Adapting to change : on the mechanism of type I-E CRISPR-Cas defence
Künne, Tim A. - \ 2017
Wageningen University. Promotor(en): J. van der Oost, co-promotor(en): S.J.J. Brouns. - Wageningen : Wageningen University - ISBN 9789463436649 - 239
immunity - defence mechanisms - rna - bacteria - escherichia coli - analytical methods - priming - immuniteit - verdedigingsmechanismen - rna - bacteriën - escherichia coli - analytische methoden - zaadbevochtiging
Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery.
Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times.
Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis.
Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders.
In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.
In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming.
Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.
How to measure health improvement? : assessment of subtle shifts in metabolic phenotype
Fazelzadeh, Parastoo - \ 2017
Wageningen University. Promotor(en): A.H. Kersten; J.P.M. van Duynhoven, co-promotor(en): M.V. Boekschoten. - Wageningen : Wageningen University - ISBN 9789463430739 - 187
health promotion - improvement - measurement - metabolic profiling - elderly - obesity - microarrays - rna - peripheral blood mononuclear cells - gezondheidsbevordering - verbetering - meting - metabolische profilering - ouderen - obesitas - microarrays - rna - perifere mononucleaire bloedcellen
Human health is impacted by a complex network of interactions between biological pathways, mechanisms, processes, and organs, which need to be able to adapt to a continuously changing environment to maintain health. This adaptive ability is called ‘phenotypic flexibility’. It is thought that health is compromised and diseases develop when these adaptive processes fail. As the product of interactions between several factors such as genetic makeup, diet, lifestyle, environment and the gut microbiome, the ‘metabolic phenotype’ provides a readout of the metabolic state of an individual. Understanding these relationships will be one of a major challenges in nutrition and health research in the next decades. To address this challenge, the development of high-throughput omics tools combined with the application of elaborate statistical analyses will help characterize the complex relationship of (bio) chemicals in human systems and their interaction with other variables including environment and lifestyle to produce the measured phenotype. An important aim of this thesis was to identify phenotype shifts by looking at effect of prolonged resistance-type exercise training on skeletal muscle tissue in older subjects and the possible shift toward the features of younger subjects as a reference for a healthier phenotype. A second aim was to identify phenotype shifts by looking at the response to a challenge in obese subjects and the possible shift toward lean subjects as a reference for a healthier phenotype.
Chapter 2 and 3 of this thesis show how the significant remaining plasticity of ageing skeletal muscle can adapt to resistance-type exercise training. The data indicate that frail and healthy older subjects have two distinct phenotypes according to the skeletal muscle tissue metabolite profiles and that exercise training shifts aged muscle towards a younger phenotype. We showed that the effect of exercise on amino acid derived acylcarnitines (AAAC’s) in older subjects points towards decreased branched chain amino acid catabolism, likely due to compromised activation of the branched chain α-keto acid hydrogenase (BCKDH) complex. Furthermore, we found that the protocadherin gamma gene cluster might be involved in aged-muscle denervation and re-innervation. Finally, plasma was found to be a poor indicator of muscle metabolism, emphasizing the need for direct assessment of metabolites in muscle tissue.
Chapter 4 of this thesis examines whether a mixed meal challenge response provides a readout for a shift in phenotype upon weight loss in obese male subjects. We concluded that weight loss moderately affects the mixed meal challenge response of both plasma metabolome and transcriptome of peripheral blood mononuclear cells in obese subjects. Measurements at the fasted and postprandial state also provide us with a different type of information.
In Chapter 5 it is demonstrated that the global testing of pathways could provide a concise summary of the multiple univariate testing approach used in Chapter 4. In Chapter 6 it is discussed how the findings of this thesis increase our understanding of how to measure phenotypic flexibility as a proxy of health. In this thesis it is shown that the correlations between tissue and plasma metabolites are rather weak, emphasising the need to perform organ-specific studies. Availability of less invasive/painful sampling techniques and the use of small amounts of tissue would enable larger scale human studies on adipose tissue and skeletal muscle to more accurately define phenotypical shifts due to diet or lifestyle interventions. With respect to the assessment of phenotypical flexibility by omics approaches, significant complications can be expected in trying to relate plasma metabolism to PBMC gene expression. Organ-focussed approaches that integrate multiple omics levels using system biology approaches are considered to be a lot more promising.
From species to trait evolution in Aethionema (Brassicaceae)
Mohammadin, Setareh - \ 2017
Wageningen University. Promotor(en): M.E. Schranz. - Wageningen : Wageningen University - ISBN 9789463431385 - 125
brassicaceae - evolution - rna - genomes - genetic diversity - phytogeography - glucosinolates - quantitative trait loci - next generation sequencing - brassicaceae - evolutie - rna - genomen - genetische diversiteit - plantengeografie - glucosinolaten - loci voor kwantitatief kenmerk - next generation sequencing
The plant family Brassicaceae (or crucifers) is an economically important group that includes many food crops (e.g. cabbages and radishes), horticultural species (e.g. Draba, Iberis, Lunaria), and model plant species (particularly Arabidopsis thaliana). Because of the fundamental importance of A. thaliana to plant biology, it makes the Brassicaceae an ideal system for comparative genomics and to test wider evolutionary, ecological and speciation hypotheses. One such hypothesis is the ‘Whole Genome Duplication Radiation Lag Time’ (WGD-RLT) model for the role of polyploidy on the evolution of important plant families such as the Brassicaceae. The WGD-RLT model indicates a higher rate of diversification of a core-group compared to its sister group, due to a lag time after a whole genome duplication event that made it possible for novel traits or geo- or ecological events to increase the core groups diversification rate.
