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Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major.

ABSTRACT: A cell-free system based on washed Leishmania major membranes was labelled with GDP-[3H]Man in the presence of synthetic glucosaminyl-phosphatidylinositol (GlcN-PI) and N-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI). In both cases, the major radiolabelled products were Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1, 2-dipalmitoylglycerol) and Man alpha 1-4GlcN alpha 1-6myo-inositol1-HPO4- (sn-1-palmitoyl-2-lyso-glycerol), to which an additional d-mannose residue was added when a chase with an excess of GDP-Man was performed. The L. major cell-free system can therefore be used to observe the actions of four enzymes, namely GlcNAc-PI de-N-acetylase, Dol-P-Man-GlcN-PI alpha 1-4-mannosyltransferase, a phospholipase A2-like activity and a second alpha-mannosyltransferase activity. The substrate specificities of the first two of these enzymes were studied using a series of substrate analogues. GlcNAc-PI de-N-acetylase was tested against a variety of N-acylated GlcN-PI substrates and was able to cleave N-acetyl and N-propyl groups but not larger groups such as N-butyl, N-isobutyl, N-pentyl and N-hexyl. The Dol-P-Man-GlcN-PI alpha1-4-mannosyltransferase activity required the amino group of the glucosamine residue and the d-configuration of the myo-inositol residue of the GlcN-PI acceptor substrate.

SUBMITTER: Smith TK

PROVIDER: S-EPMC1218683 | biostudies-other | 1997 Sep

REPOSITORIES: biostudies-other

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Project description:Visceral leishmaniasis is a deadly infectious disease caused by Leishmania donovani, a kinetoplastid parasite for which no licensed vaccine is available. To identify potential vaccine candidates, we systematically identified genes encoding putative cell surface and secreted proteins essential for parasite viability and host infection. We identified a protein encoded by LdBPK_061160 which, when ablated, resulted in a remarkable increase in parasite adhesion to tissue culture flasks. Here, we show that this phenotype is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored surface molecules and that LdBPK_061160 encodes a noncatalytic component of the L. donovani GPI-mannosyltransferase I (GPI-MT I) complex. GPI-anchored surface molecules were rescued in the LdBPK_061160 mutant by the ectopic expression of both human genes PIG-X and PIG-M, but neither gene could complement the phenotype alone. From further sequence comparisons, we conclude that LdBPK_061160 is the functional orthologue of yeast PBN1 and mammalian PIG-X, which encode the noncatalytic subunits of their respective GPI-MT I complexes, and we assign LdBPK_061160 as LdPBN1. The LdPBN1 mutants could not establish a visceral infection in mice, a phenotype that was rescued by constitutive expression of LdPBN1. Although mice infected with the null mutant did not develop an infection, exposure to these parasites provided significant protection against subsequent infection with a virulent strain. In summary, we have identified the orthologue of the PBN1/PIG-X noncatalytic subunit of GPI-MT I in trypanosomatids, shown that it is essential for infection in a murine model of visceral leishmaniasis, and demonstrated that the LdPBN1 mutant shows promise for the development of an attenuated live vaccine. IMPORTANCE Visceral leishmaniasis is a deadly infectious disease caused by the parasites Leishmania donovani and Leishmania infantum. It remains a major global health problem, and there is no licensed highly effective vaccine. Molecules that are displayed on the surface of parasites are involved in host-parasite interactions and have important roles in immune evasion, making vaccine development difficult. One major way in which parasite surface molecules are tethered to the surface is via glycophosphatidylinositol (GPI) anchors; however, the enzymes required for all the biosynthetic steps in these parasites are not known. Here, we identified the enzyme required for an essential step in the GPI anchor-biosynthetic pathway in L. donovani, and we show that while parasites lacking this gene are viable in vitro, they are unable to establish infections in mice, a property we show can be exploited to develop a live genetically attenuated parasite vaccine.

Project description:Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection.

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Early steps in glycosylphosphatidylinositol biosynthesis in Leishmania major.

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