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Early-life bacterial colonisation can be of particular importance to the overall growth and health of an animal, especially influencing intestinal and immune system development with long-term implications. This is especially relevant in pig production where post-weaning enteric infection is one of the major concerns related to the gut health of pigs, and is associated with economic losses and welfare problems. Commercial pig production systems involves early and abrupt weaning, which contrasts with the gradual transition from mother’s milk to solid feed in nature. Due to such abrupt weaning, a piglet is challenged with multiple stressors (including environmental-, nutritional- and psychological-) which is usually associated with changes in gut microbiota and a high incidence of diarrhoea. Modulating intestinal microbiota to reduce weaning-associated problems in pigs, is getting increasing scientific and commercial interest, as microbial dysbiosis (or imbalance) has been identified as a leading cause of post-weaning intestinal infections. The overall aim of this thesis was to understand the molecular effects of early-life feeding (pre-weaning provision of fibrous feed), associated with the pattern of intestinal microbiota colonisation and gut maturation. We hypothesised that early feeding of a fibrous diet containing both soluble and insoluble fermentable fibres will affect the microbiota and physiological development of neonatal or suckling piglets, facilitating a better preparation for the weaning transition.First, we focussed on assessing an optimal sampling method to study microbiota development in neonatal piglets. We showed that rectal swabs are a suitable alternative sample type to study the porcine microbiome development in early-life, when faecal sampling is challenging. Although rectal swab samples bring a certain degree of variability with the presence of mucosa-adhered population, they also provide the opportunity to assess the impact of an intervention on the mucosa-adhered populations. The second aim of this thesis was to investigate the age-related gut microbiota colonisation and the impact of fibrous feed on the gut microbiota colonisation as well as intestinal physiology in neonatal piglets. We performed a longitudinal study (using rectal swabs) to evaluate the impact of early feeding (fibrous feed) on the microbiota colonisation at pre- and post-weaning time-points. The results revealed that the early-fed (EF) piglets had an accelerated maturation of the microbiota, compared to the control (CON) piglets that consumed milk exclusively. Accelerated maturation at pre-weaning time-points were characterised by the simultaneous emergence of typical post-weaning-associated microbial groups (such as Prevotella, Roseburia, Faecalibacterium, Ruminococcus, Megasphaera, Subdoligranulum) and a more rapid decline of typical early-life/pre-weaning microbial genera (e.g., Fusobacterium, Finegoldia, Bacteroides, Eschechichia-Shigella). Moreover, we found a quantitative association between eating behaviour of EF piglets (video scores) and their microbiota signature, indicating that the piglets who spent more time at the feeding trough had a higher abundance of ‘accelerated’ microbial groups. Furthermore, early feeding altered the colonic microbiota composition, increased microbial fermentation products (SCFA) in the colon and modulated intestinal development i.e., increased weights and lengths of several intestinal tract segments, as well as a decreased villus-crypt ratio in jejunal mucosa and an increased abundance of proliferative cells in colon mucosa, just before weaning. The third aim of this thesis was to evaluate the host mucosa transcriptome response due to pre-weaning fibrous feed. Maximum impact of early feeding was detected at weaning (day29; compared to other time-points), which was followed by convergence of the transcriptome three weeks post-weaning (day+21) in EF and CON piglets. We showed that early feeding not only accelerates the gut microbiota, but also the host transcriptome maturation at weaning. The results revealed that in the EF group, oxidative phosphorylation, cholesterol biosynthesis and oxidative stress-related pathways were significantly enriched, whereas sirtuin signalling and immune response pathways were downregulated in the colon mucosa at day29. Remarkably, three days after weaning, the EF piglets displayed a stronger mucosal responsiveness compared to the CON piglets, reflected in the increased expression of genes (transcriptomics) related to immune activation, epithelial migration and “wound-repair" like processes necessary to maintain gut barrier integrity (during weaning transition) as well as jejunal morphometry. In the last chapter, we explored fundamental aspects of host-microbe interactions, by evaluating the associations between the intestinal microbiota and behaviour of suckling piglets. We aimed to tentatively reveal associations between the intestinal microbiota composition and piglet behaviour in a test for coping style, (i.e. a personality trait), as well as anxiety- and exploration-related behaviour in a novel environment test. A number of microbial groups such as Coprococcus, CAG-873, Atopobium and Prevotella were identified to be associated with anxiety- and/or exploration-related behaviour, although these results are premature and need further validation for their biological relevance. Overall, the findings of this thesis indicate that EF piglets show an “alerted system” with an enhanced responsiveness to external stimuli of feed and microbiome development, compared to the CON group, thus exemplifying the potential of early-life programming to modulate intestinal development in piglets. Furthermore, the results of this thesis indicate that early feeding of fibrous feed has considerable potential to better prepare young piglets for the weaning transition.

Recent insights have shown that there is a triangular relationship between the diet, immune system and intestinal microbiome, in which the gut microbiota is a substantial contributor to human metabolism and health. A better understanding of the relationship between dietary components, including food processing, and the activity of metabolites produced by the gut microbiota on the host, is an essential step in designing food that benefits human health. The nutrient composition of a diet is obviously of importance to health, but the way food is designed also plays an important role in how much reaches the colon, where it is metabolised by the microbiota. Therefore, this thesis describes the effects of different dietary components, (fibres, tryptophan, glucosinolates and polyphenols) on immune modulation during human digestion and fermentation with a main focus on activation of the Aryl hydrocarbon Receptor. The results give insight into the effects of food preparation and microbial fermentation on the potential health properties of food compounds.

The intestinal microbiota plays a crucial role in the homeostasis of the human gastrointestinal tract by maintaining an anti-inflammatory status. Microbial imbalance in the gut, which is often referred to as ‘dysbiosis’, is known to be one of the major contributors to many human diseases, including inflammatory bowel disease (IBD). IBD is characterized by a chronic inflammation, but the current available treatment with anti-inflammatory drugs is often not effective. Therefore, the overarching aim of this thesis was to try to find bacterial strains or bacterial metabolites that have an immunomodulatory (i.e. an anti-inflammatory) function and may therefore be used as a therapy or treatment of IBD. In collaboration with the Rowett Institute, University of Aberdeen in Scotland, UK and the Department of Medical Microbiology, at the University Medical Centre Groningen, The Netherlands, a large number of colonic anaerobic bacteria were isolated from healthy patients. In our lab, we cultured more than 100 of these different strains of bacteria under strictly anaerobic conditions. We observed their growth characteristics and investigated their immunomodulatory properties. A high ratio between secreted anti-and pro-inflammatory cytokines (IL-10/IL-12 ratio) has been reported to be an indicator of positive correlation between in vitro trials and the attenuation of clinical symptoms in in vivo mouse models of colitis. Our initial screening therefore started with stimulation of peripheral mononuclear blood lymphocytes (PBMCs) with standardized concentrations of the bacteria or their culture supernatant. We determined the cytokine secretion of these PBMC after bacterial stimulation and determined the IL-10/IL-12 ratio. Additionally we checked if the viability of the PBMC was not affected by the bacterial strains or their metabolites which were secreted in the supernatant. As IBD is characterized by periods of remission and occasional flare-ups (periods of higher inflammation), we investigated both healthy and disease situations. To achieve this, we added heat inactivated bacteria (HIB) to our PBMC culture. These HIB triggered a strong induction of both pro-and anti- inflammatory cytokines, which is reminiscent of actual inflammation. Co-stimulation with both HIB and our bacterial strains enabled us to investigate the effect of the bacterial strains ‘during inflammation’. Additionally, we checked whether the bacterial strains were able to trigger NF-κB signalling via Toll like receptor (TLR) activation, which is one of the most known mechanisms to modulate immune response of the host. Finally, to assess oxidative stress, which is known to occur during flare-ups and damages the epithelial cells, we investigated whether the different bacterial strains were able to modulate nitric oxide (NO) secretion by a mouse macrophage (RAW 264.7) cell line. Again we were able to mimic an inflammatory situation by addition of bacterial lipopolysaccharides (LPS) to these cells and could thereby also investigate the effect of bacteria in an already inflamed gut.In Chapter 2 we summarize the characterisation of 68 different colonic anaerobic bacteria tested. Most importantly, we found that there is a large variation among the tested strains in their immunomodulatory properties. The variation in induction of cytokine and NO secretion as well as in the ability to trigger NF-κB signalling between strains was significant. Interestingly, the bacterially induced immune profiles were highly strain dependent and not characteristic for a specific bacterial species. We must therefore conclude that generalizations cannot be made easily and any newly discovered strain needs to be individually investigated to determine its immunomodulatory properties. However, we could identify three different immune profiles, resulting from bacterial stimulation. The first ‘immunostimulatory’ profile was characterized by strongly inducing cytokine secretion in PBMCs. Most of these strains also elicited relatively high concentrations of NO secretion and strong NF-κB signalling after TLR activation. The second ‘immunomodulatory’ profile was characterized by induction of only moderate amounts of cytokine secretion. However, when an inflammatory status was mimicked (addition of HIB), these strains were able to attenuate the secretion of pro- inflammatory cytokines. The final ‘immuno suppressive’ or ‘silent’ profile was characterized by a low capacity to induce cytokine or NO secretion. More importantly, when HIB was added as an inflammatory stimulus, these strains attenuated the resulting pro-inflammatory cytokine response.Several studies showed that there is a negative correlation between the relative abundance of F. prausnitzii and the disease severity of IBD, therefore a causal connection has been suggested. Indeed, several in vivo studies have shown that addition of certain strains of F. prausnitzii could attenuate clincal symptoms of colitis in mice. We therefore focused on another 28 bacterial strains that all belonged to the species of F. prausnitzii in Chapter 3. After thorough in vitro investigation we have to conclude that the immunomodulatory properties are really strain specific, as the tested properties of the strains (cytokine, NO secretion and NF-κB signaling via TLR activation) do not correlate with genomic phylogenetic clusters. Among the different strains tested, we found all three different immune profiles that were observed in Chapter 2. Moreover, the general assumption that all F. prausnitzii strains induce strong IL-10 secretion was found to not be universally true, as there were some ‘silent’ strains that hardly induced any IL-10 secretion. Interestingly, one of the strains that was the strongest activator of NF-κB signaling, hardly induced any cytokine secretion, which suggest some specific mechanism to prevent downstream effects. Indeed this strain manifested a ‘silent’ profile and as such was considered to be of interest for further in vivo trials.During the in vitro screening procedures described above we observed that the culture supernatant of all strains tested was able to attenuate HIB induced cytokine secretion. Further investigations showed that the original growth medium (without any bacteria present), and especially the short chain fatty acids (SCFA) in the medium also triggered this attenuation effect. SCFA were reported to have immunomodulatory effects, but the studies were not all consistent. Most studies showed that butyrate induced IL-10 secretion by immune cells and thereby triggered regulatory T cell differentiation. In Chapter 4 we comparedthe effects of acetate and butyrate on different immune cell mechanisms. We found that butyrate decreased cell viability when administered at higher concentrations, whereas similar, or even higher concentrations of acetate did not affect cell viability. Interestingly, although both acetate and butyrate were able to attenuate HIB induced secretion of pro- inflammatory cytokines, only acetate was able to increase the anti-inflammatory cytokine IL-10. More importantly, butyrate actually decreased the secretion of the anti-inflammatory IL-10 in vitro in both PBMC as well as CD14+ monocytes. This decreased IL-10 secretion could result in an overall more inflammatory response (lower IL-10/IL-12 ratio) compared to the response triggered by acetate. To investigate how butyrate and acetate elicited their effects, we tried to determine the mechanism by which they affected cytokine secretion. As G protein-coupled receptors (GPCR) are known to mediate the effect of SCFA, we blocked the expression of GPCR in monocyte-derived dendritic cells (MDDC) with specific inhibitors. The attenuating effects of SCFA on HIB induced cytokine secretion were still observed, suggesting that modulation of cytokine secretion used an GPCR-independent mechanism. Butyrate (and to a lesser extent acetate) are also known to affect histone acetylation and could thereby modulate gene transcription. We found that butyrate indeed increased histone acetylation, which may point to a mechanism used in modulation of cytokines secretion. Interestingly, NF- κB activation was also found to be differentially modulated by acetate compared to butyrate, although the underlying mechanism has not yet been elucidated.As we found clear effects of both acetate and butyrate on cytokine secretion by PBMC and CD14+ monocyte, we wondered if other biological pathways would be affected as well. We therefore stimulated CD14+ monocytes with acetate and butyrate (with or without co- stimulation with HIB) and investigated the resulting transcription profile in Chapter 5. Our monocytes showed the same attenuation of pro-inflammatory cytokine secretion in HIB induced samples as was seen in the PBMC and CD14+ monocyte. Acetate did not cause major effects in the number of regulated genes, but butyrate significantly affected the regulation of many different (immune) pathways and genes therein.To compare the effects that SCFA might have on epithelial cells with the effects we found on immune cells, we investigated the effect of SCFA on an ex vivo 3D porcine ileum organoid model in Chapter 6. We exposed the organoid cells to acetate and butyrate and performed a transcriptomic analysis. Similar to the results of the transcriptomic analysis of CD14+ monocyte, butyrate proved to elicit greater changes in gene expression compared to acetate and substantially affected apoptosis and cell-cycle related pathways. In contrast, acetate mainly affected cellular metabolism process- related pathways, suggesting a less damaging effect on gut epithelial function compared to butyrate.To conclude our studies, we tested several bacterial strains that appeared promising in the in vitro trials, in an in vivo mouse DSS-induced colitis model in Chapter 7. We observed attenuation of the clinical symptoms after addition of these ‘silent’ strains, which confirm our hypothesis that the bacterial strains that induced an ‘silent’ profile would be able to reduce colonic inflammation.In the last chapter, Chapter 8, we summarize and discuss the combined results from this thesis in the context of other studies regarding host- microbe interactions, probiotics and the effects of SCFA. We explain how these findings contribute to a better understanding of the immunomodulatory properties of colonic anaerobic bacterial strains and their metabolites and provide suggestions for future research, and reflect on the overall aim of this thesis.

