Leishmaniasis is the second largest parasitic killer in the world (after malaria) responsible for an estimated 500,000 cases each year worldwide. Visceral leishmaniasis infections (i.e. internal organs) are more complicated to diagnose due to the lack of visible symptoms. Current tests of the disease look for antibodies against the parasite, but these antibodies are expressed long after the infection. It has been found the Leishmania parasite excretes a unique phosphoglycan repeat unit: By testing for this repeat unit, the diagnostic test will be looking for an active infection in the patient. It has been found that there are 4 monoclonal antibodies to detect this uniquely excreted phosphoglycan. This provides a basis for a test kit, the biomarker (i.e. the phosphoglycan repeats), and the way to detect the biomarker. For the construction of a dipstick style test or an ELISA format assay, a synthetic repeat unit phosphoglycan is needed to act as a positive control in a dipstick test or as a known comparison in a ELISA. The synthetic structure must also be anchored to a solid surface for testing. Biotin will be used as the anchor, it has shown many different uses in biotechnology and its very high affinity to the avidin proteins make it very useful. A 6-aminohexanol spacer arm will also be included. After many attempts to introduce the biotin spacer moiety to the repeat unit at the D-mannose 1-phosphate, it was decided to change the strategy and integrate the biotin-spacer moiety as a phosphate at the D-galactose unit. This meant performing chain elongation from the reducing end, with the first disaccharide unit being capped at the D-mannose anomeric position with a methyl group. Below are examples of the targeted biotinylated phosphoglycan structures to be prepared in this project.

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The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote’s life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one L. donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild-type but similar in size to LPG from wild-type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically-grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% are the disaccharide Glc(β1,2)Man, trisaccharide Gal(β1,4)[Glc(β1,2)]Man, and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man in addition to wild-type Gal(β1,4)Man and Man(α1,2)Man and Gal(β1,4)[Man(α1,2)]Man terminating caps, These glucose containing isoforms were absent in stationary parasites. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a two-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. The β-glucosyltransferase in both JABBA and wild-type in vitro produced a β-glucosidase sensitive and β-galactosidase sensitive trisaccharide, indicative of the mannose of repeating units and caps being used in substrate, novel in comparison to other Leishmania species. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms, and has a unique β-glucosyltransferase not active in vivo in wild-type parasites.

Leishmania donovani is a blood-borne tropicial parasite, which infects humans through bites by Phlebotomus sandflies. The parasite survives and multiplies inside macrophages in inner organs, and causes the deadly disease visceral leishmaniasis (Kala-Azar). Macrophages and dendritic cells (DC) are professional antigen-presenting cells involved in the initiation of immune responses. Immature DC are present in all tissues where they internalise and process antigen, in response to which they migrate from tissue, into draining lymphoid organs, undergo maturation and present antigens to lymphocytes. Control measures for leishmaniasis include testing of new diagnostics and development of affordable and effective vaccines for humans. Lipophosphoglycan (LPG) is the major surface component of Leishmania donovani promastigotes. LPG comprises a membrane-anchoring lysophosphatidylinositol part and an extracellular chain of disaccharide phosphates. These repetitions are crucial for parasite survival inside macrophages following phagocytosis. LPG has several specific effects on the host cell including inhibition of protein kinase C (PKC) activity, and inhibition of phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin. Confocal microscopy and image analysis were used to follow F-actin dynamics in single macrophages during phagocytosis of L. donovani promastigotes and LPG-coated particles. F-actin did not depolymerize, but instead progressively polymerized around phagosomes with LPG-containing prey. This correlated with reduced translocation of PKCα to the phagosome and blocked phagosomal maturation. LPG also inhibited cortical actin turnover, which could be the underlying cause of the reduced uptake of LPG-containing prey. Extracellular- and intracellular calcium was necessary for phagocytosis, periphagosomal F-actin breakdown and phagosomal maturation in macrophages interacting with unopsonized prey,and for the action of LPG. We also studied F-actin turnover in macrophages overexpressing dominant-negative (DN) PKCα. DN PKCα macrophages showed increased amounts of cortical F-actin, decreased phagocytic capacity, inhibition of periphagosomal F-actin breakdown and defective phagosomal maturation. When DN PKCα macrophages interacted with LPG-containing prey, phagocytosis was almost completely blocked. Moreover, we found that Leishmania promastigotes and particularly LPG inhibit DC maturation and detachment from distinct surfaces. Thus, LPG from Leishmania donovani could directly inhibit DC migration to lymphoid organs, antigen-presentation and development of immunity.