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Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive  Escherichia coli  ( EIEC  HN280 and  EIEC  4608-58) and  Salmonella enterica  Serovar Typhimurium ( Stm  14028) replicating in epithelial colorectal adenocarcinoma cells (Caco-2). To this aim, we supplied [U- 13 C 6 ]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The  13 C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that  EIEC  HN280 growing in the cytosol of the host cells, as well as  Stm  14028 replicating in the  Salmonella -containing vacuole (SCV) utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates.  EIEC  4608-58 used C 3 -compound(s) in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C 3 -substrate(s) accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers) from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of  13 C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.

Carbon Metabolism of Enterobacterial Human Pathogens Growing in Epithelial Colorectal Adenocarcinoma (Caco-2) Cells