scholarly journals Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma

2020 ◽  
Vol 295 (22) ◽  
pp. 7743-7752 ◽  
Author(s):  
Xiaohan Liang ◽  
Jianmin Cui ◽  
Xuke Yang ◽  
Ningbo Xia ◽  
Yaqiong Li ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Pyruvate Dehydrogenase ◽  
Acyl Carrier Protein ◽  
Pyruvate Dehydrogenase Complex ◽  
Acid Synthesis ◽  
Lytic Cycle ◽  
Growth Defect ◽  
Wide Range

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9–assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo. Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

On the Specificity of the Herbicide Chlorsulfuron in Intact Spinach Chloroplasts

1985 ◽  
Vol 40 (11-12) ◽  
pp. 917-918 ◽  
Author(s):  
Uwe Homeyer ◽  
D. Schulze-Siebert ◽  
G. Schultz
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Acid Synthesis ◽  
Spinach Chloroplasts ◽  
Transamination Reaction ◽  
Dehydrogenase Complex

Abstract In vitro incubation of intact spinach chloroplasts with 1 mᴍ Pyruvate was used to study the specificity of action of the herbicide Chlorsulfuron on the synthesis of valine, alanine and fatty acids. As a result, increasing concentrations of the herbicide strongly inhibited valine synthesis while fatty acid synthesis via pyruvate dehydrogenase complex (PDC) and alanine formation by transamination reaction was promoted.

scholarly journals The Two Functional Enoyl-Acyl Carrier Protein Reductases of Enterococcus faecalis Do Not Mediate Triclosan Resistance

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lei Zhu ◽  
Hongkai Bi ◽  
Jincheng Ma ◽  
Zhe Hu ◽  
Wenbin Zhang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Enterococcus Faecalis ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Content Type ◽  
Enoyl Acp Reductase ◽  
Triclosan Resistance

ABSTRACTEnoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. TheEnterococcus faecalisgenome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of anEscherichia colifabItemperature-sensitive mutant strain under nonpermissive conditions.In vitroassays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression ofE. faecalis fabKconfers toE. colihigh levels of resistance to the antimicrobial triclosan, deletion offabKfrom theE. faecalisgenome showed that FabK does not play a detectable role in the inherent triclosan resistance ofE. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion offabKgrow normally without fatty acid supplementation, whereasfabIdeletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs.IMPORTANCEThe finding that exogenous fatty acids support growth ofE. faecalisstrains defective in fatty acid synthesis indicates that inhibitors of fatty acid synthesis are ineffective in counteringE. faecalisinfections because host serum fatty acids support growth of the bacterium.

scholarly journals Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

2016 ◽  
Vol 84 (12) ◽  
pp. 3597-3607 ◽  
Author(s):  
Jiangwei Yao ◽  
Megan E. Ericson ◽  
Matthew W. Frank ◽  
Charles O. Rock
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Carrier Protein ◽  
Type Ii ◽  
Intracellular Growth ◽  
Content Type ◽  
Bacterial Fatty Acid

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogenListeria monocytogenesencode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype ofEscherichia colistrain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate ofL. monocytogenesin laboratory medium. Robust exogenous fatty acid incorporation was not detected inL. monocytogenesunless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth ofL. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth ofL. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth ofL. monocytogenes.

scholarly journals Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

1972 ◽  
Vol 128 (5) ◽  
pp. 1089-1096 ◽  
Author(s):  
H. Kather ◽  
M. Rivera ◽  
K. Brand
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Pentose Phosphate ◽  
Fat Cells ◽  
Pentose Phosphate Cycle ◽  
Wide Range

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-14C]-, [1-14C]- or [6-14C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of 14C from differently labelled [14C]glucose into CO2, fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.

scholarly journals Response of Bacillus subtilis to Cerulenin and Acquisition of Resistance

2001 ◽  
Vol 183 (10) ◽  
pp. 3032-3040 ◽  
Author(s):  
Gustavo E. Schujman ◽  
Keum-Hwa Choi ◽  
Silvia Altabe ◽  
Charles O. Rock ◽  
Diego de Mendoza
Keyword(s):  
Bacillus Subtilis ◽  
Fatty Acid ◽  
Reporter Gene ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Acid Synthesis ◽  
Covalent Modification ◽  
Chain Elongation ◽  
Growth Defect

