT-2 cells infected with distinct SI isolates. Fragments of LPG were used to show that inhibition of syncytia formation was dependent on the length of the LPG fragment. Treatment of CD4+ cells or HIV-1 isolates with LPG inhibited infection in vitro. Furthermore, LPG inhibited the replication of SI viral isolates in CD4+ T cells in vitro. LPG had no toxic effects on peripheral blood mononuclear cells at the highest concentrations used in these assays. Further, LPG rapidly associated with the surface membrane of a human T cell line and subsequently disassociated over a 24-h period. The development of compounds capable of inhibiting HIV-induced syncytia formation should provide novel therapeutic approaches to control the spread of virus and disease progression.

Animals↗
PubMed1995

A specialized pathway affecting virulence glycoconjugates of Leishmania.

For virulence and transmission, the protozoan parasite Leishmania must assemble a complex glycolipid on the cell surface, the lipophosphoglycan (LPG). Functional complementation identified the gene LPG2, which encodes an integral Golgi membrane protein implicated in intracellular compartmentalization of LPG biosynthesis. Ipg2- mutants lack only characteristic disaccharide-phosphate repeats, normally present on both LPG and other surface or secreted molecules considered critical for infectivity. In contrast, a related yeast gene, VAN2/VRG4, is essential and required for general Golgi function. These results suggest that LPG2 participates in a specialized virulence pathway, which may offer an attractive target for chemotherapy.

Amino Acid Sequence↗
PubMed1995

Potent inhibition of viral fusion by the lipophosphoglycan of Leishmania donovani.

Lipophosphoglycan (LPG) is an amphiphile produced by Leishmania. Its chemical structure consists of a hydrophilic flexible polymer of repeating PO4-6Gal beta 1-4Man alpha 1 units (on average 16 units) linked via a hexasaccharide core to a lyso-1-O-alkyl-P1 membrane anchor. In the study of viral fusion we report in this paper, we have introduced LPG into human erythrocyte ghost (HEG) membranes, with the purpose of understanding how the LPG-induced surface-structural changes may modulate the interactions between a viral envelope and the HEG membranes. We have found that LPG, when incorporated at very low concentrations into intact human erythrocyte membranes, strongly inhibits Sendai virus-induced hemolysis. When incorporated into HEGs, it reduces the binding of both Sendai and influenza viruses to HEGs; furthermore, it strongly inhibits the overall viral fusion to HEGs, being among the most potent known inhibitors. We have also shown that LPG stabilizes the bilayer structure of phosphatidylethanolamine against the formation of an inverted-hexagonal structure. We suggest that LPG may give rise to an effective "steric repulsion" between the viral and HEG membranes, thereby modulating some specific modes of interaction between viral-target membranes in the overall fusion process; LPG may also modulate the bending rigidity and the spontaneous curvature of the HEG membrane in the direction of making the destabilization and rearrangement of the underlying lipid bilayer more difficult.

Animals↗
PubMed1995

Stage-specific binding of Leishmania donovani to the sand fly vector midgut is regulated by conformational changes in the abundant surface lipophosphoglycan.

The life cycle of Leishmania parasites within the sand fly vector includes the development of extracellular promastigotes from a noninfective, procyclic stage into an infective, metacyclic stage that is uniquely adapted for transmission by the fly and survival in the vertebrate host. These adaptations were explored in the context of the structure and function of the abundant surface lipophosphoglycan (LPG) on Leishmania donovani promastigotes. During metacyclogenesis, the salient structural feature of L. donovani LPG is conserved, involving expression of a phosphoglycan chain made up of unsubstituted disaccharide-phosphate repeats. Two important developmental modifications were also observed. First, the size of the molecule is substantially increased because of a twofold increase in the number of phosphorylated disaccharide repeat units expressed. Second, there is a concomitant decrease in the presentation of terminally exposed sugars. This later property was indicated by the reduced accessibility of terminal galactose residues to galactose oxidase and the loss of binding by the lectins, peanut agglutinin, and concanavalin A, to metacyclic LPG in vivo and in vitro. The loss of lectin binding was not due to downregulation of the capping oligosaccharides as the same beta-linked galactose or alpha-linked mannose-terminating oligosaccharides were present in both procyclic and metacyclic promastigotes. The capping sugars on procyclic LPG were found to mediate procyclic attachment to the sand fly midgut, whereas these same sugars on metacyclic LPG failed to mediate metacyclic binding. And whereas intact metacyclic LPG did not inhibit procyclic attachment, depolymerized LPG inhibited as well as procyclic LPG, demonstrating that the ligands are normally buried. The masking of the terminal sugars is attributed to folding and clustering of the extended phosphoglycan chains, which form densely distributed particulate structures visible on fracture-flip preparations of the metacyclic surface. The exposure and subsequent masking of the terminal capping sugars explains the stage specificity of promastigote attachment to and release from the vector midgut, which are key events in the development of transmissible infections in the fly.