Oral health depends on a complex interplay between the mucosal tissues, physicochemical and microbial components present in the oral cavity. Maintenance of a stable ecosystem is an essential determinant of oral health. However, as a result of a major change in the ecosystem, the stability can be disturbed leading to an increased risk for development of diseases. Epithelial integrity and barrier function are key aspects to support oral health. The oral cavity consists of stratified squamous epithelium that provides a physical barrier that protects the underlying tissues from mechanical or chemical damage, loss of fluids, and invasion from pathogenic bacteria. In order to maintain barrier function, the oral mucosa undergoes constant renewal and repair. When the epithelial integrity is compromised, prompt re-epithelialization is required to restore homeostasis by re-establishment of cell-cell contacts. This thesis describes the development of a quantitative high-throughput scratch assay to study oral re-epithelialization in vitro as well as the influence of different components of the oral ecosystem (i.e. oral commensal bacteria and salivary metabolites) on gingival re-epithelialization. In Chapter 2, we describe the development and optimization of an automated high-throughput scratch assay that uses a mathematical model to extract biologically relevant parameters that describe re-epithelialization kinetics. The assay was used to screen 39 lactic acid bacteria (LAB) for their influence in re-epithelialization and therefore, assess their potential to promote gingival epithelial re-epithelialization. The results revealed that exposure of the cells to different Streptococcus salivarius strains leads to enhanced re-epithelialization kinetics. In particular, we showed that S. salivarius MS-oral-D6 is able to stimulate re-epithelialization through a secreted serine protease. Furthermore, in Chapter 3, we developed and implemented the Kinetic Re-Epithelialization Analysis Pipeline (KREAP) into a toolbox in Galaxy, providing an open-source, web-based platform for reproducible image processing and data analysis of high-throughput scratch assays. In addition, Chapter 4 provides a detailed procedure of the assay using cell lines originating from oral mucosa as well as from skin, which expands the application of the assay to other research fields. In Chapter 5, we explored the role of the salivary metabolome on oral re-epithelialization using our scratch assay and the metabolite profiles of a subset of 63 unstimulated saliva samples collected from a healthy cohort (n = 61) during a two-week experimental gingivitis study. Elastic net regression with stability selection led to the identification of a metabolite signature consisting of 10 metabolites that were related to the re-epithelialization kinetics observed in vitro. Using this signature, we were able to predict the re-epithelialization capacity of the remaining 242 saliva samples collected during the clinical study. Higher concentrations of certain plasmalogens, diacylglycerol, and amino acid derivatives in saliva were associated with enhanced re-epithelialization capacity. Moreover, the predicted re-epithelialization capacity of the saliva samples was positively correlated with the gingival bleeding scores determined for the participants during the experimental gingivitis challenge. These scores are used as a clinical parameter to assess the inflammatory state of the periodontal tissues and thus, we propose that the identified metabolite signature reflects the intensity of the mucosal interactions with the resident microbiota. In addition, we found that individuals who displayed larger variation of the metabolite signature over time were associated with a higher increase in gingival bleeding scores during the experimental gingivitis challenge, implying that unstable host-microbe interactions and higher variability of the associated metabolite signature may be an indicator for stronger responses under a challenge. In Chapter 6, we employed elastic net regression with stability selection to identify operational taxonomical units (OTUs) that were positively and negatively associated with gingival bleeding scores in the participants of a cross-sectional study (n = 268). Representative species of the identified OTUs were screened for their influence in oral re-epithelialization using the high-throughput scratch assay. The results revealed that the group of bacteria that had a negative association with gingival bleeding significantly increased re-epithelialization kinetics in comparison with the positive associated group. Importantly, most of these associations were confirmed in a different cohort that participated in a two-week experimental gingivitis challenge study. Higher relative abundances of Actinomyces oris/viscosus/naeslundii, Rothia dentocariosa and Veillonella dispar were strongly correlated with lower gingival bleeding scores, whereas higher relative abundances of Porphyromonas catoniae, Selenomonas sputigena, Leptrotichia buccalis and Streptococcus anginosus were associated with increased gingival bleeding. Additionally, we showed that microbial signatures in saliva can potentially be used to assess the risk of an individual to develop oral diseases, such as gingivitis. Chapter 7 explored the differential modulatory mechanisms exerted by Actinomyces oris, Actinomyces viscosus, Veillonella parvula and Fusobacterium nucleatum subsp. animalis on gingival re-epithelialization kinetics. Cell-based assays and transcriptomic analysis of scratched gingival cells revealed that treatment with A. oris, A. viscosus or V. parvula enhanced re-epithelialization kinetics by increasing cell survival through downregulation of the p53 signalling pathway. In contrast, the increased re-epithelialization kinetics observed with F. nucleatum subsp. animalis was characterized by a stronger induction of regulatory genes involved in cell proliferation, apoptosis and innate immunity responses. Lastly, we showed that the stimulatory effects observed on cell migration by A. viscosus and F. nucleatum subsp. animalis depend on direct bacteria-host cell contact, whereas A. oris and V. parvula probably secrete soluble compounds that act as chemoattractants for gingival cells. Chapter 8, summarizes and discusses the key findings of this thesis and provides new avenues for further research. This thesis provides insight in the complex interplay between the oral mucosa and different components of the oral ecosystem that may lead to the development of innovative strategies that could support oral health through the maintenance of epithelial integrity and barrier function. Such strategies may involve the development of novel diagnostic tools to predict disease risk as well as the generation of next-generation probiotics or bioactive compounds to stimulate re-epithelialization in patients with impaired wound healing.

