scholarly journals Functional Characterization of a Mucus-Specific LPXTG Surface Adhesin from Probiotic Lactobacillus rhamnosus GG

2011 ◽  
Vol 77 (13) ◽  
pp. 4465-4472 ◽  
Author(s):  
Ingemar von Ossowski ◽  
Reetta Satokari ◽  
Justus Reunanen ◽  
Sarah Lebeer ◽  
Sigrid C. J. De Keersmaecker ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Functional Characterization ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Surface Proteins ◽  
Adhesive Interaction ◽  
Content Type ◽  
Intestinal Mucus ◽  
Mucosal Surfaces

ABSTRACTIn spite of the wealth of clinical evidence supporting the health benefits ofLactobacillus rhamnosusGG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover,L. rhamnosusGG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) inL. rhamnosusGG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction betweenL. rhamnosusGG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean thatL. rhamnosusGG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic.

scholarly journals Manufacturing process influences properties of probiotic bacteria

2010 ◽  
Vol 105 (6) ◽  
pp. 887-894 ◽  
Author(s):  
Łukasz Grześkowiak ◽  
Erika Isolauri ◽  
Seppo Salminen ◽  
Miguel Gueimonde
Keyword(s):  
Human Subjects ◽  
Lactobacillus Rhamnosus ◽  
Clinical Intervention ◽  
Probiotic Strain ◽  
Adhesion Properties ◽  
Influence Model ◽  
Intestinal Mucus ◽  
Probiotic Strains ◽  
Micro Organisms

Production and manufacturing methods and the food carrier may influence the properties of probiotic strains, and have an impact on the outcome of clinical intervention studies. The aim of the present study was to establish whether the properties of a specific probiotic strain,Lactobacillus rhamnosusGG, may differ depending on the product and source of the strain. In total, fifteen differentL. rhamnosusisolates, among them fourteen labelled asL.rhamnosusGG, were isolated from specific probiotic products. The micro-organisms were phenotypically and genotypically characterised. Their adhesion properties were compared using the human intestinal mucus model, and the ability of the isolates to influence model pathogen adhesion to human colonic mucus was assessed. AllL. rhamnosusisolates used were confirmed as members of the speciesL. rhamnosus. Except the reference strain OL, allL.rhamnosusisolates showed randomly amplified polymorphic DNA, enterobacterial repetitive intergenic consensus and pulsed-field gel electrophoresis profiles identical to that ofL. rhamnosusGG (ATCC 53103). AllL.rhamnosusisolates showed similar tolerance to acid and were able to bind to human colonic mucus. However, pathogen exclusion by inhibition and competition varied significantly among the differentL. rhamnosusisolates and pathogens tested. The results suggest that different sources of the same probiotic may have significantly altered strain properties. This should be considered inin vivostudies on human subjects and also for quality control of probiotic products.

scholarly journals The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

2014 ◽  
Vol 197 (5) ◽  
pp. 882-892 ◽  
Author(s):  
Jessica R. Galloway-Peña ◽  
Xiaowen Liang ◽  
Kavindra V. Singh ◽  
Puja Yadav ◽  
Chungyu Chang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Bile Salt ◽  
Enterococcus Faecium ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Type I ◽  
Gram Positive ◽  
Content Type

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6Enterococcus faeciumproteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence inE. faeciumand possibly other Gram-positive bacterial species.

scholarly journals Mucosal Adhesion Properties of the Probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED Pilin Subunits

2010 ◽  
Vol 76 (7) ◽  
pp. 2049-2057 ◽  
Author(s):  
Ingemar von Ossowski ◽  
Justus Reunanen ◽  
Reetta Satokari ◽  
Satu Vesterlund ◽  
Matti Kankainen ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Binding Capacity ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Gene Clusters ◽  
Probiotic Bacterium ◽  
Lactobacillus Rhamnosus Gg ◽  
Adhesion Properties ◽  
Intestinal Mucus ◽  
Pilin Subunit

ABSTRACT Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.

Adaptation factors of the probiotic Lactobacillus rhamnosus GG

2010 ◽  
Vol 1 (4) ◽  
pp. 335-342 ◽  
Author(s):  
S. Lebeer ◽  
J. Vanderleyden ◽  
S. De Keersmaecker
Keyword(s):  
Current Knowledge ◽  
Lactobacillus Rhamnosus ◽  
Health Benefit ◽  
Probiotic Strain ◽  
Probiotic Bacteria ◽  
Host Immune System ◽  
Lactobacillus Rhamnosus Gg ◽  
Probiotic Lactobacilli ◽  
Micro Organisms ◽  
Performance Adaptation

Probiotic bacteria are administered as live micro-organisms to provide a health benefit to the host. Knowledge on adaptation factors that promote the survival and persistence of probiotics in the intestine is key to understand and improve their ecological and probiotic performance. Adaptation factors include adhesins, molecules conferring stress tolerance and nutritional versatility, antimicrobial products against competing microbes, and factors promoting resistance against the host immune system. Here, we present an overview of the current knowledge on adaptation factors of probiotic lactobacilli, with focus on the prototypical and widely documented probiotic strain Lactobacillus rhamnosus GG.

Challenges in translational research on probiotic lactobacilli: from in vitro assays to clinical trials

2013 ◽  
Vol 4 (1) ◽  
pp. 83-100 ◽  
Author(s):  
M. Meijerink ◽  
A. Mercenier ◽  
J.M. Wells
Keyword(s):  
Translational Research ◽  
Selection Criteria ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Intestinal Barrier ◽  
Probiotic Strain ◽  
In Vitro Assays ◽  
Intestinal Barrier Function

Beneficial effects of certain probiotic strains have been established in the treatment and prevention of various immune and intestinal disorders in humans, including allergic diseases, chronic inflammatory diseases and diarrhoea. The proposed mechanisms underlying the immunomodulatory effects of probiotics in humans are not understood in precise detail but include enhancement of intestinal barrier function, altered epithelial signalling, competition with pathogens and effects on immune cells and immunity depending on the probiotic strain. The publication of controversial or inconclusive probiotic studies in humans highlights the need for a better understanding of the mechanisms and improved strain selection criteria. This review focuses on the immunomodulatory properties of lactobacilli and bifidobacteria in vitro and in vivo, current knowledge concerning the mechanisms in vivo and challenges in translational research on probiotics. A better understanding of the molecular mechanisms of probiotics, the effect of probiotic mixtures versus single strains, the effect of formulation of probiotics and the fate of ingested probiotics should help to clarify the value of immune assays as selection criteria for probiotics.

scholarly journals Piliation of Lactobacillus rhamnosus GG Promotes Adhesion, Phagocytosis, and Cytokine Modulation in Macrophages

2015 ◽  
Vol 81 (6) ◽  
pp. 2050-2062 ◽  
Author(s):  
Cynthia E. Vargas García ◽  
Mariya Petrova ◽  
Ingmar J. J. Claes ◽  
Ilke De Boeck ◽  
Tine L. A. Verhoeven ◽  
...  
Keyword(s):  
Close Contact ◽  
Lactobacillus Rhamnosus ◽  
Interleukin 10 ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Phagocytic Cells ◽  
Content Type ◽  
Intestinal Mucus ◽  
Anti Inflammatory

ABSTRACTRecently,spaCBA-encoded pili on the cell surface ofLactobacillus rhamnosusGG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus ofL. rhamnosusGG in the interaction with macrophagesin vitroby comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.

scholarly journals Genomic Characterization of Non-Mucus-Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis

2014 ◽  
Vol 80 (22) ◽  
pp. 7001-7009 ◽  
Author(s):  
Pia Rasinkangas ◽  
Justus Reunanen ◽  
François P. Douillard ◽  
Jarmo Ritari ◽  
Virva Uotinen ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Lactobacillus Rhamnosus ◽  
Illumina Miseq ◽  
Class Iii ◽  
Dot Blot ◽  
Content Type ◽  
Intestinal Mucus ◽  
Pilus Biogenesis ◽  
Dot Blot Analysis ◽  
The Impact

ABSTRACTLactobacillus rhamnosusGG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed thatL. rhamnosusGG harbors a genomic island including thespaCBA-srtC1gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis inL. rhamnosusGG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied toL. rhamnosusGG 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 thesrtC1gene; class II, with a deletion containing thespaCBA-srtC1gene cluster; and class III, with mutations in thespaAgene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficientsrtC1gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.

scholarly journals Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Yvan Caspar ◽  
Claire Siebert ◽  
Vivien Sutera ◽  
Corinne Villers ◽  
Alexandra Aubry ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Molecular Mechanisms ◽  
Hot Spot ◽  
Functional Characterization ◽  
Health Concern ◽  
Content Type ◽  
Francisella Novicida ◽  
High Level

ABSTRACT Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC50) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.

scholarly journals Propionibacterium freudenreichii: General Characteristics and Probiotic Traits

2021 ◽  
Author(s):  
Vinícius de Rezende Rodovalho ◽  
Diego Lucas Neres Rodrigues ◽  
Gwénaël Jan ◽  
Yves Le Loir ◽  
Vasco Ariston de Carvalho Azevedo ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Bacterial Species ◽  
Short Chain Fatty Acids ◽  
Surface Proteins ◽  
Propionibacterium Freudenreichii ◽  
Long Time ◽  
Beneficial Traits

Propionibacterium freudenreichii is a Gram-positive dairy probiotic bacterial species that has been used as a ripening starter in the production of Swiss-type cheese for a long time. It has been exploited for the optimization of cheese production, including ripening capacities and aroma compounds production, but also for the production of vitamin B12 and organic acids. Furthermore, it has emerged in the probiotics landscape owing to several beneficial traits, including tolerance to stress in the gastrointestinal tract, adhesion to host cells, anti-pathogenic activity, anticancer potential and immunomodulatory properties. These beneficial properties have been confirmed with in vitro and in vivo investigations, using several omics approaches that allowed the identification of important molecular actors, such as surface proteins, short-chain fatty acids and bifidogenic factors. The diversity within the species was shown to be an important aspect to take into consideration, since many of these properties were strain-dependent. New studies should dive further into the molecular mechanisms related to the beneficial properties of this species and of its products, while considering the complexities of strain diversity and the interactions with the host and its microbiota. This chapter reviews current knowledge on the possible impact of P. freudenreichii on human health.

scholarly journals Origin and Impact of Nitric Oxide in Pseudomonas aeruginosa Biofilms

2015 ◽  
Vol 198 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Francesca Cutruzzolà ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Model Organism ◽  
Bacterial Cells ◽  
Chronic Infections ◽  
Content Type ◽  
Bacterial Physiology ◽  
Planktonic Bacteria

The formation of the organized bacterial community called biofilm is a crucial event in bacterial physiology. Given that biofilms are often refractory to antibiotics and disinfectants to which planktonic bacteria are susceptible, their formation is also an industrially and medically relevant issue.Pseudomonas aeruginosa, a well-known human pathogen causing acute and chronic infections, is considered a model organism to study biofilms. A large number of environmental cues control biofilm dynamics in bacterial cells. In particular, the dispersal of individual cells from the biofilm requires metabolic and morphological reprogramming in which the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) plays a central role. The diatomic gas nitric oxide (NO), a well-known signaling molecule in both prokaryotes and eukaryotes, is able to induce the dispersal ofP. aeruginosaand other bacterial biofilms by lowering c-di-GMP levels. In this review, we summarize the current knowledge on the molecular mechanisms connecting NO sensing to the activation of c-di-GMP-specific phosphodiesterases inP. aeruginosa, ultimately leading to c-di-GMP decrease and biofilm dispersal.

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