von Emloh, Louise
(2024)
Investigating the bile acid mediated control of Clostridioides difficile infection.
PhD thesis, University of Nottingham.
Abstract
Clostridioides difficile is a spore forming, anaerobic Gram-positive bacterium. If ingested, spores can cause C. difficile associated disease (CDAD), with symptoms ranging from diarrhoea to pseudomembranous colitis. Germination of spores in the gut is both induced and inhibited by primary and secondary hepatic bile acids, respectively, and the biotransformation between the two structures by 7α-dehydroxylation, encoded by bile acid inducible (bai) genes, is therefore key in the onset of CDAD. Colonic bacteria capable of 7α-dehydroxylation are limited and are mainly from the Clostridia family. These species, particularly Clostridium scindens, have been implicated in bile acid-mediated colonisation resistance against C. difficile. Unfortunately, research into C. scindens has been hindered by its genetic intractability.
This study investigated the use of genome editing tools in the native 7α-dehydroxylating species Peptacetobacter hiranonis, with the aim of generating loss-of-function mutations in bai genes. Gene transfer into P. hiranonis was established and current editing tools were assessed, but their lack of function required an expansion of the genetic toolkit in this novel organism. Reporter assays were established and utilised to harness the functionality of native promoters, in addition to the development of inducible promoters through the use of riboswitches. Whilst a reproducible CRISPR-Cas system was unable to be developed, a ΔbaiCD strain was produced and was shown to be incapable of 7α-dehydroxylation. This strain was further studied in in vitro competition assays with C. difficile to explore bile acid-mediated colonisation resistance.
Clostridium butyricum, a gut commensal, was chosen as a chassis strain to study the 7α-dehydroxylation pathway. The genome editing tools allele-exchange and RiboCas, an inducible CRISPR-Cas9 system, were first established in the strain and used to develop a triple auxotrophic knockout capable of cargo insertion at three loci. Mobilisation of the bai genes from C. scindens into C. butyricum was investigated, with expression driven by promoters characterised for use in C. butyricum in this study. Whilst it was not possible to do so for the full pathway, the bai genes suggested to be essential for 7α-dehydroxylation were inserted into the C. butyricum genome, and gene expression was confirmed. Bile acid-mediated inhibition of C. difficile germination by the strain was not demonstrated, but assessment of its ability to 7α-dehydroxylate is ongoing.
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