Method development for the establishment of diverse microbes as advanced microbiome therapeutics

Carmen Sands

Research output: Book/ReportPh.D. thesis

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Abstract

In recent years the gut microbiome has become associated with an ever increasing range of health conditions. As a result there is a growing interest in treating the gut microbiome as a potential therapeutic target. One avenue by which the gut microbiome can be modulated is through the use of advanced microbiome therapeutics (AMTs). AMTs are a form of probiotic, comprising live microbes that have been engineered for a specific therapeutic function, in general producing a therapeutic biomolecule directly in the gut. Drug delivery by AMT has several advantages over traditional methods of drug delivery, such as localised delivery, which in turn allows lower doses to be administered. Furthermore, the unique
probiotic qualities of the AMT chassis can have therapeutic effects in their own right, which can be harnessed during AMT development.

In this thesis three methods are presented to aid in the development of future AMTs, across three manuscripts. The first of these methods is developed for the probiotic yeast Saccharomyces boulardii. Here, a method for assessing promoter activity in vivo is presented, together with an assessment of the translatability of various in vitro characterisation conditions to in vivo conditions. Characterisation of 12 promoters across 5 carbon sources shows that promoter expression is highly carbon-source dependent, while the translatability assessment found relative promoter expression in vivo to be strongly correlated with expression in sucrose.

The second method presented is developed for the gut bacterium and obligate anaerobe Anaerobutyricum soehngenii. Here, a method for the transfer of heterologous DNA via conjugal transfer is documented. The method utilises E. coli CA434 as a conjugal donor and results in a conjugation efficiency of 3.1x10−6. The rate of plasmid loss was also determined and was found to be 1% per generation on average.

The final manuscript of this thesis demonstrates how experimental and computational approaches can be blended to develop a method for predicting the behaviour of probiotic consortia. The study investigates 85 bacterial strains and the yeast S. boulardii. Pair-wise growth experiments between a single bacterial strain with S. boulardii identified 5 bacterial strains with positive growth interactions with S. boulardii. Species metabolic interaction analysis was successfully employed to predict 3- to 5-strain consortia compositions with potential positive interactions, which were validated in vitro. The study highlights the role of S. boulardii in enhancing positive inter-species metabolic interactions and increasing
anti-inflammatory effects in probiotic consortia.

Overall the work presented in this thesis contributes new knowledge and methods for the development of AMTs, a novel method by which to treat disease.
Original languageEnglish
Number of pages142
Publication statusPublished - 2023

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