VIRK, BHUPINDER,KAUR (2013) Excessive folate synthesis in Escherichia coli and its influence on Caenorhabditis elegans ageing. Doctoral thesis, Durham University.
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A previous discovery showed that a spontaneous mutant in the aroD gene of the RNAi bacteria HT115(DE3), caused a significant increase in C. elegans longevity. In this thesis, I aimed to confirm this finding and attempt to determine whether chemical inhibition of folate synthesis in other E. coli strains would also increase C. elegans lifespan, and uncover the mechanism via which inhibited folate synthesis increases animal lifespan.
In order to confirm that the aroD mutant E. coli increase lifespan I investigated effects of using different media types. Data show that the aroD mutant E. coli shows variable effects on C. elegans lifespan with different media types due to the varying folate content of media used. I found that the aroD mutant E. coli effect on lifespan was maximal using a high purity agar with peptone. I then attempted to mimic the effect on other E. coli bacterial strains, such as OP50 commonly used as the E. coli food source for C. elegans, by using sulfamethoxazole as a chemical intervention to inhibit folate synthesis. Sulfamethoxazole increased C. elegans lifespan in a dose dependent manner. Interestingly, even though SMX is used as an antibiotic, the highest concentration used did not appear to decrease growth of the E. coli lawn substantially. Liquid chromatography coupled to mass spectrometry was used to measure and confirm the folate levels in both E. coli and C. elegans.
In order to uncover the specific mechanism by which inhibition of folate synthesis increases C. elegans lifespan, I needed to distinguish whether folate status of E. coli itself, folate status of C. elegans, or an interaction of folate status in both organisms was responsible for the increase in lifespan. The drug methotrexate (MTX) and the C. elegans gcp-2.1 mutant were used to investigate the effect of inhibiting the worm folate cycle, and impeding animal folate uptake. Results suggest that animal folate status does not play a role in the extended longevity. SMX and kanamycin have similar but not additive effects on lifespan, suggesting a common mechanism for both drugs. This is surprising as kanamycin prevents bacterial proliferation, whilst SMX does not. Bacterial accumulation assays suggested that bacterial accumulation may be a marker of ageing not a cause in this system.
Together the data presented here show that it may be possible to use chemical interventions to treat the mammalian gut flora, inhibiting excessive microbial folate synthesis and potentially slow ageing, without disrupting microbial ecology.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||C. elegans; E. coli; folate; SMX|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||15 Jul 2014 09:34|