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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

Depletion of USP in L. major ugp-/-usp-/c mutant and effect on glycoconjugates biosynthesis.(A) Western blot of Log phase wild type or ugp-/-usp-/c promastigotes lysates probed with an anti-USP antiserum (upper panel) or the anti-LPG monoclonal antibody WIC79.3 (middle panel). Parasites were cultivated in medium containing 1, 0.05, 0.005 µM or no FK506 as indicated. Loading was assessed by Coomassie staining of an identically loaded SDS-Page ran separately (lower panel). (B) In vitro conversion of Glc-1P (black bars) or Gal1P (white bars) into nucleotide sugars by cell lysates of wild type and ugp-/-usp-/c mutant cultivated in medium containing various FK506 concentrations as indicated. Values represent the mean ± SEM from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant difference (*p < 0.01), One-way ANOVA with Tukey post test. (C) Negative-ion MALDI spectra of glycosylinositolphospholipids (GIPLs) isolated from wild type and ugp-/-usp-/c mutant grown in presence of 1 or 0.01 μM FK506. Each prominent peak is annotated with its m/z value, the letter a, b, c, d or e referring to the structure depicted above. The lipid moiety consists of a 1-alkyl-2-lyso-phosphatidylinositol or 1-alkyl-2-acyl-phosphatidylinositol. The length of the alkyl and acyl chains is indicated above each peak; C- indicates absence of an alkyl chain.
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pntd.0004205.g002: Depletion of USP in L. major ugp-/-usp-/c mutant and effect on glycoconjugates biosynthesis.(A) Western blot of Log phase wild type or ugp-/-usp-/c promastigotes lysates probed with an anti-USP antiserum (upper panel) or the anti-LPG monoclonal antibody WIC79.3 (middle panel). Parasites were cultivated in medium containing 1, 0.05, 0.005 µM or no FK506 as indicated. Loading was assessed by Coomassie staining of an identically loaded SDS-Page ran separately (lower panel). (B) In vitro conversion of Glc-1P (black bars) or Gal1P (white bars) into nucleotide sugars by cell lysates of wild type and ugp-/-usp-/c mutant cultivated in medium containing various FK506 concentrations as indicated. Values represent the mean ± SEM from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant difference (*p < 0.01), One-way ANOVA with Tukey post test. (C) Negative-ion MALDI spectra of glycosylinositolphospholipids (GIPLs) isolated from wild type and ugp-/-usp-/c mutant grown in presence of 1 or 0.01 μM FK506. Each prominent peak is annotated with its m/z value, the letter a, b, c, d or e referring to the structure depicted above. The lipid moiety consists of a 1-alkyl-2-lyso-phosphatidylinositol or 1-alkyl-2-acyl-phosphatidylinositol. The length of the alkyl and acyl chains is indicated above each peak; C- indicates absence of an alkyl chain.

Mentions: The absence of UGP had been demonstrated in the parental strain by Western blot using a rabbit polyclonal anti-UGP serum [13]. To confirm the functionality of the degradation system in the different ugp-/-usp-/c clones, we analysed the USP protein level in lysates of Log phase promastigotes using a rabbit polyclonal anti-USP serum [16]. As shown in Fig 2A (upper panel), in presence of 1 μM FK506, ddUSP was clearly detectable at approximately 81 kDa (USP: ~69 kDa; destabilising domain: ~12 kDa) whereas in wild type parasites, native USP migrated at approximately 69 kDa. The amount of ddUSP in the different clones stabilised with 1μM FK506 was estimated to 50–60% of wild type from the signal intensity. As anticipated, at lower FK506 concentrations of 0.05 μM, only traces of ddUSP were still discernible and the enzyme was essentially absent if the FK506 concentration was reduced to 0.005 μM (Fig 2A, upper panel, right). As expected USP level was not affected by the FK506 concentration in wild type parasites (Fig 2A, upper panel, left).


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)

Depletion of USP in L. major ugp-/-usp-/c mutant and effect on glycoconjugates biosynthesis.(A) Western blot of Log phase wild type or ugp-/-usp-/c promastigotes lysates probed with an anti-USP antiserum (upper panel) or the anti-LPG monoclonal antibody WIC79.3 (middle panel). Parasites were cultivated in medium containing 1, 0.05, 0.005 µM or no FK506 as indicated. Loading was assessed by Coomassie staining of an identically loaded SDS-Page ran separately (lower panel). (B) In vitro conversion of Glc-1P (black bars) or Gal1P (white bars) into nucleotide sugars by cell lysates of wild type and ugp-/-usp-/c mutant cultivated in medium containing various FK506 concentrations as indicated. Values represent the mean ± SEM from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant difference (*p < 0.01), One-way ANOVA with Tukey post test. (C) Negative-ion MALDI spectra of glycosylinositolphospholipids (GIPLs) isolated from wild type and ugp-/-usp-/c mutant grown in presence of 1 or 0.01 μM FK506. Each prominent peak is annotated with its m/z value, the letter a, b, c, d or e referring to the structure depicted above. The lipid moiety consists of a 1-alkyl-2-lyso-phosphatidylinositol or 1-alkyl-2-acyl-phosphatidylinositol. The length of the alkyl and acyl chains is indicated above each peak; C- indicates absence of an alkyl chain.
© Copyright Policy
Related In: Results  -  Collection

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

pntd.0004205.g002: Depletion of USP in L. major ugp-/-usp-/c mutant and effect on glycoconjugates biosynthesis.(A) Western blot of Log phase wild type or ugp-/-usp-/c promastigotes lysates probed with an anti-USP antiserum (upper panel) or the anti-LPG monoclonal antibody WIC79.3 (middle panel). Parasites were cultivated in medium containing 1, 0.05, 0.005 µM or no FK506 as indicated. Loading was assessed by Coomassie staining of an identically loaded SDS-Page ran separately (lower panel). (B) In vitro conversion of Glc-1P (black bars) or Gal1P (white bars) into nucleotide sugars by cell lysates of wild type and ugp-/-usp-/c mutant cultivated in medium containing various FK506 concentrations as indicated. Values represent the mean ± SEM from n = 3 independent cultures; for the ugp-/-usp-/c mutant, each culture represents a different clone. Significant difference (*p < 0.01), One-way ANOVA with Tukey post test. (C) Negative-ion MALDI spectra of glycosylinositolphospholipids (GIPLs) isolated from wild type and ugp-/-usp-/c mutant grown in presence of 1 or 0.01 μM FK506. Each prominent peak is annotated with its m/z value, the letter a, b, c, d or e referring to the structure depicted above. The lipid moiety consists of a 1-alkyl-2-lyso-phosphatidylinositol or 1-alkyl-2-acyl-phosphatidylinositol. The length of the alkyl and acyl chains is indicated above each peak; C- indicates absence of an alkyl chain.
Mentions: The absence of UGP had been demonstrated in the parental strain by Western blot using a rabbit polyclonal anti-UGP serum [13]. To confirm the functionality of the degradation system in the different ugp-/-usp-/c clones, we analysed the USP protein level in lysates of Log phase promastigotes using a rabbit polyclonal anti-USP serum [16]. As shown in Fig 2A (upper panel), in presence of 1 μM FK506, ddUSP was clearly detectable at approximately 81 kDa (USP: ~69 kDa; destabilising domain: ~12 kDa) whereas in wild type parasites, native USP migrated at approximately 69 kDa. The amount of ddUSP in the different clones stabilised with 1μM FK506 was estimated to 50–60% of wild type from the signal intensity. As anticipated, at lower FK506 concentrations of 0.05 μM, only traces of ddUSP were still discernible and the enzyme was essentially absent if the FK506 concentration was reduced to 0.005 μM (Fig 2A, upper panel, right). As expected USP level was not affected by the FK506 concentration in wild type parasites (Fig 2A, upper panel, left).

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