ABSTRACT Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein (ACP) synthases. The Bacillus subtilis fabF (yjaY) gene (fabFb ) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which B. subtilis adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the fabF gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in fabF reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the B. subtilis fabF locus, and sequence analysis of the mutant fabF allele showed that a single base change resulted in the synthesis of FabFb[I108F]. The purified FabFb and FabFb[I108F] proteins had similar specific activities with myristoyl-ACP as the substrate. FabFb exhibited a 50% inhibitory concentration (IC50) of cerulenin of 0.1 μM, whereas the IC50 for FabFb[I108] was 50-fold higher (5 μM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain.

scholarly journals The regulation of glyceride synthesis in isolated white-fat cells. The effects of palmitate and lipolytic agents

1972 ◽  
Vol 128 (5) ◽  
pp. 1057-1067 ◽  
Author(s):  
E. D Saggerson
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acids ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Fat Cells ◽  
Glyceride Synthesis ◽  
Glucose Incorporation ◽  
High Concentrations ◽  
Stimulation Of

1. 0.5mm-Palmitate stimulated incorporation of [U-14C]glucose into glyceride glycerol and fatty acids in normal fat cells in a manner dependent upon the glucose concentration. 2. In the presence of insulin the incorporation of 5mm-glucose into glyceride fatty acids was increased by concentrations of palmitate, adrenaline and 6-N-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate up to 0.5mm, 0.5μm and 0.5mm respectively. Higher concentrations of these agents produced progressive decreases in the rate of glucose incorporation into fatty acids. 3. The effects of palmitate and lipolytic agents upon the measured parameters of glucose utilization were similar, suggesting that the effects of lipolytic agents are mediated through increased concentrations of free fatty acids. 4. In fat cells from 24h-starved rats, maximal stimulation of glucose incorporation into fatty acids was achieved with 0.25mm-palmitate. Higher concentrations of palmitate were inhibitory. In fat cells from 72h-starved rats, palmitate only stimulated glucose incorporation into fatty acids at high concentrations of palmitate (1mm and above). 5. The ability of fat cells to incorporate glucose into glyceride glycerol in the presence of palmitate decreased with increasing periods of starvation. 6. It is suggested that low concentrations of free fatty acids stimulate fatty acid synthesis from glucose by increasing the utilization of ATP and cytoplasmic NADH for esterification of these free fatty acids. When esterification of free fatty acids does not keep pace with their provision, inhibition of fatty acid synthesis occurs. Provision of free fatty acids far in excess of the esterification capacity of the cells leads to uncoupling of oxidative phosphorylation and a secondary stimulation of fatty acid synthesis from glucose.

scholarly journals Synthesis of fatty acids in the perfused mouse liver

1974 ◽  
Vol 142 (3) ◽  
pp. 611-618 ◽  
Author(s):  
D. Michael W. Salmon ◽  
Neil L. Bowen ◽  
Douglas A. Hems
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
De Novo ◽  
Saturated Fatty Acids ◽  
Carbon Sources ◽  
Acid Synthesis ◽  
Hepatic Glycogen ◽  
Total Rate ◽  
Glucose Carbon

1. Fatty acid synthesis de novo was measured in the perfused liver of fed mice. 2. The total rate, measured by the incorporation into fatty acid of3H from3H2O (1–7μmol of fatty acid/h per g of fresh liver), resembled the rate found in the liver of intact mice. 3. Perfusions with l-[U-14C]lactic acid and [U-14C]glucose showed that circulating glucose at concentrations less than about 17mm was not a major carbon source for newly synthesized fatty acid, whereas lactate (10mm) markedly stimulated fatty acid synthesis, and contributed extensive carbon to lipogenesis. 4. The identification of 50% of the carbon converted into newly synthesized fatty acid lends further credibility to the use of3H2O to measure hepatic fatty acid synthesis. 5. The total rate of fatty acid synthesis, and the contribution of glucose carbon to lipogenesis, were directly proportional to the initial hepatic glycogen concentration. 6. The proportion of total newly synthesized lipid that was released into the perfusion medium was 12–16%. 7. The major products of lipogenesis were saturated fatty acids in triglyceride and phospholipid. 8. The rate of cholesterol synthesis, also measured with3H2O, expressed as acetyl residues consumed, was about one-fourth of the basal rate of fatty acid synthesis. 9. These results are discussed in terms of the carbon sources of hepatic newly synthesized fatty acids, and the effect of glucose, glycogen and lactate in stimulating lipogenesis, independently of their role as precursors.

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