Phenylketonuria (PKU) is a genetic disease characterized by the inability to convert dietary phenylalanine to tyrosine by phenylalanine hydroxylase. Given the importance of gut microbes in digestion, a genetically engineered microbe could potentially degrade some ingested phenylalanine from the diet prior to absorption. To test this, a phenylalanine lyase gene from Anabaena variabilis (AvPAL) was codon-optimized and cloned into a shuttle vector for expression in Lactobacillus reuteri 100-23C (pHENOMMenal). Functional expression of AvPAL was determined in vitro, and subsequently tested in vivo in homozygous PAHenu2 (PKU model) mice. Initial trials of two PAHenu2 homozygous (PKU) mice defined conditions for freeze-drying and delivery of bacteria. Animals showed reduced blood phe within three to four days of treatment with pHENOMMenal probiotic, and blood phe concentrations remained significantly reduced (P < 0.0005) compared to untreated controls during the course of experiments. Although pHENOMMenal probiotic could be cultured from fecal samples at four months post treatment, it could no longer be cultivated from feces at eight months post treatment, indicating eventual loss of the microbe from the gut. Preliminary screens during experimentation found no immune response to AvPAL. Collectively these studies provide data for the use of a genetically engineered probiotic as a potential treatment for PKU.

Mercury is a global contaminant, which may be microbially transformed into methylmercury (MeHg), which bioaccumulates. This results in potentially toxic body burdens in high trophic level organisms in aquatic ecosystems and maternal transfer to offspring. We previously demonstrated effects on developing fish including hyperactivity, altered time-to-hatch, reduced survival, and dysregulation of the dopaminergic system. A link between gut microbiota and central nervous system function in teleosts has been established with implications for behavior. We sequenced gut microbiomes of fathead minnows exposed to dietary MeHg to determine microbiome effects. Dietary exposures were repeated with adult CD-1 mice. Metabolomics was used to screen for metabolome changes in mouse brain and larval fish, and results indicate effects on lipid metabolism and neurotransmission, supported by microbiome data. Findings suggest environmentally relevant exposure scenarios may cause xenobiotic-mediated dysbiosis of the gut microbiome, contributing to neurotoxicity. Furthermore, small-bodied teleosts may be a useful model species for studying certain types of neurodegenerative diseases, in lieu of higher vertebrates.

The Pahenu2mutation in C57BL/6J mice is a well characterized model for studying phenylketonuria (PKU), with Pahenu2homozygotes displaying heightened blood phenylalanine and other characteristics of PKU. Pahenu2 homozygous females do not successfully rear young on any conventional diet due to their disease status. The most commonly used successful breeding strategy is crossing Pahenu2heterozygous females with homozygous mutant males; producing litters of 50% homozygous and 50% heterozygous animals. In many treatment studies the heterozygous pups produced are not useable, but add to experimental costs and total animals used. To this end our lab created a dietary regimen with reduced phenylalanine and increased large neutral amino acid content, enabling homozygous mating. Phenylalanine levels in homozygous females and males on the new diet are significantly lower than that of homozygous females and males on traditional diets (p = 1.35x10-4 and p = 1.5x10-5 respectively). Litters born to Pahenu2homozygous mothers on this diet demonstrate no significant difference in litter size compared to litters born to heterozygous mothers (p > 0.75). No observable defects were noted in litters born from homozygous crosses. This dietary regimen enables litter production of 100% Pahenu2homozygous animals for investigators who wish to rapidly expand their Pahenu2 mouse colony size or do not require heterozygous littermate controls in their PKU studies.

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