Research Article: Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

Date Published: November 3, 2015

Publisher: Public Library of Science

Author(s): Sebastian Damerow, Carolin Hoppe, Giulia Bandini, Patricia Zarnovican, Falk R. Buettner, Carsten G. K. Lüder, Michael A. J. Ferguson, Françoise H. Routier, Alvaro Acosta-Serrano.

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.

Partial Text: 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]. According to the World Health Organisation, more than 1 million new cases of cutaneous leishmaniasis and at least 30 000 deaths due to visceral leishmaniasis occur annually. Current treatments are far from ideal and the need to develop new treatments against leishmaniasis is generally recognised [2].

In eukaryotes, the biosynthesis of glycans, such as those creating a protective cell surface glycocalyx or cell wall, requires the activation of monosaccharides in the form of nucleotide sugars. These high-energy compounds may be formed from appropriate monosaccharides or be generated from interconversion of existing nucleotide sugars. For example, depending on the cell type, UDP-Gal, UDP-galacturonic acid, UDP-glucuronic acid, UDP-xylose and UDP-arabinose may be generated from UDP-Glc. In Leishmania parasites, however, the variety of nucleotide sugars synthesised is limited to UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man, GDP-Fuc and GDP-arabinose [23]. Previous works have shown that in this protozoan parasite, UDP-Gal is synthesised by a de novo pathway involving UGP [13,14] and by a salvage pathway mediated by USP [15,16]. Deficiency of UGP alone does not abolish biosynthesis of UDP-Gal and its immediate precursor UDP-Glc [13] since, as we demonstrate here, their biosynthesis is partially compensated by USP. Targeted deletion of both UGP and USP was however unsuccessful suggesting that the metabolites UDP-Glc and/or UDP-Gal are essential for Leishmania in vitro growth. To demonstrate the importance of the UDP-Glc/UDP-Gal biosynthesis, we therefore deleted one of the two USP alleles in the UGP deficient strain (ugp-/-) and used a degron system enabling conditional degradation of the enzyme produced by the remaining USP allele (Fig 1). Addition of 1μM FK506 to the growth medium enabled stabilisation of the ddUSP protein product and supported restricted UDP-Glc and UDP-Gal biosynthesis, which was nonetheless sufficient to maintain parasite growth. With low FK506 concentrations, ddUSP was destabilised and the UDP-Glc/UDP-Gal biosynthesis could be reduced to minimal level leading to growth arrest and cell death, allowing us to demonstrate that UDP-Gal and/or UDP-Glc are essential metabolites for L. major promastigotes.



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