Filling in gaps of Drosophila melanogaster urate degradation metabolic pathway using metabolomics approaches: towards the core metabolome of the fruit fly

The primary goal of systems biology is to integrate complex omics data, and data obtained from traditional experimental studies in order to provide a holistic understanding of organismal function. One way of achieving this aim is to generate genome-scale metabolic models (GEMs), which contain information on all metabolites, enzyme-coding genes, and biochemical reactions in a biological system. Drosophila melanogaster GEM has not been reconstructed to date. Constraint-free genome-wide metabolic model of the fruit fly has been reconstructed in our lab, identifying gaps, where no enzyme was identified and metabolites were either only produced or consume. The main focus of the work presented in this thesis was to develop a pipeline for efficient gap filling using metabolomics approaches combined with standard reverse genetics methods, using 5-hydroxyisourate hydrolase (5-HIUH) as an example. 5-HIUH plays a role in urate degradation pathway. Inability to degrade urate can lead to inborn errors of metabolism (IEMs) in humans, including hyperuricemia. Based on sequence analysis Drosophila CG30016 gene was hypothesised to encode 5- HIUH. CG30016 knockout flies were examined to identify Malpighian tubules phenotype, and shortened lifespan might reflect kidney disorders in hyperuricemia in humans. Moreover, LC-MS analysis of mutant tubules revealed that CG30016 is involved in purine metabolism, and specifically urate degradation pathway. However, the exact role of the gene has not been identified, and the complete method for gap filling has not been developed. Nevertheless, thanks to the work presented here, we are a step closer towards the development of a gap-filling pipeline in Drosophila melanogaster GEM. Importantly, the areas that require further optimisation were identified and are the focus of future research. Moreover, LC-MS analysis confirmed that tubules rather than the whole fly were more suitable for metabolomics analysis of purine metabolism. Previously, Dow/Davies lab has generated the most complete tissue-specific transcriptomic atlas for Drosophila – FlyAtlas.org, which provides data on gene expression across multiple tissues of adult fly and larva. FlyAtlas revealed that transcripts of many genes are enriched in specific Drosophila tissues, and that it is possible to deduce the functions of individual tissues within the fly. Based on FlyAtlas data, it has become clear that the fly (like other metazoan species) must be considered as a set of tissues, each 2 with its own distinct transcriptional and functional profile. Moreover, it revealed that for about 30% of the genome, reverse genetic methods (i.e. mutation in an unknown gene followed by observation of phenotype) are only useful if specific tissues are investigated. Based on the FlyAtlas findings, we aimed to build a primary tissue-specific metabolome of the fruit fly, in order to establish whether different Drosophila tissues have different metabolomes and if they correspond to tissue-specific transcriptome of the fruit fly (FlyAtlas.org). Different fly tissues have been dissected and their metabolome elucidated using LC-MS. The results confirmed that tissue metabolomes differ significantly from each other and from the whole fly, and that some of these differences can be correlated to the tissue function. The results illustrate the need to study individual tissues as well as the whole organism. It is clear that some metabolites that play an important role in a given tissue might not be detected in the whole fly sample because their abundance is much lower in comparison to other metabolites present in all tissues, which prevent the detection of the tissue-specific compound

Mapping an atlas of tissue-specific drosophila melanogaster metabolomes by high resolution mass spectrometry

Metabolomics can provide exciting insights into organismal function, but most work on simple models has focussed on the whole organism metabolome, so missing the contributions of individual tissues. Comprehensive metabolite profiles for ten tissues from adult Drosophila melanogaster were obtained here by two chromatographic methods, a hydrophilic interaction (HILIC) method for polar metabolites and a lipid profiling method also based on HILIC, in combination with an Orbitrap Exactive instrument. Two hundred and forty two polar metabolites were putatively identified in the various tissues, and 251 lipids were observed in positive ion mode and 61 in negative ion mode. Although many metabolites were detected in all tissues, every tissue showed characteristically abundant metabolites which could be rationalised against specific tissue functions. For example, the cuticle contained high levels of glutathione, reflecting a role in oxidative defence; the alimentary canal (like vertebrate gut) had high levels of acylcarnitines for fatty acid metabolism, and the head contained high levels of ether lipids. The male accessory gland uniquely contained decarboxylated S-adenosylmethionine. These data thus both provide valuable insights into tissue function, and a reference baseline, compatible with the FlyAtlas.org transcriptomic resource, for further metabolomic analysis of this important model organism, for example in the modelling of human inborn errors of metabolism, aging or metabolic imbalances such as diabetes

Relatively abundant polar metabolites in male accessory glands.

<p>*In source fragment for methylthioadenosine. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<1×10³).</p

Major metabolites in cuticle.

<p>(A) Relatively abundant polar metabolites in cuticle. Data are presented as a heat-map of peak areas, from red (>2×10⁷) to blue (<5×10³). (B) Relatively abundant lipids in cuticle. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<5×10³).</p

Relatively abundant lipids in crop.

<p>PA = phosphatidic acid. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<5×10³).</p

Extracted ion chromatograms showing GSH and its precursors extracted from Drosophila cuticle.

<p>Extracted ion chromatograms showing GSH and its precursors extracted from Drosophila cuticle.</p

Major metabolites in ovaries.

<p>(A) Relatively abundant polar metabolites in Drosophila ovaries. *In source GSH fragment formed. **In source GSH fragment formed. Data are presented as a heat-map of peak areas, from red (>2×10⁷) to blue (<5×10³). (B) Relatively abundant lipids in <i>Drosophila</i> ovary. PG = phosphoglycerol; GPG = glyceryl phosphoglycerol; PI = phosphoinositol; Cer = ceramide. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<1×10³).</p

Major metabolites in testes.

<p>(A) Testes relatively abundant polar metabolites. *Gives guanine as an in source fragment ion. **Isomeric with adenosine which but elutes earlier gives guanine as an in source fragment ion. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<5×10³). (B) Relatively abundant lipids in testes. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<1×10³).</p

Major metabolites in midgut.

<p>(A) Relatively abundant metabolites in midgut. Data are presented as a heat-map of peak areas, from red (>1×10⁷) to blue (<5×10³). (B) Relatively abundant lipids in the midgut. Hist = acyl histamine. Red = area >10⁷ Yellow area >2×10⁴ Blue <10³.</p