Metabolites for which MIDs could be quantified, were reliable using our standard isolation protocol (S2E Fig), were used to determine pathway activity (Figs 3A and S3B), were used for isotope tracing (Fig 6), or were included in the metabolic model (Figs 4 and S4); n.d. stands for not determined.

Metabolites for which MIDs could be quantified, were reliable using our standard isolation protocol (S2E Fig), were used to determine pathway activity (Figs 3A and S3B), were used for isotope tracing (Fig 6), or were included in the metabolic model (Figs 4 and S4); n.d. stands for not determined.</p

Bacterial isolation and optimal sampling time point allow robust quantification of metabolites.

(A) Uninfected host RAW264.7 cells (denoted by “-”), and host cells infected with a replication-deficient mutant (ΔssaV), or the wt were separated at 20 hpi by SDS-PAGE alongside serially diluted STm grown in monoculture (dilutions denoted above blot). Immunoblot was performed using an antibody against the bacterial RecA protein (38 kDa) for the quantification of bacterial material among host material in the infection assay by densitometry (numbers (a.u.) on the blot) via a standard curve. The experiment was performed in biological duplicates (S1 Raw Image). (B) Enrichment/depletion after filtration, and washing/concentration applied to infected RAW264.7 cells. Fold protein levels relative to the level before the filtration are shown for bacteria (anti-RecA), cytosolic proteins (anti-GAPDH), mitochondria (anti-VDAC1), histones from nuclei (anti-H3), and the endoplasmic reticulum (ER, anti-Calreticulin). Mitochondria and nuclei were removed by the filtering step, and solubilized ER and cytosolic proteins by centrifuging and washing the bacterial-containing flow-through. Bars are the averages of triplicates. Quantification by densitometry from immunoblots (Methods). (C) Same as in (B) but with the bacterial enrichment protocol applied to infected HeLa cells. Using the same protocol, bacterial enrichment from HeLa cells is less strong compared to RAW264.7 cells, reflecting physiological differences between the 2 different cell lines. Bars are the averages from biological duplicates. (D) Same data as in Fig 2D, but ratios of peak areas of control over the sample are separately depicted here for each metabolite. The solid, dashed, and dotted lines indicate the height at which the control reaches 100%, 50%, or 20%, respectively, of the peak area of the sample. Data are representative of 2 independent experiments in infected host RAW264.7 cells. (E) Total fractional labeling enrichment (TFLE, n = 38) from bacterial monocultures fed with 50% labeled mannitol compared between the standard and the fast enrichment protocol. Green dots indicate metabolites with significantly different (adjusted p-value STm inside HeLa cells in a mtl-containing medium. Line is the average of biological triplicates. (I) Absolute metabolite concentrations in the cell culture medium during STm replication inside RAW264.7 cells and HeLa cells. Lines are the average of biological duplicates. The black line on the right side of each figure indicates the concentration range covered by the standard curve. (J) Fractional 13C labeling enrichment for 31 metabolites (their 244 isotopologues are compared) from bacteria isolated from RAW264.7 cells compared between 12 and 16 hpi (r is the Pearson correlation). Dots are averages from biological duplicates. The data underlying this figure can be found in S1 Data. (TIF)</p

Mannitol is not metabolized by host cells, but internalized and used by intracellular <i>S</i>Tm.

(A) Experimental concept: Can 13C-labeled mannitol, supplied to infected host cells, traverse across mammalian cell membranes without degradation and be taken up by intracellular STm (yellow)? (B) Fractional 13C enrichment of hexose-phosphates and alanine from RAW264.7 cells, determined 48 h after the addition of U-13C mannitol (mtl) or U-13C glucose (glc) into the glucose-containing cell culture medium (DMEM with 1 g/L glucose, Methods). 13C from labeled glucose is incorporated (labeled fraction present), but not from mannitol (no labeled fraction present). Graphs show averages from biological triplicates. (C) Mannitol metabolism in STm: Mannitol enters and is phosphorylated via MtlA; mannitol 1P (toxic when accumulating) is oxidized by MtlD to fructose 6P, where it enters glycolysis. The uptake and metabolization of mannitol are subject to glucose repression via the dephosphorylated phosphocarrier protein HPr, which enhances the activity of the repressor MtlR [32]. (D) Growth yield of STm wild type (wt), ∆mtlA, and ∆mtlD, in MOPS medium with amino acids (Methods), and with combinations of glucose (glc), glycerol (glyc), and mannitol (mtl), as the main carbon sources. Bars depict the averages of technical duplicates and data are representative of 2 independent experiments. (E) Intracellular STm wt and ∆mtlD isolated from RAW264.7 macrophages 20 hpi in a gentamicin protection assay (MOI = 100) supplemented +/− mannitol (mtl), and then serially diluted and spotted on an LB agar plate. The image is representative of 2 independent experiments in biological triplicates. The data underlying this figure can be found in S1 Data. glc, glucose; MOI, multiplicity of infection; STm, Salmonella Typhimurium.</p

Original high-resolution images of the western blot membrane depicted in S2 Fig, in 2 replicates.

The data underlying these images can be found in S1 Data. (PDF)</p

Mixing plots in model space (varying in reaction bidirectionalities, all replicates).

To certify that the replicates did not get stuck in different regions of model space, all replicates are shown. Each subplot represents 1 independent MCMC run. The plots show that the sampler mixes well in the model space and that the mixing is very reproducible for all 10 replicate chains. (A) Low glucose concentration, (B) high glucose concentration. (TIF)</p

Purines and pyrimidines have only their ribose unit labeled.

(A) Purine base structure and origin of C-atoms. The 13C-labeled C-atoms theoretically stem from formate, glycine, bicarbonate, or the ribose unit via the PPP. (B) Pyrimidine base structure and origin of C-atoms. The 13C-labeled C-atoms theoretically stem from aspartate, bicarbonate, and the ribose unit via the PPP. (C, D) Fractional 13C enrichment of AMP and UMP (nucleotides, with the ribose units) in bacteria isolated from RAW264.7 macrophages. Bars are averages of biological triplicates. Data are corrected for the natural 13C abundance and the 12C isotopic impurity of the fully labeled 13C mannitol. (E, F) Fractional 13C enrichment of adenine and uracil (without the ribose units) in bacterial RNA isolated from RAW264.7 macrophages. Bars are averages of biological duplicates. The data underlying this figure can be found in S1 Data. PPP, pentose phosphate pathway; TFLE, total fractional labeling enrichment.</p

Fractional 13C enrichment of <i>S</i>Tm isolated from RAW264.7 cells for low and high glucose medium.

Fractional 13C enrichment of STm isolated from RAW264.7 cells for low and high glucose medium.</p

Metabolite levels (a.u.) quantified from <i>S</i>Tm monocultures growing in MOPS glucose or mannitol medium.

Metabolite levels (a.u.) quantified from STm monocultures growing in MOPS glucose or mannitol medium.</p

PSRF from the Markov chains.

A value below 1.1 indicates proper convergence. (XLSX)</p

Fraction of forward and backward fluxes being active.

For each potentially bidirectional reaction, it is counted how often the forward and backward flux samples had non-zero values in the MCMC runs. If the value is above 95%/below 5% the direction is assumed to be active (denoted by “+”)/inactive (denoted by “-”), otherwise, the data is inconclusive (denoted by “?”). The principal direction is inferred from 95% net flux credible intervals. (XLSX)</p