Animals↗
PubMed1995

Identification of genes mediating lipophosphoglycan biosynthesis by functional complementation of Leishmania donovani mutants.

A powerful approach for identifying the genes involved in the infectious cycle of pathogens is functional genetic complementation. Here, the current status of this technology in Leishmania is reviewed, focusing on the genes involved in the biosynthesis of the unique parasite surface glycolipid, lipophosphoglycan (LPG). LPG plays multiple roles in the Leishmania infectious cycle, in both the sand fly vector and in establishing successful intracellular parasitism within the vertebrate macrophage. The emerging methods for generating LPG mutations and for recovering the affected gene(s) by complementation with an episomal genomic Leishmania DNA library are reviewed. The properties and probable roles of the first two genes identified by this methodology are discussed. These methods also show great promise in the search for genes affecting other virulence factors of Leishmania as well as in the identification of new drug-resistance loci.

Animals↗
PubMed1995

Activation of human immunodeficiency virus type 1 in monocytoid cells by the protozoan parasite Leishmania donovani.

In this study, we demonstrated that the protozoan parasite Leishmania donovani and one of its major surface molecules, the lipophosphoglycan (LPG), can induce human immunodeficiency virus type 1 (HIV-1) expression in U1 and OM-10.1, two cell lines of monocytoid origin latently infected with HIV-1. Treatment of U1 cells with various concentrations of LPG (1, 5, and 10 microM) resulted in a dose-dependent secretion of tumor necrosis factor alpha (TNF-alpha). Suppression of LPG-induced HIV-1 expression by polyclonal anti-TNF-alpha antibodies further confirmed the involvement of this cytokine. Results from these studies indicate that the protozoan parasite L. donovani can induce the secretion of TNF-alpha that will function in an autocrine or paracrine manner to upregulate HIV-1 expression. Our data suggest for the first time that this protozoan parasite can be viewed as a potential cofactor in the pathogenesis of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan.

Phlebotomine vectors can in some instances transmit only certain species of Leishmania. Comparison of a large number of vector/parasite pairs revealed that species-specific differences in vectorial competence were in every case directly correlated with the ability of promastigotes to attach to the sand-fly midgut, the variable outcomes of which were controlled by structural polymorphisms in the surface lipophosphoglycan (LPG) of the parasite. The ability of Phlebotomus papatasi to transmit only Leishmania major could be attributed to the unique, highly substituted nature of L. major LPG that provides for multiple terminally exposed beta-linked galactose residues for binding. While the relatively unsubstituted LPGs of other Leishmania species were unable to mediate promastigote attachment to P. papatasi, they could mediate binding to midguts of Phlebotomus argentipes, which was found to be a potentially competent vector for every Leishmania species examined. The data suggest that at least some phlebotomine vectors differ with respect to the parasite recognition sites which they express and that midgut adhesion is a sufficiently critical component of vectorial competence as to provide the evolutionary drive for LPG structural polymorphisms.

Animals↗
PubMed1994

Phosphatidylinositol-anchored molecules and inducible lipopolysaccharide binding sites of human and mouse bone marrow cells.

We have previously established that lipopolysaccharide (LPS) induces the expression of new specific LPS-binding sites (LpsR) in mouse bone marrow cells (BMC). We now show that exposure of human BMC to LPS elicits the production of both CD14 molecules (detectable with monoclonal antibody My4) and LpsR (detectable with fluorescein isothiocyanate-LPS). Pretreatment of stimulated human BMC with My4 inhibited the binding of fluorescein isothiocyanate-LPS. The stimulation of human BMC, but not mouse BMC, required the presence of serum. Other characteristics of mouse and human BMC examined were very similar. Their inducible LpsR interacted with the lipid moieties of LPS and Leishmania donovani lipophosphoglycan and with a soluble preparation of peptidoglycan. Moreover, mouse and human LpsR were susceptible to treatment with a phosphatidylinositol-specific phospholipase C (PI-PLC), thus suggesting that both are PI-anchored CD14 molecules. Neither LpsR appeared able to interact with a synthetic LPS antagonist (compound PPDm2) structurally related to the lipid region of LPS. However, PPDm2 blocked LPS-induced expression of LpsR in both BMC. Furthermore, in both species, pretreatment of BMC with PI-PLC did not prevent the cells from expressing LpsR in response to LPS. The results support the hypothesis that the elicited LpsR of mouse and human BMC is an inducible form of CD14, whereas the putative "signaling LPS receptor" of these cells is not CD14 or any other PI-anchored molecule.

Acute-Phase Proteins↗
PubMed1994

Leishmania donovani infection enhances macrophage viability in the absence of exogenous growth factor.

Bone marrow-derived macrophages rapidly die in the absence of macrophage growth factor (M-CSF). However, as demonstrated here, bone marrow-derived macrophages infected with Leishmania donovani exhibit increased viability in the absence of exogenous growth factor. Forty-eight hours after inoculation with promastigotes or amastigotes, infected cell cultures contained 180 and 95% more cells, respectively, than control cultures. This effect was specific to Leishmania infection, as uptake of latex beads or avirulent promastigotes by macrophages did not enhance cell viability. L. donovani-infected macrophages also displayed increased phagocytic capacity, as compared with control macrophages and macrophages grown continuously in M-CSF-containing medium. Supernatants collected from infected cells elaborated a factor(s) that enhanced macrophage viability but did not stimulate macrophage DNA synthesis. This activity of L. donovani-infected cell-conditioned medium could be abrogated by preincubation of macrophages with cycloheximide before inoculation with the parasite, implying that macrophage protein synthesis is required for the elaboration of this factor(s).

Animals↗
PubMed1994

The role of the lipophosphoglycan of Leishmania in vector competence.

The surface lipophosphoglycans (LPG) of Leishmania promastigotes express stage- and species-specific polymorphisms that are defined by variations in the type and number of phosphorylated oligosaccharide repeats. We have studied how these polymorphic structures control the development of transmissible infections in the sandfly vector as well as the species-specificity of vectorial competence. Procyclic promastigotes displayed an inherent capacity to bind to midgut epithelial cells of a competent vector. This capacity was lost during their transformation of metacyclic promastigotes, permitting the selective release and anterior migration of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release were found to be controlled by developmental modifications in terminally exposed saccharides on LPG, which, depending on the species of Leishmania, involved either substitution or capping of terminal side-chain sugars, loss of terminal side-chain sugars, substitution or loss of neutral capping sugars. The stage-specific terminal sugars involved in midgut adhesion are, in some cases, also species-specific, and the extent to which these differences affect midgut attachment, forcefully predicted vectorial competence.

Animals↗
PubMed1993

Defective galactofuranose addition in lipophosphoglycan biosynthesis in a mutant of Leishmania donovani.

