Figure 1.
1-L-MT treatment abolishes the IDO-mediated antibacterial effect.
HeLa cells were left unstimulated or stimulated with IFN-γ (500 U/mL) for 72 h. IFN-γ-stimulated cells were additionally treated with 1-L-MT or 1-D-MT (330 µg/mL each). Thereafter, cell supernatants were infected with S. aureus and bacterial growth was detected after additional 24 h (A) Cell culture supernatants were infected with bacteria and bacterial growth was determined photometrically. (B) The addition of L-tryptophan (L-trp) or 1-L-MT (C) to the supernatants at the time point of infection allowed bacterial growth in all experimental groups. Data are given as bacterial growth, determined by optical density at 620 nm, +/− SEM of three independent experiments with each experiment performed in triplicates. Asterisks indicate significant inhibition of bacterial growth (p<0.05).
Figure 2.
Bacterial growth in tryptophan-free medium with additional L-tryptophan or 1-L-MT.
(A) Staphylococcus aureus was cultured in medium with denoted supplementation of L-trp (black), D-trp (blue), 1-L-MT (red) or 1-D-MT (green) (100 µg/mL each). After 18 h, bacterial growth was determined photometrically. Asterisks indicate significant increase of bacterial growth compared to the 1-L-MT group (p<0.05). (B) Long-term culture of Staphylococcus aureus. Bacteria were cultured within tryptophan-free culture medium with or without L-tryptophan or 1-L-MT (100 µg/mL each) and bacterial growth within each culture was determined after 18 h photometrically. Thereafter, the bacterial culture was diluted in PBS serially and 10 µl were used to infect new tryptophan-free culture medium, according to 10–100 CFU/well. Asterisks mark no significant difference (p<0.05) in the 1-MT group, compared to tryptophan. (C) Different bacterial strains were grown in tryptophan-free medium with additional L-tryptophan or 1-L-MT (100 µg/mL each) for 18 h. Asterisks mark no significant difference (p<0.05) in the 1-MT group, compared to tryptophan.. All data are given as bacterial growth, determined by optical density at 620 nm, +/− SEM of triplicates of three independent experiments (A) or +/− SD of duplicates of one representative experiment (B, C).
Figure 3.
Analysis of 1-L-MT dissolved in tryptophan-free cell culture medium.
(A) Exemplary HPLC chromatogram (lot L24579) showing 1-L-MT peak (at retention time 7.6 min) and “contamination” peak (at retention time 4.2 min). (B) Exemplary HPLC chromatogram of 1-L-MT lot L24579 with supplemented L-tryptophan (L-trp). Note that the L-trp peak appears at the same retention time as the “contamination” peak in A. For both analyses 3-nitrotyrosine (retention time 3.5 min) served as internal control. (C) MS analysis of 1-L-MT lot L24579 dissolved in tryptophan-free cell culture medium. Additional to 1-L-MT with the m/z of 219, also tryptophan with the m/z of 205 was detected. n = 1. (D) Table showing the tryptophan contamination (µg/mg 1-MT) in different 1-MT lots. (E) Diagram revealing the growth of Staphylococcus aureus in tryptophan-free medium in presence of different 1-MT lots. Asterisks indicate that the bacterial growth is significantly higher in the presence of MKBF4000V as compared to L24579.
Figure 4.
MS/MS spectra for a tryptophan-containing peptide from S. aureus cell lysate.
S. aureus was grown in the presence of L-tryptophan (A) or 1-L-MT (B). From the spectra shown, the peptide SVVIAYEPIWAIGTGK from S. aureus Triosephosphate isomerase (Uniprot ID: A7WZS8) was identified with a mascot score of 63 (L-tryptophan) and 47 (1-L-MT). The peptide contains L-Tryptophan and not 1-L-MT in both samples. Fragment ions identifying the tryptophan residue are clearly visible and are marked with an asterisk.
Table 1.
Peptides identified in bacteria with LC-MS/MS.
Figure 5.
Impact of tryptophan contamination in 1-L-MT on kynurenine detection and determination of IDO activity.
(A) Measurement of kynurenine in the supernatant of IFN-γ-stimulated (1000 U/mL) 86HG39 glioblastoma cells cultured in IMDM medium with additional 100 µg/mL L-tryptophan for 72 h. During this stimulation period, the cells were treated with different 1-L-MT lots (grey bars) or 1-D-MT lots (shaded bars) (200 µg/mL each). (B) Kynurenine production within different 1-L-MT lots. 86HG39 cells were cultured in custom-made, tryptophan-free cell culture medium and stimulated with IFN-γ (1000 U/mL) for 72 h in the presence of 1-MT or L-trp (200 µg/mL). In both experimental groups the kynurenine content in the cell culture supernatants was determined by optical density at 492 nm +/− SEM, using Ehrlich's reagent. A significant inhibition of kynurenine production (A) and a significant increase in kynurenine production (B) (p<0.05) as compared to the negative control is marked with an asterisk (*) n = 3.
Figure 6.
1-L-MT serves as tryptophan replacement for Toxoplasma gondii and human T cells.
(A) Toxoplasma gondii growth in 86HG39 cells and (B) human T cell growth was observed in tryptophan-depleted medium in the presence of different amounts of L-tryptophan or 1-L-MT (0–300 µg/mL for T. gondii and 0–75 µg/mL for T cells). Parasites as well as human cells were unable to proliferate in the absence of tryptophan, but retained their ability to proliferate in the presence of L-tryptophan and, importantly, of 1-L-MT. Data are given as T. gondii growth, measured by [3H] uracil incorporation and as T-cell proliferation, measured by [3H] thymidine incorporation +/− SEM of three (T cells) or four (parasites) independent experiments with each experiment performed in triplicates. * = increased growth as compared to the negative control (p<0.05). ** = significant decrease as compared to the 0,75 µg/mL tryptophan group (p<0.05).
Figure 7.
MS/MS spectra for a tryptophan-containing peptide from 86HG39 cell culture lysate.
86HG39 cells were grown in the presence of L-tryptophan (A) or 1-L-MT (B). From the spectra shown, the peptide SADTLWDIQK from human L-lactate dehydrogenase B chain was identified with a mascot score of 54 in both samples. For cells grown in the presence of L-tryptophan or 1-L-MT, the spectra appear almost identical. Fragment ions identifying the tryptophan residue are clearly visible and are marked with an asterisk.
Table 2.
Peptides identified in human cells with LC-MS/MS.
Table 3.
Sources of used substances.