Various types of dietary fatty acids have different effects on human health. The aim of this thesis was to increase our understanding of the molecular mechanisms underlying the effects of dietary fatty acids. To do this, we examined changes in whole genome gene expression profiles upon both acute as well as longer term dietary fatty acid interventions. Furthermore, from previous research, it is clear that large inter-individual differences in the response to dietary fatty acids exist. We used whole genome gene expression analyses to increase our understanding of the mechanisms underlying some of these inter-individual differences. Many modifiable and non-modifiable factors can be the cause of these inter-individual differences. In chapter 2, we reviewed all studies that examined differences in the transcriptional response to dietary interventions based on the presence of one of these factors. These include gender, age, BMI, body composition, blood lipid levels and gut microbial composition. We conclude that transcriptome analyses are well-suited for studying the underlying mechanisms behind these differences in the response to diet. Nevertheless, the number of studies that use this approach remains limited. Another factor that may modify the response to a dietary intervention is genetics, e.g. the apolipoprotein E4 (APOE4) variant. People who carry the APOE4 allele have an increased risk of cardiovascular disease. Fish-oil supplementation may help in the prevention of cardiovascular disease, though inter-individual differences in the response to n-3 polyunsaturated fatty acids on gene expression profiles have been observed. In chapter 3, we aimed to assess the impact of APOE4 on peripheral blood mononuclear cell (PBMC) whole genome gene expression at baseline and following a 6-month fish-oil intervention. We observed increased gene expression of IFN signaling and cholesterol biosynthesis pathways in APOE4 carriers, which might explain part of the association between APOE4 and CVD. Furthermore, fish-oil supplementation may be beneficial by decreasing interferon signalling-related gene expression in APOE4 carriers. Another long-term dietary intervention with fatty acids was studied in chapter 4. We examined the effect of a 12-week high medium-chain saturated fatty acid diet on subcutaneous adipose tissue gene expression profiles. We observed increased expression of genes involved in oxidative energy metabolism and decreased inflammation-related gene expression due to the high medium-chain saturated fatty acid intake. Considering the role of the adipose tissue in sustaining the low-grade inflammation that is associated with obesity, these findings may be indicative of a more anti-inflammatory phenotype of the adipose tissue. We concluded that medium-chain saturated fatty acids may potentially have beneficial effects on adipose tissue functioning. Besides studying the effects of long-term interventions with fatty acids on whole genome gene expression, we also examined the effects of acute high-fat challenges. In chapter 5, we determined the additional value of determining whole genome gene expression changes in response to a high-fat challenge compared to assessment at fasting only. In addition, we aimed to identify whether a 4 week high-fat high-calorie diet can induce a shift in gene expression profiles in healthy subjects towards a metabolic syndrome-like gene expression profile. We found that fasting whole blood whole genome gene expression profiles are highly responsive to a 4-week high-fat high-calorie diet, with changes in in the direction of a metabolic syndrome-like gene expression profile. High-fat challenge responses in healthy subjects show only minimal changes in gene expression upon the dietary intervention and a marginal shift in the direction of the metabolic syndrome. We concluded that fasting gene expression profiles are more responsive compared to high-fat challenge responses to a 4-week high-fat high-calorie diet. Besides chapter 5, several other studies have also examined changes in whole genome gene expression in blood cells induced by high-fat challenges. In chapter 6, we combined microarray data from four high-fat challenge studies varying in study population, challenge composition and research laboratory. By performing this meta-analysis, we showed a general PBMC whole genome gene expression response to a high-fat challenge. We concluded that a meta-analysis provides added value for the discovery of consistently differentially expressed genes and pathways compared to selecting only those genes and pathways that are identified in all separate studies. In conclusion, in this thesis we showed differences in the whole genome gene expression response to fish-oil supplementation in PBMCs of APOE4 carriers vs non-carriers. Furthermore, the effects on whole genome gene expression of the two long-term dietary interventions, i.e. the fish-oil supplementation in PBMCs of APOE4 carriers and the high medium-chain saturated fatty acid diet in adipose tissue, may be beneficial by downregulation of gene expression related to inflammation. We also showed that whole genome gene expression responses to high-fat challenges are affected by a 4-week high-fat high-calorie diet, though changes in fasting gene expression profiles are much more pronounced. Finally, we showed the value of meta-analysis of microarray data in high-fat challenge studies for identifying the general response to a high-fat challenge.

Q fever is a worldwide zoonotic infectious disease caused by the bacterium Coxiella burnetii. During 2007-2010, the largest Q fever outbreak was reported in The Netherlands, where more than 4000 human cases were registered showing a serious burden of the disease. During this outbreak, goats harboring predominantly the CbNL01 genotype strain were identified as the major source of disease in humans and drastic measures such as mass culling of infected goats were implemented to reduce the spread of the pathogen and control the disease. In order to minimize such complications in the future, it is crucial to have a thorough understanding of the disease causing pathogen and to develop effective Q fever vaccines. The causes of the large Dutch outbreak are not well-understood and one of the main reasons speculated were the hyper-virulent behavior of the circulating C. burnetii isolates. The research described in this thesis focuses on the characterization of C. burnetii outbreak strains isolated from infected goats, cattle, sheep and human clinical materials. Our studies were initiated to better understand the bacterial pathogenesis, virulence, evolution, adaptations in various environments, host immune responses and to identify pathogen related factors that have modulated the disease outbreak. We specifically aimed to identify the virulence factors and mechanisms that contributed to the increased zoonotic potential of the strain associated with the Dutch Q fever outbreak. The studies presented in this thesis majorly applied Pathogenomic approaches at the genome and transcriptome level to decipher host-pathogen interactions and to develop new tools to study C. burnetii infections. A transcriptome analysis of the outbreak C. burnetii strain of the CbNL01 genotype grown under in vivo and in vitro conditions resulted in the identification of distinct metabolic adaptations and virulence mechanisms of the bacterium. Detailed comparative analysis of complete genome sequences of C. burnetii strains showed a high similarity between strains of the same genotype. Genome sequences of the Dutch outbreak CbNL01 genotype strains were more divergent than the genome sequences of the less prevalent CbNL12 genotype strains and the NM reference strain. The analysis also showed that the high virulence of the outbreak strains was not associated with acquiring novel virulence-related genes arguing against the idea that the Dutch outbreak was due to emergence of hyper-virulent strains though horizontal gene transfer. Among the prominent genetic differences in the CbNL01 outbreak strains compared to CbNL12 and NM, were the presence of several point mutations and increased transposon mediated genome plasticity, which might have contributed to its epidemic potential. Point mutations, especially in a large number of membrane proteins, could also have contributed to the increased zoonotic potential of CbNL01 strains allowing this clone to escape the host immune responses in goats and humans. In addition, mutations in critical genes involved in virulence and evasion of the host immune system could be potentially involved in the increased virulence of the CbNL01 outbreak strains. On the contrary, studies on host immune responses in an in vivo (experimental infections in mice) and an in vitro (human PBMC’s stimulation) model did not show any difference associated with the strain genotype. However, differences in immune responses were found to be associated with the host-origin of the C. burnetii strains. Among different host-origin strains, strains derived from goats and humans generated significantly lower innate and adaptive immune responses than strains derived from cattle, whereas no differences in immune responses were observed when strains were grouped based upon their genotype. These observations support immune evasions as a major virulence strategy of goat and human strains in hosts and further suggest that bacteria originating from goats have a greater potential to cause outbreaks in humans. This indicates that for Q fever prevention purposes goats should be efficiently monitored for the presence of C. burnetii. Taken together, the results described in this thesis suggest that the virulence potential of C. burnetii strains is not only based on genetic differences, but also on other host-adaptation mechanisms such as transposition of genomic elements and/or differential regulation of gene expression. Finally, the results from this thesis provide a framework for future studies in the development of vaccines and diagnostic tools for Q fever.

The increasing prevalence of antibiotic resistance in pathogenic bacteria and the potential future implications for human and animal morbidity and mortality, health-care costs and economic losses pose an urgent worldwide problem. As a result, exploration of alternative strategies to combat antibiotic resistant bacteria have intensified over the last decades. The work described in this thesis focused on the study of naturally occurring antimicrobial peptides (AMPs) and other bioactive molecules produced by bacteria as potential alternatives to prevent or treat infections with pathogenic bacteria. A large part of the thesis aimed to increase knowledge about the role of the microbiota (the collection of microbes present at a certain location of the body) of the oral cavity or small intestine in the abundance of Streptococcus suis, a pathogenic bacteria that mostly causes disease in young pigs. We identified commensal bacteria that displayed strong and selective antagonism against this S. suis. Several bacteria that showed strong growth inhibition of S. suis in the lab through the production of AMPs were isolated and characterised. This thesis increased the understanding of the role of host- and microbiota-derived biologically active small molecules in microbe-microbe and microbe-host interplay. Such knowledge may contribute to the development of novel therapeutic solutions to treat antibiotic resistant bacteria, such as beneficial microbial communities (i.e. next-generation probiotics) or biotechnological applications of natural or modified AMPs.

Streptococcus suis is Gram-positive bacterium and its natural habitat is the upper respiratory tract of pigs, and in particular the tonsils and nasal cavity. Although it is considered to be a normal member of the adult pig microbiome, it can cause serious diseases in pigs and humans. S. suis is in fact one of the most important swine pathogens world-wide, causing a wide variety of diseases in pigs including septicemia, arthritis, endocarditis, and meningitis that leads often to a rapid death within 1-2 days. Although most human infections are considered the consequence of occupational exposure, in the last years the number of human cases has increased and isolates with multi-resistance genes have been isolated. Human infection caused by S. suis are characterized by a similar symptomatology as in pigs. Despite the economic loss in the pork industry due to S. suis infection and its importance as emerging zoonotic agent, experimental studies of S. suis virulence and pathology have been hampered by the lack of efficient methods for genetic transformation and the lack of a simple, cost-effective model to investigate S. suis virulence. In some streptococcal species, genetic transformation can be carried out very efficiently as these species can be experimentally induced to take-up and recombine homologous extracellular DNA. The discovery of natural competence in some streptococci and the potential of opening up new avenues for genetic analysis of S. suis, was the motivation for investigating natural competence in this important pathogen. In Chapter 2 we showed that a peptide pheromone induces competence in S. suis. The induction was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon; the SigX-inducing peptide (XIP); and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. This has resulted in the development of a novel methodology that will enable diverse research groups to accelerate discovery of novel features of S. suis ecology and pathology, especially with respect to virulence. In Chapter 3 we investigated the genetic regulation of competence in S. suis and we provided a hypothetical model of the S. suis transformasome. We verified the essential role of the S. suis major pilin, and CinA for efficient competence development, supporting the notion that our predicted multi-protein transformasome indeed appears to function as described for other streptococci. We have also characterised the differential metabolic states that enable competence, and the metabolic state associated with competence exit (Chapter 4). In Chapter 5 we investigated for the first time the use a zebrafish larvae model to assess the relative virulence of S. suis strains in porcine infections. Because of its convenience and cost-effectiveness, this model may be used to assay virulence of environmental S. suis strains, in particularly those of clinical relevance to infection of pigs and humans. Furthermore, a large number of bacterial mutants and strains can be screened for their virulence and in vivo pathogenicity, opening up new avenues to investigate the so far undiscovered pathways mediating successful host infection by S. suis. In Chapter 6 we applied these two innovative methods (the competence system and the zebrafish larval model) to characterize two different two-component systems (TCS) of S. suis. TCS are important players in the regulation of bacterial adaptation to changes in environmental conditions, including those encountered in the host during infection. In this study, we studied the role of the two TCS of S. suis 2 strain S10 in virulence and in the survival of the bacteria in the bloodstream and host tissue. Chapter 7 summarizes and discusses the key results and the future prospective of the thesis research.

This publication describes his work as a PhD student in the Host-Microbe Interactomics Chair group at Wageningen University within the Gastrointestinal Health theme. It has been completed under the supervision of Prof. Dr Jerry M Wells, Dr Jan Dekker and the TIFN project leader, Prof. Dr Paul de Vos. Mucus serves as a protective layer between the intestinal content and the intestinal wall. It facilitates the passage of the luminal content through the intestine, reducing the risk of mechanical damage to the intestinal epithelium. The overarching goal of this thesis was to investigate the role of mucus in the maintenance of the intestinal immune barrier and the effects of ageing and gender differences on mucus production and the gut barrier. We found by using a mouse model that decreased mucus production leads to changes in microbiota and mucosal stress responses, without the appearance of pathology, demonstrating the importance of mucus in intestinal homeostasis. The mucus barrier was shown to deteriorate during aging but this could be prevented with specific probiotics. Furthermore gender-specific differences in the effects of ageing on the mucosal barrier were found. Increased knowledge on these mechanisms might contribute significantly to disease prevention and treatment, for instance by optimizing gender-specific dietary and pharmacological requirements. The study presented in this thesis was performed within the framework of Top Institute Food and Nutrition, within the GH002 project.

Summary The microbiota of the gastrointestinal (GI) tract plays a key role in the digestion of our food. The human gut microbiota can be studied using in vitro and animal models. In this thesis the mouse model is used to study the microbiota interaction with the diet and the host in different regions along the GI tract. These interacting microbes in the GI tract of humans and other mammals yield a wide range of metabolites, among which the short chain fatty acids (SCFA), in particular butyrate, acetate, and propionate, are the most abundant products of carbohydrate fermentation. Fermentable carbohydrates can modify the composition of the gut microbiota and change the SCFA concentrations in the gut. Opportunities for increasing specific SCFA by targeting their producers with carbohydrates are discussed. Five different fibres – resistant starch, inulin, fructooligosaccharides, arabinoxylan and guar gum – are tested for their modification of the mucosal tissue transcriptome, luminal microbiota composition and SCFA concentrations in the murine colon. The fibres inulin, fructooligosaccharides, arabinoxylan and guar gum led to increased SCFA concentrations and induced similar changes in relative abundance of microbial groups as determined by the MITChip, a phylogenetic microarray targeting the 16S ribosomal RNA of mouse intestinal microorganisms. Furthermore, these four fibres induced regulation of overlapping sets of genes in the mouse intestinal mucosa, where the transcription factor PPARγ was predicted to be a prominent upstream regulator of these processes. Multivariate data integration revealed strong correlations between the expression of genes involved in energy metabolism and the relative abundance of bacteria belonging to Clostridium cluster XIVa. Similar analyses were done for the caeca of the same mice, and were complemented with metatransciptome analyses. To comprehensively analyse RNAseq data of complex natural microbial communities, a de novo metatranscriptome assembly pipeline was developed and applied to unravel the activity profiles of the microbiota residing in the mouse cecum. This revealed distinct contributions of bacterial families to the fermentation of fibres into SCFA, involving the Bifidobacteriaceae, Lachnospiraceae, Clostridiaceae, Bacteroidaceae, Erysipelotrichaceae and Ruminococcaceae in some or all stages of the overall fibre fermentation activity. All families expressed genes encoding enzymes involved in the production of SCFA in different ratios. Specifically, butyrate producing bacteria correlated with a set of host genes involved in processes such as energy metabolism, transcriptional regulation and the mucosal immune system. In addition to complex carbohydrates, amino acids derived from dietary proteins can also serve as substrates for SCFA formation, leading to expansion of the fermentation end-product palet by including branched-SCFA. The long-term effects of high protein-diets on microbial community composition and activity were analysed. The caecal microbiota composition was changed by the high dietary protein. Most of the gene functions detected by metatranscriptomics in these caecal samples were assigned to the Lachnospiraceae, Erysipelotrichaceae and Clostridiaceae. High protein diets induced a decrease of Lachnospiraceae activity, but stimulated the activity of the Erysipelotrichaceae, while the Clostridiaceae appeared to express the broadest range of amino acid metabolism associated pathways. In conclusion, this thesis describes dietary interventions to modulate the mouse intestinal microbiota and mucosa. The data provides expansion of the knowledge on interactions between the diet, microbiota and host. This information can be used to optimize the design and validation on dietary intervention studies in humans.

Lactococcus lactis is an important lactic acid bacteria (LAB) species that is used for the manufacture of dairy products, such as cheese, buttermilk, and other fermented products. The predominant function of this bacterium in dairy fermentation is the production of lactic acid, as its major fermentation end-product that contributes to preservation and microbial safety of the product. Moreover, L. lactis is frequently encountered in natural ecosystems such as in (rotting) plant material. Due to restricted energy source availability, natural microbial communities commonly live in a situation that can be characterized as ‘hunger’, which is different from strict nutrient-starvation. As a consequence, environmental microbes commonly grow at very low-growth rates as compared to laboratory cultures. Analogously, microorganisms can experience such nutrient-poor conditions in diverse industrial fermentation applications. For example, LAB encounter extreme low or no energy source availability during the extended ripening process of cheeses or dry sausages, which can take months. Despite these harsh environmental conditions, many LAB are able to remain viable in these processes for months and sustain a low-level metabolic activity, which plays an important role in their contribution to flavor and aroma formation in the product matrix. In this thesis, the quantitative physiology of L. lactis at near-zero specific growth rates was studies, employing both metabolic and genome-wide transcriptome studies in an experimental set-up of carbon-limited retentostat cultivation. Chapter 2 describes how retentostat cultivation enables uncoupling of growth and non-growth related processes in L. lactis, allowing the quantitative analysis of the physiological adaptations of this bacterium to near-zero growth rates. In chapter 3, transcriptome and metabolome analyses were integrated to understand the molecular adaptation of L. lactis to near-zero specific growth rate, and expand the studies in chapter 2 towards gene regulations patterns that play a profound role in zero-growth adaptation. Chapter 4 describes the enhanced robustness to several stress conditions of L. lactis after its adaptation to extremely low-specific growth rate by carbon-limited retentostat cultivation. In this chapter correlations were modelled that quantitatively and accurately describe the relationships between growth-rate, stress-robustness, and stress-gene expression levels, revealing correlation coefficients for each of the varieties involved. Chapter 5 evaluates the distinction between the transcriptome responses to extended carbon-limited growth and severe starvation conditions, where the latter condition was elicited by switching off the medium supply of the retentostat cultures described in chapter 1. Chapter 6 highlights the comparison of the physiological and molecular adaptations of industrially important microorganisms towards carbon-limited retentostat conditions. In conclusion, this thesis describes the quantitative physiological, metabolic, and genome-wide transcriptional adaptations of L. lactis at near-zero specific growth rates induced by carbon source limited retentostat cultivation, and compares these molecular adaptations to those elicited by strict carbon-starvation conditions.

Discovery of Inhibitors of Bacterial Histidine Kinases Summary The thesis is on novel antibacterial drug discovery (http://youtu.be/NRMWOGgeysM). Using structure-based and fragment-based drug discovery approach, we have identified small-molecule histidine-kinase inhibitors with antibacterial effect against multi-drug resistant strains, including clinical isolates of multi-drug resistant bacteria such as MRSA. Furthermore, we have shown broadening of the antibacterial spectrum and lowering the toxicity of the histidine-kinase inhibitors using nanoparticles. The results open up exciting possibilities for development of novel antibacterial(nano)medicines.