Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts.

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(ß1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one ß-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.

Leishmania major-Phlebotomus duboscqi interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes.

OBJECTIVES: To assess the effect of monoclonal antibodies (MABS) raised against L. major derived LPG on L. major development in vitro and in its natural vector P. duboscqi. Also determine whether LPG molecule and the sand fly the gut lysates have shared epitopes. DESIGN: A laboratory based study. SETTING: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions. METHODS: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x 10(6) amastigotes in 20 microl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100 microl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100 microl of 1:100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37 degrees C and growth at 26 degrees C, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms. RESULTS: 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and no MABS supported full parasite development up to the metacyclic stage. Results also showed that flies, which had fed on MABS, showed low parasitemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5). CONCLUSIONS: These findings showed that anti-LPG MABS were effective in reducing sand fly infections. This study also showed that P. duboscqi gut lysates and proteins present in L. major-derived LPG share two common proteins of molecular weights 105 kDa and 106 kDa. Further analysis of these individual proteins from the gut should be studied with a view of determining their vaccine potential.

Galactofuranose metabolism: a potential target for antimicrobial chemotherapy.

Galactofuranose-containing glycoconjugates are present in numerous microbes, many of which are pathogenic for humans. Metabolic aspects of the monosaccharide have proven difficult to elucidate, because galactofuranose metabolites and glycoconjugates are relatively unstable during analyses. Recent advances in biochemical and genetic approaches, however, have facilitated a better understanding of galactofuranose metabolism. This review summarizes our current information on its metabolism and a few selected glycoconjugates containing this furanose.

Leishmania donovani-derived lipophosphoglycan plus BCG induces a Th1 type immune response but does not protect Syrian golden hamsters (Mesocricetus auratus) and BALB/c mice against Leishmania donovani.

The efficacy of Leishmania donovani-derived lipophosphoglycan (LPG) plus Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine candidate against visceral leishmaniosis in susceptible BALB/c mouse and Syrian golden hamster (Mesocricetus auratus) models was investigated. Following a triple vaccination with a total dose of 150 microl BCG plus 60 microg or 30 microg of LPG for hamsters and BALB/c mice respectively, there were no noticeable side effects both locally and systemically; implying that the molecule was safe at this dosage level. Vaccinated animals demonstrated an activation of both the humoral as well as cell-mediated responses to LPG, which correlated with resistance against the disease. Protection by LPG plus BCG, was however, poor as the remaining immunized animals showed disease progression leading to severity of the disease as illustrated by emaciation, mass loss and heavy splenic parasitaemia in hamsters. These data nevertheless suggest that it may be rewarding to further evaluate the potential of LPG as a vaccine candidate in leishmaniosis using other adjuvants, which may enhance its immunogenicity.

Transmission blocking vaccine studies in leishmaniasis: II. Effect of immunisation using Leishmania major derived 63 kilodalton glycoprotein, lipophosphoglycan and whole parasite antigens on the course of L. major infection in BALB/c mice.

BACKGROUND: Safe, effective and inexpensive vaccines may be the most practical tool for control of any form of leishmaniasis. Leishmaniasis produces a state of pre-immunition which is the underlying mechanism for prolonged immunity to re-infection. Low doses of parasites has been shown to be able to induce protection in mice. It is not known, however, how immune sera from a susceptible host immunised with Leishmania-derived antigens when taken in by the sandfly affects the development and the subsequent transmission of the parasite to naive hosts. OBJECTIVE: To monitor the course of disease in BALB/c mice following challenge using L. major infected P. duboscqi which had previously fed on immunised mice. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG) and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. On the seventh day these sandflies were used to infect naive BALB/c mice and the course of infection followed for a period of at least three months. RESULTS: Mice infected using sandflies which had previously fed on WPA or rgp63-immunized mice showed disease exacerbation as the infection progressed, whereas those infected using sandflies which had previously fed on LPG-immunised mice had the least lesion sizes compared to control mice infected using sandflies which had fed on saline immunised mice (p < 0.05). CONCLUSIONS: Results from this study indicate that the course of L. major infection in BALB/c mice was dependent on the infective dose of parasites transmitted by the sandflies. Results from this study suggests that sub-infective doses of the parasite from sandflies previously fed on animals immunised with Leishmania-derived antigens needs to be evaluated for their potential in vaccine development against Leishmania infections.

Transmission blocking vaccine studies in leishmaniasis: I. Lipophosphoglycan is a promising transmission blocking vaccine molecule against cutaneous leishmaniasis.

BACKGROUND: New strategies for control of leishmaniasis is needed as chemotherapy using antimonial drugs is prolonged, expensive, associated with side effects and relapses. Vector control has limitations and a vaccine which may be the best approach is not available. OBJECTIVES: To assess the level of inhibition of promastigote development and gut morphology in infected Phlebotomus duboscqi sandflies fed on different groups of BALB/c mice immunised with rgp63, lipophosglycan (LPG) or their cocktail and whole parasite antigens prepared from L. major culture-derived promastigotes. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), LPG and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. Some of the sandflies were dissected on days 2, 4 and 6 after feeding and observed using the light and the transmission electron microscopy for any changes in their gut morphology. The remaining sandflies were all dissected on the sixth day post-feeding and examined for procyclics, nectomonads, haptomonads and metacyclic promastigote forms of Leishmania. RESULTS: Sandflies which had previously fed on WPA, LPG plus rgp63 cocktail and LPG-immunised mice showed the lowest infection rates compared to control sandflies fed on saline immunised mice (p < 0.05). A significant number of procyclic promastigotes, the first developmental form of the parasite in culture as well as in the sandfly was observed in sandflies which fed on LPG-immunised mice (p < 0.05). The dominant parasite form in sandflies which fed on rgp63 or LPG-immunised mice was the nectomonad form but very few of the infective metacyclic forms (p < 0.05). Control sandflies fed on saline immunised or infected mice alone displayed a normal pattern of parasite development up to the metacyclic stage. Studies showed that two possible mechanisms through which immune sera from immunised mice may cause inhibition of parasite development is by exflagellation of nectomonad forms and degeneration of the sandfly midgut epithelium as revealed by light and electron microscopy studies respectively. CONCLUSIONS: This study has shown that immune-mediated transmission blocking may be applied to Leishmania infections. Based on observation of the procyclic promastigotes, the dominance of the nectomonad forms, low infectivity rates in sandflies fed on LPG-immunised mice, we concluded that LPG stands out to be a promising transmission blocking vaccine candidate in leishmaniasis.

Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events.

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue.

Glycoconjugate structures of parasitic protozoa.

Glycoconjugates are abundant and ubiquitious on the surface of many protozoan parasites. Their tremendous diversity has implicated their critical importance in the life cycle of these organisms. This review highlights our current knowledge of the major glycoconjugates, with particular emphasis on their structures, of representative protozoan parasites, including Leishmania, Trypanosoma, Giardia, Plasmodia, and others.

Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species.

Lipophosphoglycan is a prominent member of the phosphoglycan-containing surface glycoconjugates of Leishmania. Genetic tests enable confirmation of its role in parasite virulence and permit discrimination between the roles of lipophosphoglycan and related glycoconjugates. When two different lipophosphoglycan biosynthetic genes from Leishmania major were knocked out, there was a clear loss of virulence in several steps of the infectious cycle but, with Leishmania mexicana, no effect on virulence was found. This points to an unexpected diversity in the reliance of Leishmania species on virulence factors, a finding underscored by recent studies showing great diversity in the host response to Leishmania species.

Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin.

A critical step in the infectious cycle of Leishmania is the differentiation of parasites within the sand fly vector to the highly infective metacyclic promastigote stage. Here, we establish tetrahydrobiopterin (H4B) levels as an important factor controlling the extent of metacyclogenesis. H4B levels decline substantially during normal development, and genetic or nutritional manipulations showed that low H4B caused elevated metacyclogenesis. Mutants lacking pteridine reductase 1 (PTR1) had low levels of H4B, remained infectious to mice, and induced larger cutaneous lesions (hypervirulence). Thus, the control of pteridine metabolism has relevance to the mechanism of Leishmania differentiation and the limitation of virulence during evolution.

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.

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.

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.

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.

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.

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.

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.

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.

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.