Key to the success of intracellular pathogens is the ability to sense and respond to a changing host cell environment. Macrophages exposed to microbial products undergo metabolic changes that drive inflammatory responses. However, the role of macrophage metabolic reprogramming in bacterial adaptation to the intracellular environment has not been explored. Here, using metabolic profiling and dual RNA sequencing, we show that succinate accumulation in macrophages is sensed by intracellular Salmonella Typhimurium (S. Tm) to promote antimicrobial resistance and type III secretion. S. Tm lacking the succinate uptake transporter DcuB displays impaired survival in macrophages and in mice. Thus, S. Tm co-opts the metabolic reprogramming of infected macrophages as a signal that induces its own virulence and survival, providing an additional perspective on metabolic host–pathogen cross-talk.

A transepithelial pathway delivers succinate to macrophages, thus perpetuating their proinflammatory metabolic state Graphical abstract Highlights d Succinate uptake is elevated in macrophages to perpetuate their pro-inflammatory state d Na + -dependent transporters mediate transepithelial succinate delivery into macrophages d Succinate concentrations are elevated in the serum and feces of IBD patients d Succinate-metabolizing bacteria are altered in IBD patients

Background In the kidney, low urinary citrate increases the risk for developing kidney stones, and elevation of luminal succinate in the juxtaglomerular apparatus increases renin secretion, causing hypertension. Although the association between stone formation and hypertension is well established, the molecular mechanism linking these pathophysiologies has been elusive. Methods To investigate the relationship between succinate and citrate/oxalate levels, we assessed blood and urine levels of metabolites, renal protein expression, and BP (using 24-hour telemetric monitoring) in male mice lacking slc26a6 (a transporter that inhibits the succinate transporter NaDC-1 to control citrate absorption from the urinary lumen). We also explored the mechanism underlying this metabolic association, using coimmunoprecipitation, electrophysiologic measurements, and flux assays to study protein interaction and transport activity. Results Compared with control mice, slc26a6 2/2 mice (previously shown to have low urinary citrate and to develop calcium oxalate stones) had a 40% decrease in urinary excretion of succinate, a 35% increase in serum succinate, and elevated plasma renin. Slc26a6 2/2 mice also showed activity-dependent hypertension that was unaffected by dietary salt intake. Structural modeling, confirmed by mutational analysis, identified slc26a6 and NaDC-1 residues that interact and mediate slc26a6's inhibition of NaDC-1. This interaction is regulated by the scaffolding protein IRBIT, which is released by stimulation of the succinate receptor SUCNR1 and interacts with the NaDC-1/slc26a6 complex to inhibit succinate transport by NaDC-1. Conclusions These findings reveal a succinate/citrate homeostatic pathway regulated by IRBIT that affects BP and biochemical risk of calcium oxalate stone formation, thus providing a potential molecular link between hypertension and lithogenesis.

IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl−-sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl− (Cl−in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl−in. Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl−in. The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl−in. Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl−in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl−in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3− secretion.

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IRBIT is a multifunctional protein that also controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters, including NBCe1‐B, CFTR and Slc26a6, by unknown mechanisms. With the ubiquitous NBCe1‐B, IRBIT interacts with the autoinhibitory domain (AID), exposing two cryptic GXXXP motifs to confer regulation by Cl−in. Here, we report that IRBIT‐dependent combinatorial dephosphorylation of S232/S233/S235 controls NBCe1‐B close and open states by determining autoinhibition and regulation by Cl−in. The pS233/pS235 conformer is fully active, no longer activated by IRBIT or inhibited by Cl−in. The pS232/pS235 conformer is similar to wild‐type NBCe1‐B, while the pS232/pS233 conformer is partially active, but retains inhibition by Cl−in. IRBIT then recruits the kinases SPAK and CaMKII to control phosphorylation of S65 and S12, respectively, which determine inhibition by Cl−in at the 32GXXXP36 and 194GXXXP198 motifs. Recruitment by IRBIT of PP1 and calcineurin dephosphorylates S65 and S12, respectively, and relieves inhibition by Cl−in. These findings reveal how multiple kinase/phosphatase pathways use multiple phosphorylation sites to fine tune a transport function, which has important implications for epithelial fluid and HCO3− secretion.Support or Funding InformationAll the work was supported by NIH fundingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cl− is the major anion and important osmolyte in most living cells and has a prominent role in cellular homeostasis and intracellular chloride (Cl−in) affect the activity of several transporters. IRBIT is a multifunctional protein that controls the activity of IP3 receptors and multiple Cl− and HCO3− transporters by unknown mechanism. In the case of the ubiquitous NBCe1‐B, IRBIT interacts with the NBCe1‐B N terminus autoinhibitory domain (AID), exposing two cryptic Cl‐interacting sites to confer regulation of NBCe1‐B by Cl−in. Here, we report that IRBIT do so by recruiting a panel of phosphatases and kinases that both regulate signaling by Cl−in and modulate NBCe1‐B conformation to open the transport pathway. Thus, IRBIT recruits the phosphatase PP1 and the kinase SPAK to control phosphorylation of S65 to control regulation of NBCe1‐B by Cl−in at a 32GXXXP36 site. IRBIT recruits the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of S12 to control regulation of NBCe1‐B by Cl−in at a 195GXXXP199 site. IRBIT then acts on S232, S233, S235 to generate multiple conformations with either dephosphorylated S232/S233, S232/S235 or S233/S235. Each conformation has different activation by IRBIT and regulation by Cl−in. S232/S233/S235 are conserved in several members of the NBC transporters. Mutation of serine to alanine resulted in a fully activated conformation, while mutation of serine to phosphomimetic aspartate inhibited transport and eliminated IRBIT mediated activation, suggesting that S232/S233/S235 determine whether NBC transporter is in an opened or closed conformation. These findings together with structural modeling of NBCe1‐B uncover an intricate mechanism by Cl−in signals through IRBIT‐mediated recruitment of kinases and phosphatases to regulate key transporters involved in epithelial fluid and electrolyte secretion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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