Aethionema is the species-poor sister genus of the core Brassicaceae and hence is at an important comparative position to analyse trait and genomic evolution of the species-rich core group. Aethionema species occur mainly in the western Irano-Turanian region, which is concordantly the biodiversity hotspot of the Brassicaceae family. Moreover comparing Aethionema to the Brassicaceae core group can help us to understand and test the ‘WGD-RLT’ model. However to be able to do so we first need to know more about Aethionema. In this thesis, I investigated various levels of evolutionary change (from macro, to micro to trait evolution) within the genus Aethionema, with a major focus the emerging model species Aethionema arabicum.
Next generation sequencing has made it possible to use the genomes of many species in a comparative framework. However, the formation of proteins and enzymes, and in the end the phenotype of the whole plant, relies on transcription from particular regions of the genome including genes. Hence, the transcriptome makes it possible to assess the functional parts of the genome. However, the functional part of the genome not only relies on the protein coding genes. Gene regulatory elements like promoters and long non-coding RNAs function as regulators of gene expression and hence are involved in increasing or decreasing transcription. In Chapter 2 I used the transcriptome of four different Aethionema species to understand the lineage specificity of these long non-coding RNAs. Moreover in a comparison with the Brassicaceae core group and Brassicaceae’s sister family the Cleomaceae I show that although the position of long non-coding RNAs can be conserved, their sequences do not have to be.
Most of the Aethionema species occur in the Irano-Turanian region, a politically instable region, making it hard for scientist to collect from. However the natural history collections made throughout the last centuries are a great resource. Combing these collections with the newest sequencing techniques, e.g. next generation sequencing, have allowed me to infer the phylogeny of ~75% of the known Aethionema species in a time calibrated and historical biogeographical framework. Hence, I was able to establish that Aethionema species likely originated from the Anatolian Diagonal and that major geological events like the uplift of the Turkish and Iranian plateau have had a hand in their speciation (Chapter 3).
To examine species-level processes I sequenced and analysed transcriptomes of eight Ae. arabicum accessions coming from Cyprus, Iran and Turkey to investigate population structure, genetic diversity and local adaptation (Chapter 4). The most prominent finding was a ploidy difference between the Iranian and Turkish/Cypriotic lines, whereby the former were (allo)tetraploid and the latter diploid. The tetraploid Iranian lines seem to have one set of alleles from the Turkish/Cypriotic gene-pool. However we do not know where the other alleles come from. In addition to the differences in ploidy level there are also differences in glucosinolate defence compounds between these two populations (Iranian vs Turkish/Cypriotic), with the Iranian lines lacking the diversity and concentration of indolic glucosinolates that the Turkish/Cypriotic lines have. This chapter serves as a good resource and starting point for future research in the region, maybe by using the natural history collections that are at hand.
Glucosinolates (i.e. mustard oils) are mainly made by Brassicales species, with their highest structural diversity in the Brassicaceae. In Chapter 5, I examined two Ae. arabicum lines (CYP and TUR) and their recombinant inbred lines to assess glucosinolate composition in different tissues and throughout the plants development. The levels of glucosinolates in the leaves changed when Ae. arabicum went from vegetative to a reproductive state. Moreover, a major difference in glucosinolate content (up to 10-fold) between CYP and TUR indicates a likely regulatory pathway outside of the main glucosinolate biosynthesis pathway. Multi-trait and multi-environment QTL analyses based on leaves, reproductive tissues and seeds identified a single major QTL. Fine mapping this region reduced the interval to only fifteen protein coding genes, including the two most intriguing candidates: FLOWERING LOCUS C (FLC) and the sulphate transporter SULTR2;1. These findings show an interesting correlation between development and defence.
Finally, Chapter 6 gives a final discussion of this thesis and its results. It brings the different topics together, put them in a bigger picture and look forward to new research possibilities.
Mining the human intestinal microbiota for biomarkers associated with metabolic disorders
Hermes, Gerben - \ 2016
Wageningen University. Promotor(en): Hauke Smidt; Erwin Zoetendal. - Wageningen : Wageningen University - ISBN 9789462579514 - 205
gastrointestinal microbiota - metabolic disorders - biomarkers - obesity - intestinal microorganisms - antibiotics - dna sequencing - rna - ribosomal rna - microbiota van het spijsverteringskanaal - stofwisselingsstoornissen - biomarkers - obesitas - darmmicro-organismen - antibiotica - dna-sequencing - rna - ribosomaal rna
After birth, our gastrointestinal (GI) tract is colonized by a highly complex assemblage of microbes, collectively termed the GI microbiota, that develop intimate interactions with our body. Recent evidence indicates that the GI microbiota and its products may contribute to the development of obesity and related diseases. This, coupled with the current worldwide epidemic of obesity, has moved microbiome research into the spotlight of attention. Although the main cause of obesity and its associated metabolic complications is excess caloric intake compared with expenditure, differences in GI tract microbial ecology between individuals might be an important biomarker, mediator or even new therapeutic target. Nevertheless, it is currently still unclear which bacterial groups play a role in the development of the metabolic syndrome in humans. This might partly be explained by: 1. Biological factors such as the heterogeneity in genotype, lifestyle, diet; and the often complex aetiology of human disease of which the metabolic syndrome is no exception. 2. Technological factors, such as the use of miscellaneous incompatible methods to assess the gut microbiota, often enumerating specific groups rather than using broad 16S rRNA gene surveys or metagenomics. 3. Studies vary greatly in the populations considered, their designs, and the degree of control for potential confounding factors such as lifestyle and diet. Nevertheless, recent research on this matter has shown a conceptual shift by focusing on more homogenous subpopulations, based on stricter control over variables such age range or through the use of both anthropometric (weight, total body fat) as well as biochemical variables (insulin resistance, hyperlipidaemia) to define groups.
