Network analysis of temporal functionalities of the gut induced by perturbations in new-born piglets
Benis, N. ; Schokker, D. ; Suarez Diez, M. ; Martins dos Santos, V.A.P. ; Smidt, H. ; Smits, M.A. - \ 2015
BMC Genomics 16 (2015). - ISSN 1471-2164
protein-interaction network - intestinal microbiota - early-life - gastrointestinal-tract - immune-system - antibiotic exposure - adaptive immunity - resistant starch - microarray experiments - ruminococcus-bromii
Background Evidence is accumulating that perturbation of early life microbial colonization of the gut induces long-lasting adverse health effects in individuals. Understanding the mechanisms behind these effects will facilitate modulation of intestinal health. The objective of this study was to identify biological processes involved in these long lasting effects and the (molecular) factors that regulate them. We used an antibiotic and the same antibiotic in combination with stress on piglets as an early life perturbation. Then we used host gene expression data from the gut (jejunum) tissue and community-scale analysis of gut microbiota from the same location of the gut, at three different time-points to gauge the reaction to the perturbation. We analysed the data by a new combination of existing tools. First, we analysed the data in two dimensions, treatment and time, with quadratic regression analysis. Then we applied network-based data integration approaches to find correlations between host gene expression and the resident microbial species. Results The use of a new combination of data analysis tools allowed us to identify significant long-lasting differences in jejunal gene expression patterns resulting from the early life perturbations. In addition, we were able to identify potential key gene regulators (hubs) for these long-lasting effects. Furthermore, data integration also showed that there are a handful of bacterial groups that were associated with temporal changes in gene expression. Conclusion The applied systems-biology approach allowed us to take the first steps in unravelling biological processes involved in long lasting effects in the gut due to early life perturbations. The observed data are consistent with the hypothesis that these long lasting effects are due to differences in the programming of the gut immune system as induced by the temporary early life changes in the composition and/or diversity of microbiota in the gut.
The first thousand days – intestinal microbiology of early life: establishing a symbiosis
Wopereis, H. ; Oozeer, R. ; Knipping, K. ; Belzer, C. ; Knol, J. - \ 2014
Pediatric Allergy and Immunology 25 (2014)5. - ISSN 0905-6157 - p. 428 - 438.
gradient gel-electrophoresis - infant gut microbiota - fecal microbiota - atopic-dermatitis - allergic diseases - immune-system - galacto-oligosaccharides - fructo-oligosaccharides - reduced diversity - human-milk
The development of the intestinal microbiota in the first years of life is a dynamic process significantly influenced by early-life nutrition. Pioneer bacteria colonizing the infant intestinal tract and the gradual diversification to a stable climax ecosystem plays a crucial role in establishing host–microbe interactions essential for optimal symbiosis. This colonization process and establishment of symbiosis may profoundly influence health throughout life. Recent developments in microbiologic cultivation-independent methods allow a detailed view of the key players and factors involved in this process and may further elucidate their roles in a healthy gut and immune maturation. Aberrant patterns may lead to identifying key microbial signatures involved in developing immunologic diseases into adulthood, such as asthma and atopic diseases. The central role of early-life nutrition in the developmental human microbiota, immunity, and metabolism offers promising strategies for prevention and treatment of such diseases. This review provides an overview of the development of the intestinal microbiota, its bidirectional relationship with the immune system, and its role in impacting health and disease, with emphasis on allergy, in early life.
Functional characterization of probiotic surface layer protein-carrying Lactobacillus amylovorus strains
Hynönen, U. ; Kant, R. ; Lähteinen, T. ; Pietilä, T.E. ; Beganovic, J. ; Smidt, H. ; Uroic, K. ; Åvall-Jääskeläinen, S. ; Palva, A. - \ 2014
BMC Microbiology 14 (2014). - ISSN 1471-2180 - 16 p.
enterotoxigenic escherichia-coli - human intestinal mucus - lactic-acid bacteria - human dendritic cells - s-layer - in-vitro - acidophilus ncfm - aeromonas-salmonicida - epithelial-cells - immune-system
Background - Adhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) –layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins. Results - Three of the L. amylovorus strains studied adhered to IPEC-1 cells, while four strains inhibited the adherence of E. coli, indicating additional mechanisms other than competition for binding sites being involved in the inhibition. None of the strains bound to porcine mucus. The culture supernatants of all of the strains exerted inhibitory effects on the growth of E. coli, Salmonella, Listeria and Yersinia, and a variable, strain-dependent induction was observed of both pro- and anti-inflammatory cytokines in human DCs. L. amylovorus DSM 16698 was shown to carry two S-layer-like proteins on its surface in addition to the major S-layer protein SlpA. In contrast to expectations, none of the major S-layer proteins of the IPEC-1 -adhering strains mediated bacterial adherence. Conclusions - We demonstrated adhesive and significant pathogen inhibitory efficacies among the swine intestinal L. amylovorus strains studied, pointing to their potential use as probiotic feed supplements, but no independent role could be demonstrated for the major S-layer proteins in adherence to epithelial cells. The results indicate that many intestinal bacteria may coexist with and confer benefits to the host by mechanisms not attributable to adhesion to epithelial cells or mucus.
Immunomodulatory Properties of Streptococcus and Veillonella Isolates from the Human Small Intestine Microbiota
Bogert, B. van den; Meijerink, M. ; Zoetendal, E.G. ; Wells, J.M. ; Kleerebezem, M. - \ 2014
PLoS ONE 9 (2014)12. - ISSN 1932-6203 - 20 p.
segmented filamentous bacteria - tight junction proteins - patch dendritic cells - gut microbiota - lactobacillus-plantarum - ly6c(hi) monocytes - genome sequence - oral tolerance - immune-system - in-vivo
The human small intestine is a key site for interactions between the intestinal microbiota and the mucosal immune system. Here we investigated the immunomodulatory properties of representative species of commonly dominant small-intestinal microbial communities, including six streptococcal strains (four Streptococcus salivarius, one S. equinus, one S. parasanguinis) one Veillonella parvula strain, one Enterococcus gallinarum strain, and Lactobacillus plantarum WCFS1 as a bench mark strain on human monocyte-derived dendritic cells. The different streptococci induced varying levels of the cytokines IL-8, TNF-a, and IL-12p70, while the V. parvula strain showed a strong capacity to induce IL-6. E. gallinarum strain was a potent inducer of cytokines and TLR2/6 signalling. As Streptococcus and Veillonella can potentially interact metabolically and frequently co-occur in ecosystems, immunomodulation by pair-wise combinations of strains were also tested for their combined immunomodulatory properties. Strain combinations induced cytokine responses in dendritic cells that differed from what might be expected on the basis of the results obtained with the individual strains. A combination of (some) streptococci with Veillonella appeared to negate IL-12p70 production, while augmenting IL-8, IL-6, IL-10, and TNF-a responses. This suggests that immunomodulation data obtained in vitro with individual strains are unlikely to adequately represent immune responses to mixtures of gut microbiota communities in vivo. Nevertheless, analysing the immune responses of strains representing the dominant species in the intestine may help to identify immunomodulatory mechanisms that influence immune homeostasis.
Protein transport across the small intestine in food hypersensitivity
Reitsma, M. ; Westerhout, J. ; Wichers, H.J. ; Wortelboer, H. ; Verhoeckx, K.C.M. - \ 2014
Molecular Nutrition & Food Research 58 (2014)1. - ISSN 1613-4125 - p. 194 - 205.
transepithelial antigen transport - undegraded dietary antigen - simulated gastric fluid - human peyers-patches - dendritic cells - macromolecular transport - in-vitro - beta-lactoglobulin - immune-system - epithelial exosomes
In view of the imminent deficiency of protein sources for human consumption in the near future, new protein sources need to be identified. However, safety issues such as the risk of allergenicity are often a bottleneck, due to the absence of predictive, validated and accepted methods for risk assessment. The current strategy to assess the allergenic potential of proteins focuses mainly on homology, stability and cross-reactivity, although other factors such as intestinal transport might be of added value too. In this review, we present an overview of the knowledge of protein transport across the intestinal wall and the methods currently being used to measure this. A literature study reveals that protein transport in sensitised persons occurs para-cellularly with the involvement of mast cells, and trans-cellularly via enterocytes, while in non-sensitised persons micro-fold cells and enterocytes are considered most important. However, there is a lack of comparable systematic studies on transport of allergenic proteins. Knowledge of the multiple protein transport pathways and which model system can be useful to study these processes may be of added value in the risk assessment of food allergenicity.
