The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids - PubMed (original) (raw)
The Trypanosoma brucei dihydroxyacetonephosphate acyltransferase TbDAT is dispensable for normal growth but important for synthesis of ether glycerophospholipids
Rachel Zufferey et al. PLoS One. 2017.
Abstract
Glycerophospholipids are the most abundant constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans and nagana in cattle. They are essential cellular components that fulfill various important functions beyond their structural role in biological membranes such as in signal transduction, regulation of membrane trafficking or control of cell cycle progression. Our previous studies have established that the glycerol-3-phosphate acyltransferase TbGAT is dispensable for growth, viability, and ester lipid biosynthesis suggesting the existence of another initial acyltransferase(s). This work presents the characterization of the alternative, dihydroxyacetonephosphate acyltransferase TbDAT, which acylates primarily dihydroxyacetonephosphate and prefers palmitoyl-CoA as an acyl-CoA donor. TbDAT restores the viability of a yeast double null mutant that lacks glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. A conditional null mutant of TbDAT in T. brucei procyclic form was created and characterized. TbDAT was important for survival during stationary phase and synthesis of ether lipids. In contrast, TbDAT was dispensable for normal growth. Our results show that in T. brucei procyclic forms i) TbDAT but not TbGAT is the physiologically relevant initial acyltransferase and ii) ether lipid precursors are primarily made by TbDAT.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Fig 1. TbDAT complements the lethal phenotype of a yeast double null mutant scgat1Δscgat2Δ.
(A) Growth on galactose or glucose-containing medium. 1, scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:TbDAT] clone 1; 2, scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:TbDAT] clone 2; 3, scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1]; 4, scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:LmDAT] [25]. (B) PCR analysis on S. cerevisiae strains scgat1Δscgat2Δ [GAL1:_ScGAT1]_[32] (1), scgat1Δscgat2Δ [GAL1:ScGAT1][ADH1:TbDAT] (2) and scgat1Δscgat2Δ [ADH1:TbDAT] (3). Primers 652 (
5’-ATACGAAGGGCTGTGTAGG-3’
) and 653 (
5’-TCAACACCGATTTCACCG-3’
), 120 (
5’-CGGGATCCTCTAGACTACATCCTTGATGCCCGCTTG-3’
) and 131 (
5’-AGCTAAGATGTTGTGGCTCCGTG-3’
), and 654 (
5’-CATTGCTTGTCACACTTGG-3’
) and 655 (
5’- TCACTTCTTTCCCCACGGTAC-3’
), were used to amplify ScGAT1, TbDAT and ScHNM1 (positive control), respectively. (C) GPAT and DHAPAT activities of scgat1Δscgat2Δ [ADH1:TbDAT]. The GPAT and DHAPAT assays were performed with 0.2 and 1.5 mg proteins derived from whole cell lysates, respectively, and 150 μM palmitoyl-CoA. 100% DHAPAT activity corresponds to 66 pmol/minxmg. (D) Fatty acyl-CoA specificity of _Tb_DAT expressed in scgat1Δscgat2Δ [ADH1:TbDAT]. The values were normalized to the best fatty acyl-CoA donor palmitoyl-CoA. 100% DHAPAT corresponds to 66 pmol/minxmg. (E) Fatty acyl-CoA specificity of DHAPAT activity present in T. brucei whole cell extracts. The values were normalized to the best fatty acyl-CoA donor oleoyl-CoA. 100% DHAPAT activity corresponds to 5.16 nmol/minxmg. (C)(D)(E) The enzymatic assays were carried out at least twice in duplicate and standard deviations are shown.
Fig 2. TbDAT complements the slow growth phenotype and restores survival during the stationary phase of growth of the L. major null mutant Δlmdat/Δlmdat.
(A) Growth curves were carried out as described in Methods’ section. White circles, null mutant Δlmdat/Δlmdat [25]; black circles, complemented line Δlmdat/Δlmdat [LmDAT NEO]; grey circles, Δlmdat/Δlmdat [TbDAT NEO]. This assay was performed twice in duplicate and standard deviations are shown. (B) Western blot analysis in the presence of lipophosphoglycan specific antibody WIC79.3 [35]. Equivalent of 2x106 cells was loaded in each lane. 1, complemented line Δlmdat/Δlmdat [LmDAT NEO]; 2, Δlmdat/Δlmdat; 3, Δlmdat/Δlmdat [TbDAT NEO].
Fig 3. V5:_Tb_DAT localizes to the glycosome.
A V5-tagged version of _Tb_DAT was expressed in the null mutant Δtbdat/Δtbdat background. Immunofluorescence assay in the presence of anti-V5 monoclonal antibodies (ii) and glycosome specific antiserum [37] (iii). i, DIC; iv, overlap of ii and iii. The bar represents 10 μm.
Fig 4. Generation of a conditional null mutant of TbDAT in T. brucei.
(A) Wild-type organization of the TbDAT allele. (B) Genotype of the conditional null mutant Δtbdat/Δtbdat/TetO:V5:TbDAT that expresses a V5-tagged (grey oval) version of _Tb_DAT that is regulated by the TetO promotor (white oval) and inserted into the intergenic region of the ribosomal RNA locus. Both TbDAT genes were replaced with the resistance cassettes blasticidin (BSD, dark grey rectangle) and puromycin (PAC, light grey rectangle). (C) Agarose gel electrophoresis of PCR analysis of the conditional null mutant Δtbdat/Δtbdat/TetO:V5:TbDAT. The position of the primers used here is depicted in (A) and (B). (D) RT-PCR analysis of strain Δtbdat/Δtbdat/TetO:V5:TbDAT. RNA was isolated from strain Δtbdat/Δtbdat/TetO:V5:TbDAT grown in the presence (+) or absence (−; 10 days) of doxycycline (doxy) that was (+) or was not (−) subjected to the reverse transcriptase reaction (RT). Amplification of the 18S rRNA cDNA was used as a positive control. (E) Western blot analyses in the presence of anti-V5 (Thermo Fisher Scientific, Waltham, MA) to reveal V5:_Tb_DAT and arginase specific antibodies (ARG; [43]) as a loading control. Approximately 1x107 cell equivalent was loaded in each lane. The protein marker is shown on the left. The Western blot analysis was carried out twice and a representative experiment is shown.
Fig 5. _Tb_DAT is dispensable for normal growth but is important for survival during the stationary phase.
(A) Growth curves in the presence (black circles) or absence (white circles) of 1 μM doxycycline. (B) Growth curves in the presence (black circles, black triangles) or absence (white circles, white triangles) of 1 μM doxycycline. Dead cells were revealed by Zombie green staining. (C) Quantification of dead cells expressed in percentage of total cells as a function of time. (D) DIC of two days stationary phase of growth cells grown in the presence (left panel) or absence (right panel) of doxycycline. The scale bar represents 10 μm. (E) Growth curve of Δtbdat/Δtbdat/TetO:V5:TbDAT grown in the presence (black circles) or absence (white circles) of doxycycline in delipidated medium. (A)(B)(C)(E) Growth curves were performed at least twice in duplicate and standard deviations are shown.
Fig 6. _Tb_DAT is the main DHAPAT enzyme in procyclic trypanosomes.
(A) The GPAT and DHAPAT assays were performed with 0.2 and 1.5 mg proteins derived from whole cell lysates of cells grown in the presence or absence (10 days) of doxycycline, respectively. 100% of DHAPAT and GPAT activity corresponds to 5.48 nmol/minxmg and 1.31 pmol/minxmg, respectively. (B) Fatty acyl-CoA specificity of remaining DHAPAT activity in Δtbdat/Δtbdat/TetO:V5:TbDAT grown in the absence of doxycycline for 10 days. The values were normalized to the best fatty acyl-CoA donor oleoyl-CoA and 100% DHAPAT corresponds to 0.47 nmol/minxmg. (C) GPAT and DHAPAT activity quantification of Δlmgat/Δlmgat (Δ) and Δlmgat/Δlmgat [TbGAT NEO] (TbGAT) were carried out in the presence of 100 μM and 150 μM oleoyl-CoA, respectively. *, p value < 0.05. (D) DHAPAT assay with Δlmdat/Δlmdat (1, [25]), Δlmdat/Δlmdat/TbGAT (2), and Δlmdat/Δlmdat/LmDAT (3, [25]). The assay was performed as described in Materials and methods section in the presence of 150 μM oleoyl-CoA and 100% DHAPAT activity corresponds to 1.9 pmol/minxmg. *, p value < 0.05. (A)(B)(C)(D) The activity assays were carried out at least twice in duplicate and standard deviations are shown.
Fig 7. Contribution of _Tb_GAT and _Tb_DAT to glycerophospholipid biosynthesis.
_Tb_DAT acylates preferentially DHAP and is involved in the biosynthesis of ether and of ester glycerophospholipids (dotted arrow). In contrast, _Tb_GAT acylates both DHAP and G3P but is implicated mainly in ester glycerophospholipid biosynthesis (solid arrow) [31].
References
- Patnaik PK, Field MC, Menon AK, Cross GA, Yee MC, Butikofer P. Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms. Mol Biochem Parasitol. 1993;58(1):97–105. . - PubMed
- Smith TK, Butikofer P. Lipid metabolism in Trypanosoma brucei. Mol Biochem Parasitol. 2010;172(2):66–79. doi: 10.1016/j.molbiopara.2010.04.001 ; PubMed Central PMCID: PMCPMC3744938. - DOI - PMC - PubMed
- Zhang K, Beverley SM. Phospholipid and sphingolipid metabolism in Leishmania. Mol Biochem Parasitol. 2010;170(2):55–64. doi: 10.1016/j.molbiopara.2009.12.004 ; PubMed Central PMCID: PMCPMC2815228. - DOI - PMC - PubMed
- Coppolino MG, Dierckman R, Loijens J, Collins RF, Pouladi M, Jongstra-Bilen J, et al. Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ialpha impairs localized actin remodeling and suppresses phagocytosis. J Biol Chem. 2002;277(46):43849–57. doi: 10.1074/jbc.M209046200 . - DOI - PubMed
- Krauss M, Kukhtina V, Pechstein A, Haucke V. Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes. Proc Natl Acad Sci U S A. 2006;103(32):11934–9. doi: 10.1073/pnas.0510306103 ; PubMed Central PMCID: PMCPMC1567676. - DOI - PMC - PubMed
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources