Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates
Fernández Ramírez, M.D. ; Smid, E.J. ; Abee, T. ; Nierop Groot, M.N. - \ 2015
International Journal of Food Microbiology 207 (2015). - ISSN 0168-1605 - p. 23 - 29.
lactic-acid bacteria - enterococcal surface protein - listeria-monocytogenes - pseudomonas-putida - bacillus-subtilis - starter cultures - genetic-analysis - rhamnosus gg - resistance - industry
Lactobacillus plantarum has been associated with food spoilage in a wide range of products and the biofilm growth mode has been implicated as a possible source of contamination. In this study we analysed the biofilm forming capacity of L. plantarum WCFS1 and six food spoilage isolates. Biofilm formation as quantified by crystal violet staining and colony forming units was largely affected by the medium composition, growth temperature and maturation time and by strain specific features. All strains showed highest biofilm formation in Brain Heart Infusion medium supplemented with manganese and glucose. For L. plantarum biofilms the crystal violet (CV) assay, that is routinely used to quantify total biofilm formation, correlates poorly with the number of culturable cells in the biofilm. This can in part be explained by cell death and lysis resulting in CV stainable material, conceivably extracellular DNA (eDNA), contributing to the extracellular matrix. The strain to strain variation may in part be explained by differences in levels of eDNA, likely as result of differences in lysis behaviour. In line with this, biofilms of all strains tested, except for one spoilage isolate, were sensitive to DNase treatment. In addition, biofilms were highly sensitive to treatment with Proteinase K suggesting a role for proteins and/or proteinaceous material in surface colonisation. This study shows the impact of a range of environmental factors and enzyme treatments on biofilm formation capacity for selected L. plantarum isolates associated with food spoilage, and may provide clues for disinfection strategies in food industry.
Interactions between formulation and spray drying conditions related to survival of Lactobacillus plantarum WCFS1
Perdana, J.A. ; Fox, M.B. ; Siwei, C. ; Boom, R.M. ; Schutyser, M.A.I. - \ 2014
Food Research International 56 (2014). - ISSN 0963-9969 - p. 9 - 17.
glass-transition temperature - membrane phase-behavior - lactic-acid bacteria - flow-cytometry - industrial applications - dairy ingredients - osmotic-stress - water activity - rhamnosus gg - gel phase
Protective solid carriers are commonly added to probiotic cultures prior to drying. Their formulation is not trivial and depends on the drying conditions applied. In this study, we systematically investigated the influence of formulation parameters on the survival of Lactobacillus plantarum WCFS1 after drying. Low molecular weight carbohydrates (less than 2 kDa) with high glass transition temperatures provided the highest level of protection at both low (25 degrees C) and high (50 degrees C or higher) drying temperatures. Low molecular weight carbohydrates may provide stabilization by closely interacting with the lipid bilayer of the cell membranes. Meanwhile, carbohydrates with high glass transition temperatures probably provide stabilization via fixation of the cells in a glassy powder. Furthermore, adequate amounts of solid carrier are required to sufficiently stabilize the cells during drying. During drying, crystallization of solid carriers may occur. Depending on the crystal geometry, crystallization can be either beneficial (e.g. with mannitol or sorbitol) or detrimental (e.g. with lactose) to cell survival. Finally, the effect of formulation on cell viability during storage was studied. A decimal reduction time of approximately 300 days was observed when spray dried L. plantarum WCFS1 was stored at temperatures below 40 degrees C. The outcome of this study was used as a basis to construct a generalized diagram to indicate the combinations of formulation and drying conditions to maximally retain viability and operate dryers at high efficiency. (C) 2013 Elsevier Ltd. All rights reserved.
GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum
Lee, I.C. ; Swam, I.I. van; Tomita, S. ; Morsomme, P. ; Rolain, T. ; Hols, P. ; Kleerebezem, M. ; Bron, P.A. - \ 2014
Journal of Bacteriology 196 (2014)9. - ISSN 0021-9193 - p. 1671 - 1682.
complete genome sequence - lactic-acid bacteria - escherichia-coli - campylobacter-jejuni - acidophilus ncfm - epithelial-cells - surface protein - rhamnosus gg - glycoproteins - binding
Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
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.
