Dataset Information

Differential Requirements for L-Citrulline and L-Arginine during Antimycobacterial Macrophage Activity.

ABSTRACT: Microbicidal NO production is reliant on inducible NO synthase-mediated L-arginine metabolism in macrophages (M?s). However, L-arginine supply can be restricted by arginase activity, resulting in inefficient NO output and inhibition of antimicrobial M? function. M?s circumvent this by converting L-citrulline to L-arginine, thereby resupplying substrate for NO production. In this article, we define the metabolic signature of mycobacteria-infected murine M?s supplied L-arginine, L-citrulline, or both amino acids. Using liquid chromatography-tandem mass spectrometry, we determined that L-arginine synthesized from L-citrulline was less effective as a substrate for arginase-mediated L-ornithine production compared with L-arginine directly imported from the extracellular milieu. Following Mycobacterium bovis bacillus Calmette-Guérin infection and costimulation with IFN-?, we observed that M? arginase activity did not inhibit production of NO derived from L-citrulline, contrary to NO inhibition witnessed when M?s were cultured in L-arginine. Furthermore, we found that arginase-expressing M?s preferred L-citrulline over L-arginine for the promotion of antimycobacterial activity. We expect that defining the consequences of L-citrulline metabolism in M?s will provide novel approaches for enhancing immunity, especially in the context of mycobacterial disease.

SUBMITTER: Rapovy SM

PROVIDER: S-EPMC6432794 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Differential Requirements for L-Citrulline and L-Arginine during Antimycobacterial Macrophage Activity.

Rapovy Shannon M SM Zhao Junfang J Bricker Rebecca L RL Schmidt Stephanie M SM Setchell Kenneth D R KD Qualls Joseph E JE

Journal of immunology (Baltimore, Md. : 1950) 20150826 7

Microbicidal NO production is reliant on inducible NO synthase-mediated L-arginine metabolism in macrophages (MΦs). However, L-arginine supply can be restricted by arginase activity, resulting in inefficient NO output and inhibition of antimicrobial MΦ function. MΦs circumvent this by converting L-citrulline to L-arginine, thereby resupplying substrate for NO production. In this article, we define the metabolic signature of mycobacteria-infected murine MΦs supplied L-arginine, L-citrulline, or b ...[more]

PMID: 26311904

Similar Datasets

Project description:L-citrulline and L-arginine supplementation has been shown to have several beneficial effects on the cardiovascular system. Nitric oxide (NO) protects against the progression of atherosclerosis and is synthesized by nitric oxide synthase (NOS), which converts L-arginine (L-Arg) into L-citrulline (L-Cit). Our previous study revealed that chronic administration of a combination of L-Cit and L- Arg has a better therapeutic effect on high cholesterol-induced atherosclerosis in rabbits. We investigated how L-Arg and L-Cit affect endothelial function, aging and atherosclerosis. Following a 3-day stimulation of human umbilical venous endothelial cells (HUVECs) with high glucose (HG: 22 mM) and L-Arg (300 μM), L-Cit (300 μM) or L-Arg plus L-Cit (LALC: each 150 μM) supplementation, endothelial senescence and function were evaluated. These amino acids were also administered to dyslipidemic type 2 diabetic (ZDFM) rats fed a high cholesterol diet. They were fed L-Arg or L-Cit or LALC for four weeks. Aortic senescence was investigated by measuring senescence-associated ß-galactosidase (SA-ß-gal), telomerase activity, DNA damage and p16INK4a protein expression. Only L-Cit and LALC supplementation retarded the HG-induced endothelial senescence, as evaluated by SA-ß-gal activity, a widely used marker of cellular senescence, p16INK4a expression, a senescence-related protein, and DNA damage. Under HG conditions, L-Cit and LCLA restored telomerase activity to levels observed under normal glucose (NG) conditions. Under HG conditions, L-Cit decreased ROS production, as measured by CM-H2DCFDA and the expression of p67phox, a major component of NADPH oxidase. Under HG conditions, L-Cit and LALC increased NO production, as measured by DAF-2AM. Endothelial NO synthase (eNOS) and phosphorylated eNOS were decreased under HG conditions and L-Cit and LALC significantly increased these levels. Arginase 2 protein expression increased under the HG conditions, and L-Cit and LALC significantly attenuated this effect. In ZDFM rats, SA-ß-gal activity was detected on the aortic endothelial surface; however, L-Cit and LALC reduced these levels. L-Cit and LALC both decreased the proportion of senescent cells. Furthermore, treatment with LALC for 4 weeks increased plasma NO production. Therefore conclusively, L-citrulline supplementation rescued NO levels better than L-arginine supplementation by inhibiting ROS production and arginase 2 protein expression. Consequently, L-Cit and LCLA supplementation retaeded HG-induced endothelial senescence.

Project description:BackgroundImpaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia.Methodology/principal findingsTo study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot.Conclusion/significanceL-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Project description:Toward the creation of a robust and efficient producer of L-arginine and L-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92(up), and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (Delta argR), the production was further increased. The best mutation set, Delta argR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical L-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefold-increased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38 degrees C.

Dataset Information

Differential Requirements for L-Citrulline and L-Arginine during Antimycobacterial Macrophage Activity.

Publications

Differential Requirements for L-Citrulline and L-Arginine during Antimycobacterial Macrophage Activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets