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Polyphosphate (poly-P) biosynthesis in bacteria has been linked to many physiological processes and has been characterized as an interesting functional molecule involved in intestinal homeostasis. We determined the capacity for poly-P production of 18 probiotic strains mainly belonging to Bifidobacterium and former Lactobacillus genera, showing that poly-P synthesis is a strain-specific trait dependent on the availability of phosphate and the growth phase. Bifidobacteria were especially capable of poly-P synthesis and poly-P kinase (ppk) genes were identified in their genomes together with a repertoire of genes involved in phosphate transport and metabolism. In Bifidobacterium longum KABP042, the strain we found with highest poly-P production, variations in ppk expression were linked to growth conditions and presence of phosphate in the medium. Moreover, the strain produced poly-P in presence of breast milk and human milk oligosaccharide lacto-N-tetraose increased the amount of poly-P synthesized. Compared to KABP042 supernatants low in poly-P, exposure of Caco-2 cells to KABP042 supernatants rich in poly-P resulted in decreased epithelial permeability and increased barrier resistance, induction of epithelial protecting factors such as HSP27 and enhanced expression of tight junction protein genes. These results highlight the role of bifidobacteria-derived poly-P as a strain-dependent functional factor acting on epithelial integrity.

Humans are mainly exposed to mercury (Hg) through contaminated foodstuffs. However, the effects of Hg on the intestinal tract have received little attention. We performed a subchronic exposure to inorganic mercury or methylmercury in mice through drinking water (1, 5 or 10 mg/L for four months) to evaluate their intestinal impact. Histological, biochemical and gene expression analyses showed that both Hg species induced oxidative stress in small intestine and colon, while inflammation was mainly detected in the colon. Increased fecal albumin content indicated a compromised epithelial barrier. Mucus production was possibly also affected, as an increase in Muc2 expression was detected. However, differential effects were detected between both Hg species. Activation of p38 MAPK and increased crypt depth were detected in colon only with MeHg. Minor differences in microbiota composition were detected between unexposed and exposed mice. Although significant differences were detected between both Hg species at 10 mg/L, only the relative abundances of low abundance taxa were affected. Concentrations of microbial-derived short-chain fatty acids were decreased, suggesting an effect on microbial metabolism or increased demand by the intestinal epithelium. Results obtained confirm previous in vitro studies and highlights the intestinal mucosa as an initial target of Hg., This study was funded by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (RTI2018-098071-B-I00) and it was co-funded by the European Commission through the European Regional Development Fund (Multiregional operative program for Spain 2014–2020).

The gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-l-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fucosidase genes were selected for substrate specificity characterization. The α-l-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on α1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-α-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on α1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on α1,6-linked l-fucose, and Fuc5372 showed a preference for α1,2 linkages. Fuc2358 exhibited a broad substrate specificity releasing l-fucose from all the tested free histo-blood group antigens, HMOs, and 6FN. This latest enzyme also displayed activity in glycoconjugates carrying lacto-N-fucopentaose II (Lea) and lacto-N-fucopentaose III (Lex) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed l-fucose from neoglycoproteins and human α-1 acid glycoprotein. These results give insight into the great diversity of α-l-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition. IMPORTANCE α-l-Fucosyl residues are frequently present in many relevant glycans, such as human milk oligosaccharides (HMOs), histo-blood group antigens (HBGAs), and epitopes on cell surface glycoconjugate receptors. These fucosylated glycans are involved in a number of mammalian physiological processes, including adhesion of pathogens and immune responses. The modulation of l-fucose content in such processes may provide new insights and knowledge regarding molecular interactions and may help to devise new therapeutic strategies. Microbial α-l-fucosidases are exoglycosidases that remove α-l-fucosyl residues from free oligosaccharides and glycoconjugates and can be also used in transglycosylation reactions to synthesize oligosaccharides. In this work, α-l-fucosidases from the GH29 family were identified and characterized from the metagenome of fecal samples of breastfed infants. These enzymes showed different substrate specificities toward HMOs, HBGAs, naturally occurring glycoproteins, and neoglycoproteins. These novel glycosidase enzymes from the breast-fed infant gut microbiota, which resulted in a good source of α-l-fucosidases, have great biotechnological potential., This work was supported by grant PID2020-115403RB (C21 and C22) from the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI) (10.13039/501100011033).

Exposure to mercury (Hg) mostly occurs through diet, where it is mainly found as inorganic Hg [Hg(II)] or methylmercury (MeHg). In vivo studies have linked its exposure with neurological and renal diseases, however, its toxic effects upon the gastrointestinal tract are largely unknown. In order to evaluate the effect of Hg on intestinal mucosa, a bicameral system was employed with co-cultures of Caco-2 and HT29-MTX intestinal epithelial cells and THP-1 macrophages. Cells were exposed to Hg(II) and MeHg (0.1, 0.5, 1 mg/L) during 11 days. The results evidenced a greater pro-inflammatory response in cells exposed to Hg with increments of IL-8 (15-126%) and IL-1β release (39-63%), mainly induced by macrophages which switched to a M1 phenotype. A pro-oxidant response was also observed in both cell types with an increase in ROS/RNS levels (44-140%) and stress proteins expression. Intestinal cells treated with Hg displayed structural abnormalities, hypersecretion of mucus and defective tight junctions. An increased paracellular permeability (123-170%) at the highest concentrations of Hg(II) and MeHg and decreased capacity to restore injuries in the cell monolayer were also observed. All these toxic effects were governed by various inflammatory signalling pathways (p38 MAPK, JNK and NF-κB)., This study was funded by MCIN/AEI (RTI2018-098071-B-I00) and it was co-funded by the European Union through the ERDF (Multiregional operative program for Spain 2014–2020)

A bicameral model consisting of Caco-2 and HT29-MTX intestinal epithelial cells and THP-1-derived macrophages has been used to test the ability of two strains of Lactobacillus to protect from damage caused by mercury. Exposure to 1 mg/ml mercury [Hg(II) or methyl-Hg] for seven days in this model resulted in an inflammatory and pro-oxidant response mainly driven by macrophages. This led to an impairment in the intestinal barrier, defective tight-junctions, increased permeability and mucus hypersecretion. In addition, the wound-healing capacity of the epithelial monolayer was also diminished. However, the presence of heat-killed Lactobacillus intestinalis or Lactobacillus johnsonii cells during Hg exposure reverted these effects, and most of the parameters recovered values similar to control cells. Both lactobacilli showed the capacity to bind Hg(II) and methyl-Hg under the cell culture conditions. This points to Hg sequestration as a likely mechanism that counteracted Hg toxicity. However, differences in the Hg binding capacity and in the effects between both strains suggest that other probiotic-mediated mechanisms may play a role in the alleviation of the damage elicited by Hg. These results show the potential of the bicameral intestinal epithelial model for screening of effective strains for their use in later in vivo studies., This study was funded by MCIN/AEI (RTI2018-098071-B-I00) and it was co-funded by the European Union through the ERDF (Multiregional operative program for Spain 2014–2020).

The original publication is available at https://doi.org/10.3920/BM2019.0184, Lipoteichoic acid (LTA) is a key component of the cell wall of most Gram-positive bacteria and plays many structural and functional roles. In probiotic lactobacilli, the function of LTA in mediating bacteria/host cross-talk has been evidenced and it has been postulated that, owing to its anionic nature, LTA may play a role in toxic metal sequestration by these bacteria. However, studies on this last aspect employing strains unable to synthesise LTA are lacking. We have inactivated the LTA polymerase encoding gene ltaS in two different Lactobacillus plantarum strains. Analysis of LTA contents in wild-type and ltaS mutant strains corroborated the role of this gene as a major contributor to LTA synthesis in L. plantarum. The mutant strains displayed strain-dependent anomalous cell morphologies that resulted in elongated or irregular cells with aberrant septum formation. They also exhibited higher sensitivity to several stresses (osmotic and heat) and to antimicrobials that target the cell wall. The toxicity of inorganic [(Hg(II)] and organic mercury (methyl-Hg) was also increased upon ltaS mutation in a strain-dependent manner. However, the mutant strains showed 0 to 50% decrease in their capacity of Hg binding compared to their corresponding parental strains. This result suggests a partial contribution of LTA to Hg binding onto the cell surface that was dependent on the strain and the Hg form., This work has been financed by research grants AGL2015‐68920‐R and RTI2018‐098071‐B‐I00 from the Agencia Estatal de Investigación from Spain (AEI) that were partially funded by the European Regional Development Fund. A.C. was hired by a contract from the Ayudas para la Promoción de Empleo Joven from the Spanish Ministry of Economy, partially funded by the European Social Fund.

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies., This research was supported by Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/ 501100011033, and by “ERDF A way of making Europe” grants AGL2017-84165-C2-2-R and PID2020-115403RB-C22 to J.R.-D., C.S.-B. is the recipient of a predoctoral grant RE2018-083315 funded by MCIN/AEI/ 10.13039/501100011033 and by “ESF Investing in your future”. This work was also supported by Valencian Government grant IDIFEDER/2018/056. N.P.-G. is the recipient of a predoctoral grant from the Valencian Government ACIF/2020/085. R.G.-R. is the recipient of a postdoctoral grant from the Valencian Government APOST/2017/037.

Intestinal microbiota-virus-host interaction has emerged as a key factor in mediating enteric virus pathogenicity. With the aim of analyzing whether human gut bacteria improve the inefficient replication of human rotavirus in mice, we performed fecal microbiota transplant (FMT) with healthy infants as donors in antibiotic-treated mice. We showed that a simple antibiotic treatment, irrespective of FMT, resulted in viral shedding for 6 days after challenge with the human rotavirus G1P[8] genotype Wa strain (RVwa). Rotavirus titers in feces were also significantly higher in antibiotic-treated animals with or without FMT but they were decreased in animals subject to self-FMT, where a partial re-establishment of specific bacterial taxons was evidenced. Microbial composition analysis revealed profound changes in the intestinal microbiota of antibiotic-treated animals, whereas some bacterial groups, including members of Lactobacillus, Bilophila, Mucispirillum, and Oscillospira, recovered after self-FMT. In antibiotic-treated and FMT animals where the virus replicated more efficiently, differences were observed in gene expression of immune mediators, such as IL1β and CXCL15, as well as in the fucosyltransferase FUT2, responsible for H-type antigen synthesis in the small intestine. Collectively, our results suggest that antibiotic-induced microbiota depletion eradicates the microbial taxa that restrict human rotavirus infectivity in mice., This research was funded by Spanish Government (Ministerio de Economía y Competitividad) grants AGL2017-84165-C2-1-R to MJY, AGL2017-84165-C2-2-R to JRD. The study was also supported by Valencian Government grant IDIFEDER/2018/056 and by European FEDER founds. RGR is the recipient of a postdoctoral grant from the Valencian Government (APOST/2017/037). CSB is the recipient of a FPI predoctoral grant from the Spanish Government (RE2018-083315). SVV is the recipient of a predoctoral grant from the Valencian Government (ACIF/2016/437).

Chronic exposure to inorganic arsenic (As) [As(III) + As(V)], which affects millions of people, increases the incidence of some kinds of cancer and other non-carcinogenic pathologies. Although the oral pathway is the main form of exposure, in vivo studies have not been conducted to verify the intestinal toxicity of this metalloid. The aim of this study is to perform an in vivo evaluation of the intestinal toxicity of inorganic As, using female BALB/c mice exposed through drinking water to various concentrations of As(III) (20, 50, and 80 mg/L) for 2 months. An increase was observed in oxygen and/or nitrogen reactive species, and in gene and protein expression of pro-inflammatory cytokines (IL-1β, IL-2, IL-6) at concentrations equal to or greater than 50 mg/L. These changes were accompanied by a profound remodeling of the intestinal microbial profile in terms of diversity and global composition, which could be at the basis or exacerbate As(III) toxic effects. The histological study showed that there was moderate inflammation of the mucosa and submucosa, accompanied by hyperplasia of crypts at the highest administered dose. In addition, all the treatments with As(III) resulted in a decreased expression of Muc2, which encodes one of the main components of the intestinal layer of mucus. The effects described are compatible with the increased intestinal permeability observed at concentrations equal to or greater than 50 mg/L, indicative of loss of barrier function., This work was supported by the Spanish Ministry of Science, Innovation and Universities (AGL2015-68920-R), for which the authors are deeply indebted. Gabriela M. Chiocchetti received a fellowship from the Brazilian Government (CAPES BEX1086/14-6). We thank M.C. Collado, C. Alcántara, M. Selma, M. Dzidic and A. Boix for their advice in metagenome analyses. We also thank Inmaculada Noguera, from the Animal Production Service from the University of Valencia, for her assistance in the animal experiments.

In lactobacilli, CcpA is known to modulate the expression of genes involved in sugar metabolism, stress response and aerobic adaptation. This study aimed to evaluate a ccpA mutant of Lacticaseibacillus casei BL23 to increase lactic acid production using cheese whey. The ccpA derivative (BL71) showed better growth than the L. casei wild-type in the whey medium. In a stirred tank reactor, at 48 h, lactate production by BL71 was eightfold higher than that by BL23. In batch fermentations, the final values reached were 44.23 g L−1 for BL71 and 27.58 g L−1 for BL23. Due to a decrease in the delay of lactate production in the mutant, lactate productivity increased from 0.17 g (L.h)−1 with BL23 to 0.80 g (L.h)−1 with BL71. We found that CcpA would play additional roles in nitrogen metabolism by the regulation of the proteolytic system. BL71 displayed higher activity of the PepX, PepQ and PrtP enzymes than BL23. Analysis of prtP expression confirmed this deregulation in BL71. Promoter analysis of the prtP gene revealed CcpA binding sites with high identity to the cre consensus sequence and the interaction of CcpA with this promoter was confirmed in vitro. We postulate that deregulation of the proteolytic system in BL71 allows a better exploitation of nitrogen resources in cheese whey, resulting in enhanced fermentation capacity. Therefore, the ccpA gene could be a good target for future technological developments aimed at effective and inexpensive lactate production from dairy industrial wastes.

The gut microbiota has emerged as a key factor in the pathogenesis of intestinal viruses, including enteroviruses, noroviruses and rotaviruses (RVs), where stimulatory and inhibitory effects on infectivity have been reported. With the aim of determining whether members of the microbiota interact with RVs during infection, a combination of anti-RV antibody labeling, fluorescence-activated cell sorting and 16S rRNA amplicon sequencing was used to characterize the interaction between specific bacteria and RV in stool samples of children suffering from diarrhea produced by G1P[8] RV. The genera Ruminococcus and Oxalobacter were identified as RV binders in stools, displaying enrichments between 4.8- and 5.4-fold compared to samples nonlabeled with anti-RV antibodies. In vitro binding of the G1P[8] Wa human RV strain to two Ruminococcus gauvreauii human isolates was confirmed by fluorescence microscopy. Analysis in R. gauvreauii with antibodies directed to several histo-blood group antigens (HBGAs) indicated that these bacteria express HBGA-like substances on their surfaces, which can be the target for RV binding. Furthermore, in vitro infection of the Wa strain in differentiated Caco-2 cells was significantly reduced by incubation with R. gauvreauii. These data, together with previous findings showing a negative correlation between Ruminococcus levels and antibody titers to RV in healthy individuals, suggest a pivotal interaction between this bacterial group and human RV. These results reveal likely mechanisms of how specific bacterial taxa of the intestinal microbiota could negatively affect RV infection and open new possibilities for antiviral strategies., This research was supported by Spanish Government (Ministerio de Economía y Competitividad) grants AGL2017-84165-C2-1-R to M.J.Y. and AGL2017-84165-C2-2-R and RYC-2013-12442 to J.R.-D. This work was also supported by Valencian Government grant IDIFEDER/2018/056. R.G.-R. is the recipient of a postdoctoral grant from the Valencian Government APOST/2017/037. C.S.-B. is the recipient of a predoctoral grant FPI from the Spanish Government RE2018-083315. S.V.-V. is the recipient of a predoctoral grant from the Valencian Government ACIF/2016/437.

The survival of commensal bacteria in the human gut partially depends on their ability to metabolize host-derived molecules. The use of the glycosidic moiety of N-glycoproteins by bacteria has been reported, but the role of N-glycopeptides or glycoamino acids as the substrates for bacterial growth has not been evaluated. We have identified in Lactobacillus casei strain BL23 a gene cluster (alf-2) involved in the catabolism of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn (6′FN-Asn), a constituent of the core-fucosylated structures of mammalian N-glycoproteins. The cluster consists of the genes alfHC, encoding a major facilitator superfamily (MFS) permease and the α-l-fucosidase AlfC, and the divergently oriented asdA (aspartate 4-decarboxylase), alfR2 (transcriptional regulator), pepV (peptidase), asnA2 (glycosyl-asparaginase), and sugK (sugar kinase) genes. Knockout mutants showed that alfH, alfC, asdA, asnA2, and sugK are necessary for efficient 6′FN-Asn utilization. The alf-2 genes are induced by 6′FN-Asn, but not by its glycan moiety, via the AlfR2 regulator. The constitutive expression of alf-2 genes in an alfR2 strain allowed the metabolism of a variety of 6′-fucosyl-glycans. However, GlcNAc-Asn did not support growth in this mutant background, indicating that the presence of a 6′-fucose moiety is crucial for substrate transport via AlfH. Within bacteria, 6′FN-Asn is defucosylated by AlfC, generating GlcNAc-Asn. This glycoamino acid is processed by the glycosylasparaginase AsnA2. GlcNAc-Asn hydrolysis generates aspartate and GlcNAc, which is used as a fermentable source by L. casei. These data establish the existence in a commensal bacterial species of an exclusive metabolic pathway likely to scavenge human milk and mucosal fucosylated N-glycopeptides in the gastrointestinal tract. IMPORTANCE The gastrointestinal tract accommodates more than 1014 microorganisms that have an enormous impact on human health. The mechanisms enabling commensal bacteria and administered probiotics to colonize the gut remain largely unknown. The ability to utilize host-derived carbon and energy resources available at the mucosal surfaces may provide these bacteria with a competitive advantage in the gut. Here, we have identified in the commensal species Lactobacillus casei a novel metabolic pathway for the utilization of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn, which is present in the core-fucosylated N-glycoproteins from mammalians. These results give insight into the molecular interactions between the host and commensal/probiotic bacteria and may help to devise new strategies to restore gut microbiota homeostasis in diseases associated with dysbiotic microbiota., This work was financed by funds of the Spanish Ministry for Economy and Competitiveness (MINECO)/FEDER through projects AGL2014-52996-C2 (1-R and 2-R) and AGL2017-84165-C2 (1-R and 2-R). J.E.B. was supported by a Santiago Grisolía predoctoral fellowship from the Valencian Government. J.R.-D. was supported by a Ramon y Cajal contract by the Spanish Ministry for Economy and Competitiveness. R.G.-R. was the recipient of a postdoctoral fellowship from the Generalitat Valenciana that was cofounded by the European Social Fund. Part of the equipment employed in this work has been funded by the Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020).

Chemical contaminants that are present in food pose a health problem and their levels are controlled by national and international food safety organizations. Despite increasing regulation, foods that exceed legal limits reach the market. In Europe, the number of notifications of chemical contamination due to pesticide residues, mycotoxins and metals is particularly high. Moreover, in many parts of the world, drinking water contains high levels of chemical contaminants owing to geogenic or anthropogenic causes. Elimination of chemical contaminants from water and especially from food is quite complex. Drastic treatments are usually required, which can modify the food matrix or involve changes in the forms of cultivation and production of the food products. These modifications often make these treatments unfeasible. In recent years, efforts have been made to develop strategies based on the use of components of natural origin to reduce the quantity of contaminants in foods and drinking water, and to reduce the quantity that reaches the bloodstream after ingestion, and thus, their toxicity. This review provides a summary of the existing literature on strategies based on the use of lactic acid bacteria or yeasts belonging to the genus Saccharomyces that are employed in food industry or for dietary purposes., This work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2015-68920), for which the authors are deeply indebted. Gabriela de Matuoka e Chiocchetti received a fellowship from the Brazilian Government (CAPES-proceso BEX1086/14-6) and Carlos Jadán-Piedra received a Personnel Training Grant from SENESCYT (Ecuadorian Ministry of Higher Education, Science, Technology and Innovation).

Metal sequestration by bacteria has been proposed as a strategy to counteract metal contamination in foodstuffs. Lactobacilli can interact with metals, although studies with important foodborne metals such as inorganic [Hg(II)] or organic (CH3 Hg) mercury are lacking. Lactobacilli were evaluated for their potential to bind these contaminants and the nature of the interaction was assessed by the use of metal competitors, chemical and enzymatical treatments, and mutants affected in the cell wall structure.; Results: Lactobacillus strains efficiently bound Hg(II) and CH3 Hg. Mercury binding by Lactobacillus casei BL23 was independent of cell viability. In BL23, both forms of mercury were cell wall bound. Their interaction was not inhibited by cations and it was resistant to chelating agents and protein digestion. Lactobacillus casei mutants affected in genes involved in the modulation of the negative charge of the cell wall anionic polymer lipoteichoic acid showed increased mercury biosorption. In these mutants, mercury toxicity was enhanced compared to wild-type bacteria. These data suggest that lipoteichoic acid itself or the physicochemical characteristics that it confers to the cell wall play a major role in mercury complexation.; Conclusion: This is the first example of the biosorption of Hg(II) and CH3 Hg in lactobacilli and it represents a first step towards their possible use as agents for diminishing mercury bioaccessibility from food at the gastrointestinal tract. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

The capacity of two LAB strains to inhibit inorganic [Hg(II)] and organic (methyl-Hg; MeHg) mercury translocation through monolayers of co-cultures of NCM460 and HT29-MTX colonic cells was evaluated. Lactobacillus casei BL23 and Lactobacillus acidophilus ATCC4356 reduced the permeability of Hg(II) and MeHg from aqueous solutions through NCM460/HT29-MTX monolayers (20–94% reduction). However, assays using the bioaccessible (soluble) Hg fraction obtained by in vitro gastrointestinal digestion of Hg-contaminated swordfish only showed a reduction (42%) with the BL23 strain. In vivo experiments carried out in mice receiving an acute dose of Hg(II) or MeHg (0.5 mg/kg body weight/day) with or without lactobacilli resulted in significant decreases of the bioavailability of MeHg with both strains and increased excretion of Hg in feces after treatment with the lactobacilli. However, Hg(II) bioavailability or excretion was not affected. Hg accumulation in liver and kidney remained similar in LAB-treated or non-treated animals. This is the first study of the impact of LAB on Hg(II) and MeHg toxicokinetics and shows that some LAB strains have potential to diminish MeHg bioavailability. Furthermore, it has established the basis for new studies on the protective effect of LAB under conditions resembling subchronic and chronic Hg exposures., This work was supported by the Spanish Ministry of Science, Universities and Innovation (AGL2015-68920-R). Carlos Jadán Piedra received a Personnel Training Grant from SENESCYT (Ecuadorian Ministry of Higher Education, Science, Technology and Innovation) to carry out this study. Álvaro Crespo had a Contrato de Garantía Juvenil from the Spanish Government which was partially funded by the European Social Fund.

A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups.

