A pathogen-specific isotope tracing approach reveals metabolic activities and fluxes of intracellular Salmonella

Abstract

Pathogenic bacteria proliferating inside mammalian host cells need to rapidly adapt to the intracellular environment. How they achieve this and scavenge essential nutrients from the host has been an open question due to the difficulties in distinguishing between bacterial and host metabolites in situ. Here, we capitalized on the inability of mammalian cells to metabolize mannitol to develop a stable isotopic labeling approach to track Salmonella enterica metabolites during intracellular proliferation in host macrophage and epithelial cells. By measuring label incorporation into Salmonella metabolites with liquid chromatography–mass spectrometry (LC–MS), and combining it with metabolic modeling, we identify relevant carbon sources used by Salmonella, uncover routes of their metabolization, and quantify relative reaction rates in central carbon metabolism. Our results underline the importance of the Entner–Doudoroff pathway (EDP) and the phosphoenolpyruvate carboxylase for intracellularly proliferating Salmonella. More broadly, our metabolic labeling strategy opens novel avenues for understanding the metabolism of pathogens inside host cells.

Extracellular ATP is an environmental cue in bacteria

“…While eATP is neither transported into E. coli 20,55 nor serves to phosphorylate bacterial molecules 20 , its components may be internalised after ATP hydrolysis. Indeed, carbon flux mapping in intracellular S. Typhimurium showed that ribose and nucleobases partly and fully originate from the host, respectively, rather than from de novo synthesis 80 . Altogether, our data reveal that the eATP response presents the characteristics of bacterial gene regulatory networks and metabolic fluxes.…”

Succinate utilisation by Salmonella is inhibited by multiple regulatory systems

“…An important element of the pathogenic lifestyle of S. Typhimurium involves the hijacking of macrophages, and the intracellular proliferation of the bacteria within Salmonella-containing vacuoles (SCVs) [7]. Recently, it has been discovered that macrophages undergo metabolic reprogramming during bacterial infection, leading to the build-up of tricarboxylic acid (TCA) cycle intermediates, including succinate [8,9]. This C 4 -dicarboxylate acts as an important proinflammatory molecule that is also involved in hypoxic and metabolic signalling [10,11].…”