Perturbations in microbial diversity and community structure in adults with overweight and obesity may be partly due to long-term dietary habits or physiological changes in these subjects. As such, exploring the association between the gut microbiota and variation in BMI and weight in early life, prior to or close to the onset of overweight, might provide additional insights into these processes. Therefore, we studied the fecal microbiota of 295 six-seven year old children from the KOALA Birth Cohort, living in the south of the Netherlands. This age range is relatively uncharted microbiota territory. We found that its composition seems to conform to tot same ecosystem rules as that of adults. The bimodal distribution pattern of several bacterial groups as well as their co-correlating groups that were reported previously, including Uncultured Clostridiales II (UCII), Prevotella spp. and Dialister were confirmed. Furthermore, one of the previously described bimodal groups (Uncultured Clostridiales I) was shown before to exhibit very clear shifting state probabilities associated with ageing, where the high abundance state was mainly observed above 40 years of age. This was corroborated as no support for bimodality of this group was observed in the children included in the study described here. A large part of the variation in microbiota composition was explained by the abundance of aforementioned groups in contrast to the anthropometric outcomes, suggesting that in this group of healthy children within a relatively normal weight range, weight and associated parameters were not major drivers of overall genus-level microbial composition or vice versa. Hereafter, multiple linear and logistic regression models with rigorous adjustment for confounders were applied to investigate individual microbiota features association with weight related anthropometric outcomes. Previously reported parameters such as diversity, richness and Bacteroidetes to Firmicutes ratio, were not significantly associated with any of the outcomes. Nevertheless, the abundance of several specific bacterial taxa; Akkermansia, Sutterella wadsworthia et rel. and Bryantella formatexigens et rel. and the dichotomous abundance state of the bi-modally distributed UCII was consistently associated with weight-related outcomes.
Other biochemical features of the metabolic syndrome have been associated with the gut microbiome. Mainly rodent studies have indicated that antibiotic treatment may improve glucose homeostasis and metabolic impairments. Therefore, the effects of gut microbiota manipulation by antibiotics (7d administration of amoxicillin, vancomycin or a placebo) on tissue-specific insulin sensitivity, energy metabolism, gut permeability and inflammation in 57 obese, pre-diabetic men from the same geographical region, were investigated. Vancomycin decreased bacterial diversity and significantly reduced well known butyrate- producing Firmicutes from Clostridium clusters IV and XIVa and bacterial groups involved in bile acid metabolism. These changes occurred concomitantly with altered plasma and fecal concentrations of these metabolites. In adipose tissue, gene expression of oxidative pathways was upregulated by antibiotics, whereas immune-related pathways were downregulated by vancomycin. However, antibiotic treatment had no significant effects on tissue-specific insulin sensitivity, energy/substrate metabolism, postprandial hormones and metabolites, systemic inflammation, gut permeability and adipocyte size. Importantly, despite a still considerably altered microbial composition at eight weeks follow-up, energy harvesting, adipocyte size and whole-body insulin sensitivity (HOMA-IR) remained unaltered. Overall these data indicate that interference with adult microbiota by antibiotic treatment for 7 days had no clinically relevant impact on metabolic health in obese humans. These data are in contrast with several rodent studies as well as a human intervention. The present study, which was well-powered and placebo-controlled, indicates that the previously reported vancomycin-induced effects on human peripheral insulin sensitivity are probably of minor physiological significance.
The aforementioned group that was relatively homogeneous with regards to phenotype was combined with another cohort with similar phenotypical characteristics (obese, male and pre-diabetic) from another region of the Netherlands, to investigate whether tissue specific insulin sensitivity, as measured by the golden standard hyperinsulinemic-euglycemic clamp technique, is related to a specific microbial pattern. Remarkably, despite the fact that both cohorts were constructed based on comparable recruitment strategies, the average microbiota composition in both cohorts showed pronounced differences. Firstly, we found no consistent and significant association between liver, adipose tissue or skeletal muscle insulin sensitivity and the microbiota in both cohorts. Nevertheless, Random Forests classifiers using microbiota composition as predictors revealed taxa associated with fasting glucose concentrations and HbAc1 but only in one cohort. The top microbial features distinguishing classes were different Proteobacteria and groups involved in butyrogenesis, such as Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium rectale and related species, for fasting glucose levels. For HbAc1 these taxa were Oscillospira guillermondii, Sporobacter termitidis, Lactobacillus gasseri and Peptococcus niger and related species. The striking cohort-specific observations suggest that the relation between microbiota composition and type 2 diabetes mellitus as well as other characteristics of the metabolic syndrome is very dependent on the selected cohort of patients and their respective baseline microbiota composition. Similar observations have been made by other researchers as well. It could be that differences in microbiota composition are not associated with the insulin resistance phenotype when the overweight and/or obese state of the patient is already established, as is the case for our metabolic syndrome patients. In the latter case we cannot exclude that the composition of the fecal microbiota may play a role in the worsening of insulin sensitivity in an early stage in the development from a lean towards an overweight/obese phenotype. Furthermore, the observation of a subgroup- specific microbiota only observed in one of the cohorts might indicate an alternative state of microbiota composition driven by yet unknown forces. Nevertheless, this study clearly demonstrated that cohort-specific microbiota differences hamper finding a consensus biological interpretation between cross-sectional studies. This, combined with the complexity of individual disease pathogenesis, as well as the individual-specific differences in microbiota composition, may explain the inconsistency in observations between different studies concerning the identification of signature microbes for obesity, irritable bowel syndrome and other diseases.
Besides the biological drivers for cohort specific inconsistencies in identified microbial biomarkers, there are also technological factors. Although high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes, evidence is increasing that methodology rather than the biological variation is responsible for observed sample composition and distribution. Large meta-analyses would aid in elucidating whether the basis for these observed inconsistencies is biological, technical or maybe a combination of both. To facilitate these meta-analyses of microbiota studies we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different Mock Communities (MC). NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies. The analysis of α- and β-diversity of these MC confirmed conclusions guided by biology rather than the methodological aspects. This pipeline was applied to biological samples to monitor the developing communities an in vitro gut model (TIM-2) fed either with a normal diet, or modified versions from which the carbohydrate (MPLC) or protein fraction was diluted (LPMC) for 72h. In combination with global metatranscriptomics and metabolomics this revealed that each diet produced distinct microbial communities and temporal patterns and ratios of metabolites. The microbiota in reactors fed diets containing normal carbohydrate levels were enriched in members of the genera Prevotella, Subdoligranulum, Blautia and Bifidobacterium, all associated with carbohydrate fermentation. In turn, the microbiota in the reactors fed the MPLC diet, containing ten-fold less carbohydrates, was enriched in the genus Bacteroides, which is associated with diets rich in protein and animal fat. This setup allows researchers to study the (trophic) interactions and task division within a community and how they are impacted by diet-related factors under controlled conditions, which may assist in defining causal links between specific diet-derived parameters microbial groups and their activities.
In conclusion, currently it seems that GI microbiota based biomarkers associated with metabolic impairments and anthropometric variables associated with the metabolic syndrome are cohort specific or possibly individual, which could partly be due to the use of incompatible analytical approaches. Nevertheless, there is growing evidence that human health is a collective property of the human body and its associated microbiome and thus requires to study the interface of two very complex systems, i.e. on one side the extraordinary coding capacity, high inter-individuality and complex dynamics of the microbiome and on the other side the multifactorial individual nature of human disease. In light of these observations the manifestation of individual dynamics of the microbiota with the host when homeostasis is lost seems plausible and likely.
Epigenetic inheritance in apomictic dandelions : stress-induced and heritable modifications in DNA methylation and small RNA
Preite, V. - \ 2016
Wageningen University. Promotor(en): Wim van der Putten, co-promotor(en): K.J.F. Verhoeven. - Wageningen : Wageningen University - ISBN 9789462578715 - 152
taraxacum officinale - epigenetics - inheritance - apomixis - dna methylation - rna - heritability - stress - taraxacum officinale - epigenetica - overerving - apomixis - dna-methylering - rna - heritability - stress
Epigenetic variation, such as changes in DNA methylations, regulatory small RNAs (sRNAs) and chromatin modifications can be induced by environmental stress. There is increasing information that such induced epigenetic modifications can be transmitted to offspring, potentially mediating adaptive transgenerational responses to environmental changes. However, it is unclear if this phenomenon is common and relevant for adaptation under natural conditions. My thesis study aimed to examine epigenetic inheritance in common and widespread apomictic dandelions (Taraxacum officinale Wig.). Due to their asexual reproduction mode by producing clonal seeds offspring from seeds are genetically uniform and thus suitable to investigate epigenetic effects that are not confounded with genetic variation.
I exposed apomictic dandelion lineages to drought and salicylic acid (SA) stress, which induces plant defense responses following pathogen attack, and found effects on patterns of DNA methylation up to two stress-free offspring generations after exposure. However, a heritable stress signal was not present in all tests and was stress- and lineage-dependent. Drought stress triggered a weak and lineage-dependent signal that was lost again in the second offspring generation. SA treatment revealed a stress-related increased rate of DNA methylation changes in the two offspring generations, but no stress signal was found in the stressed generation itself. I also observed changes in small RNA production due the drought and SA stress experienced two generations ago. These transgenerational sRNA effects showed association with gene functions related to grandparental drought and SA stress, which suggests functional relevance of the transgenerational effects.
I used a reciprocal transplantation field experiment to investigate whether exposing dandelions to natural field stresses also triggers DNA methylation changes. The experiment revealed evidence of adaptive divergence between the populations, suggesting that non-native habitats are experienced as more stressful. However, under these field conditions no induction-based DNA methylation changes were found that persisted into offspring.
By using AFLP and MS-AFLP screening of natural apomictic dandelion populations across a north-south transect in Europe I examined if natural, heritable DNA methylation variation reflects underlying genetic variation, or if it shows patterns that are not predictable from underlying genetics. I found that a large part of heritable DNA methylation differentiation along the north-south transect was correlated with genetic differentiation. However, a fraction of differentiation in heritable DNA methylation was independent from genetic variation. This suggests a potential of epigenetics to play an evolutionary role independently, at least to some extent, from underlying genetics. Overall, I found indications of epigenetic inheritance in apomictic dandelions. Whether epigenetic variation would result in adaptive phenotypic variation in nature and whether it would persist long enough to play a relevant role in adaptation remains unclear and requires further study.
Tospovirus : induction and suppression of RNA silencing
Hedil, Marcio - \ 2016
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462577848 - 137
tospovirus - rna - plants - immunity - gene silencing - biochemical pathways - rna interference - viral proteins - plant viruses - tospovirus - rna - planten - immuniteit - uitschakelen van genexpressie - biochemische omzettingen - rna-interferentie - viruseiwitten - plantenvirussen
While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants, tospoviruses have evolved a silencing suppressor function via its NSs protein. This viral protein has previously been shown to bind dsRNA that likely arises from secondary RNA folding structures in viral RNAs. The aim of the present research was to further investigate the interaction between tospoviruses and the plant antiviral RNA silencing response, including the target sequences in the viral RNA and the further role of the NSs protein as part of the tospovirus counterdefence strategy.
In order to identify the target and inducer for RNA silencing against tospoviruses, small RNAs purified from plants infected with three tospoviral species, tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), were probed against the viral RNA segments of these three different tospoviruses (Chapter 3). Virus-derived siRNAs (vsiRNAs) were found to be derived from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) from where hardly any vsiRNAs were found. Similar profiles were observed with the closely related GRSV as well as the distantly related TYRV. Dicer cleavage assays using Drosophila melanogaster embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′-UTR did not show more rapid/strong silencing, and profiling of the corresponding siRNAs generated outside the context of a viral infection still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP region/structure is a weak inducer of RNA silencing and plays a minor role in the amplification of a strong antiviral RNA silencing response.
Next, a biochemical analysis was performed using E. coli-expressed and purified NSs from GRSV and TYRV. The binding of both purified NSs proteins to small and long dsRNA indicated that this is likely a generic feature of all tospoviral NSs proteins (Chapter 4). Binding of siRNAs to NSs furthermore revealed two shifts on polyacrylamide gels i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When the NSs protein of TSWV resistant breaker (RB) isolates (of Tsw-gene based resistance), which lack RSS activity when transiently expressed, were analyzed using extracts from infected plants still a major (second) shift of siRNAs was observed, similar to the case with extracts containing TSWV resistant inducer (RI) isolates. In contrast, plant extracts containing transiently expressed NSs proteins alone (no infection) showed only the smaller, first shift for NSsRI but no shift for NSsRB.
The ability of NSs to suppress systemic silencing is demonstrated for the NSs proteins of TSWV, GRSV and TYRV, and their relative strengths to suppress local and systemic silencing were compared (Chapter 5). A system was developed to quantify suppression via GFP silencing constructs, allowing comparison of relative RNA silencing suppressor strength. In this case NSs proteins of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Moreover, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream of the biogenesis of siRNAs and their sequestration.
In summary, evidence is presented showing that sequences from all three genomic segments from tospovirus are targeted by the plant RNA silencing machinery. The predicted hairpin sequence in the IGR is poorly targeted. Biochemical experiments with purified NSs proteins further support the view that binding to small and long dsRNA is a characteristic common to all tospovirus NSs proteins. Furthermore, tospovirus NSs proteins suppress systemic silencing and there are indications that local and systemic silencing suppression can be uncoupled in NSs. Collectively, these results add to our current understanding of the tospovirus-plant interaction involving antiviral RNA silencing and the viral counter-defence (NSs protein). Lastly, the results of the research presented in this thesis are discussed in light of the current knowledge on RNA silencing and to present some future perspectives and questions that remain open and/or resulted from this thesis (Chapter 6).
Two Distinct DNA Binding Modes Guide Dual Roles of a CRISPR-Cas Protein
Blosser, T.R. ; Loeff, L. ; Westra, E.R. ; Vlot, M. ; Künne, T.A. ; Sobota, M. ; Dekker, C. ; Brouns, S.J.J. ; Joo, C. - \ 2015
Molecular Cell 58 (2015)1. - ISSN 1097-2765 - p. 60 - 70.
bacterial immune-system - in-vitro reconstitution - memory b-cells - surveillance complex - crystal-structure - streptococcus-thermophilus - adaptive immunity - escherichia-coli - seed sequence - rna
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets
Reduced seed germination in Arabidopsis over-expressing SWI/SNF2 ATPase genes
Leeggangers, H.A.C.F. ; Folta, A. ; Muras, A. ; Nap, J.P.H. ; Mlynarova, L. - \ 2015
Physiologia Plantarum 153 (2015)2. - ISSN 0031-9317 - p. 318 - 326.
thaliana - dormancy - transcription - maturation - stress - arrest - growth - rna
In the life of flowering plants, seed germination is a critical step to ensure survival into the next generation. Generally the seed prior to germination has been in a dormant state with a low rate of metabolism. In the transition from a dormant seed to a germinating seed, various epigenetic mechanisms play a regulatory role. Here, we demonstrate that the over-expression of chromatin remodeling ATPase genes (AtCHR12 or AtCHR23) reduced the frequency of seed germination in Arabidopsis thaliana up to 30% relative to the wild-type seeds. On the other hand, single loss-of-function mutations of the two genes did not affect seed germination. The reduction of germination in over-expressing mutants was more pronounced in stress conditions (salt or high temperature), showing the impact of the environment. Reduced germinations upon over-expression coincided with increased transcript levels of seed maturation genes and with reduced degradation of their mRNAs stored in dry seeds. Our results indicate that repression of AtCHR12/23 gene expression in germinating wild-type Arabidopsis seeds is required for full germination. This establishes a functional link between chromatin modifiers and regulatory networks towards seed maturation and germination.
Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3
Gong, B. ; Shin, M. ; Sun, J. ; Jung, C.H. ; Bolt, E.L. ; Oost, J. van der; Kim, J.S. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)46. - ISSN 0027-8424 - p. 16359 - 16364.
bacterial immune-system - in-vitro reconstitution - escherichia-coli - antiviral defense - adaptive immunity - crystal-structure - structural basis - rna - complex - cascade
Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.
Comparative efficacy of two next-generation Rift Valley fever vaccines
Kortekaas, J.A. ; Oreshkova, N. ; Keulen, L.J.M. van; Kant, J. ; Bosch, B.J. ; Bouloy, M. ; Moulin, V. ; Goovaerts, D. ; Moormann, R.J.M. - \ 2014
Vaccine 32 (2014). - ISSN 0264-410X - p. 4901 - 4908.
nss protein - down-regulation - mp-12 vaccine - virus-vaccine - safety - transcription - sheep - immunogenicity - attenuation - rna
Rift Valley fever virus (RVFV) is a re-emerging zoonotic bunyavirus of the genus Phlebovirus. A natural isolate containing a large attenuating deletion in the small (S) genome segment previously yielded a highly effective vaccine virus, named Clone 13. The deletion in the S segment abrogates expression of the NSs protein, which is the major virulence factor of the virus. To develop a vaccine of even higher safety, a virus named R566 was created by natural laboratory reassortment. The R566 virus combines the S segment of the Clone 13 virus with additional attenuating mutations on the other two genome segments M and L, derived from the previously created MP-12 vaccine virus. To achieve the same objective, a nonspreading RVFV (NSR-Gn) was created by reverse-genetics, which not only lacks the NSs gene but also the complete M genome segment. We have now compared the vaccine efficacies of these two next-generation vaccines and included the Clone 13 vaccine as a control for optimal efficacy. Groups of eight lambs were vaccinated once and challenged three weeks later. All mock-vaccinated lambs developed high fever and viremia and three lambs did not survive the infection. As expected, lambs vaccinated with Clone 13 were protected from viremia and clinical signs. Two lambs vaccinated with R566 developed mild fever after challenge infection, which was associated with low levels of viral RNA in the blood, whereas vaccination with the NSR-Gn vaccine completely prevented viremia and clinical signs.
Fine mapping of the tomato yellow leaf curl virus resistance gene Ty-2 on chromosome 11 of tomato
Yang, X. ; Caro Rios, C.M. ; Hutton, S.F. ; Scott, J.W. ; Guo, Y. ; Wang, Xiaoxuan ; Rashid, H. ; Szinay, D. ; Jong, J.H.S.G.M. de; Visser, R.G.F. ; Bai, Y. ; Du, Y. - \ 2014
Molecular Breeding 34 (2014)2. - ISSN 1380-3743 - p. 749 - 760.
cultivated tomato - rna - introgression - recombination - transcription - replication - encodes - locus
Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato (Solanumlycopersicum) fromwild tomato accessions. A major gene, Ty-2 from S. habrochaites f. glabratum accession ‘‘B6013,’’ that confers resistance to TYLCV was previously mapped to a 19-cMregion on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flankingmarkersC2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized Ty-2 to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the Ty-2 region is likely not responsible for the Ty-2-conferred resistance and that two candidate genes might play a role in the Ty-2-conferred resistance. Severalmarkers very tightly linked to the Ty-2 locus are presented and useful for marker-assisted selection in breeding programs to introgress Ty-2 for begomovirus resistance.
DNA-guided DNA interference by a prokaryotic Argonaute
Swarts, D.C. ; Jore, M.M. ; Westra, E.R. ; Zhu, Y. ; Janssen, J.H. ; Snijders, A.P. ; Wang, Y. ; Patel, D.J. ; Berenguer, J. ; Brouns, S.J.J. ; Oost, J. van der - \ 2014
Nature 507 (2014)7491. - ISSN 0028-0836 - p. 258 - 261.
thermophile thermus-thermophilus - silencing complex - structural basis - rna - archaebacteria - recognition - eubacteria - cleavage - sequence - protein
RNA interference is widely distributed in eukaryotes and has a variety of functions, including antiviral defence and gene regulation. All RNA interference pathways use small single-stranded RNA (ssRNA) molecules that guide proteins of the Argonaute (Ago) family to complementary ssRNA targets: RNA-guided RNA interference. The role of prokaryotic Ago variants has remained elusive, although bioinformatics analysis has suggested their involvement in host defence. Here we demonstrate that Ago of the bacterium Thermus thermophilus (TtAgo) acts as a barrier for the uptake and propagation of foreign DNA. In vivo, TtAgo is loaded with 5'-phosphorylated DNA guides, 13-25 nucleotides in length, that are mostly plasmid derived and have a strong bias for a 5'-end deoxycytidine. These small interfering DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite structural homology to its eukaryotic counterparts, TtAgo functions in host defence by DNA-guided DNA interference
|De bouwstenen van het leven : een introductie tot de moleculaire celbiologie
Prinsen, J.A.M.M. ; Leij, F.R. van der - \ 2014
Wageningen : Wageningen Academic Publishers - ISBN 9789086862412 - 470
cellen - moleculaire biologie - celbiologie - atomen - moleculen - eiwitten - dna - rna - metabolisme - celdeling - studieboeken - cells - molecular biology - cellular biology - atoms - molecules - proteins - dna - rna - metabolism - cell division - textbooks
In De bouwstenen van het leven wordt - ook voor leken - op een toegankelijke wijze een overzicht gegeven van de basale kennis van de biochemie en de moleculaire celbiologie (ook wel Life Sciences genoemd). De wereld van DNA, RNA en eiwitten, van energiemetabolisme tot en met genetica, is door Jan Prinsen en Feike van der Leij op een overzichtelijke manier begrijpelijk gemaakt. Niet alleen voor Bachelor-studenten een goed alternatief naast Engelstalige standaardwerken, maar voor iedereen die wil weten hoe de processen in ons lichaam en in de natuur om ons heen op moleculair niveau verklaard kunnen worden. De auteurs hebben daarbij een klassieke kijk gecombineerd met de laatste inzichten en maken op een heldere manier gebruik van de soms onvermijdelijke vaktaal. Het is daarmee oprecht een introductie tot de moleculaire celbiologie te noemen. De informatie wordt in lagen aangereikt: de lezer kan zelf de mate van diepgang en detail bepalen.
Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition
Westra, E.R. ; Semenova, E. ; Datsenko, K.A. ; Jackson, R.N. ; Wiedenheft, B. ; Severinov, K. ; Brouns, S.J.J. - \ 2013
Plos Genetics 9 (2013)9. - ISSN 1553-7404
bacterial immune-system - processes pre-crrna - escherichia-coli - streptococcus-thermophilus - antiviral defense - seed sequence - foreign dna - invader dna - h-ns - rna
Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism
Molecular biology - New tool for genome surgery
Oost, J. van der - \ 2013
Science 339 (2013)6121. - ISSN 0036-8075 - p. 768 - 770.
adaptive immunity - dna cleavage - rna - endonuclease - bacteria
Gene therapy is the holy grail of human medicine. Many diseases are caused by a defective gene, sometimes with a mutation as subtle as a single-nucleotide variation. Before restoration of such a mutation in a patient's genome can take place, the target nucleotide sequence has to be cleaved at a single position, out of 3 billion possibilities. This degree of precise surgery requires an enzyme with highly selective target recognition. Successful editing of eukaryotic genomes has been accomplished with DNA nucleases designed to bear a unique site that binds to a specific DNA sequence. A major drawback of these protein-guided systems to "engineer" genomes, however, is that each new target sequence requires laboriously adjusting the specificity of the nuclease's DNA binding site. On pages 819 and 823 of this issue, Cong et al. (1) and Mali et al. (2) describe efficient genome editing in human cells based on an RNA-guided system
Selection and characterization of DNA aptamers
Ruigrok, V.J.B. - \ 2013
Wageningen University. Promotor(en): John van der Oost; Hauke Smidt. - S.l. : s.n. - ISBN 9789461735645 - 152
aptameren - dna - selectie - nucleotiden - technieken - eiwit - rna - aptamers - dna - selection - nucleotides - techniques - protein - rna
This thesis focusses on the selection and characterisation of DNA aptamers and the various aspects related to their selection from large pools of randomized oligonucleotides. Aptamers are affinity tools that can specifically recognize and bind predefined target molecules; this ability, however, is not exclusively associated with aptamers. Antibodies are the most successful affinity tools used today, but alternative affinity tools such as aptamers, engineered binding proteins and molecular imprinted polymers are emerging as sound alternatives. A comparison of their properties is described in Chapter 1. The strength and specificity of the interaction between an affinity tool and its target molecule is an important feature. Generally, an affinity tool should have a high affinity for its target and should be highly specific in order to be useful for research or commercial purposes. One highly advanced method to characterise the interaction between an affinity tool and its target molecule makes use of a Surface Plasmon Resonance (SPR)-based biosensor. Although SPR is an optical phenomenon, in depth knowledge of the physics behind this phenomenon is not required to operate an SPR-based biosensor. Experiments should be performed in a correct way, and therefore it is important to understand how experimental parameters, such as flow rate, ligand density, surface preparation, and reagent quality either improve or adversely affect data quality. Experimental considerations, as well as methods for proper data analysis are discussed in Chapter 2. Data generated within the framework of the 2011 Global Label-free Interaction Benchmark study serves as a typical example.
The ability of aptamers to bind a specific target originates from an intricate interplay between the oligonucleotide sequence and the three dimensional structure that this sequence allows to form. In Chapter 3 this is illustrated by the selection and characterisation of streptavidin-binding aptamers. Five aptamer families were identified, sharing a similar secondary structure. Although slight variations at the actual sequence level are present, two guanines are completely conserved. Using site-specific mutagenesis it was demonstrated that these guanines are essential for streptavidin binding. Binding kinetics and the dissociation constant of each aptamer was determined by SPR and were all within the range of 35-375 nM. Two aptamers can bind one streptavidin tetramer at the same time, as was shown by native mass spectrometry analysis. In addition, the three dimensional structure of the most abundant aptamer was modelled and manually docked to the streptavidin structure, in order to gain more insight in the molecular basis of the interaction. To extend this knowledge even further, crystallisation trails, aiming to obtain a co-crystal structure for the streptavidin-aptamer complex, were performed, and are described in Chapter 4. Unfortunately, these trials did only yield protein crystals, instead of the desired streptavidin-aptamer complex. Therefore, alternative experimental and computational approaches were investigated that could be used to study aptamer-protein interactions. Combining techniques as SPR, small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), and Dynamic light scattering (DLS) could be considered as an alternative to X-ray crystallography. In addition, some of these techniques may provide information on the dynamics of complex formation, whereas crystallography gives a time- and position-averaged image.
Besides streptavidin, another protein, SpaC, was subjected to aptamer selection in this thesis. SpaC is a subunit of pili present on the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG and contains a binding domain for human-mucus. Presence of this binding domain is considered an advantage, because it is already designed to interact with other molecules. Successful production and purification of recombinant SpaC protein is described in Chapter 5, as well as the characterisation of DNA oligonucleotides enriched during subsequent selection rounds. Sequence analysis revealed that specific oligonucleotides are indeed enriched. Furthermore, results of pilot SPR experiments indicated that they bind specifically to SpaC, but more detailed experiments are required to unambiguously demonstrate this.
The dynamics of aptamer enrichment are poorly understood. To address this issue and to gain a more fundamental insight in the aptamer selection process, a multiplexed high throughput sequencing effort was started, which is described in Chapter 6. In this approach samples of 70 selection rounds, derived from 8 distinct aptamer selection experiments, were barcoded, pooled together and sequenced; over 84 million paired-end reads were obtained and analysed. Samples enriched to bind streptavidin show a decrease in α-diversity across subsequent selection rounds. Interestingly, large differences were found between the composition of fractions enriched by affinity elution and thermal elution. Moreover, a small scale comparison of two clone libraries showed that affinity elution, which is expected to enrich more specific binders, also specifically enriches rapid binders.
Supportive SPR experiments have made an important contribution throughout this thesis. The main focus in Chapter 7, however, is on a new application of SPR. The development of a capture approach for supercoiled plasmid DNA, using a triple helix forming oligonucleotide, is described. It could be demonstrated that plasmid DNA can indeed be captured and that SPR can subsequently be used to derive kinetic parameters of a specific interaction with a plasmid. In this particular case the interaction between Lac repressor and its plasmid-based operator was characterised, showing that the association and dissociation rates are ~18 times lower, but that the affinity is the same, when compared to binding to linear operator DNA. This difference underscores the importance of using a DNA substrate with a physiologically relevant topology for studying DNA-protein interactions.
Adaptive & heritable immunity in bacteria : on the regulation and mechanism of CRISPR defense
Westra, E.R. - \ 2013
Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Stan Brouns. - S.l. : s.n. - ISBN 9789461735003 - 254
bacteriën - immuniteit - immuunsysteem - verdedigingsmechanismen - rna - prokaryoten - bacteria - immunity - immune system - defence mechanisms - rna - prokaryotes - cum laude
cum laude graduation (with distinction)
A large-scale evaluation of computational protein function prediction
Radivojac, P. ; Clark, W.T. ; Oron, T.R. ; Schnoes, A.M. ; Wittkop, T. ; Kourmpetis, Y.A.I. ; Dijk, A.D.J. van; Friedberg, I. - \ 2013
Nature Methods : techniques for life scientists and chemists 10 (2013). - ISSN 1548-7091 - p. 221 - 227.
gene ontology - sequence - rna - annotation - database - network - genomes - gold
Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based critical assessment of protein function annotation (CAFA) experiment. Fifty-four methods representing the state of the art for protein function prediction were evaluated on a target set of 866 proteins from 11 organisms. Two findings stand out: (i) today's best protein function prediction algorithms substantially outperform widely used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is considerable need for improvement of currently available tools.
Modest Nonadherence to Antiretroviral Therapy Promotes Residual HIV-1 Replication in the Absence of Virological Rebound in Plasma
Pasternak, A.O. ; Bruin, M. de; Jurriaans, S. ; Bakker, M. ; Berkhout, B. ; Prins, J.M. ; Lukashov, V.V. - \ 2012
The Journal of Infectious Diseases 206 (2012)9. - ISSN 0022-1899 - p. 1443 - 1452.
protease inhibitor therapy - highly sensitive methods - t-cell-activation - infected individuals - viral suppression - immune activation - drug-resistance - viremia blips - adherence - rna
Background. Modern antiretroviral therapy (ART) regimens are widely assumed to be forgivable to modest non-adherence, as virological suppression in plasma is common at adherence levels >70%. Yet, it is unknown whether HIV-1 replication is completely suppressed at these levels of adherence. Methods. We longitudinally quantified levels of cell-associated HIV-1 RNA and DNA in forty patients (median time on successful ART before the study initiation, 46 months), whose one-week adherence to therapy prior to the sampling moments was measured electronically. Results. Patients were either constantly 100% adherent, demonstrated improving adherence in time, or neither of the above (“poor adherers”). Adherence never fell below 70% in any patient, and no virological rebound in plasma was observed. Nevertheless, poor adherence, but not optimal or improving adherence, caused a significant longitudinal increase in cell-associated HIV RNA levels (P=0.006). Time-weighted changes and regression slopes of viral RNA were significantly higher in poor adherers than in optimal adherers (P
Cascade-mediated binding and bending of negatively supercoiled DNA
Westra, E.R. ; Nilges, B. ; Erp, P.B. ; Oost, J. van der; Dame, R.T. ; Brouns, S.J.J. - \ 2012
RNA Biology 9 (2012)9. - ISSN 1547-6286 - p. 1134 - 1138.
crispr-cas systems - immune-system - structural basis - rna - bacteria - archaea - defense - interference - recognition - mechanism
Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop