Tryptophan degradation as a systems phenomenon in inflammation - an analysis across 13 chronic inflammatory diseases.

BACKGROUND: Chronic inflammatory diseases (CIDs) are systems disorders that affect diverse organs including the intestine, joints and skin. The essential amino acid tryptophan (Trp) can be broken down to various bioactive derivatives important for immune regulation. Increased Trp catabolism has been observed in some CIDs, so we aimed to characterise the specificity and extent of Trp degradation as a systems phenomenon across CIDs. METHODS: We used high performance liquid chromatography and targeted mass spectrometry to assess the serum and stool levels of Trp and Trp derivatives. Our retrospective study incorporates both cross-sectional and longitudinal components, as we have included a healthy population as a reference and there are also multiple observations per patient over time. FINDINGS: We found reduced serum Trp levels across the majority of CIDs, and a prevailing negative relationship between Trp and systemic inflammatory marker C-reactive protein (CRP). Notably, serum Trp was low in several CIDs even in the absence of measurable systemic inflammation. Increases in the kynurenine-to-Trp ratio (Kyn:Trp) suggest that these changes result from increased degradation along the kynurenine pathway. INTERPRETATION: Increases in Kyn:Trp indicate the kynurenine pathway as a major route for CID-related Trp metabolism disruption and the specificity of the network changes indicates excessive Trp degradation relative to other proteogenic amino acids. Our results suggest that increased Trp catabolism is a common metabolic occurrence in CIDs that may directly affect systemic immunity. FUNDING: This work was supported by the DFG Cluster of Excellence 2167 "Precision medicine in chronic inflammation" (KA, SSchr, PR, BH, SWa), the BMBF (e:Med Juniorverbund "Try-IBD" 01ZX1915A and 01ZX2215, the e:Med Network iTREAT 01ZX2202A, and GUIDE-IBD 031L0188A), EKFS (2020_EKCS.11, KA), DFG RU5042 (PR, KA), and Innovative Medicines Initiative 2 Joint Undertakings ("Taxonomy, Treatments, Targets and Remission", 831434, "ImmUniverse", 853995, "BIOMAP", 821511).

Amino acid auxotrophies in human gut bacteria are linked to higher microbiome diversity and long-term stability.

Amino acid auxotrophies are prevalent among bacteria. They can govern ecological dynamics in microbial communities and indicate metabolic cross-feeding interactions among coexisting genotypes. Despite the ecological importance of auxotrophies, their distribution and impact on the diversity and function of the human gut microbiome remain poorly understood. This study performed the first systematic analysis of the distribution of amino acid auxotrophies in the human gut microbiome using a combined metabolomic, metagenomic, and metabolic modeling approach. Results showed that amino acid auxotrophies are ubiquitous in the colon microbiome, with tryptophan auxotrophy being the most common. Auxotrophy frequencies were higher for those amino acids that are also essential to the human host. Moreover, a higher overall abundance of auxotrophies was associated with greater microbiome diversity and stability, and the distribution of auxotrophs was found to be related to the human host's metabolome, including trimethylamine oxide, small aromatic acids, and secondary bile acids. Thus, our results suggest that amino acid auxotrophies are important factors contributing to microbiome ecology and host-microbiome metabolic interactions.

Regeneration of ergothioneine after reaction with singlet oxygen.

Ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. Important antioxidants are typically regenerated from their oxidized products, to prevent the interceptors from being lost after a single chemical reaction with a reactive oxygen species. However, no mechanism for the complete regeneration of ET has yet been uncovered. Here we define a non-enzymatic multi-step cycle for the regeneration of ET after reaction with singlet oxygen (1O2). All reaction steps were verified by density functional theory computations. Four molecules of GSH are used per turn to detoxify 1O2 to water. Pure 1O2 was generated by thermolysis at 37 °C of the endoperoxide DHPNO2. Addition of 1 mM ET to 10 mM DHPNO2 and 10 mM GSH increased the production of oxidized GSH (GSSG), measured by LC-MS/MS, by a factor of 26 (water) and 28 (D2O), respectively. In the same assay, the ring of ET alone was able to drive the cycle at equal speed; thus, the zwitterionic amino acid backbone was not involved. Our data suggest that ET reacts at least 4-fold faster with 1O2 than ascorbic acid. ET must now be viewed as tightly linked with the GSH/GSSG redox couple. The necessary thiol foundation is present in all mammalian and vertebrate cells, and also in all species that generate ET, such as cyanobacteria, mycobacteria, and fungi. Regeneration provides a decisive advantage for ET over other reactive, but non-recoverable, compounds. Our findings substantiate the importance of ET for the eradication of noxious 1O2.

Ergothioneine stands out from hercynine in the reaction with singlet oxygen: Resistance to glutathione and TRIS in the generation of specific products indicates high reactivity.

The candidate vitamin ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. However, the precise physiological role of ET is still unresolved. Here, we investigated in vitro the hypothesis that ET serves specifically to eradicate noxious singlet oxygen (1O2). Pure 1O2 was generated by thermolysis at 37°C of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide 1,4-endoperoxide (DHPNO2). Assays of DHPNO2 with ET or hercynine (= ET minus sulfur) at pH 7.4 were analyzed by LC-MS in full scan mode to detect products. Based on accurate mass and product ion scan data, several products were identified and then quantitated as a function of time by selected reaction monitoring. All products of hercynine contained, after a [4+2] cycloaddition of 1O2, a carbonyl at position 2 of the imidazole ring. By contrast, because of the doubly bonded sulfur, we infer from the products of ET as the initial intermediates a 4,5-dioxetane (after [2+2] cycloaddition) and hydroperoxides at position 4 and 5 (after Schenck ene reactions). The generation of single products from ET, but not from hercynine, was fully resistant to a large excess of tris(hydroxymethyl)aminomethane (TRIS) or glutathione (GSH). This suggests that 1O2 markedly favors ET over GSH (at least 50-fold) and TRIS (at least 250-fold) for the initial reaction. Loss of ET was almost abolished in 5mM GSH, but not in 25mM TRIS. Regeneration of ET seems feasible, since some ET products - by contrast to hercynine products - decomposed easily in the MS collision cell to become aromatic again.