Colin Dolphin

Beyond “detox”: defining endogenous roles of worm FMOs

Background

Flavin-monooxygneases (FMOs) are ‘mixed-function oxidases’ that reduce flavins, speed their reaction with oxygen, and stabilize a C4a-oxygen adduct long enough to use this reactive species to transfer an oxygen atom to a substrate. Due to involvement in drug and foreign compound metabolism FMOs have, along with CYP450s, been traditionally classified as ‘drug-metabolizing enzymes’. However, there is increasing evidence that these highly adaptable, ubiquitous enzymes, by catalyzing key steps in metabolic pathways, are also involved in conserved, endogenous activities in plants and animals. The Dolphin lab is investigating these in the model animal C. elegans.

C. elegans is a small (1mm) free-living, non-parasitic soil roundworm that feeds on microbes such as bacteria. C. elegans grows quickly – from embryo to adult in three days – is easy to culture, and can be stored frozen. Although primitive, C. elegans nonetheless shares many essential characteristics common to human biology. In addition, worms are ‘genetically tractable’ – the genome can be easily modified to create mutant strains. The worm (plus some scientists) has won the Nobel prize twice!

C. elegans has five discrete genes, fmo-1 to -5, each encoding a different FMO. We, and others, have discovered that some of these worm FMOs are associated with a variety of critical biochemical processes including hypoxic response and osmoregulation. It is likely that better understanding the functional roles of FMOs in C. elegans will also provide insight the roles – beyond foreign compound metabolism – FMOs play in human physiology and biochemistry. The lab is applying a range of genetic and biochemical methodologies to probe these relationships including –

• recombineering to build seamless fmo::reporter fusion constructs directly from fosmid-based genomic clones
• CRISPR-based genome editing to generate both fmo::reporter sequences at the endogenous loci and to create targeted, subtle mutations in fmo-x genes. Some of these mutations will copy those identified in human FMO3 to be the cause of the inherited disorder fish-odour syndrome (see below)
• LC-MS- and NMR-based metabolomic approaches to identify changes in the metabolome of worm strains lacking single fmo-x genes

Methods and techniques

Molecular biology & genetics: PCR, genetic crossing, DNA sequencing (Sanger & ‘next gen’), western blotting, recombineering, CRISPR-based genome editing, etc

Protein production: recombinant protein expression in E. coli and/or insect cells

Metabolomics: LC-MS- and NMR-based metabolomics of worm extracts

Fish-odour syndrome (trimethylaminuria): metagenomics of the intestinal microbiota

TMAu (“fish-odour syndrome”) is a debilitating metabolic disorder characterized by an objectionable fish malodour due to excessive trimethylamine (TMA) excretion. Although it is gaining recognition, TMAu has, for many years, been somewhat dismissed by the clinical community. However TMAu is not benign – sufferers are severely disabled by the powerful odour that permeates their lives. Many become socially withdrawn and display severe psychosocial reactions, including depression and suicidal tendencies.

TMA is derived from intestinal bacterial decomposition of two dietary precursors: TMA N-oxide (TMAO), present in seafood, and choline. Once generated in the lower intestine TMA is absorbed and subject to extensive, first-pass N-oxidation, catalyzed exclusively by hepatic flavin-monooxygnease 3 (FMO3), followed by excretion of the non-odorous TMAO. Two major forms of TMAu exist. TMAu1, characterized by early presentation, is due to homozygosity for inactivating FMO3 mutations. In addition, heterozygosity for a null FMO3 allele or homozygosity for reduced-function FMO3 alleles may result in transient TMAu1. In TMAu2, patients present with odour and high urinary TMA but lack (apparent) FMO3 mutations. The aetiology of TMAu2 likely involves a combination of bacterial overgrowth or dysbiosis, generating excess TMA, coupled with reduced FMO3 activity.

The study of those microorganisms that live in or on the human host has become a significant new avenue for biomedical research that is increasingly providing significant and novel insights into how these organisms influence both host health and well-being and promote pathogenic conditions. Many studies have focused on investigating the relationship between the intestinal microbiota and gastreointestinal disorders, including inflammatory bowel and neoplastic disease. As TMA production is generated solely via the host’s intestinal bacteria the relationship between the microbiota and TMAu is direct and linear. As such, the patient’s intestinal bacteria – more specifically those species responsible for TMA generation – become potential therapeutic targets in TMAu.

As a first step, we are planning to explore the compare and contrast the composition of the intestinal microbiota between TMAu patients and control subjects by applying ‘deep sequencing’ approaches to identify and partially functionally characterize faecal bacterial species. Both 16s rRNA phylogenetic and metagenomic approaches will be utilized.

At present funds are being sought for this project.

• Exploring the roles of flavin-monooxygenases using the model animal C. elegans
• Fish-odour syndrome: dissecting the role of the intestinal microbiota in trimethylaminuria
• Extrending CRISPR-based genome editing approaches