Shining light on food microbiology; applications of luciferase-tagged microorganisms in the food industry

Bioluminescence is the production of light by living organisms as a result of a number of enzyme catalysed reactions caused by enzymes termed luciferases. The lux genes responsible for the emission of light can be cloned from one bioluminescent microorganism into one that is not bioluminescent. The light emitted can be monitored and quantified and will provide information on the metabolic activity, quantity and location of cells in a particular environment, in real-time. The primary aim of this thesis was to investigate and identify several food industry related applications of lux-tagged microorganisms. The first aim was to monitor a lux-tagged Cronobacter sakazakii in reconstituted infant milk formula, in realtime. The second aim was to investigate a bioluminescent-based early warning system for starter culture disruption by bacteriophages and antibiotic residues. The third of this thesis was to examine the use of a bioluminescent-based assay to test the activity of bioengineered Nisin derivatives M21V and S29A against foodborne pathogens in laboratory media and selected foods.

Investigating the role of Fas (CD95) signalling in the modification of innate immune induced inflammation

Background/Aim: It has been demonstrated that a number of pathologies occur as a result of dysregulation of the immune system. Whilst classically associated with apoptosis, the Fas (CD95) signalling pathway plays a role in inflammation. Studies have demonstrated that Fas activation augments TLR4-mediated MyD88-dependent cytokine production. Studies have also shown that the Fas adapter protein FADD is required for RIG-I-induced IFNβ production. As a similar signalling pathway exists between RIG-I, TLR3 and the MyD88- independent of TLR4, we hypothesised that Fas activation may modulate both TLR3- and TLR4-induced cytokine production. Results: Fas activation reduced poly I:C-induced IFNβ, IL-8, IL-10 and TNFα production whilst augmenting poly I:C-, poly A:U- and Sendai virus-induced IP-10 production. TLR3-, RIG-I- and MDA5-induced IP-10 luciferase activation were inhibited by the Fas adapter protein FADD using overexpression studies. Poly I:C-induced phosphorylation of p-38 and JNK MAPK were reduced by Fas activation. Overexpression of FADD induced AP-1 luciferase activation. Point mutations in the AP-1 binding site enhanced poly I:C-induced IP- 10 production. LPS-induced IL-10, IL-12, IL-8 and TNFα production were enhanced by Fas activation, whilst reducing LPS-induced IFNβ production. Absence of FADD using FADD-/- MEFs resulted in impaired IFNβ production. Overexpression studies using FADD augmented TLR4-, MyD88- and TRIF-induced IFNβ luciferase activation. Overexpression studies also suggested that enhanced TLR4-induced IFNβ production was independent of NFκB activation. Conclusion: Viral-induced IP-10 production is augmented by Fas activation by reducing the phosphorylation of p-38 and JNK MAPKs, modulating AP-1 activation. The Fas adapterprotein FADD is required for TLR4-induced IFNβ production. Studies presented here demonstrate that the Fas signalling pathway can therefore modulate the immune response. Our data demonstrates that this modulatory effect is mediated by its adapter protein FADD, tailoring the immune response by acting as a molecular switch. This ensures the appropriate immune response is mounted, thus preventing an exacerbated immune response.