A mutant cell line of Leishmania donovani (R2D2), previously selected for resistance to the cytotoxic lectin ricin agglutinin, was found to be totally deficient in the synthesis and expression of lipophosphoglycan, a dominant surface virulence factor. The metabolic defect in R2D2 parasites responsible for its lipophosphoglycan (LPG-) phenotype was investigated in this study. Following metabolic labeling of R2D2 parasites with either [3H]galactose or [3H]mannose, the main glycosylphosphatidylinositide product that accumulated was Glc-PO4-Man-Man-GlcN-lyso-1-O-alkylphosphatidylinositol (PI). The metabolic defect was further defined using a cell-free glycosylation system. When membrane preparations from wild-type cells were incubated with UDP-[3H]galactose and unlabeled GDP-mannose in the absence of exogenous acceptors, radiolabeled lipophosphoglycan was synthesized. The addition of exogenous Man-Man-GlcN-PI or Galf-Man-Man-GN-PI stimulated lipophosphoglycan synthesis in vitro. In contrast, when membrane preparations from R2D2 cells were incubated with exogenous Man-Man-GlcN-PI as an acceptor or in the absence of exogenous acceptor, the truncated glycosylphosphatidylinositide Glc-PO4-Man-Man-GlcN-PI was the main radioactive product synthesized. However, when exogenous Galf-Man-Man-GN-PI was added to the R2D2 in vitro system, radioactive lipophosphoglycan was synthesized. Collectively, these results indicate that the mutant R2D2 cells are unable to complete the assembly of the glycan core of LPG because of a defect in the synthesis of the "activated" galactofuranosyl donor or the lack of a functional galactofuranosyltransferase.

Animals↗
PubMed1993

Isolation of virulence genes directing surface glycosyl-phosphatidylinositol synthesis by functional complementation of Leishmania.

Trypanosomatid parasites of the genus Leishmania cause a spectrum of widespread tropical diseases. In the vertebrate host they reside within the macrophage phagolysosome; however, the mechanisms employed in this remarkable survival strategy are not well understood. Recent advances in the molecular genetics of these parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant lipophosphoglycan, an abundant glycosyl-phosphatidylinositol-anchored polysaccharide. LPG1, the gene product identified by complementation of the R2D2 mutant, appears to be a glycosyltransferase responsible for the addition of galactofuranosyl residues to the nascent lipophosphoglycan chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism.

Agglutination↗
PubMed1993

Reversion to virulence in Leishmania major correlates with expression of surface lipophosphoglycan.

An attenuated clone of Leishmania major was produced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and was biochemically characterized to determine the reason(s) for its loss of virulence. We found that the degree of virulence of L. major did not correlate with either the level of expression of promastigote surface protease (PSP) or with the enzymatic activity of the molecule. In contrast, the levels of lipophosphoglycan (LPG) expressed by the attenuated clone were found to be at least 6-fold less than those of virulent L. major. When the attenuated L. major was injected into BALB/c mice and allowed to revert to virulence, the degree of reversion to virulence that the parasites underwent correlated directly with the amount and form (metacyclic) of LPG expressed by the parasites. Thus, these results further implicate LPG as an important Leishmania virulence factor.

Animals↗
PubMed1993

The lipophosphoglycan of Leishmania and macrophage protein kinase C.

Lipophosphoglycan (LPG), the major cellsurface glycoconjugate of Leishmania promastigotes, is on essential virulence determinant. One feature of LPG resides in its strong inhibitory effect on the activity of purified protein kinase C (PKC). In this article, Albert Descoteaux and Salvatore Turco briefly review the evidence that LPG effectively inhibits PKC activity in the macrophage, and discuss the implication of such inhibition on Leishmania intramacrophoge survival.

Journal Article↗
PubMed1993

An avirulent lipophosphoglycan-deficient Leishmania major clone induces CD4+ T cells which protect susceptible BALB/c mice against infection with virulent L. major.

An avirulent clone of Leishmania major was used to immunize susceptible BALB/c mice against challenge with virulent L. major. By using the immunized animals as a source of cells, CD4+ parasite-specific T-cell lines could be generated in vitro which, when adoptively transferred to naive BALB/c recipients, conferred marked protection against challenge with virulent L. major. Compared with CD4+ parasite-specific T-cell lines generated from nonimmunized BALB/c mice infected with L. major, the protective T-cell lines generated from immunized mice produced substantially less interleukin-4 and substantially more tumor necrosis factor and interleukin-2. Interestingly, the protective CD4+ T cells did not mediate L. major-specific delayed-type hypersensitivity in vivo and proliferated in vitro only in response to living L. major and not to frozen-and-thawed antigen preparations of the parasite. Finally, the avirulent clone of L. major was found to express the major surface glycolipid of L. major, lipophosphoglycan, at a level that was sixfold less than expression of this molecule by virulent L. major. In addition, lipophosphoglycan of the avirulent parasite failed to mature into the larger, or metacyclic, form of the molecule.

Animals↗