The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wildtype mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2 ؊/؊ mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.-Krumpochova, P., Sapthu, S., Brouwers, J. F., de Haas, M., de Vos, R., Borst, P., van de Wetering, K. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays. FASEB J. 26, 738 -747 (2012). www.fasebj.org Key Words: metabolomics ⅐ LC/MS ⅐ phytoestrogens ⅐ knockout mice ATP-binding cassette (ABC) transporters translocate a wide variety of compounds through membranes at the cost of ATP hydrolysis and have important roles in human physiology, pathology, toxicology, pharmacology, and disease (1). The functions of several ABC transporters are incompletely understood, due to the lack of knowledge about the substrates transported in vivo.ABCC2 [also known as multidrug resistance protein 2 (MRP2)] is a multispecific organic anion transporter, and its substrates include several anionic drugs, bile acids, and compounds conjugated to glutathione-, sulfate-, or glucuronic acid (i.e., phase II metabolites; refs. 1-4). ABCC2 has an apical subcellular localization and is present in the body at sites of uptake and elimination, such as enterocytes, hepatocytes, and cells of the proximal tubules of the kidney (2). Its apical localization in these cells allows ABCC2 to mediate hepatobiliary and urinary elimination and to function as a barrier to uptake from the gut lumen. ABCC2 function is missing in patients with the Dubin-Johnson syndrome (5-8), which leads to the impaired secretion of bilirubinglucuronides into bile and results in conjugated hyperbilirubinemia. Despite the identification of several ABCC2 substrates, its substrate spectrum remains incompletely characterized. Vesicular transport by ABC transporters in inside-out vesicles is often used to characterize their substrate specificity. This approach has several drawbacks, however: one needs upfront hypotheses about the compounds transpo...

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Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis. Despite widespread vaccination, its incidence has been rising alarmingly, and yet, the physiology of B. pertussis remains poorly understood. We combined genome-scale metabolic reconstruction, a novel optimization algorithm, and experimental data to probe the full metabolic potential of this pathogen, using B. pertussis strain Tohama I as a reference. Experimental validation showed that B. pertussis secretes a significant proportion of nitrogen as arginine and purine nucleosides, which may contribute to modulation of the host response. We also found that B. pertussis can be unexpectedly versatile, being able to metabolize many compounds while displaying minimal nutrient requirements. It can grow without cysteine, using inorganic sulfur sources, such as thiosulfate, and it can grow on organic acids, such as citrate or lactate, as sole carbon sources, providing in vivo demonstration that its tricarboxylic acid (TCA) cycle is functional. Although the metabolic reconstruction of eight additional strains indicates that the structural genes underlying this metabolic flexibility are widespread, experimental validation suggests a role of strain-specific regulatory mechanisms in shaping metabolic capabilities. Among five alternative strains tested, three strains were shown to grow on substrate combinations requiring a functional TCA cycle, but only one strain could use thiosulfate. Finally, the metabolic model was used to rationally design growth media with >2-fold improvements in pertussis toxin production. This study thus provides novel insights into B. pertussis physiology and highlights the potential, but also the limitations, of models based solely on metabolic gene content.IMPORTANCE The metabolic capabilities of Bordetella pertussis, the causative agent of whooping cough, were investigated from a systems-level perspective. We constructed a comprehensive genome-scale metabolic model for B. pertussis and challenged its predictions experimentally. This systems approach shed light on new potential host-microbe interactions and allowed us to rationally design novel growth media with >2-fold improvements in pertussis toxin production. Most importantly, we also uncovered the potential for metabolic flexibility of B. pertussis (significantly larger range of substrates than previously alleged; novel active pathways allowing growth in minimal, nearly mineral nutrient combinations where only the carbon source must be organic), although our results also highlight the importance of strain-specific regulatory determinants in shaping metabolic capabilities. Deciphering the underlying regulatory mechanisms appears to be crucial for a comprehensive understanding of B. pertussis's lifestyle and the epidemiology of whooping cough. The contribution of metabolic models in this context will require the extension of the genome-scale metabolic model to integrate this regulatory dimension.

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