APC Microbiome Ireland - Doctoral Theses
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Item Discovery and characterisation of bacteriocins from microbiomes(University College Cork, 2024) Hourigan, David; Ross, R. Paul; Hill, Colin; Science Foundation IrelandBacteria are social microorganisms that inhabit every corner of the world. They rarely live in isolation and are key members of the microbiome along with viruses, archaea, protozoa and algae. Microbiome research has exploded over the past 20 years, but we are only starting to disentangle the complexity and dynamic nature it holds. Bacteriocins are antimicrobial peptides produced by bacteria across the bacterial kingdom. They have gained attention as a suitable alternative or adjunct to traditional antibiotics due to low levels of documented resistance. However, the roles they play in shaping the microbiome or acting as selfish genetic elements are often overlooked when studying them through such a narrow scope. While it is widely accepted that these peptides can be exploited to target specific bacterial species, their abundance and diversity is often under explored. These factors will have a huge impetus on their suitability to be used as antimicrobials. In Chapter 1, we perform a literature review describing recent advancements in enterococcal genomics and how it’s advancements can aid in therapeutic strategy. Clinically problematic Enterococcus faecium are promiscuous with respect to horizontal gene transfer and are becoming a global healthcare issue. The ability to expand its own genome and rapidly mutate has made this bacterium resistant to multiple antibiotics. Therefore we explore non-antibiotic interventions, including bacteriocins, to tackle these pathogenic bacteria. However, in Chapter 2 we show that E. faecium APC1031 rapidly becomes resistant to the potent class IIa bacteriocin avicin A. We generated mutants at a frequency of approximately 1e-06 suggesting that class IIa bacteriocins alone may not be suitable to target such a “hardy” and adaptable bacterium. Over the past century bacteriocin research has been skewed towards the Bacillota. However, the Actinomycetota are gathering interest as an underexploited bacteriocin-producing phylum. In Chapter 3 we discover novel aureocin A53-like bacteriocins found within this phylum. We also show they are found in microbiomes. We then synthetise the first aureocin A53-like bacteriocins from Actinomycetota and show that they have antimicrobial activity. Chapter 4 explores the rumen microbiome as a source of novel bacteriocins by genome mining the Hungate1000 culture collection of rumen strains. We discover that between 30-70% of bacteria found in the rumen produce at least one bacteriocin which is more than double previous estimates of the bacteria found in mammalian gastrointestinal tract. Nisin is the most well studied bacteriocin. It is gathering attention as a suitable alternative to antibiotics. However, little is known of how widespread the genes responsible for its production are. In Chapter 5 we discover widespread nisin-like bacteriocin production genes and show they are on mobile genetic elements and present in bacteria of aetiological concern. We also show that some of these peptides can be heterologously expressed and that they have antimicrobial activity. We then show that nisin VP is a novel nisin variant from an anaerobic bacterium isolated from the pig gut. Chapter 6 shows that two Streptococcus devriesei strains have the genetic capability to produce a conserved novel circular bacteriocin. This novel bacteriocin streptocyclin BTW is an amylocyclicin-like bacteriocin found within the genus streptococcus. Both strains with the genetic capacity to produce this bacteriocin are found in the oral microbiota. The role of bacteriocins as antimicrobial peptides is a well-studied trait. However, little is known of the genetic systems that localise near them within genomes. In Chapter 7, we search for genes encoding protein families that have co-localised near lanthipeptide biosynthetic gene clusters and discover an enrichment of anti-phage defence systems. This suggests lanthipeptide production may be attribute to bacteriophage-bacteria interactions. This thesis explored the diversity of bacteriocin gene clusters that are found in microbiomes. We have shown that these peptides have antimicrobial activity in vitro. However, their abundance and diversity implies they play multiple roles in complex microbiomes.Item Novel insights into the role of the gut microbiome on hippocampal synaptic plasticity(University College Cork, 2024) Collins, Michael K.; Cryan, John; O'Riordan, Ken; Science Foundation Ireland; Irish Research CouncilSynaptic plasticity especially within the hippocampus is a central process of the learning machinery of the brain. The microbiota gut-brain axis has emerged as an important regulator of hippocampal function yet there exists a paucity of information on how and when this axis can affect hippocampal synaptic plasticity. New strides need to be taken to develop successful interventions for human diseases which feature dysregulation of synaptic plasticity and to understand when normal synaptic plasticity may be susceptible to altered regulation from the microbiota-gut brain axis. In this thesis we focused primarily on how adult synaptic plasticity in the hippocampus may be affected by microbiome interventions. We also assessed the microbiome’s role in altering the developmental trajectory of the hippocampal transcriptome with a special focus on plasticity-related pathways. Firstly, we used germ free mice to examine how growing up without a gut microbiome has altered synaptic plasticity with hippocampal slice electrophysiology. While basal synaptic efficacy and pre-synaptic short-term plasticity appear normal, we found a deficit of hippocampal long-term potentiation in slices from male germ-free mice whereas slices from female animals produced this plasticity process relatively normally. However, the spike output of these neuronal populations remained normal, possibly indicating an altered excitation-inhibition balance or a homeostatic compensatory mechanism. Following this, we began to interrogate whether the microbiota’s ability to alter synaptic plasticity is developmentally wired or remains susceptible in adulthood. To this end we depleted the microbiome in adult life using either an antibiotic cocktail or a single antibiotic. This two-week microbiota intervention in adulthood produced only subtle effects on short-term plasticity and left LTP unchanged, demonstrating the robustness of hippocampal plasticity to the effects of microbiome depletion in adulthood. We then investigated how a local exposure to short-chain fatty acids (SCFAs), an abundantly produced class of microbial metabolite, may affect hippocampal synaptic plasticity in adulthood. Three SCFAs were tested, acetate, propionate, and butyrate at physiologically relevant concentrations. While no effect of acetate or propionate was found, butyrate-treated slices produced a greater degree of LTP in slices from both male and female mice. We hypothesized this effect was occurring through butyrate’s action at free fatty acid receptor 3 (FFAR3). However, when slices were pre-treated with an inhibitor of this receptor only slices from female mice reverted to producing normal levels of LTP, showing once again a sex-specific response to microbiome signals. Finally, we returned to the GF mouse model, examining four putative critical windows in early life, to establish how the microbiota changes the hippocampal transcriptome and metabolome. Metabolomic analysis revealed post-natal day (P) 21 to be the most differentially regulated in GF mice regardless of sex. GF status also significantly altered the hippocampal transcriptome with effects culminating at the oldest examined timepoint, P21. This effect was mirrored in a targeted enrichment analysis of pathways relating to synaptic plasticity. Unlike the metabolomic analysis however, transcriptomic analysis revealed a strong interaction of microbiome and sex with females showing the greatest differences despite a similarly altered metabolome. Overall, the results of this thesis provide novel insights into how the microbiome may alter hippocampal plasticity in adulthood and early life, with microbiome-induced effects often emerging in a sex-specific manner.Item Effects of the biotic and abiotic environment on ecology and evolution within novel in vitro and ex vivo models of the human gut microbiome(University College Cork, 2024) Pennycook, Joseph; Scanlan, Pauline; Claesson, Marcus; Science Foundation IrelandLeveraging the precision and replicability of in vitro and ex vivo models, we investigated the ecology and evolution of the human gut microbiota in a variety of environmental conditions. We developed a novel system for culturing microbiota communities and first used it to study the effects of the biotic and abiotic environment on the relationship between a gut-derived bacteriophage and its host. We found that an interaction between the nutrient medium and the broader community of bacteria led to the extinction of phage populations via competitive exclusion of the host and acidification of the medium. After reviewing the existing body of literature regarding the effects of antibiotic treatment on human gut microbiota, we tested the effects of sub-clinical antibiotic exposure on ex vivo faecal communities. Exposure to sub-clinical concentrations of clindamycin led to less diverse communities, more prevalent antibiotic resistance, and a greatly reduced presence of the Actinobacteria phylum, while exposure to amoxicillin and ciprofloxacin had less clear effects. Metabolic assays revealed that exposure to sub-inhibitory antibiotics promoted faster metabolism in Bifidobacterium, although not a faster growth rate, and also that the presence of an antibiotic often prevented the inhibition of metabolism by other environmental stressors. This work has demonstrated the value of laboratory models in the study of the human gut microbiota, while highlighting the importance of environmental context in understanding the system's ecology and evolution.Item Proliferation and evolution of lactococcal bacteriophages in cheese fermentations(University College Cork, 2024) Yu, Jun-Hyeok; van Sinderen, Douwe; Mahony, Jennifer; Nauta, Arjen; FrieslandCampina; Irish Research CouncilLactococcus is a widely exploited genus of the lactic acid bacteria in global dairy fermentations. However, bacterial strains that are routinely applied in these fermentations are susceptible to (bacterio)phage infection during the fermentation process which in certain case may have a significant negative economic impact. Although various strategies to prevent phage proliferation during fermentations have been developed, phages remain a major industrial challenge. In this thesis, an undefined mesophilic starter culture employed in the production of Dutch-type cheeses was dissected to elucidate its complex microbial community. Various lactococcal strains were isolated from this undefined mesophilic starter and used as potential bacterial hosts of phages present in associated samples in order to assess the presence and prevalence of phages during various stages of the cheese production process. Metagenome- and cultivation-based analysis of the starter culture revealed the dominant presence of Lactococcus species, particularly Lactococcus cremoris and Lactococcus lactis, in addition to minor populations of Leuconostoc mesenteroides. The intraspecies differentiation of L. cremoris/lactis isolates revealed a substantial strain-level diversity with regards to cell wall polysaccharide (CWPS) types, phage sensitivity profiles, and plasmid content. Additionally, the potential of isolated Lactococcus laudensis strains for future applications in the dairy industry was assessed by examining their genomic and phenotypic characteristics. DNA-based methods were used to track Lactococcus throughout the cheese production process using the corresponding starter culture, demonstrating significant shifts in both the relative abundance of the different component cwps genotypes and the overall bacterial population. In parallel, the proliferation and diversity of lactococcal phages during this process was analysed. Virome analysis of 