Genotypic adaptations associated with prolonged persistence of Lactobacillus plantarum in the murine digestive tract
Bokhorst-van de Veen, H. van; Smelt, M.J. ; Wels, M. ; Hijum, S.A.F.T. van; Vos, P. de; Kleerebezem, M. ; Bron, P.A. - \ 2013
Biotechnology Journal 8 (2013)8. - ISSN 1860-6768 - p. 895 - 904.
gastrointestinal-tract - rhamnosus gg - human flora - mice - host - strains - protein - genome - genes - identification
Probiotic bacteria harbor effector molecules that confer health benefits, but also adaptation factors that enable them to persist in the gastrointestinal tract of the consumer. To study these adaptation factors, an antibiotic-resistant derivative of the probiotic model organism Lactobacillus plantarum WCFS1 was repeatedly exposed to the mouse digestive tract by three consecutive rounds of (re)feeding of the longest persisting colonies. This exposure to the murine intestine allowed the isolation of intestine-adapted derivatives of the original strain that displayed prolonged digestive tract residence time. Re-sequencing of the genomes of these adapted derivatives revealed single nucleotide polymorphisms as well as a single nucleotide insertion in comparison with the genome of the original WCFS1 strain. Detailed in silico analysis of the identified genomic modifications pinpointed that alterations in the coding regions of genes encoding cell envelope associated functions and energy metabolism appeared to be beneficial for the gastrointestinal tract survival of L. plantarum WCFS1. This work demonstrates the feasibility of experimental evolution for the enhancement of the gastrointestinal residence time of probiotic strains, while full-genome re-sequencing of the adapted isolates provided clues towards the bacterial functions involved. Enhanced gastrointestinal residence is industrially relevant because it enhances the efficacy of the delivery of viable probiotics in situ.
Regulation of intestinal homeostasis and immunity with probiotic lactobacilli
Baarlen, P. van; Wells, J. ; Kleerebezem, M. - \ 2013
Trends in Immunology 34 (2013)5. - ISSN 1471-4906 - p. 208 - 215.
wall teichoic-acid - gut microbiota - rhamnosus gg - t-cells - lipoteichoic acid - functional-analysis - epithelial barrier - controlled-trials - dendritic cell - in-vivo
The gut microbiota provide important stimuli to the human innate and adaptive immune system and co-mediate metabolic and immune homeostasis. Probiotic bacteria can be regarded as part of the natural human microbiota, and have been associated with improving homeostasis, albeit with different levels of success. Composition of microbiota, probiotic strain identity, and host genetic differences may account for differential modulation of immune responses by probiotics. Here, we review the mechanisms of immunomodulating capacities of specific probiotic strains, the responses they can induce in the host, and how microbiota and genetic differences between individuals may co-influence host responses and immune homeostasis.
Cell surface-associated compounds of probiotic lactobacilli sustain the strain-specificity dogma
Bron, P.A. ; Tomita, S. ; Mercenier, A.M.E. ; Kleerebezem, M. - \ 2013
Current Opinion in Microbiology 16 (2013)3. - ISSN 1369-5274 - p. 262 - 269.
wall teichoic-acid - lipoteichoic acid - rhamnosus gg - staphylococcus-aureus - gut microbiota - in-vitro - bacterial-peptidoglycan - acidophilus deficient - structural-variation - dendritic cell
Probiotic lactobacilli can positively impact on the health status of targeted (diseased) populations but efficacy depends strongly on the strain employed and the molecular basis for this phenomenon is poorly understood. This review discusses the current state-of-the-art in the field of molecular probiotic-host interactions, focusing on subtle strain-specific differences in the biochemical characteristics of cell surface-associated probiotic ligands and the consequences thereof for the immune responses elicited. This research is bound to enhance our understanding of strain-specificity in relation to probiotic functionality and will allow molecular science-based design of screening and characterization assays targeted to improved selection of probiotic candidate strains. Moreover, identified bioactive effector molecules could be isolated or produced for administration in a more pharmacological regime.
Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling
Remus, D.M. ; Kranenburg, R. van; Swam, I.I. van; Taverne, N. ; Bongers, R.S. ; Wels, M. ; Wells, J. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
Microbial Cell Factories 11 (2012)1. - ISSN 1475-2859 - 10 p.
lactic-acid bacteria - gene-expression omnibus - streptococcus-pneumoniae - lactococcus-lactis - exopolysaccharide biosynthesis - microarray data - subsp cremoris - rhamnosus gg - identification - strains
Background - Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. Results - The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (¿cps1A-I, ¿cps2A-J, ¿cps3A-J and ¿cps4A-J) or combinations of cps clusters (¿cps1A-3J and ¿cps1A-3I, ¿cps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The ¿cps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-¿B activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the ¿cps1A-I and ¿cps3A-J mutants but appeared slightly increased after stimulation with the ¿cps2A-J and ¿cps4A-J mutants, while the ¿cps1A-3J and ¿cps1A-3J, ¿cps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. Conclusions - Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.
An intimate tete-a-tete - How probiotic lactobacilli communicate with the host
Remus, D.M. ; Kleerebezem, M. ; Bron, P.A. - \ 2011
European Journal of Pharmacology 668 (2011)Suppl. 1. - ISSN 0014-2999 - p. S33 - S42.
intestinal epithelial-cells - innate immune-system - peptidoglycan recognition proteins - inflammatory-bowel-disease - lactic-acid bacteria - toll-like receptor-2 - nf-kappa-b - rhamnosus gg - lipoteichoic acid - escherichia-coli
Pharmaceutical agents are routinely used in the treatment of gastrointestinal disorders and their role as modulators of host cell responses is well characterized. In contrast, the understanding of the molecular mechanisms, which determine the role of probiotics, i.e. health-promoting bacteria, as host cell modulators is still in its infancy. Both in vitro and in vivo studies are just starting to reveal the capability of probiotic lactobacilli to modulate host cell-signaling networks and the associated influences on downstream regulatory pathways, including modulation of mucosal cytokine profiles that dictate host immune functions. The communication between probiotic lactobacilli and intestinal host cells is multifactorial and involves an integrative repertoire of receptors on the host side that recognize multiple effector molecules on the bacterial side, of which most have been found to be cell wall- or cell surface-associated compounds and proteins. This review describes the discovery of these bacterial effector molecules and their role in strain- and species-specific modulation of host signaling pathways. Unraveling the mechanisms responsible for probiotic-host interactions will progress this research field towards molecular science and will provide markers for probiotic product quality control as well as host-response efficacy. These developments can ultimately lead to a more dedicated, personalized application of probiotics with strong molecular and scientific support for health promotion.
Identification of Genetic Loci in Lactobacillus plantarum That Modulate the Immune Response of Dendritic Cells Using Comparative Genome Hybridization
Meijerink, M. ; Hemert, S. van; Taverne, N. ; Wels, M.W.W. ; Bron, P.A. ; Vos, P. de; Savelkoul, H.F.J. ; Bilsen, J.G.P.M. van; Kleerebeezem, M. ; Wells, J. - \ 2010
PLoS ONE 5 (2010)5. - ISSN 1932-6203 - 12 p.
lactic-acid bacteria - necrosis-factor-alpha - regulatory t-cells - intestinal inflammation - functional-analysis - probiotic bacteria - lipoteichoic acid - rhamnosus gg - immunomodulatory properties - gastrointestinal-tract
Background - Probiotics can be used to stimulate or regulate epithelial and immune cells of the intestinal mucosa and generate beneficial mucosal immunomodulatory effects. Beneficial effects of specific strains of probiotics have been established in the treatment and prevention of various intestinal disorders, including allergic diseases and diarrhea. However, the precise molecular mechanisms and the strain-dependent factors involved are poorly understood. Methodology/Principal Findings - In this study, we aimed to identify gene loci in the model probiotic organism Lactobacillus plantarum WCFS1 that modulate the immune response of host dendritic cells. The amounts of IL-10 and IL-12 secreted by dendritic cells (DCs) after stimulation with 42 individual L. plantarum strains were measured and correlated with the strain-specific genomic composition using comparative genome hybridisation and the Random Forest algorithm. This in silico “gene-trait matching” approach led to the identification of eight candidate genes in the L. plantarum genome that might modulate the DC cytokine response to L. plantarum. Six of these genes were involved in bacteriocin production or secretion, one encoded a bile salt hydrolase and one encoded a transcription regulator of which the exact function is unknown. Subsequently, gene deletions mutants were constructed in L. plantarum WCFS1 and compared to the wild-type strain in DC stimulation assays. All three bacteriocin mutants as well as the transcription regulator (lp_2991) had the predicted effect on cytokine production confirming their immunomodulatory effect on the DC response to L. plantarum. Transcriptome analysis and qPCR data showed that transcript level of gtcA3, which is predicted to be involved in glycosylation of cell wall teichoic acids, was substantially increased in the lp_2991 deletion mutant (44 and 29 fold respectively). Conclusion - Comparative genome hybridization led to the identification of gene loci in L. plantarum WCFS1 that modulate the immune response of DCs