Lactobacillus kefiri

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Maria De Los Angeles Serradell - One of the best experts on this subject based on the ideXlab platform.

  • synthesis and catalytic application of silver nanoparticles supported on Lactobacillus kefiri s layer proteins
    Nanomaterials, 2020
    Co-Authors: Patricia Araceli Bolla, Maria De Los Angeles Serradell, Sofia Huggias, Jose Fernando Ruggera, Monica Laura Casella
    Abstract:

    Research on nanoparticles obtained on biological supports is a topic of growing interest in nanoscience, especially regarding catalytic applications. Silver nanoparticles (AgNPs) have been studied due to their low toxicity, but they tend to aggregation, oxidation, and low stability. In this work, we synthesized and characterized AgNPs supported on S-layer proteins (SLPs) as bidimensional regularly arranged biotemplates. By different reduction strategies, six AgNPs of variable sizes were obtained on two different SLPs. Transmission electron microscopy (TEM) images showed that SLPs are mostly decorated by evenly distributed AgNPs; however, a drastic reduction by NaBH4 led to large AgNPs whereas a smooth reduction with H2 or H2/NaBH4 at low concentration leads to smaller AgNPs, regardless of the SLP used as support. All the nanosystems showed conversion values between 75–80% of p-nitrophenol to p-aminophenol, however, the increment in the AgNPs size led to a great decrease in Kapp showing the influence of reduction strategy in the performance of the catalysts. Density functional theory (DFT) calculations indicated that the adsorption of p-nitrophenolate species through the nitro group is the most favored mechanism, leading to p-aminophenol as the only feasible product of the reaction, which was corroborated experimentally.

  • immunostimulation by Lactobacillus kefiri s layer proteins with distinct glycosylation patterns requires different lectin partners
    Journal of Biological Chemistry, 2020
    Co-Authors: Mariano Malamud, Maria De Los Angeles Serradell, Gustavo J Cavallero, Adriana C Casabuono, Bernd Lepenies, Alicia S Couto
    Abstract:

    S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography–pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow–derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow–derived dendritic cells from C-type lectin receptor–deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

  • regular arrangement of pt nanoparticles on s layer proteins isolated from Lactobacillus kefiri synthesis and catalytic application
    Molecular Catalysis, 2020
    Co-Authors: Patricia Araceli Bolla, Maria De Los Angeles Serradell, Sofia Huggias, Jose Fernando Ruggera, Agustina Sanz, Monica Laura Casella
    Abstract:

    Fil: Bolla, Patricia Araceli. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - La Plata. Centro de Investigacion y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigacion y Desarrollo en Ciencias Aplicadas; Argentina

  • s layer glycoprotein from Lactobacillus kefiri exerts its immunostimulatory activity through glycan recognition by mincle
    Frontiers in Immunology, 2019
    Co-Authors: Paula Carasi, Mariano Malamud, Bernd Lepenies, Matias Hernan Assandri, Teresa Freire, Maria De Los Angeles Serradell
    Abstract:

    The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea. Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca2+-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR–hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3 and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signalling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle-/- or CARD9-/- mice were not capable to enhance OVA-specific response in CD4+ T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.

  • s layer glycoprotein from Lactobacillus kefiri cidca 8348 enhances macrophages response to lps in a ca 2 dependent manner
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Paula Carasi, Mariano Malamud, Maria De Los Angeles Serradell, Teresa Freire
    Abstract:

    The S-layer is a (glyco)-proteinaceous envelope constituted by self-assembled subunits that form a two-dimensional lattice covering the surface of different species of Bacteria and Archaea. It could be considered as one of the most abundant biopolymers in our planet. Because of their unique self-assembly features, exhibiting repetitive identical physicochemical properties down to the subnanometer scale, as well as their involvement in specific interactions with host cells, the S-layer proteins (SLPs) show a high potential application in different areas of biotechnology, including the development of antigen carriers or new adjuvants. The presence of a glycosylated SLP on potentially probiotic Lactobacillus kefiri strains was previously described by our research group. In this study, we aim to investigate the role of carbohydrates present in the SLP from L. kefiri CIDCA 8348 (SLP-8348) in their internalization by murine macrophages, as well as to analyze their immunomodulatory capacity and their effect on LPS-stimulated macrophages. RAW 264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca+2 chelating agent. These results correlated with the recognition of SLP-8348 by ConA lectin. We further show that while SLP-8348 was not able to induce the activation of macrophages by itself, it favored the LPS-induced response, since there was a significant increase in the expression of surface cell markers MHC-II, CD86 and CD40, as well as in IL-6 and IL-10 expression at both transcript and protein levels, in comparison with LPS-stimulated cells. The presence of EGTA completely abrogated this synergistic effect. Taken together, these results strongly suggest the involvement of both glycosidic residues and Ca+2 ions in the recognition of SLP-8348 by cellular receptors on murine macrophages. Moreover, these results suggest the potentiality of the SLP-8348 for the development of new adjuvants capable of stimulating antigen presenting cells by interaction with glycan receptors.

Paula Carasi - One of the best experts on this subject based on the ideXlab platform.

  • s layer glycoprotein from Lactobacillus kefiri exerts its immunostimulatory activity through glycan recognition by mincle
    Frontiers in Immunology, 2019
    Co-Authors: Paula Carasi, Mariano Malamud, Bernd Lepenies, Matias Hernan Assandri, Teresa Freire, Maria De Los Angeles Serradell
    Abstract:

    The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea. Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca2+-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR–hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3 and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signalling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle-/- or CARD9-/- mice were not capable to enhance OVA-specific response in CD4+ T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.

  • s layer glycoprotein from Lactobacillus kefiri cidca 8348 enhances macrophages response to lps in a ca 2 dependent manner
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Paula Carasi, Mariano Malamud, Maria De Los Angeles Serradell, Teresa Freire
    Abstract:

    The S-layer is a (glyco)-proteinaceous envelope constituted by self-assembled subunits that form a two-dimensional lattice covering the surface of different species of Bacteria and Archaea. It could be considered as one of the most abundant biopolymers in our planet. Because of their unique self-assembly features, exhibiting repetitive identical physicochemical properties down to the subnanometer scale, as well as their involvement in specific interactions with host cells, the S-layer proteins (SLPs) show a high potential application in different areas of biotechnology, including the development of antigen carriers or new adjuvants. The presence of a glycosylated SLP on potentially probiotic Lactobacillus kefiri strains was previously described by our research group. In this study, we aim to investigate the role of carbohydrates present in the SLP from L. kefiri CIDCA 8348 (SLP-8348) in their internalization by murine macrophages, as well as to analyze their immunomodulatory capacity and their effect on LPS-stimulated macrophages. RAW 264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca+2 chelating agent. These results correlated with the recognition of SLP-8348 by ConA lectin. We further show that while SLP-8348 was not able to induce the activation of macrophages by itself, it favored the LPS-induced response, since there was a significant increase in the expression of surface cell markers MHC-II, CD86 and CD40, as well as in IL-6 and IL-10 expression at both transcript and protein levels, in comparison with LPS-stimulated cells. The presence of EGTA completely abrogated this synergistic effect. Taken together, these results strongly suggest the involvement of both glycosidic residues and Ca+2 ions in the recognition of SLP-8348 by cellular receptors on murine macrophages. Moreover, these results suggest the potentiality of the SLP-8348 for the development of new adjuvants capable of stimulating antigen presenting cells by interaction with glycan receptors.

  • deleterious metabolic effects of high fructose intake the preventive effect of Lactobacillus kefiri administration
    Nutrients, 2017
    Co-Authors: Paula Carasi, Maria De Los Angeles Serradell, Maria Guillermina Zubiria, Sabrina Eliana Gambaro, Maria Amanda Rey, Andres Giovambattista
    Abstract:

    Modern lifestyle and diets have been associated with metabolic disorders and an imbalance in the normal gut microbiota. Probiotics are widely known for their health beneficial properties targeting the gut microbial ecosystem. The aim of our study was to evaluate the preventive effect of Lactobacillus kefiri (L. kefiri) administration in a fructose-rich diet (FRD) mice model. Mice were provided with tap water or fructose-added (20% w/v) drinking water supplemented or not with L. kefiri. Results showed that probiotic administration prevented weight gain and epidydimal adipose tissue (EAT) expansion, with partial reversion of the adipocyte hypertrophy developed by FRD. Moreover, the probiotic prevented the increase of plasma triglycerides and leptin, together with the liver triglyceride content. Leptin adipocyte secretion was also improved by L. kefiri, being able to respond to an insulin stimulus. Glucose intolerance was partially prevented by L. kefiri treatment (GTT) and local inflammation (TNFα; IL1β; IL6 and INFγ) was completely inhibited in EAT. L. kefiri supplementation generated an impact on gut microbiota composition, changing Bacteroidetes and Firmicutes profiles. Overall, our results indicate that the administration of probiotics prevents the deleterious effects of FRD intake and should therefore be promoted to improve metabolic disorders.

  • Lactobacillus kefiri shows inter-strain variations in the amino acid sequence of the S-layer proteins
    Antonie van Leeuwenhoek, 2017
    Co-Authors: Mariano Malamud, Paula Carasi, Sílvia Bronsoms, Sebastián A. Trejo, María De Los Angeles Serradell
    Abstract:

    The S-layer is a proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea , and it could be involved in cell recognition of microbes among other several distinct functions. In this work, both proteomic and genomic approaches were used to gain knowledge about the sequences of the S-layer protein (SLPs) encoding genes expressed by six aggregative and sixteen non-aggregative strains of potentially probiotic Lactobacillus kefiri . Peptide mass fingerprint (PMF) analysis confirmed the identity of SLPs extracted from L . kefiri , and based on the homology with phylogenetically related species, primers located outside and inside the SLP-genes were employed to amplify genomic DNA. The O-glycosylation site SASSAS was found in all L . kefiri SLPs. Ten strains were selected for sequencing of the complete genes. The total length of the mature proteins varies from 492 to 576 amino acids, and all SLPs have a calculated pI between 9.37 and 9.60. The N-terminal region is relatively conserved and shows a high percentage of positively charged amino acids. Major differences among strains are found in the C-terminal region. Different groups could be distinguished regarding the mature SLPs and the similarities observed in the PMF spectra. Interestingly, SLPs of the aggregative strains are 100% homologous, although these strains were isolated from different kefir grains. This knowledge provides relevant data for better understanding of the mechanisms involved in SLPs functionality and could contribute to the development of products of biotechnological interest from potentially probiotic bacteria.

  • impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota
    Clinical & Developmental Immunology, 2015
    Co-Authors: Paula Carasi, Silvia M Racedo, Claudine Jacquot, David Emmanuel Romanin, Maria De Los Angeles Serradell, Maria C Urdaci
    Abstract:

    The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-γ, GM-CSF, and IL-1β genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders.

Mariano Malamud - One of the best experts on this subject based on the ideXlab platform.

  • probiotic Lactobacillus kefiri prevents endotoxin induced preterm birth and stillbirth in mice
    Reproduction, 2021
    Co-Authors: Maria Silvia Ventimiglia, Natalin Valeff, Marlon Pozo Alban, Juan Manuel Paturlanne, Lorena Juriol, Florencia Quadrana, Martina Cecotti, Mariano Malamud, Marcos Javier Dibo
    Abstract:

    Preterm birth (PTB), defined as birth occurring before 37 weeks of pregnancy, affects 5-18% of pregnancies and is the leading cause of neonatal morbidity and mortality worldwide. Although PTB is considered a syndrome, infection-induced inflammation accounts up to 50% of all cases. Despite the effort to reduce the incidence of PTB, it continues rising worldwide and current approaches for preventing or treating PTB are largely unsatisfactory. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. It is well known that probiotics can modulate the host immune system exerting a potent anti-inflammatory activity. The main aim of this work was to evaluate the capacity of the probiotic Lactobacillus kefiri (Lk48) to prevent preterm birth in mice. C57BL/6 female mice were treated with Lk48 or vehicle a week before and during pregnancy and were challenged with LPS (10 µg), a dose known to induce 100% of PTB, on gestational day 16. Percentages of PTB as well as stillbirth were evaluated. We observed that oral administration of Lk48 significantly reduced the occurrence of LPS-induced PTB and stillbirth as well as improved post-natal development. This protective effect was associated with a reduction in leucocyte infiltration and reduced inflammation-induced damage in reproductive tissue. Besides, Lk48 treatment also modulated the diversity of vaginal microbiota. Our results demonstrated that prophylactic consumption of probiotic Lactobacillus kefiri prevented LPS-induced PTB and still birth in mice and open new avenues for exploring novel and promising strategies for preventing PTB in humans.

  • immunostimulation by Lactobacillus kefiri s layer proteins with distinct glycosylation patterns requires different lectin partners
    Journal of Biological Chemistry, 2020
    Co-Authors: Mariano Malamud, Maria De Los Angeles Serradell, Gustavo J Cavallero, Adriana C Casabuono, Bernd Lepenies, Alicia S Couto
    Abstract:

    S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography–pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow–derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow–derived dendritic cells from C-type lectin receptor–deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

  • s layer glycoprotein from Lactobacillus kefiri exerts its immunostimulatory activity through glycan recognition by mincle
    Frontiers in Immunology, 2019
    Co-Authors: Paula Carasi, Mariano Malamud, Bernd Lepenies, Matias Hernan Assandri, Teresa Freire, Maria De Los Angeles Serradell
    Abstract:

    The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea. Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca2+-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR–hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3 and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signalling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle-/- or CARD9-/- mice were not capable to enhance OVA-specific response in CD4+ T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.

  • s layer glycoprotein from Lactobacillus kefiri cidca 8348 enhances macrophages response to lps in a ca 2 dependent manner
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Paula Carasi, Mariano Malamud, Maria De Los Angeles Serradell, Teresa Freire
    Abstract:

    The S-layer is a (glyco)-proteinaceous envelope constituted by self-assembled subunits that form a two-dimensional lattice covering the surface of different species of Bacteria and Archaea. It could be considered as one of the most abundant biopolymers in our planet. Because of their unique self-assembly features, exhibiting repetitive identical physicochemical properties down to the subnanometer scale, as well as their involvement in specific interactions with host cells, the S-layer proteins (SLPs) show a high potential application in different areas of biotechnology, including the development of antigen carriers or new adjuvants. The presence of a glycosylated SLP on potentially probiotic Lactobacillus kefiri strains was previously described by our research group. In this study, we aim to investigate the role of carbohydrates present in the SLP from L. kefiri CIDCA 8348 (SLP-8348) in their internalization by murine macrophages, as well as to analyze their immunomodulatory capacity and their effect on LPS-stimulated macrophages. RAW 264.7 cells internalized the SLP-8348 in a process that was mediated by carbohydrate-receptor interactions since it was inhibited by glucose, mannose or EGTA, a Ca+2 chelating agent. These results correlated with the recognition of SLP-8348 by ConA lectin. We further show that while SLP-8348 was not able to induce the activation of macrophages by itself, it favored the LPS-induced response, since there was a significant increase in the expression of surface cell markers MHC-II, CD86 and CD40, as well as in IL-6 and IL-10 expression at both transcript and protein levels, in comparison with LPS-stimulated cells. The presence of EGTA completely abrogated this synergistic effect. Taken together, these results strongly suggest the involvement of both glycosidic residues and Ca+2 ions in the recognition of SLP-8348 by cellular receptors on murine macrophages. Moreover, these results suggest the potentiality of the SLP-8348 for the development of new adjuvants capable of stimulating antigen presenting cells by interaction with glycan receptors.

  • a glycoproteomic approach reveals that the s layer glycoprotein of Lactobacillus kefiri cidca 83111 is o and n glycosylated
    Journal of Proteomics, 2017
    Co-Authors: Gustavo J Cavallero, Mariano Malamud, Adriana C Casabuono, M De Los Angeles Serradell, Alicia S Couto
    Abstract:

    Abstract In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O -glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O -glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri , substituted on average with eight glucose residues and decorated with galacturonic acid and another O -glycosylated site on peptide 471–476, with a Glc 5–8 GalA 2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N -glycan structures. To our knowledge, this is the first description of the structure of N -glycans in S-layer glycoproteins from Lactobacillus species. Significance A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N -glycosidic chains in S-layer glycoprotein from Lactobacillus spp.

Kunho Seo - One of the best experts on this subject based on the ideXlab platform.

  • culture supernatant produced by Lactobacillus kefiri from kefir inhibits the growth of cronobacter sakazakii
    Journal of Dairy Research, 2018
    Co-Authors: Donghyeon Kim, Dana Jeong, Il-byeong Kang, Kwang-young Song, Hyunsook Kim, Kunho Seo
    Abstract:

    Cronobacter sakazakii is a life-threatening foodborne pathogen found in powdered infant formula and dairy products. Kefir is a dairy probiotic product and its antimicrobial activity against C. sakazakii was reported in our previous study. To identify key microorganisms that mediate growth suppression, we tested the antimicrobial activity of culture supernatants derived from lactic acid bacteria found in kefir. Lactobacillus kefiri DH5, L. kefiranofaciens DH101, and B ifidobacterium longum 720 (a commercial probiotic strain that served as a positive control) all significantly inhibited the growth of C. sakazakii ATCC 29544, delaying the initiation of exponential growth from 3 to 9 h in the nutrient broth. Among them, L. kefiri DH5 exerted the strongest antimicrobial effects against C. sakazakii, showing bactericidal effect at the addition of 300 µl of supernatant in 1 ml of nutrient broth. Interestingly, the supernatant of L. kefiri DH5 has higher pH and lower titrable acidity than that of L. kefiranofaciens DH101, suggesting metabolites produced by heterofermentation of L. kefiri acted more effectively to antagonise the growth of C. sakazakii. In addition, the supernatant of L. kefiri DH5 induced the leakage of cytoplasmic materials including nucleic acid and proteins, suggesting L. kefiri DH5 disrupted the cellular membrane integrity of C. sakazakii. Considering that pH neutralisation reduced the L. kefiri-dependent growth suppression, it is inferred that this activity is mainly due to organic acids produced during the fermentation process.

  • dual function of Lactobacillus kefiri dh5 in preventing high fat diet induced obesity direct reduction of cholesterol and upregulation of ppar α in adipose tissue
    Molecular Nutrition & Food Research, 2017
    Co-Authors: Donghyeon Kim, Dana Jeong, Il-byeong Kang, Kwang-young Song, Hyunsook Kim, Kunho Seo
    Abstract:

    cope Kefir consumption inhibits the development of obesity and non-alcoholic fatty liver disease in mice fed 60% high-fat diet (HFD). To identify the key contributor of this effect, we isolated lactic acid bacteria (LAB) from kefir and examined their anti-obesity properties from in vitro screening and in vivo validation. Methods and results Thirteen kefir LAB isolates were subjected to survivability test using artificial gastrointestinal environment and cholesterol-reducing assay. Lactobacillus kefiri DH5 showed 100% survivability in gastrointestinal environments and reduced 51.6% of cholesterol; thus, this strain was selected for in vivo experiment. Compared to the HFD-saline group, the HFD-DH5 group showed significantly lower body weight (34.68 vs. 31.10 g; p < 0.001), epididymal adipose tissue weight (1.39 vs. 1.05 g; p < 0.001), blood triglyceride (38.2 vs. 31.0 mg/dL; p < 0.01) and LDL-cholesterol levels (19.4 vs. 15.7 mg/dL; p < 0.01). In addition, L. kefiri DH5 administration significantly modulated gut microbiota of HFD-fed mice. The hepatic steatosis was significantly milder (Lesion score, 2.1 vs. 1.2; p < 0.001) and adipocyte diameter was significantly smaller (65.1 vs. 42.2 μm; p < 0.001) in the HFD-DH5 group. L. kefiri DH5 upregulated PPARα, FABP4, and CPT1 expression in the epididymal adipose tissues (2.29-, 1.77-, and 2.05-fold change, respectively), suggesting a reduction in adiposity by stimulating fatty acid oxidation. Conclusion L. kefiri DH5 exerts anti-obesity effects by direct reduction of cholesterol in the lumen and upregulation of PPARα gene in adipose tissues. This article is protected by copyright. All rights reserved

  • development of rapid and highly specific taqman probe based real time pcr assay for the identification and enumeration of Lactobacillus kefiri in kefir milk
    International Dairy Journal, 2016
    Co-Authors: Donghyeon Kim, Dana Jeong, Il-byeong Kang, Kwang-young Song, Hyunsook Kim, Hongseok Kim, Sookyoung Lee, Kunho Seo
    Abstract:

    Abstract Lactobacillus kefiri is one of the key functional lactic acid bacteria in kefir milk. We designed a novel real-time PCR primer/probe set, LK_508 targeting the recA gene, for the rapid identification and enumeration of L. kefiri. In inclusivity and exclusivity test using standard strains and kefir isolates, only the 9 tested L. kefiri strains were positive, with the remaining 38 closely related microorganisms testing negative, thus indicating 100% sensitivity and specificity of the assay. The population of L. kefiri was 3.77, 4.30, 4.79, and 5.63 log cfu mL−1 of kefir milk fermented at 25 °C with 5% grain-milk ratio for 12, 24, 36, and 48 h, respectively. The newly developed qPCR assay could be applied to investigate the quantitative relationship of kefir microbiota in fermentation process.

Serradell, María De Los Ángeles - One of the best experts on this subject based on the ideXlab platform.

  • S-Layer Glycoprotein from Lactobacillus kefiri Exerts Its Immunostimulatory Activity Through Glycan Recognition by Mincle
    2020
    Co-Authors: Malamud Mariano, Carasi Paula, Assandri, Matías Hernán, Freire Teresa, Lepenies Bernd, Serradell, María De Los Ángeles
    Abstract:

    The development of new subunit vaccines has promoted the rational design of adjuvants able to induce a strong T-cell activation by targeting specific immune receptors. The S-layer is a (glyco)-proteinaceous envelope constituted by subunits that self-assemble to form a two-dimensional lattice that covers the surface of different species of Bacteria and Archaea. Due to their ability to self-assemble in solution, they are attractive tools to be used as antigen/hapten carriers or adjuvants. Recently, we have demonstrated that S-layer glycoprotein from Lactobacillus kefiri CIDCA 8348 (SLP-8348) enhanced the LPS-induced response on macrophages in a Ca2+-dependent manner, but the receptors involved in these immunomodulatory properties remain unknown. Therefore, we aim to determine the C-type lectin receptors (CLRs) recognizing this bacterial surface glycoprotein as well as to investigate the role of glycans in both the immunogenicity and adjuvant capacity of SLP-8348. Here, using a mild periodate oxidation protocol, we showed that loss of SLP-8348 glycan integrity impairs the cell-mediated immune response against the protein. Moreover, our data indicate that the adjuvant capacity of SLP-8348 is also dependent of the biological activity of the SLP-8348 glycans. In order to evaluate the CLRs involved in the interaction with SLP-8348 an ELISA-based method using CLR–hFc fusion proteins showed that SLP-8348 interacts with different CLRs such as Mincle, SingR3, and hDC-SIGN. Using BMDCs derived from CLR-deficient mice, we show that SLP-8348 uptake is dependent of Mincle. Furthermore, we demonstrate that the SLP-8348-induced activation of BMDCs as well as its adjuvant capacity relies on the presence of Mincle and its signaling adaptor CARD9 on BMDCs, since SLP-8348-activated BMDCs from Mincle−/− or CARD9−/− mice were not capable to enhance OVA-specific response in CD4+ T cells purified from OT-II mice. These findings significantly contribute to the understanding of the role of glycans in the immunomodulation elicited by bacterial SLPs and generate a great opportunity in the search for new adjuvants derived from non-pathogenic microorganisms.Instituto de Estudios Inmunológicos y Fisiopatológico

  • Impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota
    2019
    Co-Authors: Carasi Paula, Racedo S.m., Jacquot C., Romanin, David Emmanuel, Serradell, María De Los Ángeles, Urdaci M.c.
    Abstract:

    The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-γ, GM-CSF, and IL-1β genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders.Facultad de Ciencias ExactasInstituto de Estudios Inmunológicos y Fisiopatológico

  • Deleterious Metabolic Effects of High Fructose Intake: The Preventive Effect of Lactobacillus kefiri Administration
    2019
    Co-Authors: Zubiría, María Guillermina, Carasi Paula, Serradell, María De Los Ángeles, Gambaro, Sabrina Eliana, Rey, María Amanda, Giovambattista Andrés
    Abstract:

    Modern lifestyle and diets have been associated with metabolic disorders and an imbalance in the normal gut microbiota. Probiotics are widely known for their health beneficial properties targeting the gut microbial ecosystem. The aim of our study was to evaluate the preventive effect of Lactobacillus kefiri (L. kefiri) administration in a fructose-rich diet (FRD) mice model. Mice were provided with tap water or fructose-added (20% w/v) drinking water supplemented or not with L. kefiri. Results showed that probiotic administration prevented weight gain and epidydimal adipose tissue (EAT) expansion, with partial reversion of the adipocyte hypertrophy developed by FRD. Moreover, the probiotic prevented the increase of plasma triglycerides and leptin, together with the liver triglyceride content. Leptin adipocyte secretion was also improved by L. kefiri, being able to respond to an insulin stimulus. Glucose intolerance was partially prevented by L. kefiri treatment (GTT) and local inflammation (TNFα; IL1β; IL6 and INFγ) was completely inhibited in EAT. L. kefiri supplementation generated an impact on gut microbiota composition, changing Bacteroidetes and Firmicutes profiles. Overall, our results indicate that the administration of probiotics prevents the deleterious effects of FRD intake and should therefore be promoted to improve metabolic disorders.Facultad de Ciencias Exacta

  • Safety Characterization and Antimicrobial Properties of Kefir-Isolated Lactobacillus kefiri
    2018
    Co-Authors: Carasi Paula, Díaz Mariángeles, Racedo, Silvia M., De Antoni, Graciela Liliana, Urdaci, María C., Serradell, María De Los Ángeles
    Abstract:

    Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

  • Regular arrangement of Pt nanoparticles on S-layer proteins isolated from Lactobacillus kefiri: synthesis and catalytic application
    'Elsevier BV', 2018
    Co-Authors: Bolla, Patricia Araceli, Serradell, María De Los Ángeles, Sanz Agustina, Huggias Sofia, Ruggera, José Fernando, Casella, Mónica Laura
    Abstract:

    In this work, platinum bionanocatalysts using S-layer proteins isolated from two strains of Lactobacillus kefiri as template were obtained. TEM and STEM images evidenced that Pt/CBS8348 shows a P6-array while Pt/CBS83111 does not show any defined array. Noteworthy, nanometric and subnanometric Pt clusters were observed in both Pt/CBS8348 and Pt/CBS83111 catalysts, with an average metal particle size estimated by TEM of 3.62 and 3.88 nm, respectively. Also, Pt/CBS8348 showed a higher number of dispersed Pt single atoms than Pt/CBS83111. Both Pt bionanocatalysts showed an excellent performance in the reduction of p-nitrophenol, reaching conversion values close to 65-70% in 15 min. Besides, both Pt/CBS8348 and Pt/CBS83111 were successfully reused nine times, maintaining conversion values between 75 and 90%.Fil: Bolla, Patricia Araceli. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Ciencias Aplicadas; ArgentinaFil: Sanz, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Ciencias Aplicadas; ArgentinaFil: Huggias, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Ciencias Aplicadas; ArgentinaFil: Ruggera, José Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Ciencias Aplicadas; ArgentinaFil: Serradell, María de los Ángeles. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Cátedra de Microbiología; Argentina. Universidad Nacional Arturo Jauretche. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Casella, Mónica Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Ciencias Aplicadas "Dr. Jorge J. Ronco". Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Ciencias Aplicadas; Argentin

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