Abstract
Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.
Highlights
Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania and transmitted by the bite of a sandfly
Since treatments against all forms of leishmaniasis are limited in number and efficacy, many efforts are made to identify potential drug targets and develop new therapies
We used a combination of gene deletion and conditional protein destabilization to demonstrate that biosynthesis of the nucleotide sugar UDP-glucose and its derivative UDP-galactose is essential for parasite growth
Summary
Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania and transmitted by the bite of a sandfly. The severity of the diseases depends on parasite species as well as the immune status of the host and ranges from self-healing cutaneous lesions to fatal visceral infections [1]. A system for conditional destabilisation of protein has been described This original system involves fusion of a mutated FK506 binding protein destabilising domain to the protein of interest and its stabilisation by addition of a specific ligand [5]. This system has not yet been applied to essential proteins
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