Influence of Tryptophan Contained in 1-Methyl-Tryptophan on Antimicrobial and Immunoregulatory Functions of Indoleamine 2,3-Dioxygenase

<div><p>Indoleamine 2,3-dioxygenase (IDO) has been identified as an important antimicrobial and immunoregulatory effector molecule essential for the establishment of tolerance by regulating local tryptophan (Trp) concentrations. On the other hand, the immunosuppressive capacity of IDO can have detrimental effects for the host as it can lead to deleterious alterations of the immune response by promoting tolerance to some types of tumors. To suppress this disadvantageous IDO effect, the competitive inhibitor 1-Methyl-Tryptophan (1-MT) is being tested in clinical trials. However, it remains inconclusive which stereoisomer of 1-MT is the more effective inhibitor of IDO-mediated immunosuppression. While IDO enzyme activity is more efficiently inhibited by 1-L-MT in cell-free or <em>in vitro</em> settings, 1-D-MT is superior to 1-L-MT in the enhancement of anti-tumor responses <em>in vivo</em>.</p> <p>Here, we present new data showing that commercially available 1-L-MT lots contain tryptophan in amounts sufficient to compensate for the IDO-mediated tryptophan depletion <em>in vitro</em>. The addition of 1-L-MT abrogated IDO-mediated antimicrobial effects and permitted the growth of the tryptophan-auxotroph microorganisms <em>Staphylococcus aureus</em> and <em>Toxoplasma gondii.</em> Consistent with this, the tryptophan within 1-L-MT lots was sufficient to antagonize IDO-mediated inhibition of T cell responses. Mass spectrometry (MS) analysis revealed not only tryptophan within 1-L-MT, but also the incorporation of this tryptophan in bacterial and human proteins that were generated in the presence of 1-L-MT in otherwise tryptophan-free conditions. In summary, these data reveal that tryptophan within 1-L-MT can affect the results of <em>in vitro</em> studies in an L-stereospecific and IDO-independent way.</p> </div

Bacterial growth in tryptophan-free medium with additional L-tryptophan or 1-L-MT.

<p>(A) <i>Staphylococcus aureus</i> 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 <i>Staphylococcus aureus</i>. 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).</p

Sources of used substances.

*<p>based on elemental analysis.</p

Peptides identified in bacteria with LC-MS/MS.

<p>Total numbers and numbers of tryptophan (trp) containing peptides are given for protein samples from different bacteria grown either with L-tryptophan or 1-L-MT as unique available tryptophan source. For all analyses, tryptophan methylation was used as a variable modification, but none of the peptides identified contained methylated tryptophan.</p

1-L-MT serves as tryptophan replacement for <i>Toxoplasma gondii</i> and human T cells.

<p>(A) <i>Toxoplasma gondii</i> 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 <i>T. gondii</i> 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 <i>T. gondii</i> 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).</p

MS/MS spectra for a tryptophan-containing peptide from <i>S. aureus</i> cell lysate.

<p><i>S. aureus</i> was grown in the presence of L-tryptophan (A) or 1-L-MT (B). From the spectra shown, the peptide SVVIAYEPIWAIGTGK from <i>S. aureus</i> 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.</p

Peptides identified in human cells with LC-MS/MS.

<p>Total numbers and numbers for tryptophan (trp) containing peptides are given for protein samples from different human tumor cells grown either with L-tryptophan or 1-L-MT as the only available tryptophan source. For all analyses, tryptophan methylation was used as a variable modification, but none of the identified peptides contained methylated tryptophan.</p