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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Biotechnology of health-promoting bacteria
Douillard, François P. ; Vos, Willem M. de - \ 2019
Biotechnology Advances 37 (2019)6. - ISSN 0734-9750
Applied genomics - Gut microbiota - Next-generation therapeutic bacteria - Probiotics

Over the last decade, there has been an increasing scientific and public interest in bacteria that may positively contribute to human gut health and well-being. This interest is reflected by the ever-increasing number of developed functional food products containing health-promoting bacteria and reaching the market place as well as by the growing revenue and profits of notably bacterial supplements worldwide. Traditionally, the origin of probiotic-marketed bacteria was limited to a rather small number of bacterial species that mostly belong to lactic acid bacteria and bifidobacteria. Intensifying research efforts on the human gut microbiome offered novel insights into the role of human gut microbiota in health and disease, while also providing a deep and increasingly comprehensive understanding of the bacterial communities present in this complex ecosystem and their interactions with the gut-liver-brain axis. This resulted in rational and systematic approaches to select novel health-promoting bacteria or to engineer existing bacteria with enhanced probiotic properties. In parallel, the field of gut microbiomics developed into a fertile framework for the identification, isolation and characterization of a phylogenetically diverse array of health-promoting bacterial species, also called next-generation therapeutic bacteria. The present review will address these developments with specific attention for the selection and improvement of a selected number of health-promoting bacterial species and strains that are extensively studied or hold promise for future food or pharma product development.

Comparative genomic analysis of the multispecies probiotic-marketed product VSL#3
Douillard, François P. ; Mora, Diego ; Eijlander, Robyn T. ; Wels, Michiel ; Vos, Willem M. De - \ 2018
PLoS ONE 13 (2018)2. - ISSN 1932-6203
Several probiotic-marketed formulations available for the consumers contain live lactic acid bacteria and/or bifidobacteria. The multispecies product commercialized as VSL#3 has been used for treating various gastro-intestinal disorders. However, like many other products, the bacterial strains present in VSL#3 have only been characterized to a limited extent and their efficacy as well as their predicted mode of action remain unclear, preventing further applications or comparative studies. In this work, the genomes of all eight bacterial strains present in VSL#3 were sequenced and characterized, to advance insights into the possible mode of action of this product and also to serve as a basis for future work and trials. Phylogenetic and genomic data analysis allowed us to identify the 7 species present in the VSL#3 product as specified by the manufacturer. The 8 strains present belong to the species Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium breve and B. animalis subsp. lactis (two distinct strains). Comparative genomics revealed that the draft genomes of the S. thermophilus and L. helveticus strains were predicted to encode most of the defence systems such as restriction modification and CRISPR-Cas systems. Genes associated with a variety of potential probiotic functions were also identified. Thus, in the three Bifidobacterium spp., gene clusters were predicted to encode tight adherence pili, known to promote bacteria-host interaction and intestinal barrier integrity, and to impact host cell development. Various repertoires of putative signalling proteins were predicted to be encoded by the genomes of the Lactobacillus spp., i.e. surface layer proteins, LPXTG-containing proteins, or sortase-dependent pili that may interact with the intestinal mucosa and dendritic cells. Taken altogether, the individual genomic characterization of the strains present in the VSL#3 product confirmed the product specifications, determined its coding capacity as well as identified potential probiotic functions.
Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system
Nissilä, Eija ; Douillard, François P. ; Ritari, Jarmo ; Paulin, Lars ; Järvinen, Hanna M. ; Rasinkangas, Pia ; Haapasalo, Karita ; Meri, Seppo ; Jarva, Hanna ; Vos, Willem M. De - \ 2017
PLoS ONE 12 (2017)5. - ISSN 1932-6203
Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well-studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005-2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system.
The Variable Regions of Lactobacillus rhamnosus Genomes Reveal the Dynamic Evolution of Metabolic and Host-Adaptation Repertoires
Ceapa, C.D. ; Davids, M. ; Ritari, Jarmo ; Lambert, J. ; Wels, M. ; Douillard, François P. ; Smokvina, Tamara ; Vos, Willem M. de; Knol, J. ; Kleerebezem, M. - \ 2016
Genome Biology and Evolution 8 (2016)6. - ISSN 1759-6653 - p. 1889 - 1905.
Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes (core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer (variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total (the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides (EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat (CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core (shared) proteins matched with the clade distribution obtained from the presence–absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains’ core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification
Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens
Bui, Thi Phuong Nam ; Shetty, Sudarshan Anand ; Lagkouvardos, Ilias ; Ritari, Jarmo ; Chamlagain, Bhawani ; Douillard, François P. ; Paulin, Lars ; Piironen, Vieno ; Clavel, Thomas ; Plugge, Caroline M. ; Vos, Willem M. de - \ 2016
Environmental Microbiology Reports 8 (2016)6. - ISSN 1758-2229 - p. 1024 - 1037.

Intestinimonas is a newly described bacterial genus with representative strains present in the intestinal tract of human and other animals. Despite unique metabolic features including the production of butyrate from both sugars and amino acids, there is to date no data on their diversity, ecology, and physiology. Using a comprehensive phylogenetic approach, Intestinimomas was found to include at least three species that colonize primarily the human and mouse intestine. We focused on the most common and cultivable species of the genus, Intestinimonas butyriciproducens, and performed detailed genomic and physiological comparison of strains SRB521T and AF211, isolated from the mouse and human gut respectively. The complete 3.3-Mb genomic sequences of both strains were highly similar with 98.8% average nucleotide identity, testifying to their assignment to one single species. However, thorough analysis revealed significant genomic rearrangements, variations in phage-derived sequences, and the presence of new CRISPR sequences in both strains. Moreover, strain AF211 appeared to be more efficient than strain SRB521T in the conversion of the sugars arabinose and galactose. In conclusion, this study provides genomic and physiological insight into Intestinimonas butyriciproducens, a prevalent butyrate-producing species, differentiating strains that originate from the mouse and human gut.

Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili : Evidence for a Novel and Heterospecific Probiotic Mechanism
Tytgat, Hanne L.P. ; Douillard, François P. ; Reunanen, Justus ; Rasinkangas, Pia ; Hendrickx, Antoni P.A. ; Laine, Pia K. ; Paulin, Lars ; Satokari, Reetta ; Vos, Willem M. de - \ 2016
Applied and Environmental Microbiology 82 (2016)19. - ISSN 0099-2240 - p. 5756 - 5762.

Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections.

Polymorphisms, chromosomal rearrangements, and mutator phenotype development during experimental evolution of Lactobacillus rhamnosus GG
Douillard, François P. ; Ribbera, Angela ; Xiao, Kun ; Ritari, Jarmo ; Rasinkangas, Pia ; Paulin, Lars ; Palva, Airi ; Hao, Yanling ; Vos, Willem M. de - \ 2016
Applied and Environmental Microbiology 82 (2016)13. - ISSN 0099-2240 - p. 3783 - 3792.

Lactobacillus rhamnosus GG is a lactic acid bacterium widely marketed by the food industry. Its genomic analysis led to the identification of a gene cluster encoding mucus-binding SpaCBA pili, which is located in a genomic island enriched in insertion sequence (IS) elements. In the present study, we analyzed by genome-wide resequencing the genomic integrity of L. rhamnosus GG in four distinct evolutionary experiments conducted for approximately 1,000 generations under conditions of no stress or salt, bile, and repetitive-shearing stress. Under both stress-free and salt-induced stress conditions, the GG population (excluding the mutator lineage in the stress-free series [see below]) accumulated only a few single nucleotide polymorphisms (SNPs) and no frequent chromosomal rearrangements. In contrast, in the presence of bile salts or repetitive shearing stress, some IS elements were found to be activated, resulting in the deletion of large chromosomal segments that include the spaCBA-srtC1 pilus gene cluster. Remarkably, a high number of SNPs were found in three strains obtained after 900 generations of stress-free growth. Detailed analysis showed that these three strains derived from a founder mutant with an altered DNA polymerase subunit that resulted in a mutator phenotype. The present work confirms the stability of the pilus production phenotype in L. rhamnosus GG under stress-free conditions, highlights the possible evolutionary scenarios that may occur when this probiotic strain is extensively cultured, and identifies external factors that affect the chromosomal integrity of GG. The results provide mechanistic insights into the stability of GG in regard to its extensive use in probiotic and other functional food products.

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG
Douillard, François P. ; Rasinkangas, Pia ; Bhattacharjee, Arnab ; Palva, Airi ; Vos, Willem M. De - \ 2016
PLoS ONE 11 (2016)4. - ISSN 1932-6203

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoidshaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.

Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili
Tytgat, Hanne ; Teijlingen, N.H. van; Sullan, R.M. ; Douillard, F.P. ; Rasinkangas, P. ; Messing, M. ; Reunanen, J. ; Satokari, R. ; Vanderleyden, J. ; Dufrêne, Y.F. ; Geijtenbeek, T.B.H. ; Vos, W.M. de; Lebeer, S. - \ 2016
PLoS ONE 11 (2016)3. - ISSN 1932-6203
Mapping of the microbial molecules underlying microbiota-host interactions is key to understand how microbiota preserve mucosal homeostasis. A pivotal family of such bacterial molecules are pili. Pili are proteinaceous cell wall appendages with a well-documented role in adhesion, whilst their role in immune interaction with the host is less established. Gram-positive pili are often posttranslationally modified by sortase-specific cleavage reactions and the formation of intramolecular peptide bonds. Here we report glycosylation as a new level of posttranslational modification of sortase-dependent pili of a beneficial microbiota species and its role in immune modulation. We focused on the SpaCBA pili of the model probiotic and beneficial human gut microbiota isolate Lactobacillus rhamnosus GG. A unique combination of molecular techniques, nanoscale mechanical and immunological approaches led to the identification of mannose and fucose residues on the SpaCBA pili. These glycans on the pili are recognized by human dendritic cells via the C-type lectin receptor DC-SIGN, a key carbohydrate-dependent immune tailoring pattern recognition receptor. This specific lectin-sugar interaction is moreover of functional importance and modulated the cytokine response of dendritic cells. This provides insight into the direct role bacterial glycoproteins can play in the immunomodulation of the host. Modification of the complex heterotrimeric pili of a model probiotic and microbiota isolate with mannose and fucose is of importance for the functional interaction with the host immune lectin receptor DC-SIGN on human dendritic cells. Our findings shed light on the yet underappreciated role of glycoconjugates in bacteria-host interactions.
Complete Genome Sequence of Enterococcus faecium Commensal Isolate E1002
Tytgat, Hanne ; Douillard, F.P. ; Laine, P.K. ; Paulin, L. ; Willems, R.J.L. ; Vos, W.M. de - \ 2016
Genome Announcements 4 (2016)2. - ISSN 2169-8287
The emergence of vancomycin-resistant enterococci (VRE) has been associated with an increase in multidrug-resistant nosocomial infections. Here, we report the 2.614-Mb genome sequence of the Enterococcus faecium commensal isolate E1002, which will be instrumental in further understanding the determinants of the commensal and pathogenic lifestyle of E. faecium.
A novel consortium of Lactobacillus rhamnosus and Streptococcus thermophilus for increased access to functional fermented foods
Kort, Remco ; Westerik, Nieke ; Mariela Serrano, L. ; Douillard, François P. ; Gottstein, Willi ; Mukisa, Ivan M. ; Tuijn, Coosje J. ; Basten, Lisa ; Hafkamp, Bert ; Meijer, Wilco C. ; Teusink, Bas ; Vos, Willem de; Reid, Gregor ; Sybesma, Wilbert - \ 2015
Microbial Cell Factories 14 (2015)1. - ISSN 1475-2859
Bacterial fermentation - Consortium - Enrichment - Fermented foods - Functional foods - Lactobacillus rhamnosus GG - Lactobacillus rhamnosus yoba 2012 - Streptococcus thermophilus C106 - Yoghurt

