Carbon metabolism of enterobacterial human pathogens growing in epithelial colorectal adenocarcinoma (Caco-2) cells - PubMed (original) (raw)
Carbon metabolism of enterobacterial human pathogens growing in epithelial colorectal adenocarcinoma (Caco-2) cells
Andreas Götz et al. PLoS One. 2010.
Abstract
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Glucose consumption by the host cells and the intracellular bacteria.
Infection of Caco-2 cells with the three enterobacterial wild-type strains was carried out as described in Materials and Methods for 6 and 8 h, respectively, using 200 µmol of 14C-labelled glucose per 20 ml culture medium. The columns show the amount of 14C-label (corresponding to the consumed 14C-glucose in µmol) incorporated into the total host cells with or without bacterial infection (hatched bars), into the separated host cells (light gray bars) and into the isolated bacteria (dark grey bars).
Figure 2. 13C-Incorporation into amino acids of the intracellular bacteria and the infected Caco-2 host cells (see also Table S3).
The colours (according to a quasi logarithmic scale) indicate the amount of 13C-incorporation in each amino acid. (A) _EIEC_-1 wild-type strain, the indicated mutants of this strain (lanes a-f), and the corresponding Caco-2 cells (g-l). Colours in (a-d)/(g-j) and (e,f)/(k,l) show 13C-incorporation into amino acids of slow and faster proliferating Caco-2 cells, respectively. (B) _EIEC_-2, isogenic glucose/glucose-6P uptake mutant (m-n), and the Caco-2 cells infected with these two strains (o-p). (C) Stm and the indicated mutants; (q-β) show the 13C-incorporation into amino acids of the bacterial strains and the infected Caco-2 cells in the same order as in (A). The small white stars mark values with high standard deviations (see also Tables S3 and S4). White boxes indicate amino acids that could not be detected.
Figure 3. 13C-Isotopologue distribution in the amino acids de novo synthesised in intracellular _EIEC_-1 (A), _EIEC_-2 (C) and Stm (D) replicating in slower proliferating Caco-2 cells as well as _EIEC_-1 (B) and Stm (E) replicating in faster proliferating Caco-2 cells (see text for details); Caco-2 cells infected with _EIEC_-1 (F) (coloured bars, see also Table S4).
The 13C-distribution of the glucose-6P uptake mutants is similar to wt and that of the glucose uptake mutant similar to the glucose/glucose-6P uptake mutants (Δ) and therefore not shown (but see Table S4).The coloured boxes indicate the percentage of the 13C-isotopologues (M+1 to M+9) of the different amino acids (right scales). The black bars indicate the overall amount of 13C-incorporation (in mol%) into amino acids of the wild-type strains (wt) and their corresponding glucose/glucose-6P uptake mutants (Δ) as well as in the Caco-2 cells, infected with wt and Δ strains (left scales). Note that two left scales were used in A-F to better differentiate the 13C-incorporation into the various de novo synthesised amino acids of the three intracellular bacteria and their mutants. * indicates that data from glucose uptake mutant are displayed. ** These data are not shown in the figure due to high standard deviations or missing data points (see Table S4).
Figure 4. 13C-Incorporation into amino acids of host cells (Caco-2) and intracellular bacteria (see also Table S5).
(A) 13C-labelled amino acids in Caco-2 cells (not infected) grown in the presence of 10 mM [U-13C6]glucose for 6h (lane a) and with infection by _EIEC_-2 (infection by _EIEC_-1 led to similar results) (lane b). (B) 13C-labelled amino acids in Caco-2 cells and bacteria when the Caco-2 cells were grown for 10 generations in the presence of 10 mM [U-13C6]glucose and then infected with the bacteria. The data shows the uptake of the 6 labelled host cell amino acids by the three intracellular bacterial wild-type strains (wt) and the corresponding glucose/glucose-6P uptake mutants (indicated as Δ) during the 6.5 or 8.5 h period of infection (lanes c-h). Labelled amino acids of the Caco-2 host cells 6.5 or 8.5 h after infection by the wild-type strains (wt) or their glucose/glucose-6P uptake mutants (Δ) (lanes i-n). Colours indicate the amount of 13C-incorporation (% 13C) according to a quasi logarithmic scale.
Figure 5. Proposed catabolic pathways and metabolite fluxes employed by the enterobacterial strains replicating in Caco-2 cells.
Amino acids (in part) de novo synthesised by the bacteria are encircled in green. The import of 13C-labelled amino acids from the host cell is indicated by arrows, the magnitude of which roughly reflects the relative amount of imported amino acids under the conditions (A) and (B). _EIEC_-1 wild-type strain and Stm follow the metabolic pathways shown in A (red arrows) which is mainly fed by glucose (indicated in yellow), while the glucose/glucose-6P uptake mutants essentially follow the metabolic pathways shown in B (red arrows) mainly fed by C3-substrates. _EIEC_-2 appears to combine the pathways of model A and B.
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