College of Science, Engineering and Food Science - Doctoral Theses
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Item Defining gut mediated metabolism for health and disease(University College Cork, 2024) Quilter, Karina; Joyce, Susan; Melgar Villeda, Silvia; Science Foundation IrelandWe investigated the overall concept that human produced and microbially modified bile acids could act as an indicator of health. In chapter 3 we verified disruptions to bile acid metabolism in a porcine model of metabolic syndrome towards cardiovascular disease, induced by diet. In chapter 4 we applied dietary means to address mild hypercholesterolemia and followed BA readouts to indicate health parameters, we determined that BAs could be used as indicators of return to health but only when acute fed conditions were initiated and tracked over a 6 hour time period. Furthermore we noted that baseline fold change in BAs could be applied to distinguish responder and non-responder directions in a clinical setting. In chapter 5 we examined the metabolic outputs relating to elite athletes through 3 intervention studies. Our indications were that a key subset of BAs -significant in westernized MetS and CVD induction- were reduced in elite athletes. We further noted that different sports elicited different correlations with both microbes and metabolites in these small cohorts. In chapter 6, the key BAs associated with MetS-CVD and their interchangeable intermediates were examined at the cellular level and shown to have different outcomes in cell organelle functionality and related gene expression systems depending on dietary lipid interventions. In all, certain BAs are convergent indicators of disease, they diversify in health, just like microbiome composition. A number of key BAs were identified that can indicate health and the push towards disease in this study.Item Gut-heart axis in a large animal model of metabolic syndrome and heart failure(University College Cork, 2024) Cluzel, Gaston; Caplice, Noel M.; Stanton, Catherine; SFI ManufacturingBackground The metabolic syndrome (MetS) is a pathological condition diagnosed as the combination of obesity with either hypertension, dyslipidaemia, or hyperglycaemia. MetS constitutes a deadly cocktail of cardiovascular risk factors that greatly increases patient mortality. Among the cardiovascular complications of MetS, heart failure with preserved ejection fraction (HFpEF) represents one of the greatest unmet clinical needs of the 21st century. Indeed, as HFpEF prevalence increases along with soaring MetS cases, current therapeutic strategies fail to prevent disease complications. Therefore, novel approaches are required. MetS and HFpEF are accompanied by a low-grade inflammation (LGI) state. LGI is characterised as a steady but lingering increase in circulating inflammatory factors. Inflammatory signalling is known for promoting structural and functional changes in the myocardium that may contribute to HFpEF. Thus, decreasing LGI may reduce HFpEF progression. While the precise origin of LGI is uncertain, the gut microbiome has recently emerged as a hidden organ with critical immune regulatory functions. Crucially, the gut microbiome is tightly connected to the intestinal barrier. In MetS and HFpEF, patients show alteration of the gut microbiome and of the intestinal barrier, a phenomenon called gut permeability. Gut permeability results in the translocation of bacterial antigens from the gut lumen to circulation. Circulating bacterial antigens are pro-inflammatory, that contributes to LGI and, indirectly, to cardiac structural changes and HFpEF. Therefore, therapeutic strategies aimed at the gut microbiome may effectively prevent HFpEF via reducing gut permeability and LGI. This concept is described as the “gut-heart” axis. The gut-heart axis constitutes a novel field of investigation in cardiometabolic disorders and may answer the urgent need for novel therapeutic strategies directed against HFpEF. However, more research is needed to characterise the mechanisms involved in gut-heart signalling. Aims of the project This thesis aimed at characterising the cardiac pathological mechanisms involved in gut-heart signalling, and determining whether they can be modulated by a microbiota-targeted treatment. Methodology In this project, gut-heart axis pathological signalling was characterised using a porcine model of MetS and HFpEF induced by Western diet (WD) and hypertensive corticosteroid salts (desoxycorticosterone acetate, DOCA). Then, to investigate the effects of a gut microbiome-targeted intervention on MetS and HFpEF, this model was supplemented with a synbiotic product combining soluble corn fibre and Lactobacillus mucosae. Inflammatory signalling associated with HFpEF structural changes was investigated in the four cardiac chambers. In particular, the project focused on the roles of tumour necrosis factor (TNF)-α, lipopolysaccharide (LPS) and NOD-like receptor family, pyrin domain containing 3 (NLRP3). These central inflammatory pathways may be key in transducing gut-originating LGI into cardiac pathological signalling in HFpEF. Results Upon WD and DOCA challenge, the porcine model constituted a clinically-relevant reproduction of MetS and HFpEF. MetS was characterised by increased body weight, severe hypertension, hypercholesterolemia, and hypertriglyceridemia. HFpEF was characterised by left