Importance of UDP-glucose biosynthesis for the protozoan parasite Leishmania major
Final Report Abstract
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. In the frame of this project, we have defined the pathways for UDP-galactose biosynthesis in Leishmania parasites. This nucleotide-sugar arises from a de novo pathway involving biosynthesis of UDP-glucose by UGP and its subsequent epimerisation (as other organisms) but also from an unusual galactose salvage pathway mediated by USP (Figure 1). Upon deletion of the USP encoding gene, Leishmania 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. USP is thus entirely responsible for galactose salvage but does not significantly contribute to the de novo synthesis of UDP-glucose in standard conditions. Using a mutant lacking the de novo biosynthetic pathway (due to deletion of UGP), we could furthermore show that USP may support the biosynthesis of the cell surface lipophosphoglycan from extracellular galactose. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contribute to successful vector infection. We have recently initiated a collaboration with Matt Rogers from the London Tropical School to test this hypothesis. In order to abolish the biosynthesis of UDP-galactose, we have tried to delete both UGP and USP in L. major but repeatedly failed, suggesting that the pathways mediated by these enzymes are essential. Thus deletion of UGP was here combined with conditional destabilisation of USP to control the enzyme level and deplete the cellular pools of UDP-glucose and UDP-galactose. Remarkably, stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady pool of these two nucleotide sugars, preserve most of glycoinositolphospholipids galactosylation, and support a regular parasite growth in culture. Nevertheless the UDP-glucose/UDP-galactose biosynthesis was strongly reduced as displayed by the quasi-absence of the polysaccharide lipophosphoglycan. Most importantly, in destabilising conditions, absence of both UGP and USP resulted in depletion of UDP-glucose and UDP-galactose and led to growth cessation and cell death. This study is the first to successfully demonstrate essentiality of a pathway in Leishmania parasites using conditional protein degradation. So far, the essentiality of a gene is inferred from the ability to generate a chromosomal null mutant in the presence, but not absence of an episome that express the targeted gene. Finally in collaboration with Dr. Roman Fedorov (Department of biophysical Chemistry, Hannover Medical School), we have analysed the enzymatic cycle of Leishmania UGP and identified potential sites for allosteric regulation, which are absent in human UGP. This raises the possibility of designing inhibitors specific of the trypanosomatid UGP. Based on the results obtained within this project, we were successful in obtaining funding from the European Community in the frame of the Marie Curie Training Network GlycoPar. This consortium involves renowned researchers and pharmaceutical companies such as GlykoSmithKline.
Publications
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(2013). Catalytic Mechanism and Allosteric Regulation of UDP-Glucose Pyrophosphorylase from Leishmania major. ACS Catal., 3, 2976−2985
Führing J., Cramer J.T., Routier F.H., Lamerz A-C., Baruch P., Gerardy-Schahn R and Fedorov R.
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Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis. Int.J.Parasitol., International Journal for Parasitology, Volume 45, Issue 12, October 2015, Pages 783-790
Damerow S., Hoppe C., Bandini G., Zarnovican P., Buettner F.R., Ferguson M.A.J. and Routier F.H.
