Laboratorio Of Bacterias Lacticas Y Probioticos

Valencia, Spain

Laboratorio Of Bacterias Lacticas Y Probioticos

Valencia, Spain
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Reynolds L.A.,Infection and Evolution and Institute of Immunology and Infection Research | Smith K.A.,Infection and Evolution and Institute of Immunology and Infection Research | Filbey K.J.,Infection and Evolution and Institute of Immunology and Infection Research | Harcus Y.,Infection and Evolution and Institute of Immunology and Infection Research | And 5 more authors.
Gut microbes | Year: 2014

The intestinal microbiota are pivotal in determining the developmental, metabolic and immunological status of the mammalian host. However, the intestinal tract may also accommodate pathogenic organisms, including helminth parasites which are highly prevalent in most tropical countries. Both microbes and helminths must evade or manipulate the host immune system to reside in the intestinal environment, yet whether they influence each other's persistence in the host remains unknown. We now show that abundance of Lactobacillus bacteria correlates positively with infection with the mouse intestinal nematode parasite, Heligmosomoides polygyrus, as well as with heightened regulatory T cell (Treg) and Th17 responses. Moreover, H. polygyrus raises Lactobacillus species abundance in the duodenum of C57BL/6 mice, which are highly susceptible to H. polygyrus infection, but not in BALB/c mice, which are relatively resistant. Sequencing of samples at the bacterial gyrB locus identified the principal Lactobacillus species as L. taiwanensis, a previously characterized rodent commensal. Experimental administration of L. taiwanensis to BALB/c mice elevates regulatory T cell frequencies and results in greater helminth establishment, demonstrating a causal relationship in which commensal bacteria promote infection with an intestinal parasite and implicating a bacterially-induced expansion of Tregs as a mechanism of greater helminth susceptibility. The discovery of this tripartite interaction between host, bacteria and parasite has important implications for both antibiotic and anthelmintic use in endemic human populations.


Bidart G.N.,Laboratorio Of Bacterias Lacticas Y Probioticos | Bidart G.N.,National University of San Martín of Argentina | Rodriguez-Diaz J.,Laboratorio Of Bacterias Lacticas Y Probioticos | Monedero V.,Laboratorio Of Bacterias Lacticas Y Probioticos | Yebra M.J.,Laboratorio Of Bacterias Lacticas Y Probioticos
Molecular Microbiology | Year: 2014

The probiotic Lactobacillus casei catabolizes galacto-N-biose (GNB) and lacto-N-biose (LNB) by using a transport system and metabolic routes different from those of Bifidobacterium. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC (EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTSGnb. Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-β-galactosidase) hydrolyses both disaccharides. However, GnbF (N-acetylgalactosamine-6P deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA (N-acetylglucosamine-6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose-6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose-6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes. © 2014 John Wiley & Sons Ltd.


Bidart G.N.,Laboratorio Of Bacterias Lacticas Y Probioticos | Rodriguez-Diaz J.,University of Valencia | Palomino-Schatzlein M.,Research Center Principe Felipe | Monedero V.,Laboratorio Of Bacterias Lacticas Y Probioticos | Yebra M.J.,Laboratorio Of Bacterias Lacticas Y Probioticos
Applied Microbiology and Biotechnology | Year: 2016

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants. © 2016 Springer-Verlag Berlin Heidelberg


Rodriguez-Diaz J.,Laboratorio Of Bacterias Lacticas Y Probioticos | Carbajo R.J.,Research Center Principe Felipe | Pineda-Lucena A.,Research Center Principe Felipe | Monedero V.,Laboratorio Of Bacterias Lacticas Y Probioticos | Yebra M.J.,Laboratorio Of Bacterias Lacticas Y Probioticos
Applied and Environmental Microbiology | Year: 2013

AlfB and AlfC α-L-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3-N-acetylglucosamine and fucosyl-α-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-α-L-fucopyranoside into fucosyl-α-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6-N-acetylglucosamine. © 2013, American Society for Microbiology.


PubMed | Research Center Principe Felipe, University of Valencia and Laboratorio Of Bacterias Lacticas Y Probioticos
Type: Journal Article | Journal: Applied microbiology and biotechnology | Year: 2016

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho--galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 694 and 711% of o-nitrophenyl--D-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.70.2g/l and for GNB was 10.80.3g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for 1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho--galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.


Bidart G.N.,Laboratorio Of Bacterias Lacticas Y Probioticos | Rodriguez-Diaz J.,Laboratorio Of Bacterias Lacticas Y Probioticos | Rodriguez-Diaz J.,University of Valencia | Yebra M.J.,Laboratorio Of Bacterias Lacticas Y Probioticos
Applied and Environmental Microbiology | Year: 2016

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a β-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of β-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3'-N-acetylglucosaminyl-mannose and 3'-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3'-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3'-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3'-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a β-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche. © 2016, American Society for Microbiology.


De Boeck R.,Laboratorio Of Bacterias Lacticas Y Probioticos | Sarmiento-Rubiano L.A.,Laboratorio Of Bacterias Lacticas Y Probioticos | Nadal I.,Laboratorio Of Bacterias Lacticas Y Probioticos | Monedero V.,Laboratorio Of Bacterias Lacticas Y Probioticos | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2010

Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTSGut) in BL251. These results showed that the PTSGut did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Δldh1 ΔgutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate. © 2009 Springer-Verlag.


Rodriguez-Diaz J.,Laboratorio Of Bacterias Lacticas Y Probioticos | Rubio-del-Campo A.,Laboratorio Of Bacterias Lacticas Y Probioticos | Yebra M.J.,Laboratorio Of Bacterias Lacticas Y Probioticos
Biotechnology and Bioengineering | Year: 2012

UDP-sugars are used as glycosyl donors in many enzymatic glycosylation processes. In bacteria UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from fructose-6-phosphate by four successive reactions catalyzed by three enzymes: Glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM), and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). In this work several metabolic engineering strategies, aimed to increment UDP-GlcNAc biosynthesis, were applied in the probiotic bacterium Lactobacillus casei strain BL23. This strain does not produce exopolysaccharides, therefore it could be a suitable host for the production of oligosaccharides. The genes glmS, glmM, and glmU coding for GlmS, GlmM, and GlmU activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous over-expression. The recombinant L. casei strain over-expressing simultaneously the genes glmM and glmS showed a 3.47 times increase in GlmS activity and 6.43 times increase in GlmM activity with respect to the control strain. Remarkably, these incremented activities resulted in about fourfold increase of the UDP-GlcNAc pool. In L. casei BL23 wild type strain transcriptional analyses showed that glmM and glmU are constitutively transcribed. By contrast, glmS transcription is down-regulated with a 21-fold decrease of glmS mRNA in cells cultured with N-acetylglucosamine as the sole carbon source compared to cells cultured with glucose. Our results revealed for the first time that GlmS, GlmM, and GlmU are responsible for UDP-GlcNAc biosynthesis in lactobacilli. © 2012 Wiley Periodicals, Inc.


Rodriguez-Diaz J.,Laboratorio Of Bacterias Lacticas Y Probioticos | Rubio-del-Campo A.,Laboratorio Of Bacterias Lacticas Y Probioticos | Yebra M.J.,Laboratorio Of Bacterias Lacticas Y Probioticos
Applied and Environmental Microbiology | Year: 2012

We have previously characterized from Lactobacillus casei BL23 three α-L-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3-N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the L-fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the α-L-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α-L-fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria. © 2012, American Society for Microbiology.


PubMed | Laboratorio Of Bacterias Lacticas Y Probioticos
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2016

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a -N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of -N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3-N-acetylglucosaminyl-mannose and 3-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a -N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche.

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