Background: The lactic acid bacterium Lactobacillus rhamnosus GG is the most studied probiotic bacterium with proven health benefits upon oral intake, including the alleviation of diarrhea. The mission of the Yoba for Life foundation is to provide impoverished communities in Africa increased access to Lactobacillus rhamnosus GG under the name Lactobacillus rhamnosus yoba 2012, world's first generic probiotic strain. We have been able to overcome the strain's limitations to grow in food matrices like milk, by formulating a dried starter consortium with Streptococcus thermophilus that enables the propagation of both strains in milk and other food matrices. The affordable seed culture is used by people in resource-poor communities. Results: We used S. thermophilus C106 as an adjuvant culture for the propagation of L. rhamnosus yoba 2012 in a variety of fermented foods up to concentrations, because of its endogenous proteolytic activity, ability to degrade lactose and other synergistic effects. Subsequently, L. rhamnosus could reach final titers of 1E+09CFUml-1, which is sufficient to comply with the recommended daily dose for probiotics. The specific metabolic interactions between the two strains were derived from the full genome sequences of L. rhamnosus GG and S. thermophilus C106. The piliation of the L. rhamnosus yoba 2012, required for epithelial adhesion and inflammatory signaling in the human host, was stable during growth in milk for two rounds of fermentation. Sachets prepared with the two strains, yoba 2012 and C106, retained viability for at least 2 years. Conclusions: A stable dried seed culture has been developed which facilitates local and low-cost production of a wide range of fermented foods that subsequently act as delivery vehicles for beneficial bacteria to communities in east Africa.

Lactic Acid Bacteria and the Human Intestinal Microbiome
Douillard, F.P. ; Vos, W.M. de - \ 2015
In: Biotechnology of Lactic Acid Bacteria: Novel Applications second edition / Mozzi, F., Vignolo, G.M., Raya, R.R., John Wiley and Sons - ISBN 9781118868409 - p. 120 - 133.
The great interest in the human microbiome has revived attention paid to LAB presence in the human intestine. This chapter first discusses the LAB associated with the human intestinal microbiota and their potential roles in health and diseases. It then addresses recent metagenomic studies that challenge the established belief that the human intestine is colonized by many lactobacilli and tries to offer insights into one highly relevant question: are LAB originally occurring (autochthonous) and/or just transiently passing (allochthonous) in the human intestinal tract? To further illustrate this, the chapter describes one particular case study: the well-characterized Lactobacillus rhamnosus species, which has been extensively studied at both genomic and phenotypic levels.
Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera
Sun, Z. ; Harris, H.M.B. ; McCann, A. ; Guo, C. ; Argimón, S. ; Zhang, W. ; Yang, X. ; Jeffery, I.B. ; Cooney, J.C. ; Kagawa, T.F. ; Liu, W. ; Song, Y. ; Salvetti, E. ; Wrobel, A. ; Rasinkangas, P. ; Parkhill, J. ; Rea, M.C. ; O'Sullivan, O. ; Ritari, J. ; Douillard, F.P. ; Paul Ross, R. ; Yang, R. ; Briner, A.E. ; Felis, G.E. ; Vos, W.M. de; Barrangou, R. ; Klaenhammer, T.R. ; Caufield, P.W. ; Cui, Y. ; Zhang, H. ; O'Toole, P.W. - \ 2015
Nature Communications 6 (2015). - ISSN 2041-1723 - 13 p.
Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species
Functional genomics of lactic acid bacteria: from food to health
Douillard, F.P. ; Vos, W.M. de - \ 2014
Microbial Cell Factories 13 (2014)suppl.1. - ISSN 1475-2859
lactobacillus-rhamnosus gg - human gastrointestinal-tract - sortase-dependent proteins - johnsonii strain ncc533 - gram-positive bacteria - subsp cremoris mg1363 - lactococcus-lactis - in-vitro - streptococcus-thermophilus - enterococcus-faecalis
Genome analysis using next generation sequencing technologies has revolutionized the characterization of lactic acid bacteria and complete genomes of all major groups are now available. Comparative genomics has provided new insights into the natural and laboratory evolution of lactic acid bacteria and their environmental interactions. Moreover, functional genomics approaches have been used to understand the response of lactic acid bacteria to their environment. The results have been instrumental in understanding the adaptation of lactic acid bacteria in artisanal and industrial food fermentations as well as their interactions with the human host. Collectively, this has led to a detailed analysis of genes involved in colonization, persistence, interaction and signaling towards to the human host and its health. Finally, massive parallel genome re-sequencing has provided new opportunities in applied genomics, specifically in the characterization of novel non-GMO strains that have potential to be used in the food industry. Here, we provide an overview of the state of the art of these functional genomics approaches and their impact in understanding, applying and designing lactic acid bacteria for food and health.
Genomic Characterization of Non-Mucus Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis
Rasinkangas, P. ; Reunanen, J. ; Douillard, F.P. ; Ritari, J. ; Uotinen, V. ; Palva, A. ; Vos, W.M. de - \ 2014
Applied and Environmental Microbiology 80 (2014)22. - ISSN 0099-2240 - p. 7001 - 7009.
intestinal epithelial-cells - placebo-controlled trial - gram-positive bacteria - functional-analysis - atopic disease - strain gg - adhesion - probiotics - surface - prevention
Lactobacillus rhamnosus GG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed that L. rhamnosus GG harbors a genomic island including the spaCBA-srtC1 gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis in L. rhamnosus GG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied to L. rhamnosus GG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in the srtC1 gene; class II, with a deletion containing the spaCBA-srtC1 gene cluster; and class III, with mutations in the spaA gene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficient srtC1 gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.
Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis
Douillard, F.P. ; Rasinkangas, P. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2014
Journal of Biological Chemistry 289 (2014)22. - ISSN 0021-9258 - p. 15764 - 15775.
complete genome sequence - gram-positive bacteria - enterococcus-faecium isolate - group-b streptococcus - lactic-acid bacteria - lactococcus-lactis - corynebacterium-diphtheriae - bacillus-anthracis - pilin subunit - reveals pili
In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.
Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin
Douillard, F.P. ; Kant, R. ; Ritari, J. ; Paulin, L. ; Palva, A. ; Vos, W.M. de - \ 2013
Microbial Biotechnology 6 (2013)5. - ISSN 1751-7907 - p. 576 - 587.
lactic-acid bacteria - gram-positive bacteria - rhamnosus gg - functional-analysis - cell-wall - surface-proteins - staphylococcus-aureus - controlled-trial - binding-protein - sequence
The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+ C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD-II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.
Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG
Douillard, F.P. ; Ribbera, A. ; Kant, R. ; Pietilä, T.E. ; Järvinen, H.M. ; Messing, M. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Ritari, J. ; Caggia, C. ; Lähteinen, T. ; Brouns, S.J.J. ; Satokari, R.M. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2013
Plos Genetics 9 (2013)8. - ISSN 1553-7404
lactic-acid bacteria - intestinal epithelial-cells - placebo-controlled trial - streptococcus-thermophilus - species identification - salmonella-typhimurium - gastrointestinal-tract - adaptive immunity - atopic disease - in-vitro
Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects
Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics
Douillard, F.P. ; Ribbera, A. ; Järvinen, H.M. ; Kant, R. ; Pietilä, T.E. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Caggia, C. ; Ossowski, I. von; Reunanen, J. ; Satokari, R. ; Salminen, S. ; Palva, A. ; Vos, W.M. de - \ 2013
Applied and Environmental Microbiology 79 (2013)6. - ISSN 0099-2240 - p. 1923 - 1933.
lactic-acid bacteria - binding-protein - in-vitro - carbohydrate-metabolism - sequence - gg - adhesion - bl23 - stress - mucus
Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-¿B response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities
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