atrium enlargement (LAE) and left ventricle hypertrophy (LVH). LAE was associated with tissue apoptosis, and LVH was accompanied by cardiomyocyte hypertrophy. Left atrium (LA) and left ventricle (LV) also had increased inflammatory activity with cardiac macrophage (Mφ) expansion, and activation of TNF receptor 1 (TNFR1), toll-like receptor 4 (TLR4), and NLRP3 pathways. Moreover, the increase in TNFR1, TLR4, and NLRP3 activity was colocalised with cardiac Mφ, microvascular endothelial cells, and cardiomyocytes. While no structural or pressure-induced changes were observed in right heart chambers, the right atrium and the right ventricle also exhibited prominent inflammatory signalling. Data not reported in this thesis indicated that the model exhibited LGI and features of gut permeability. Overall, the porcine model of MetS and HFpEF was characterised by inflammatory cardiac changes along with systemic and intestinal alterations. Synbiotic treatment of MetS pigs reduced LAE, LA cardiomyocyte apoptosis, and LVH, but did not affect MetS core parameters. These improvements in cardiac structural changes were associated with a reduction in cardiac Mφ expansion and in TNFR1, TLR4, and NLRP3 activity in all four cardiac chambers. Reductions in TNFR1, TLR4, and NLRP3 activity were colocalised within the cardiac Mφ, microvascular endothelial cells, and cardiomyocytes populations. Data not reported in this thesis also indicated that synbiotic treatment reduced LGI and gut permeability. Therefore, synbiotic treatment targeted at the gut microbiome reduced pathological signalling along the gut-heart axis, and effectively reduced cardiac structural changes associated with HFpEF. Discussion The porcine model of MetS and HFpEF stood out as a robust model for investigating gut-heart axis inflammatory signalling. The study also highlighted the central role of TNFR1, TLR4, and NLRP3 in driving structural changes in HFpEF through pro-apoptotic and pro-hypertrophic signalling. Crucially, synbiotic treatment targeted at the gut microbiota effectively reduced HFpEF-associated structural changes via reducing cardiac inflammatory signalling. Finally, while exempt of structural changes, the right heart reflected accurately and dynamically the systemic changes in gut-heart axis pathology and treatment. Conclusions Synbiotic targeting of the gut microbiome resulted in cardiac structural improvements in a clinically-relevant porcine model of MetS and HFpEF. This study demonstrates the critical role of gut-heart inflammatory signalling cardiometabolic disease progression.Item Unravelling the impact of early-life nutrition on microbiota-gut-brain axis signalling(University College Cork, 2024) Ratsika, Anna; Cryan, John; Codagnone, Martin; Science Foundation IrelandCurrent lifestyle habits such as aberrant diet are becoming disruptive to our health. Nutrition in early life is a key factor mediating immunity, neurodevelopment, and behavioural outcomes across the lifespan. Optimal nutrition in the first 1000 days of life, starting from preconception to early childhood, are essential for optimal growth, brain and immune system function. Growing lines of evidence suggest that gut microbiota play a role in immune system education, brain function and behaviour. Disruptions in the gut microbiota composition during critical periods of development perinatally might influence the developmental trajectory of the brain and the immune system, with implications for their function later in life. Although the effects of diet-induced microbiota alterations have been studied in the context of brain and immune system development, studies often overlook the interactions between these systems in critical time-windows of developmental opportunity. Given that external influences such as diet could be decisive for brain function there is a growing need for investigation of the effects of early-life nutrition on the microbiota-immune-brain axis during critical windows of development. In this thesis, I focused on the investigation of the influence of microbiota disruptions perinatally via 1. High-fat diet (HFD) during pregnancy and lactation, 2. C-section and 3. Early-life antibiotic administration on offspring brain and immune system development, and behaviour. Using a model of maternal HFD we disrupted the maternal microbiota, which led to increased potent neurotoxic metabolites in maternal circulation. The maternal-diet-induced microbiota disruption and associated metabolic signals had consequences for embryonic brain function and were associated with altered glutamate-related metabolites and genes in the fetal mouse brain and hyperactivity in adolescence specifically in the female offspring. Next, the impact of disruption of vertical microbiota transmission via C-section on the immune system education during weaning was investigated with outputs in the gut, the periphery and at the level of neuroimmune interactions in the juvenile brain. Our data shows that immune system priming in the gut and the periphery is aberrant in response to altered delivery mode, with implications for microglia activation in the male juvenile mouse brain. This highlights the timeframe around weaning as being a critical one for unmasking the enduring effects of early life microbiota disturbances. Lastly, the influence of postnatal early-life dietary intervention with human milk oligosaccharides (HMOs) and prebiotics was investigated on its ability to reverse the effects of antibiotic-induced microbiota depletion on brain transcriptome and behaviour in juvenile male mice. The combination of HMOs with prebiotics led to enhanced social recognition memory and learning and memory pathways in transcriptomic results in social brain areas, possibly via enriched abundance and function of the gut microbiota in male juvenile mice. Taken together, this work provides evidence that the diet and gut microbiota in critical windows of development regulate brain and immune system function. Moreover, we identified that dietary interventions targeting the gut microbiota in early life improve brain function and social behaviour, demonstrating that early-life gut microbiota is important for neurobehavioural outcomes.Item Human bifidobacterial isolates with a focus on glycan degradation and bacteriocin production by Bifidobacterium pseudocatenulatum(University College Cork, 2023) Sanchez Gallardo, Rocio; van Sinderen, Douwe; Cotter, Paul; Science Foundation Ireland; H2020 Marie Skłodowska-Curie ActionsBifidobacteria are commensal bacteria which inhabit the gastrointestinal tract of humans and other mammals, and to which various probiotic or health-promoting benefits have been attributed. Bifidobacterial strains can metabolize both host- and diet-derived glycans, and these metabolic abilities are critical for their establishment in the gut. Bifidobacteria are among the first colonisers of the neonatal gut, partially due to their ability to digest human milk oligosaccharides (HMOs), present in human milk. Members of the species Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium longum are commonly isolated from infant faecal samples and are among the most abundant species found in early life. As the infant changes its diet during weaning the microbiota evolves and therefore the bifidobacterial abundance, prevalence and composition will be subject to change. Infant-associated species become less abundant and other species such as Bifidobacterium adolescentis start to play a more prominent role as they are capable of digesting dietary plant-glycans, therefore being adult-associated strains. Some studies have indicated that Bifidobacterium pseudocatenulatum is a bifidobacterial species that remains constant across the lifetime of a person. Thus, it is expected of this species possesses an extensive enzymatic machinery to adapt to dietary changes. Chapter II of this thesis focuses on the isolation and genomic characterisation of bifidobacterial strains from breast milk. Optimization of a cultivation protocol allowed the recovery of viable Bifidobacterium from frozen human milk samples. Furthermore, the genomic characterization allowed a preliminary analysis of the potential of these strains as probiotic. Chapter III of this thesis describes the isolation and characterisation of 36 B. pseudocatenulatum strains from mother-infant dyads. Following an initial comparative genomic analysis of all strains we focussed on a selection of the B. pseudocatenulatum strains so as to explore their genomic diversity and functionality. Growth abilities of these strains on more than 30 carbohydrate substrates facilitated the correlation between the presence of specific GH-encoding genes and their ability to degrade certain glycans. A large proportion of the genomic diversity observed in these strains was shown to correspond to the presence of prophage-like elements. Chapter IV details the metabolism of LNT in the species B. pseudocatenulatum. Using a combination of transcriptomic and molecular biology techniques, the enzymatic machinery responsible for the degradation of LNT in B. pseudocatenulatum was elucidated. Furthermore, genes were identified that were shown to be responsible for certain B. pseudocatenulatum strains to degrade particular fucosylated HMOs, believed to be important for infant health. Chapter V focussed on the extracellular degradation of particular plant-glycans, in particular xylan and starch, by specific strains of B. pseudocatenulatum. The extracellular enzymes responsible for the degradation of these complex carbohydrates were identified and characterised in this set of novel strains. Furthermore, the distribution across the species of homologs of these key genes was analysed. Chapter VI describes the identification and characterisation of a gene cluster responsible for the production of a novel antimicrobial peptide, designated Pseudocin 196, by B. pseudocatenulatum MM0196. Pseudocin 196 was purified and tested for its antimicrobial activity against a range of bacteria. Pseudocin 196 is the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin and was shown to inhibit clinically relevant pathogens. The work presented in this thesis expands the knowledge of Bifidobacterium pseudocatenulatum taxon, being in particular focussed on various genomic and metabolic capabilities of this species. The findings of this thesis have generated insights into the physiological and ecological roles played by this bacterial species as a common member of the human microbiome. Understanding the symbiotic relationship between this bacterial species and its human host may generate rational approaches through the development of novel functional food products that help to support and maintain the gut homeostasis.Item Unravelling the role of Bacteroides and