Maintenance of a healthy trajectory of the intestinal microbiome during aging: A dietary approach
Candela, M. ; Biagi, E. ; Brigidi, P. ; O'Toole, P.W. ; Vos, W.M. de - \ 2014
Mechanisms of Ageing and Development 136-137 (2014). - ISSN 0047-6374 - p. 70 - 75.
gut microbiota - fecal microbiota - immune-system - elderly-patients - human longevity - age - disease - immunosenescence - intervention - enterotypes
Sharing an intense transgenomic metabolism with the host, the intestinal microbiota is an essential factor for several aspects of the human physiology. However, several age-related factors, such as changes diet, lifestyle, inflammation and frailty, force the deterioration of this intestinal microbiota-host mutualistic interaction, compromising the possibility to reach longevity. In this scenario, the NU-AGE project involves the development of dietary interventions specifically tailored to the maintenance of a healthy trajectory of the intestinal microbiome, counteracting all processes connected to the pathophysiology of the human aging
Complement C3 is inversely associated with habitual intake of provitamin A but not with dietary fat, fatty acids, or vitamin E in Middle-aged to older white adults and positively associated with intake of retinol in middle-aged to older white women
Greevenbroek, M.M. ; Arts, I.C.W. ; Kallen, C.J.H. van der; Dagnelie, P.C. ; Ferreira, I. ; Jansen, G.H.E. ; Schalkwijk, C.G. ; Feskens, E.J.M. ; Stehouwer, C.D.A. - \ 2014
The Journal of Nutrition 144 (2014)1. - ISSN 0022-3166 - p. 61 - 67.
population-based cohort - myocardial-infarction - a supplementation - serum c3 - insulin-resistance - relative validity - immune-system - disease - risk - protein
Complement factor 3 (C3) has been identified as a novel risk factor for obesity-associated cardiometabolic diseases. Data in the literature suggest that C3 concentrations may be influenced by diet. Therefore, we investigated the associations of intake of total fat, specific fatty acids, and fat-soluble vitamin E (and individual tocopherols) and vitamin A (and its dietary precursors) with circulating C3. In a white cohort [Cohort on Diabetes and Atherosclerosis Maastricht (CODAM)]; n = 501; 59.4 ± 7.1 y; 61% men], associations of habitual nutrient intake (assessed by a food-frequency questionnaire) with circulating C3 were evaluated by using cross-sectional multiple linear regression analyses. Adjustments were first performed for age, sex, glucose metabolism status (i.e., impaired glucose metabolism or type 2 diabetes), and energy intake and subsequently for BMI, waist circumference, alcohol intake, smoking behavior, and season of blood collection. No associations with C3 were observed for total dietary fat intake or intake of specific fatty acids [saturated, monounsaturated, polyunsaturated, n–6 (¿6), and n–3 (¿3) fatty acids], vitamin E, or individual tocopherols. We observed an inverse association with intake of provitamin A carotenoids a-carotene (in µg/d; regression coefficient ß = -0.075; 95% CI: -0.140, -0.010; P = 0.025) and ß-carotene (in µg/d; ß = -0.021; 95% CI: -0.044, 0.002; P = 0.068) with C3 (in mg/L). In contrast, and only in women, dietary retinol intake (in µg/d) was positively associated with C3 (ß = 0.116; 95% CI: 0.014, 0.218; P = 0.026; n = 196). In conclusion, these data suggest that fasting concentrations of C3 may, in a complex manner, be modifiable by variation in dietary provitamin A carotenoids and/or retinol content of the usual diet but most likely not by variations in fat composition and vitamin E content.
Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus
Staals, R.H.J. ; Agari, Y. ; Maki-Yonekura, S. ; Zhu, Y. ; Taylor, D.W. ; Duijn, E. van; Barendregt, A. ; Vlot, M. ; Koehorst, J.J. ; Sakamoto, K. ; Masuda, A. ; Dohmae, N. ; Schaap, P.J. ; Doudna, J.A. ; Heck, A. ; Yonekura, K. ; Oost, J. van der; Shinkai, A. - \ 2013
Molecular Cell 52 (2013)1. - ISSN 1097-2765 - p. 135 - 145.
of-flight instrument - mass-spectrometry - escherichia-coli - silencing complex - antiviral defense - immune-system - protein - interference - transcription - recognition
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes
Ontogeny of the avian intestinal immunoglobulin repertoire: Modification in CDR3 length and conserved VH-pseudogene usage
Hartog, C.G. den; Crooijmans, R.P.M.A. ; Parmentier, H.K. ; Savelkoul, H.F.J. ; Bos, N.A. ; Lammers, A. - \ 2013
Molecular Immunology 56 (2013)4. - ISSN 0161-5890 - p. 811 - 818.
segmented filamentous bacteria - b-cell repertoire - hypervariable regions - deficient mice - immune-system - t-cell - iga - mucosal - antibody - chicken
Immunoglobulins play an important role in maintenance of mucosal homeostasis in the gut. The antigen binding specificity of these immunoglobulins depends for a large part on the hypervariable CDR3 region. To gain knowledge about isotype-specific development of the CDR3 repertoire we examined CDR3 spectratypes at multiple time points between 4 and 70 days post hatch. In order to identify clonal expansions deviation from the normal distribution (SS) and the average CDR3 length was calculated. IgA-CDR3 regions were studied in more detail by DNA sequence analysis at day 7 and 70 and preferential VH pseudogene usage was estimated. The SS of CDR3 repertoires of the IgM, IgG and IgA isotypes successively increased, but for each isotype this increase was transiently. The length of the CDR3 regions decreased with age for IgM becoming similar to the CDR3 length of IgA at day 70. The IgA- and IgG-CDR3 lengths did not change with age. On average, the CDR3 length of IgA was the shortest. IgA CDR3 sequences were similar between animals aged 7 and 70 days. A limited number of pseudogenes was used, and no differences in pseudogene usage were observed between animals aged 7 and 70 days. Of the identified VH pseudogenes, half of the sequences used VH15, whilst a number of the pseudogenes were not used at all. We conclude that CDR3 spectratype profiles change during aging, whilst at the CDR3-sequence level, variation in VH pseudogene usage for ileal IgA is limited suggesting conservation during ontogeny.
Molecular signatures for the dynamic process of establishing intestinal host-microbial homeostasis: potential for disease diagnostics?
Aidy, S.F. El; Kleerebezem, M. - \ 2013
Current Opinion in Gastroenterology 29 (2013)6. - ISSN 0267-1379 - p. 621 - 627.
inflammatory-bowel-disease - gut microbiota - gene-expression - immune-system - health - diet - mucosa - twins - mice - conventionalisation
Purpose of review: The dynamic interplay of the intestinal microbiota and host has been the focus of many studies because of its impact on the health status in human life. Recent reports on the time-resolved immune and metabolic interactions between the host and microbiota, as well as the molecular signatures that mark this communication during the process of establishing a host–microbial relationship, are addressed here. Recent findings: During microbial colonization, the progressive impact of de-novo introduction of the gut microbiota on the host's physiology is tightly controlled by highly intertwined regulatory networks and achieves an efficient and balanced interplay between the host and its developing microbial community. Recent findings from germ-free mouse models have unravelled core transcriptional, metabolic and microbial signatures, which are proposed to orchestrate the molecular responses during the establishment of the multifaceted state of intestinal host–microbe homeostasis. Summary: Exploring the time-resolved dynamics of the host responses to the newly colonizing gut microbiota provides mechanistically critical understanding of the sequential host–microbe response cascades that lead to a homeostatic relationship. Thereby, these approaches can provide novel diagnostic tools and therapeutic targets, or either of the two, in humans for specific disorders associated with intestinal dysbiosis and loss of homeostasis
Intestinal colonization: How key microbial players become established in this dynamic process: Microbial metabolic activities and the interplay between the host and microbes
Aidy, S.F. El; Abbeele, P. van den; Wiele, T. van der; Louis, P. ; Kleerebezem, M. - \ 2013
Bioessays 35 (2013)10. - ISSN 0265-9247 - p. 913 - 923.
sulfate-reducing bacteria - butyrate-producing bacteria - chain fatty-acids - inflammatory bowel diseases - human gut microbiota - human colon - ulcerative-colitis - hydrogen-sulfide - immune-system - human feces
In this review, we provide an overview of the dynamic changes within the microbiota and its metabolites that are implicated in establishing and maintaining gastrointestinal homeostasis during various stages of microbial colonization. The gradual conversion of the gut microbiota toward a mutualistic microbial community involves replacement of pioneer gut colonizers with bacterial taxa that are characteristic for the adult gut. An important microbial signature of homeostasis in the adult gut is the prevalence and activity of a diverse spectrum of bacterial species that produce beneficial metabolites through metabolic interactions between microbial groups. Deciphering these microbial signatures and their metabolites that govern short and long-term equilibrium, as well as imbalances in host-microbial relationships, may provide novel diagnostic tools and/or therapeutic targets for specific disorders associated with intestinal dysbiosis and loss of homeostasis.
Genetic parameters of natural antibody isotypes and survival analysis in beak-trimmed and non-beak-trimmed crossbred laying hens
Sun, Y. ; Ellen, E.D. ; Parmentier, H.K. ; Poel, J.J. van der - \ 2013
Poultry Science 92 (2013)8. - ISSN 0032-5791 - p. 2024 - 2033.
immune-system - pineal-gland - chickens - lines - autoantibodies - repertoire - responses - antigens - disease - stress
Natural antibodies (NAb) are important humoral components of innate immunity. As the first line of defense, NAb provide protection against infection and support adaptive immunity. An earlier study indicated that serum levels of NAb isotypes IgM and IgG at a young age were predictive for survival in non-beak-trimmed purebred laying hens during the laying period. In the present study, genetic parameters of NAb isotypes were estimated and relationships between survival and NAb isotypes levels in crossbred laying hens were investigated. In total, 1,555 beak-trimmed and 1,169 non-beak-trimmed crossbred laying hens were used. Genetic parameters of IgM and IgG titers binding keyhole limpet hemocyanin at 24 wk of age were estimated with a linear animal model. The heritabilities of NAb isotypes IgG and IgM were 0.21 (SE = 0.04) and 0.26 (SE = 0.04), respectively. The genetic correlation between IgG and IgM isotypes was 0.43 (SE = 0.11). These results indicated that NAb isotype titers were heritable traits in the crossbred laying hens. Both NAb isotypes can be selected for simultaneously because the detected positive genetic correlation (0.43, SE = 0.11) between them is positive. Both row and level of the cage were indicated to be associated environmental factors for NAb isotype titers. Different from an earlier study with purebred hens, survival analysis showed no significant associations of survival with NAb isotype titers in beak-trimmed or non-beak-trimmed crossbred hens. Non-health-related causes of mortality, especially in birds with intact beaks, overruled the anticipated relationships between NAb isotype titers and survival.
Gut bacteria-host metabolic interplay during conventionalisation of the mouse germfree colon
Aidy, S. El; Derrien, M.M.N. ; Merrifield, C.A. ; Levenez, F. ; Dore, J. ; Boekschoten, M.V. ; Dekker, J. ; Holmes, E. ; Zoetendal, E.G. ; Baarlen, P. van; Claus, S.P. ; Kleerebezem, M. - \ 2013
ISME Journal 7 (2013). - ISSN 1751-7362 - p. 743 - 755.
colorectal-cancer - polysaccharide utilization - selenomonas-ruminantium - human intestine - immune-system - waste-water - microbiota - mice - mucosa - nmr
The interplay between dietary nutrients, gut microbiota and mammalian host tissues of the gastrointestinal tract is recognised as highly relevant for host health. Combined transcriptome, metabonome and microbial profiling tools were employed to analyse the dynamic responses of germfree mouse colonic mucosa to colonisation by normal mouse microbiota (conventionalisation) at different time-points during 16 days. The colonising microbiota showed a shift from early (days 1 and 2) to later colonisers (days 8 and 16). The dynamic changes in the microbial community were rapidly reflected by the urine metabolic profiles (day 1) and at later stages (day 4 onward) by the colon mucosa transcriptome and metabolic profiles. Correlations of host transcriptomes, metabolite patterns and microbiota composition revealed associations between Bacilli and Proteobacteria, and differential expression of host genes involved in energy and anabolic metabolism. Differential gene expression correlated with scyllo- and myo-inositol, glutamine, glycine and alanine levels in colonic tissues during the time span of conventionalisation. Our combined time-resolved analyses may help to expand the understanding of host-microbe molecular interactions during the microbial establishment
A defined intestinal colonization microbiota for gnotobiotic pigs
Laycock, G. ; Sait, L. ; Inman, C. ; Lewis, M. ; Smidt, H. ; Diemen, P. van; Jorgensen, F. ; Stevens, M. ; Bailey, M. - \ 2012
Veterinary Immunology and Immunopathology 149 (2012)3-4. - ISSN 0165-2427 - p. 216 - 224.
antibody repertoire development - germ-free pigs - real-time pcr - immune-system - gastrointestinal-tract - postnatal-development - filamentous bacteria - newborn piglets - oral tolerance - gut bacteria
Maximising the ability of piglets to survive exposure to pathogens is essential to reduce early piglet mortality, an important factor in efficient commercial pig production. Mortality rates can be influenced by many factors, including early colonization by microbial commensals. Here we describe the development of an intestinal microbiota, the Bristol microbiota, for use in gnotobiotic pigs and its influence on synthesis of systemic immunoglobulins. Such a microbiota will be of value in studies of the consequences of early microbial colonization on development of the intestinal immune system and subsequent susceptibility to disease. Gnotobiotic pig studies lack a well-established intestinal microbiota. The use of the Altered Schaedler Flora (ASF), a murine intestinal microbiota, to colonize the intestines of Caesarean-derived, gnotobiotic pigs prior to gut closure, resulted in unreliable colonization with most (but not all) strains of the ASF. Subsequently, a novel, simpler porcine microbiota was developed. The novel microbiota reliably colonized the length of the intestinal tract when administered to gnotobiotic piglets. No health problems were observed, and the novel microbiota induced a systemic increase in serum immunoglobulins, in particular IgA and IgM. The Bristol microbiota will be of value for highly controlled, reproducible experiments of the consequences of early microbial colonization on susceptibility to disease in neonatal piglets, and as a biomedical model for the impact of microbial colonization on development of the intestinal mucosa and immune system in neonates.
L. plantarum, L. salivarius, and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner
Smelt, M.J. ; Haan, B.J. de; Bron, P.A. ; Swam, I. van; Meijerink, M. ; Wells, J. ; Faas, M.M. ; Vos, P. de - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203
inflammatory-bowel-disease - lactobacillus-casei shirota - influenza-virus infection - placebo-controlled trial - complete genome sequence - blood mononuclear-cells - dendritic cells - double-blind - immune-system - acid bacteria
Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103+ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-¿, IL5, IL10, and IL17 production by CD4+ and CD8+ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner.
