Sustained Generation of Nitric Oxide and Control of Mycobacterial Infection Requires Argininosuccinate Synthase 1

Abstract

Nitric oxide (NO) defends against intracellular pathogens but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with < 2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.

Differential Requirements for l-Citrulline and l-Arginine during Antimycobacterial Macrophage Activity

“…NO production and antimycobacterial function of control PDMs cultured in L-citrulline mirrored those of MFs cultured in L-arginine. However, Asl flox/flox ;Tie2-cre PDMs cultured in L-citrulline did not produce NO or decrease M. bovis BCG burden, similar to our previous report analyzing MFs lacking functional Ass1 (22). These data confirm that conversion of L-citrulline to L-arginine is required for L-citrulline-mediated antimycobacterial MF function.…”

“…The following morning, medium was aspirated, and fresh R-free C-DMEM was added with L-arginine and/or L-citrulline. In all experiments, initial culture in L-arginine containing C-DMEM did not result in appreciable amounts of extracellular or intracellular L-arginine or L-citrulline after reconstituting with R-free C-DMEM (data not shown), so this method was used to conform to previously published methods (19,20,22).…”

“…We recently described that mice unable to convert L-citrulline to L-arginine in their hematopoietic compartment produce less NO and are more susceptible to M. tuberculosis and M. bovis BCG infection (22). These data led us to ask whether synthesized L-arginine had similar metabolic consequences as imported L-arginine in mycobacteriainfected MFs.…”

Dietary fat sources differentially modulate intestinal barrier and hepatic inflammation in alcohol-induced liver injury in rats

“…ASS1 is a soluble enzyme responsible for a critical biochemical reaction in the citrulline-urea and nitric oxide cycles, which catalyzes the reversible ATP-dependent ligation of citrulline and aspartate to generate argininosuccinate, and is highly conserved among species (11,36). ASS1 is expressed in many tissues, with the highest levels in the liver and kidney (11).…”

Manipulation of the Mononuclear Phagocyte System by Mycobacterium tuberculosis

“…From the host perspective, NO production is arguably one of the most important consequences mediated by IFN-g polarization of macrophages, and mice deficient for NO production readily succumb to M. tuberculosis infection (Flynn et al 1993). Moreover, M1 macrophages display a "fail-safe" mechanism to sustain NO production based on citrulline recycling via ASS1 (argininosuccinate synthase), which M2 macrophages lack (Qualls et al 2012). Conversely, M2 macrophages with enhanced levels of ARG1 display a reduced NO production and increased of iron availability, arguing that the arginine metabolism influences nutrient deprivation mechanisms in place to limit microbial growth (Kahnert et al 2006;Cairo et al 2011).…”