The role of the Kynurenine pathway in Caenorhabditis elegans gut bacterium interactions

Martin, Jack and Benedetto, Alexandre and Parry, Jackie (2023) The role of the Kynurenine pathway in Caenorhabditis elegans gut bacterium interactions. PhD thesis, Lancaster University.

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Abstract

Tryptophan is an essential amino acid whose main catabolic route is via the kynurenine pathway (KP), its metabolites are known for their immunomodulatory and neurotoxic effects. These metabolites are used by bacteria in quorum sensing and act as antibiotics. Inhibition of this pathway in C. elegans was found to increase lifespan, possibly through increasing tryptophan availability, however the role of its metabolite anthranilic acid (AA) which is stored within lysosome-related organelles (LROs) remains elusive. We hypothesise that these metabolites play a key role in the control of the C. elegans gut microbiota. Previous work in the lab showed that the growth of Enterococcus faecalis on KP metabolites was affected by many of the KP metabolites, with 3-hydroxykynurenine completely abrogating its growth. In addition, a pilot RNAseq study showed that on infection there was modulation of transcription of KP enzymes, with the upregulation of kmo-1 (encoding Kynurenine-monooxygenase 1 (KMO)) and downregulation of kynu-1 (encoding kynureninase (KYNU)) and haao-1 (encoding 3-hydroxyanthranilate 3,4-dioxygenase (HAAO). This change in transcription indicates a possible shift in metabolism leading to increased production of 3-HK. To test this hypothesis, we looked at changes in KP metabolites and the effect of mutation of the pathway on gut colonisation on infection with E. faecalis. In addition, we looked at the effect of KP metabolites on commensal bacterial growth as well as generating fluorescently tagged versions of these strains using a Tn7 transposon system to further study the effect of KP mutation on gut colonisation. To better study such interactions, we also endeavoured to develop 4 tools for us and the community by characterising commensal bacterial metabolism and beginning to develop imaging tools and methods. From this, we found that levels of KP metabolites are altered on infection with 3-HK levels increasing and that enzymes promoting resistance (kmo-1) or sensitivity (kynu-1) affected the ability for gut colonisation. The loss of kmo-1 causes an increase in E. faecalis colonisation and a loss of differential commensal gut colonisation. KP metabolites were also shown to differentially affect commensal bacterial growth. Together our results suggest that C. elegans adjusts its KP activity to regulate gut microbial populations. Showing that this is a method for controlling the colonisation of the gut and possibly linking to how bacteria within the gut evolved. Thus, supporting the eco-system on a leash model of microbial cooperation and evolution, with the host having an active investment in shaping the bacteria within its’ own microbiota.