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
The rise and fall of CRISPRs - dynamics of spacer acquisition and loss
Westra, E.R. ; Brouns, S.J.J. - \ 2012
Molecular Microbiology 85 (2012)6. - ISSN 0950-382X - p. 1021 - 1025.
immune-system - streptococcus-thermophilus - acquired-resistance - antiviral defense - escherichia-coli - seed sequence - cas systems - rna - dna - repeats
Bacteria and Archaea are continuously exposed to mobile genetic elements (MGE), such as viruses and plasmids. MGEs may provide a selective advantage, may be neutral or may cause cell damage. To protect against invading DNA, prokaryotes utilize a number of defence systems, including the CRISPR/Cas system. CRISPR/Cas systems rely on integration of invader sequences (spacers) into CRISPR loci that act as a genetic memory of past invasions. Processed CRISPR transcripts are utilized as guides by Cas proteins to cleave complementary invader nucleic acids. In this issue, two groups report on spacer acquisition and turnover dynamics of CRISPR loci in a thermoacidophilic archeon and a pathogenic bacterium. Erdmann and Garrett demonstrate that three of the six CRISPR loci of Sulfolobus solfataricus rapidly acquire new spacer sequences from a conjugative plasmid present in a virus mixture. Intriguingly, two distinct mechanisms of spacer integration are utilized: leader adjacent and internal CRISPR spacer acquisition. Lopez-Sanchez and co-workers studied the type II system of Streptococcus agalactiae and observe heterogeneity in the bacterial population. A fraction of the population lost one or more anti-mobilome spacer sequences during its cultivation, allowing the transfer of a MGE in this subpopulation and a rapid response to altering selection pressures
Dual regulatory roles of the extended N-terminus for activation of the tomato Mi-1.2 resistance protein
Lukasik-Shreepaathy, E. ; Slootweg, E.J. ; Richter, H. ; Cornelissen, B.J.C. ; Goverse, A. ; Takken, F.L.W. - \ 2012
Molecular Plant-Microbe Interactions 25 (2012)8. - ISSN 0894-0282 - p. 1045 - 1057.
secondary structure prediction - rich repeat domain - cell-death - nucleotide-binding - disease resistance - confers resistance - immune-system - coiled coils - lrr protein - arc domain
Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the NB-LRR protein family as they carry a central NB-ARC domain fused to an LRR domain. The CC domain at the N-terminus of some Solanaceous NB-LRR proteins is extended with a solanaceae domain (SD). Tomato Mi-1.2, which confers resistance against nematodes, white flies, psyllids and aphids, encodes a typical SD-CNL protein. Here, we analyzed the role of the extended N-terminus for Mi-1.2 activation. Removal of the first part of the N-terminus (Nt1) induced Mi-1.2-mediated cell death that could be suppressed by over-expression of the second half of the N-terminal region (Nt2). Yet, autoactivating NB-ARC-LRR mutants require in trans co-expression of the N-terminal region to induce cell death, indicating that the N-terminus functions both as a negative and a positive regulator. Based on secondary structure predictions we could link both functions to three distinct subdomains; a typical CC domain and two novel, structurally-conserved helical subdomains called SD1 and SD2. A negative regulatory function could be assigned to the SD1 whereas SD2 and the CC together function as positive regulators of Mi-1.2 mediated cell death
CRISPR Interference Directs Strand Specific Spacer Acquisition
Swarts, D.C. ; Mosterd, C. ; Passel, M.W.J. van; Brouns, S.J.J. - \ 2012
PLoS ONE 7 (2012)4. - ISSN 1932-6203
escherichia-coli k-12 - immune-system - streptococcus-thermophilus - crystal-structure - h-ns - dna - rna - resistance - sequence - defense
BACKGROUND: CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference. RESULTS: We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner. CONCLUSIONS: The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster - in our case the first acquired spacer - and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect
Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence
Semenova, E.V. ; Jore, M.M. ; Westra, E.R. ; Oost, J. van der; Brouns, S.J.J. - \ 2011
Proceedings of the National Academy of Sciences of the United States of America 108 (2011)25. - ISSN 0027-8424 - p. 10098 - 10103.
immune-system - escherichia-coli - dna - defense - identification - prokaryotes - cleavage - bacteriophage - recognition - archaeon
Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses