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 invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

Evaluation of a gelatin modified poly(ɛ-caprolactone) film as a scaffold for lung disease

Lung transplantation is a necessary step for the patients with the end-stage of chronic obstructive pulmonary disease. The use of artificial lungs is a promising alternative to natural lung transplantation which is complicated and is restricted by low organ donations. For successful lung engineering, it is important to choose the correct combination of specific biological cells and a synthetic carrier polymer. The focus of this study was to investigate the interactions of human lung epithelial cell line NCl-H292 that is involved in lung tissue development with the biodegradable poly(ϵ-caprolactone) before and after its chemical modification to evaluate potential for use in artificial lung formation. Also, the effect of polymer chemical modification on its mechanical and surface properties has been investigated. The poly(ϵ-caprolactone) surface was modified using aminolysis followed by immobilization of gelatine. The unmodified and modified polymer surfaces were characterized for roughness, tensile strength, and NCl-H292 metabolic cell activity. The results showed for the first time the possibility for NCI-H292 cells to adhere on this polymeric material. The Resazurin assay showed that the metabolic activity at 24 hours post seeding of 80% in the presence of the unmodified and greater than 100% in the presence of the modified polymer was observed. The roughness of the poly(ϵ-caprolactone) increased from 4 nm to 26 nm and the film strength increased from 0.01 kN to 0.045 kN when the material was chemically modified. The results obtained to date show potential for using modified poly(ϵ-caprolactone) as a scaffold for lung tissue engineering.

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

Investigations into selective metabolic aspects of bifidobacteria: carbohydrate metabolism, fatty acid biosynthesis and plasmid biology

The gastrointestinal tract (GIT) is a diverse ecosystem, and is colonised by a diverse array of bacteria, of which bifidobacteria are a significant component. Bifidobacteria are Gram-positive, saccharolytic, non-motile, non-sporulating, anaerobic, Y-shaped bacteria, which possess a high GC genome content. Certain bifidobacteria possess the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA) by a biochemical pathway that is hypothesised to be achieved via a linoleic isomerase. In Chapter two of this thesis it was found that the MCRA-specifying gene is not involved in CLA production in B. breve NCFB 2258, and that this gene specifies an oleate hydratase involved in the conversion of oleic acid into 10-hydroxystearic acid. Prebiotics are defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of bacteria in the colon. Key to the development of such novel prebiotics is to understand which carbohydrates support growth of bifidobacteria and how such carbohydrates are metabolised. In Chapter 3 of this thesis we describe the identification and characterisation of two neighbouring gene clusters involved in the metabolism of raffinose-containing carbohydrates (plus related carbohydrate melibiose) and melezitose by Bifidobacterium breve UCC2003. The fourth chapter of this thesis describes the analysis of transcriptional regulation of the raf and mel clusters. In the final experimental chapter two putative rep genes, designated repA7017 and repB7017, are identified on the megaplasmid pBb7017 of B. breve JCM 7017, the first bifidobacterial megaplasmid to be reported. One of these, repA7017, was subjected to an in-depth characterisation. The work described in this thesis has resulted in an improved understanding of bifidobacterial fatty acid and carbohydrate metabolism, Furthermore, attempts were made to develop novel genetic tools.

The relevance of bacterial isoprenoid biosynthetic pathways for host-microbe interactions

This thesis was undertaken to investigate the relevance of two bacterial isoprenoid biosynthetic pathways (Mevalonate (MVAL) and 2-C-methyl-D-erythritol 4-phosphate (MEP)) for host-microbe interactions. We determined a significant reduction in microbial diversity in the murine gut microbiota (by next generation sequencing) following oral administration of a common anti-cholesterol drug Rosuvastatin (RSV) that targets mammalian and bacterial HMG-CoA reductase (HMG-R) for inhibition of MVAL formation. In tandem we identified significant hepatic and intestinal off-target alterations to the murine metabolome indicating alterations in inflammation, bile acid profiles and antimicrobial peptide synthesis with implications on community structure of the gastrointestinal microbiota in statin-treated animals. However we found no effect on local Short Chain Fatty Acid biosynthesis (metabolic health marker in our model). We demonstrated direct inhibition of bacterial growth in-vitro by RSV which correlated with reductions in bacterial MVAL formation. However this was only at high doses of RSV. Our observations demonstrate a significant RSV-associated impact on the gut microbiota prompting similar human analysis. Successful deletion of another MVAL pathway enzyme (HMG-CoA synthase (mvaS)) involved in Listeria monocytogenes EGDe isoprenoid biosynthesis determined that the enzyme is non-essential for normal growth and in-vivo pathogenesis of this pathogen. We highlight potential evidence for alternative means of synthesis of the HMG-CoA substrate that could render mvaS activity redundant under our test conditions. Finally, we showed by global gene expression analysis (Massive Analysis of cDNA Ends (MACE RNA-seq) a significant role for the penultimate MEP pathway metabolite (E)-4-hydroxy-3-methyl-2-but-2-enyl pyrophosphate (HMBPP) in significant up regulation of genes of immunity and antigen presentation in THP-1 cells at nanomolar levels. We infected THP-1 cells with wild type or HMBPP under/over-producing L. monoctyogenes EGDe mutants and determined subtle effects of HMBPP upon overall host responses to Listeria infection. Overall our findings provide greater insights regarding bacterial isoprenoid biosynthetic pathways for host-microbe/microbe-host dialogue.

Multi-parametric metabolic assessment of cells under stress conditions

Glycolysis, glutaminolysis, the Krebs cycle and oxidative phosphorylation are the main metabolic pathways. Exposing cells to key metabolic substrates (glucose, glutamine and pyruvate); investigation of the contribution of substrates in stress conditions such as uncoupling and hypoxia was conducted. Glycolysis, O2 consumption, O2 and ATP levels and hypoxia inducible factor (HIF) signalling in PC12 cells were investigated. Upon uncoupling with FCCP mitochondria were depolarised similarly in all cases, but a strong increase in respiration was only seen in the cells fed on glutamine with either glucose or pyruvate. Inhibition of glutaminolysis reversed the glutamine dependant effect. Differential regulation of the respiratory response to FCCP by metabolic environment suggests mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function. At reduced O2 availability (4 % and 0 % O2), cell bioenergetics and local oxygenation varied depending on the substrate composition. Results indicate that both supply and utilisation of key metabolic substrates can affect the pattern of HIF-1/2α accumulation by differentially regulating iO2¬, ATP levels and Akt/Erk/AMPK pathways. Inhibition of key metabolic pathways can modulate HIF regulatory pathways, metabolic responses and survival of cancer cells in hypoxia. Hypoxia leads to transcriptional activation, by HIF, of pyruvate dehydrogenase (PDH) kinase which phosphorylates and inhibits PDH, a mitochondrial enzyme that converts pyruvate into acetyl-CoA. The levels of PDH (total and phosphorylated), PDH kinase and HIF-1α were analysed in HCT116 and HCT116 SCO2-/- (deficient in complex IV of the respiratory chain) grown under 20.9 % and 3 % O2. Data indicate that regulation of PDH can occur in a manner independent of the HIF-1/PDH kinase 1 axis, mitochondrial respiration and the demand for acetyl-CoA. Collectively these results can be applied to many diseases; reduced nutrient supply and O2 during ischemia/stroke, hypoglycaemia in diabetes mellitus and cancer associated changes in uncoupling protein expression levels.