17 dairy production samples identified Skunavirus-associated genome contigs containing distinct receptor binding proteins (RBPs). These RBPs are responsible for specific recognition and binding to the host cell surface. Phylogenetic comparison of the identified RBP amino acid sequences facilitated the prediction of their corresponding host cwps genotypes. These predictions were partially validated through host range analysis of isolated skunaviruses using the host panel isolated from the corresponding starter culture. Throughout the process, an increase in the absolute abundance of phages as well as phage compositional changes were observed. Furthermore, the isolated skunaviruses were shown to exhibit increased thermal resistance, suggesting a growing resilience against anti-phage measures employed in the production facility. Comparative genomic analysis of 18 newly and 71 previously isolated skunaviruses associated to Dutch dairy fermentation facilities revealed a correlation between their genome diversity and starter culture applied. Furthermore, a specific focus was placed on the HNH endonuclease (HNHE)-encoding genes in Skunavirus genomes, whose gene product is presumed to function as a homing endonuclease that facilitates phage evolution. The presence, diversity, and insertion location of HNHE-encoding genes were elucidated. Also, their protein sequence/structure features and enzymatic activities were elucidated. The distinct features of HNHEs at each insertion location in the structure of their C-terminal region were demonstrated. Furthermore, the correlation between used starter cultures and HNHEs were elucidated, suggesting that HNHEs in Skunavirus likely contribute to phage evolution. Among these identified HNHEs in Skunavirus genomes, the essential function of a conserved HNHE-encoding gene in phage DNA packaging was investigated. This conserved HNHE-encoding gene is located within the phage DNA packaging module in proximity to the terminase and portal protein-encoding genes. Deletion of the HNHE-encoding gene resulted in the production of defective phages, without disrupting other critical stages of the phage life cycle, such as DNA replication and structural protein assembly. This conserved HNHE possesses a typical active motif (ββα-metal fold), but exhibited non-specific endonuclease activity, highlighting the need for further studies.Item Unravelling the role of the microbiota-gut-brain axis in irritable bowel syndrome and its psychiatric comorbidities(University College Cork, 2024) Wilmes, Lars; Clarke, Gerard; Cryan, John; Irish Research CouncilIrritable bowel syndrome is a stress-related disorder of gut-brain interaction, characterised by altered bowel movements and visceral hypersensitivity, with a profound impact on quality of life. These symptoms occur in the absence of structural abnormalities or validated biomarkers. While the treatment options under consideration for irritable bowel syndrome have expanded, a significant proportion of patients fail to respond to conventional pharmacological interventions. This challenge is compounded by a subpopulation of patients with psychiatric comorbidities, including anxiety and depression, who tend to exhibit increased symptom severity and psychiatric difficulties that are harder to treat. The microbiota-gut-brain axis has emerged as a promising target for intervention, as supported by both preclinical and clinical studies. However, it remains unclear whether irritable bowel syndrome patients with psychiatric comorbidities possess distinct gut microbiota profiles compared to those without such comorbidities, potentially leading to different outcomes and requiring more precise treatment options. Using a well-validated animal model, we initially aimed to determine whether distinct gut microbiota profiles are causally linked to irritable bowel syndrome phenotypes with or without psychiatric comorbidities. In our preclinical studies, we showed that rats exposed to maternal separation could be clustered into subgroups, each representing different phenotypes: one group exhibited primarily visceral hypersensitivity, another displayed negative valence behaviour, a third had a comorbid phenotype, while a fourth group was resilient. Importantly, these subgroups showed distinct gut microbiota configurations corresponding to their specific phenotypes. Following this discovery, we conducted faecal microbiota transplants from these clusters into naïve rats, providing direct evidence that the microbiota plays a causal role in driving aspects of these phenotypes. To assess the clinical relevance of these findings, we isolated gut microbiota from three human populations: healthy individuals, irritable bowel syndrome patients, and irritable bowel syndrome patients with psychiatric comorbidities. After analysing the gut microbiota profiles, we transplanted these bacteria into naïve animals. Interestingly, while the microbiota from both IBS groups increased visceral hypersensitivity, only minor behavioural changes related to psychiatric disorders were observed in the recipient animals. Finally, we tested a novel next-generation psychobiotic, identified from patients with depression, in an animal model. Specifically, we investigated whether its psychobiotic properties exerted disease-specific effects and whether its efficacy surpassed that of traditional probiotics. However, neither the novel psychobiotic nor the traditionally selected probiotic significantly impacted gastrointestinal physiology or behaviour, highlighting the continuing challenges in translating psychobiotics between bench and bedside. Our findings underscore the critical role of the gut microbiota in driving distinct behavioural phenotypes and the potential in stratification of IBS subgroups based on the presence of psychiatric comorbidities. While the translation of these preclinical findings into effective treatments remains challenging, this suggests that targeted gut microbiota interventions may offer more precise cluster-specific therapeutic benefits. This may ultimately pave the way for more nuanced approaches in clinical IBS management where this is currently a large unmet medical need.