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

Damerow S, Hoppe C, Bandini G, Zarnovican P, Buettner FR, Lüder CG, Ferguson MA, Routier FH - PLoS Negl Trop Dis (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Analysis of nucleotide sugar pools in L. major wild type and ugp-/-usp-/c mutant.Nucleotide sugars were extracted from mid Log phase wild type (wt) and ugp-/-usp-/c promastigotes grown in presence of 1 μM (+) or 0.01 μM (-) FK506 and measured by liquid chromatography-electrospray ionisation-tandem mass spectrometry with multiple reaction monitoring. Values represent the mean ± SD from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant differences (*p < 0.01, ** p < 0.005, *** p < 0.001), paired t-test.
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pntd.0004205.g003: Analysis of nucleotide sugar pools in L. major wild type and ugp-/-usp-/c mutant.Nucleotide sugars were extracted from mid Log phase wild type (wt) and ugp-/-usp-/c promastigotes grown in presence of 1 μM (+) or 0.01 μM (-) FK506 and measured by liquid chromatography-electrospray ionisation-tandem mass spectrometry with multiple reaction monitoring. Values represent the mean ± SD from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant differences (*p < 0.01, ** p < 0.005, *** p < 0.001), paired t-test.

Mentions: The nucleotide sugar pools in wild type L. major and the ugp-/-usp-/c clones were measured by high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry using multiple reactions monitoring as previously described [23]. Surprisingly, stabilisation of the enzyme produced from a single ddUSP allele (with 1 μM FK506) was sufficient to maintain the steady-state pools of UDP-Glc and UDP-Gal (Fig 3). In all clones, a strong reduction of the UDP-Glc and UDP-Gal pools was nevertheless observed upon destabilisation of USP in medium containing 0.01 μM FK506. In contrast, the GDP-Man and GDP-Ara pools did not significantly differ from the pools measured in wild type parasites (Fig 3). Unexpectedly, the pool of UDP-N-acetylglucosamine (UDP-GlcNAc) seemed to slightly decrease in the ugp-/-usp-/c mutant upon USP destabilisation (0.01μM FK506) whereas the GDP-fucose (GDP-Fuc) pool, which is very small in wild type parasite, was strongly elevated in these conditions.


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

Damerow S, Hoppe C, Bandini G, Zarnovican P, Buettner FR, Lüder CG, Ferguson MA, Routier FH - PLoS Negl Trop Dis (2015)

Analysis of nucleotide sugar pools in L. major wild type and ugp-/-usp-/c mutant.Nucleotide sugars were extracted from mid Log phase wild type (wt) and ugp-/-usp-/c promastigotes grown in presence of 1 μM (+) or 0.01 μM (-) FK506 and measured by liquid chromatography-electrospray ionisation-tandem mass spectrometry with multiple reaction monitoring. Values represent the mean ± SD from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant differences (*p < 0.01, ** p < 0.005, *** p < 0.001), paired t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631452&req=5

pntd.0004205.g003: Analysis of nucleotide sugar pools in L. major wild type and ugp-/-usp-/c mutant.Nucleotide sugars were extracted from mid Log phase wild type (wt) and ugp-/-usp-/c promastigotes grown in presence of 1 μM (+) or 0.01 μM (-) FK506 and measured by liquid chromatography-electrospray ionisation-tandem mass spectrometry with multiple reaction monitoring. Values represent the mean ± SD from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant differences (*p < 0.01, ** p < 0.005, *** p < 0.001), paired t-test.
Mentions: The nucleotide sugar pools in wild type L. major and the ugp-/-usp-/c clones were measured by high performance liquid chromatography-electrospray ionisation-tandem mass spectrometry using multiple reactions monitoring as previously described [23]. Surprisingly, stabilisation of the enzyme produced from a single ddUSP allele (with 1 μM FK506) was sufficient to maintain the steady-state pools of UDP-Glc and UDP-Gal (Fig 3). In all clones, a strong reduction of the UDP-Glc and UDP-Gal pools was nevertheless observed upon destabilisation of USP in medium containing 0.01 μM FK506. In contrast, the GDP-Man and GDP-Ara pools did not significantly differ from the pools measured in wild type parasites (Fig 3). Unexpectedly, the pool of UDP-N-acetylglucosamine (UDP-GlcNAc) seemed to slightly decrease in the ugp-/-usp-/c mutant upon USP destabilisation (0.01μM FK506) whereas the GDP-fucose (GDP-Fuc) pool, which is very small in wild type parasite, was strongly elevated in these conditions.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany.

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.

No MeSH data available.


Related in: MedlinePlus