Phocaeicola in the human gut: exploring evolutionary dynamics, mother-to-infant vertical transmission, and functional characteristics(University College Cork, 2023) da Silva Morais, Emilene; Stanton, Catherine; Ross, R. Paul; Science Foundation Ireland; IFF - International Flavors & FragrancesThe human gut microbiota is composed of trillions of microrganims and plays a major role in human health and disease by performing a series of physiological functions, such as protection against pathogens, energy production, maintenance of intestinal barrier integrity, and regulation of the immune system. Bacteroides is one of the major genera present in the human gut. Bacteroides colonise the gut soon after birth in vaginally born babies and its abundance increases after weaning. However, different studies have shown that Bacteroides are missing or present in lower relative abundance in infants born by caesarean section (CS). Lower Bacteroides relative abundance has been associated with higher risk of atopic sensitization, especially in children of Asian heritage. Bacteroides and Phocaeicola have been linked to both health and disease. In the gut, Bacteroides is often associated with health benefits, but it can become an opportunistic pathogen in other body sites. Herein, this thesis explored a number of research topics aimed at gaining a better understanding of the role of Bacteroides and Phocaeicola in the human gut, such as production of metabolites, genetic and evolutionary features, vertical transfer, persistence and relative abundance of Bacteroides and Phocaeicola in early life. Characterization of a new species of Bacteroides and a potential Phocaeicola probiotic strain were also carried out. In Chapter 1, the current knowledge about the association of Bacteroides and Phocaeicola with health and disease was explored. Metabolic conditions associated with altered levels of these species, as well as their role as opportunistic pathogens and the challenges associated with the use of Bacteroides and Phocaeicola as probiotic were investigated. Numerous pre-clinical studies showed the benefits of strains of Bacteroides and Phocaeicola in various conditions, however, more clinical research is needed to evaluate if these benefits extend to humans. Chapter 2 explores Bacteroides and Phocaeicola in early life. Metagenomic analysis was carried out to compare the relative abundance of these species in vaginally delivered (VD) and CS-born infants during the first year of life. The presence of vertically transferred strains and strains that were persistent across different time points were also evaluated. Whole genome sequence (WGS) was carried out and the average nucleotide identity (ANI) was used to determine whether different isolates were the same strain. A blast comparison of the whole genome was also carried out. Metabolomics analyses and γ-aminobutyric acid (GABA) production was carried out in a subset of the isolates. Metagenomic analysis showed that Bacteroides are absent in CS children who were not exposed to antibiotics before birth, but present in CS-born children whose mothers were exposed to antibiotics during pregnancy and VD infants. Five groups of isolates that were vertically transferred and/or persistent across different timepoints were identified. The metabolomics profile showed that Bacteroides and Phocaeicola strains were able to produce organic acids and indole. On Chapter 3 a comparative analysis of the genomes of P. dorei and P. vulgatus were performed. Nearly 4000 assemblies were used to build the pan-genome of these species and to evaluate functional differences, like Carbohydrate-Active enZymes (CAZyme) content, antimicrobial resistance profile and mobile genetic elements. Both species have an open pan-genome and a similar antimicrobial resistance profile, but different CAZyme content, indicating they might have evolved to occupy different niches. We also hypothesised that P. dorei evolved from a larger than average subclade of P. vulgatus. Chapter 4 describes a novel Bacteroides species, Bacteroides sp. nov. (strain MSB163), which was isolated from a healthy mother 4 weeks after giving birth. The WGS, metabolomic profile, respiratory quinones, polar lipids, growth conditions and fermentation of different carbohydrate sources were analysed. Apart from differences in the genome, the main differences between MSB163 and its closest relatives, B. cellulosilyticus and B. intestinalis are the presence of phosphatidylglycerol on its cell membrane and the ability to ferment melezitose and sorbitol. In chapter 5, we characterize strain MSB026 (P. dorei), a bacteriocin producer and potential probiotic candidate. MSB026 was isolated from a VD infant, not exposed to antibiotics. WGS, growth conditions, 2’FL utilization, and production of compounds of interest were analysed. The production of health beneficial compounds, like GABA and SCFAs, associated with the strain’s technological robustness make it a potential probiotic candidate. Bacteroides and Phoceicola play a major role in the prevention and onset of different health conditions. They are present in the gut from the first days of life, producing numerous metabolites and interacting with the host in differen ways. The results outlined in this thesis expand on the knowledge of two important commensal genera of the human gut that have an important role in host health.