The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

Functional genomics of motile commensal intestinal bacteria

Flagella confer upon bacteria the ability to move and are therefore organelles of significant bacteriological importance. The innate immune system has evolved to recognise flagellin, (the major protein component of the bacterial flagellar filament). Flagellate microbes can potentially stimulate the immune systems of mammals, and thus have significant immunomodulatory potential. The flagellum-biogenesis genotype and phenotype of Lactobacillus ruminis, an autochthonous intestinal commensal, was studied. The flagellum-biogenesis genotypes of motile enteric Eubacterium and Roseburia species were also investigated. Flagellin proteins were recovered from these commensal species, their amino-termini were sequenced and the proteins were found to be pro-inflammatory, as assessed by measurement of interleukin-8 (IL-8) secretion from human intestinal epithelial cell lines. For L. ruminis, this IL-8 secretion required signalling through Toll Like Receptor 5. A model for the regulation of flagellum-biogenesis in L. ruminis was inferred from transcriptomics data and bioinformatics analyses. Motility gene expression in this species may be under the control of a novel regulator, LRC_15730. Potential promoters for genes encoding flagellin proteins in the Eubacterium and Roseburia genomes analysed were inferred in silico. Relative abundances of the target Eubacterium and Roseburia species in the intestinal microbiota of 25 elderly individuals were determined. These species were found to be variably abundant in these individuals. Motility genes from these species were variably detected in the shotgun metagenome databases generated by the ELDERMET project. This suggested that a greater depth of sequencing, or improved evenness of sequencing, would be required to capture the full diversity of microbial functions for specific target or low abundance species in microbial communities by metagenomics. In summary, this thesis used a functional genomics approach to describe flagellum-mediated motility in selected Gram-positive commensal bacteria. The regulation of flagellum biosynthesis in these species, and the consequences of flagella expression from a host-interaction perspective were also considered.

Visualising ribosome profiling and using it for reading frame detection and exploration of eukaryotic translation initiation

Ribosome profiling (ribo-seq) is a recently developed technique that provides genomewide information on protein synthesis (GWIPS) in vivo. The high resolution of ribo-seq is one of the exciting properties of this technique. In Chapter 2, I present a computational method that utilises the sub-codon precision and triplet periodicity of ribosome profiling data to detect transitions in the translated reading frame. Application of this method to ribosome profiling data generated for human HeLa cells allowed us to detect several human genes where the same genomic segment is translated in more than one reading frame. Since the initial publication of the ribosome profiling technique in 2009, there has been a proliferation of studies that have used the technique to explore various questions with respect to translation. A review of the many uses and adaptations of the technique is provided in Chapter 1. Indeed, owing to the increasing popularity of the technique and the growing number of published ribosome profiling datasets, we have developed GWIPS-viz (http://gwips.ucc.ie), a ribo-seq dedicated genome browser. Details on the development of the browser and its usage are provided in Chapter 3. One of the surprising findings of ribosome profiling of initiating ribosomes carried out in 3 independent studies, was the widespread use of non-AUG codons as translation initiation start sites in mammals. Although initiation at non-AUG codons in mammals has been documented for some time, the extent of non-AUG initiation reported by these ribo-seq studies was unexpected. In Chapter 4, I present an approach for estimating the strength of initiating codons based on the leaky scanning model of translation initiation. Application of this approach to ribo-seq data illustrates that initiation at non-AUG codons is inefficient compared to initiation at AUG codons. In addition, our approach provides a probability of initiation score for each start site that allows its strength of initiation to be evaluated.

Pregnancy-specific glycoprotein function, conservation and receptor investigation

Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt to replicate previously reported cytokine responses to PSG-treatment of immune cells and subsequently to investigate functionally important amino acids within PSG1. 2). To determine whether candidate receptor, integrin αvβ3, was a binding partner for PSG1 and to investigate whether PSG1 possessed functionality in a leukocyte-endothelial interaction assay. 3). To determine whether proteins generated from recently identified putative PSG genes in the horse shared functional properties with PSGs from other species. Outcomes: 1). Sequential domain deletion of PSG1 as well as mutation of conserved residues within the PSG1 Ndomain did not affect PSG1-induced TGF-β1. The investigated response was subsequently found to be the result of latent TGF-β1 contaminating the recombinant protein. Protein further purified by SEC to remove this showed no induction of TGF-β1. The most N-terminal glycosylation site was demonstrated to have an important role in PSG N domain secretion. PSG1 attenuated LPS-induced IL-6 and TNF-α. Investigations into signalling underpinning this proved inconclusive. 2). Integrin αvβ3 was identified as a novel PSG1 receptor mediating an as yet unknown function. Preliminary investigations into a role for PSGs as inhibitors of leukocyte endothelial interactions showed no effect by PSG1. 3). Horse PSG protein, CEACAM49, was shown to be similarly contaminated by latent TGF-β1 particle and once removed did not demonstrate TGF-β1 release. Interestingly horse PSG did show anti-platelet properties through inhibition of the plateletfibrinogen interaction as previously published for mouse and human PSGs.

Trophic role of small cetaceans and seals in Irish waters

Understanding the role of marine mammals in specific ecosystems and their interactions with fisheries involves, inter alia, an understanding of their diet and dietary requirements. In this thesis, the foraging ecology of seven marine mammal species that regularly occur in Irish waters was investigated by reconstructing diet using hard parts from digestive tracts and scats. Of the species examined, two (striped and Atlantic white-sided dolphin) can be considered offshore species or species inhabiting neritic waters, while five others usually inhabit more coastal areas (white-beaked dolphin, harbour porpoise, harbour seal and grey seal); the last species studied was the bottlenose dolphin whose population structure is more complex, with coastal and offshore populations. A total of 13,028 prey items from at least 81 different species (62 fish species, 14 cephalopods, four crustaceans, and a tunicate) were identified. 28% of the fish species were identified using bones other than otoliths, highlighting the importance of using all identifiable structures to reconstruct diet. Individually, each species of marine mammal presented a high diversity of prey taxa, but the locally abundant Trisopterus spp. were found to be the most important prey item for all species, indicating that Trisopterus spp. is probably a key species in understanding the role of these predators in Irish waters. In the coastal marine mammals, other Gadiformes species (haddock, pollack, saithe, whiting) also contributed substantially to the diet; in contrast, in pelagic or less coastal marine mammals, prey was largely comprised of planktivorous fish, such as Atlantic mackerel, horse mackerel, blue whiting, and mesopelagic prey. Striped dolphins and Atlantic white-sided dolphins are offshore small cetaceans foraging in neritic waters. Differences between the diet of striped dolphins collected in drift nets targeting tuna and stranded on Irish coasts showed a complex foraging behaviour; the diet information shows that although this dolphin forages mainly in oceanic waters it may occasionally forage on the continental shelf, feeding on available prey. The Atlantic white-sided dolphin diet showed that this species prefers to feed over the continental edge, where planktivorous fish are abundant. Some resource partitioning was found in bottlenose dolphins in Irish waters consistent with previous genetic and stable isotope analysis studies. Bottlenose dolphins in Irish waters appears to be generalist feeders consuming more than 30 prey species, however most of the diet comprised a few locally abundant species, especially gadoid fish including haddock/pollack/saithe group and Trisopterus spp., but the contribution of Atlantic hake, conger eels and the pelagic planktivorous horse mackerel were also important. Stomach content information suggests that three different feeding behaviours might occur in bottlenose dolphin populations in Irish waters; firstly a coastal behaviour, with animals feeding on prey that mainly inhabit areas close to the coast; secondly an offshore behaviour where dolphins feed on offshore species such as squid or mesopelagic fish; and a third more complex behaviour that involves movements over the continental shelf and close to the shelf edge. The other three coastal marine mammal species (harbour porpoise, harbour seal and grey seal) were found to be feeding on similar prey and competition for food resources among these sympatric species might occur. Both species of seals were found to have a high overlap (more than 80%) in their diet composition, but while grey seals feed on large fish (>110mm), harbour seals feed mostly on smaller fish (<110mm), suggesting some spatial segregation in foraging. Harbour porpoises and grey seals are potentially competing for the same food resource but some differences in prey species were found and some habitat partitioning might occur. Direct interaction (by catch) between dolphins and fisheries was detected in all species. Most of the prey found in the stomach contents from both stranded and by catch dolphins were smaller sizes than those targeted by commercial fisheries. In fact, the total annual food consumption of the species studied was found to be very small (225,160 tonnes) in comparison to fishery landings for the same area (~2 million tonnes). However, marine mammal species might be indirectly interacting with fisheries, removing forage fish. Incorporating the dietary information obtained from the four coastal species, an ECOPATH food web model was established for the Irish Sea, based on data from 2004. Five trophic levels were found, with bottlenose dolphins and grey and harbour seals occurring at the highest trophic level. A comparison with a previous model based on 1973 data suggests that while the overall Irish Sea ecosystem appears to be “maturing”, some indices indicate that the 2004 fishery was less efficient and was targeting fish at higher trophic levels than in 1973, which is reflected in the mean trophic level of the catch. Depletion or substantial decrease of some of the Irish Sea fish stocks has resulted in a significant decline in landings in this area. The integration of diet information in mass-balance models to construct ecosystem food-webs will help to understand the trophic role of these apex predators within the ecosystem.

Metabolism of host-derived carbohydrates by Bifidobacterium breve UCC2003

Bifidobacteria are Gram positive, anaerobic, typically Y-shaped bacteria which are naturally found in the digestive tract of certain mammals, birds and insects. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. The prototypical B. breve strain UCC2003 has previously been shown to utilise numerous carbohydrates of plant origin. Various aspects of host-derived carbohydrate metabolism occurring in this bacterium will be described in this thesis. Chapter II describes B. breve UCC2003 utilisation of sialic acid, a nine-carbon monosaccharide, which is found in human milk oligosaccharides (HMOs) and the mucin glycoprotein. B. breve UCC2003 was also shown to cross-feed on sialic acid released from 3’ sialyllactose, a prominent HMO, by the extracellular sialidase activity of Bifidobacterium bifidum PRL2010. Chapter III reports on the transcriptional regulation of sialic acid metabolism in B. breve UCC2003 by a transcriptional repressor encoded by the nanR gene. NanR belongs to the GntR-family of transcriptional regulators and represents the first bifidobacterial member of this family to be characterised. Chapter IV investigates B. breve UCC2003 utilisation of mucin. B. breve UCC2003 was shown to be incapable of degrading mucin; however when grown in co-culture with B. bifidum PRL2010 it exhibits enhanced growth and survival properties. A number of methods were used to investigate and identify the mucin components supporting this enhanced growth/viability phenotype. Chapter V describes the characterisation of two sulfatase-encoding gene clusters from B. breve UCC2003. The transcriptional regulation of both sulfatase-encoding gene clusters was also investigated. The work presented in this thesis represents new information on the metabolism of host-derived carbohydrates in bifidobacteria, thus increasing our understanding of how these gut commensals are able to colonise and persist in the gastrointestinal tract.