Phenotypic characterisation of wild type and mutant strains of adherent invasive Escherichia coli and Shigella, in vitro, ex vivo and in vivo models

  • Ryath Shaya Herz Al-Hemedawi

Student thesis: Doctoral Thesis


Crohn's disease (CD) is an inflammatory bowel disease (IBD) that is largely the result of aberrant immune responses, such as the increased production of proinflammatory cytokines including IL-1, TNF-ɑ, IFN- ɣ to microbial infections in genetically susceptible individuals.;Adherent-invasive Escherichia coli (AIEC) has been implicated in CD, as has Shigella sonnei due to the shared inflammatory characteristics and susceptible genetic background of Shigellosis and Crohn's disease patients, with both microorganisms showing resistance to antibiotics.;Resistance to antibiotics appears to be a factor in gastrointestinal diseases. Therefore, the present study sought to evaluate the key AIEC and Shigella sonnei virulence genes for their pathogenic roles and to address the problem of antibiotic resistance and also prevent the destruction of beneficial gut bacteria by targeting the dsbA gene (as one of the cardinal genes) using simple compounds such as the monoterpene, geraniol, which can inhibit the activity of bacteria rather than eradicate them.;This study employed a red lambda system to construct mutants of key virulence genes, such as dsbA, yadA, proQ, icsA and mxiD genes, in AIEC and Shigella. Murine cell line cultures in vitro (the RAW 264.7 macrophage cell line) and ex vivo (intestinal organoids derived from), were used together with in vivo (Galleria moth larvae) models to evaluate and ascertain the role of these genes in the pathogenesis of AIEC and Shigella.;The antimicrobial activities of geraniol were also evaluated by using the above models. Nitric oxide (NO) was estimated by measuring the production of nitrite using Griess reagent. Cell viability was assessed using MTT dye. The formation/growth rate of biofilms was also measured via a spectrophotometer to measure the optical density. Gentamicin protection assays were also used to determine the differences between wild type and mutated bacterial strains whereby RAW 264.7 cells were stimulated with cytokines in the presence of either geraniol or L-NAME.;The dsbA gene, shared by AIEC and Shigella sonnei, was one of the key genes found in both pathogens, so the deletion of this gene in the current study was found to have a major impact in terms of reducing bacterial virulence. However, although mutation of the yadA gene was not achieved in AIEC, the competitive assay found that yadA significantly reduced the death rate in moth larvae.;Moreover, Shigella sonnei genes for the Type III secretion system (TTSS), adherence, and RNA regulation also significantly reduced virulence. Geraniol was demonstrated to be effective in inhibiting bacterial infection and biofilm formation by AIEC and S. sonnei. Furthermore, it was found to reduce NO production caused by AIEC infection in RAW 264.7 cells, indicating control of inflammation characteristic of CD and shigellosis.;Differences in the reduction of adhesion were found between the dsbA mutant and wild-type strains for AIEC and Shigella in experiments using intestinal organoids directly infected. The co-culture of intestinal organoids with RAW 264.7 cells resulted in severe damage to the organoid cell structure and significant damage was also observed in response to LPS and cytokines such as TNF-ɑ and IFN-ɣ. This could have led to either the death of the organoids or protection of the organoid from bacterial induced damage when using geraniol treatment compared to L-NAME.;The data generated from these models and other experiments were consistent in that deletion of all the target genes were found to be important for virulence, with significant differences emerging between wild-type and mutated strains. This implies that these genes are important for bacterial virulence and pathogenesis in that the bacteria become harmless if they lose the genes by either mutation or inact
Date of Award26 Aug 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SupervisorJun Yu (Supervisor) & Ben Pickard (Supervisor)

Cite this