This study evaluates the potential of strains of Lactobacillus and Saccharomyces cerevisiae to reduce inorganic arsenic [iAs, As(III)+As(V)] bioavailability. Removal of iAs from aqueous solutions by these strains was evaluated and their effect on iAs bioaccessibility was analyzed by in vitro gastrointestinal digestion. The effect of the strains on iAs absorption was also tested, using intestinal cells. The results showed that iAs removal by the yeast strains was low (, This work was supported by the former Spanish Ministry of Economy and Competitiveness through project AGL2015-68920-R, for which the authors are deeply indebted.

The synthesis of the inorganic polymer polyphosphate (poly-P) in bacteria has been linked to stress survival and to the capacity of some strains to sequester heavy metals. In addition, synthesis of poly-P by certain strains of probiotic lactobacilli has been evidenced as a probiotic mechanism due to the homeostatic properties of this compound at the intestinal epithelium. We analyzed the link between poly-P synthesis, stress response, and mercury toxicity/accumulation by comparing wild-type strains of

Human milk contains a high amount of diverse oligosaccharides (HMOs) that do not provide any nutritive value to the breast-fed infant. Much evidence suggests that HMOs are associated with the protecting effect of human milk against gastrointestinal infections, through its prebiotic effect, thus stimulating beneficial bacteria growth in the colon, but also through an antiadhesive effect against enteropathogens. HMOs have also been described as direct modulators of epithelial and immune cell responses, and as a source of sialic acid, which is potentially essential for brain development and cognitive functions. Evaluation of the impact of HMOs on infant health and understanding their role on host–microbe interactions have been limited by the lack of sufficient quantities of structurally defined HMOs to conduct animal or human feeding studies. In the past years, bioengineered microorganisms and enzymatic approaches to produce specific human milk-like oligosaccharides have been developed, which could envisage potential HMOs applications for the pharmaceutical and food industry.

Rotavirus (RV) and norovirus (NoV) are the major etiological agents of viral acute gastroenteritis worldwide. Host genetic factors, the histo-blood group antigens (HBGA), are associated with RV and NoV susceptibility and recent findings additionally point to HBGA as a factor modulating the intestinal microbial composition. In vitro and in vivo experiments in animal models established that the microbiota enhances RV and NoV infection, uncovering a triangular interplay between RV and NoV, host glycobiology, and the intestinal microbiota that ultimately influences viral infectivity. Studies on the microbiota composition in individuals displaying different RV and NoV susceptibilities allowed the identification of potential bacterial biomarkers, although mechanistic data on the virus–host–microbiota relation are still needed. The identification of the bacterial and HBGA interactions that are exploited by RV and NoV would place the intestinal microbiota as a new target for alternative therapies aimed at preventing and treating viral gastroenteritis., Work at the laboratories of Vicente Monedero and Jesús Rodríguez-Díaz was supported by projects AGL2015-68920-R and AGL2014-52996-C2-2-R respectively. Jesús Rodríguez-Díaz was, as well, recipient of a Ramón y Cajal grant (RYC-2013-12442).

The host microbiota has emerged a third player in interactions between hosts and viral pathogens. This opens new possibilities to use different tools to modulate the intestinal microbial composition, aimed at reducing the risk of or treating viral enteric infections., Work at laboratories of J.R-D. and V.M. was funded by Spanish projects (RYC-2013-12442, AGL2014-52996-C2-2-R, and AGL2015-68920-R, respectively). M.C.C. acknowledges European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme (ERC starting grant, n° 639226).

Fucosyl-N-acetylglucosamine disaccharides are important core structures that form part of human mucosal and milk glyco-complexes. We have previously shown that AlfB and AlfC α-L-fucosidases from Lactobacillus casei are able to synthesize fucosyl-α-1,3--N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucosyl-α-1,6-N-acetylglucosamine (Fuc-α1,6-GlcNAc), respectively, in transglycosylation reactions. Here, these reactions were performed in a semipreparative scale, and the produced disaccharides were purified. The maximum yields obtained of Fuc-α1,3-GlcNAc and Fuc-α1,6-GlcNAc were 4.2 and 9.3 g/l, respectively. The purified fucosyl-disaccharides were then analyzed for their prebiotic effect in vitro using strains from the Lactobacillus casei/paracasei/rhamnosus group and from Bifidobacterium species. The results revealed that 6 out of 11 L. casei strains and 2 out of 6 L. rhamnosus strains tested were able to ferment Fuc-α1,3-GlcNAc, and L. casei BL87 and L. rhamnosus BL327 strains were also able to ferment Fuc-α1,6-GlcNAc. DNA hybridization experiments suggested that the metabolism of Fuc-α1,3-GlcNAc in those strains relies in an α-L-fucosidase homologous to AlfB. Bifidobacterium breve and Bibidobacterium pseudocatenolatum species also metabolized Fuc-α1,3-GlcNAc. Notably, L-fucose was excreted from all the Lactobacillus and Bifidobacterium strains fermenting fucosyl-disaccharides, except from strains L. rhamnosus BL358 and BL377, indicating that in these latest strains, L-fucose was catabolized. The fucosyl-disaccharides were also tested for their inhibitory potential of pathogen adhesion to human colon adenocarcinoma epithelial (HT29) cell line. Enteropathogenic Escherichia coli (EPEC) strains isolated from infantile gastroenteritis were used, and the results showed that both fucosyl-disaccharides inhibited adhesion to different extents of certain EPEC strains to HT29 cells in tissue culture.

16 páginas, 7 figuras, 2 tablas, Microbial colonization of different environments is enabled to a great extent by the plasticity of their sensory mechanisms, among them, the two-component signal transduction systems (TCS). Here, an example of TCS plasticity is presented: the regulation of L-malate catabolism via malic enzyme by MaeRK in Lactobacillales. MaeKR belongs to the citrate family of TCS as the Escherichia coli DcuSR system. We show that the Lactobacillus casei histidine-kinase MaeK is defective in autophosphorylation activity as it lacks a functional catalytic and ATP binding domain. The cognate response regulator MaeR was poorly phosphorylated at its phosphoacceptor Asp in vitro. This phosphorylation, however, enhanced MaeR binding in vitro to its target sites and it was required for induction of regulated genes in vivo. Elucidation of the MaeR structure revealed that response regulator dimerization is accomplished by the swapping of α4-β5-α5 elements between two monomers, generating a phosphoacceptor competent conformation. Sequence and phylogenetic analyses showed that the MaeKR peculiarities are not exclusive to L. casei as they are shared by the rest of orthologous systems of Lactobacillales. Our results reveal MaeKR as a non-canonical TCS displaying distinctive features: a swapped response regulator and a sensor histidine kinase lacking ATP-dependent kinase activity., This study was financed by grants AGL2007–60975 and AGL2010–15679 from the former Spanish Ministry of Science and by grant ACOMP2012/137 from Generalitat Valenciana to V.M. and M.Z., by grants BIO2013–42619-P and BIO2016–78571-P from Spanish Ministry of Economy and Competitiveness and PrometeoII/2014/029 from the Valencian Government to A.M and by grant BFU2016–78606-P from the Spanish Ministry of Economy and Competitiveness to P.C. P.C. is the recipient of a Ramon y Cajal contract, and L.M.-R. is the recipient of a FPI fellow contract, both from the Ministry of Economy and Competitiveness. J.M.L. was the recipient of a Juan de la Cierva contract from the former Spanish Ministry of Science and Innovation. X-ray diffraction data collection was supported by Diamond Light Source block allocation group (BAG) Proposal MX14739 and Spanish Synchrotron Radiation Facility ALBA Proposal 2015071314 and 2016071762. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (grant agreement N°283570).

Two-component systems (TCSs) are widespread signal transduction pathways mainly found in bacteria where they play a major role in adaptation to changing environmental conditions. TCSs generally consist of sensor histidine kinases that autophosphorylate in response to a specific stimulus and subsequently transfer the phosphate group to their cognate response regulators thus modulating their activity, usually as transcriptional regulators. In this review we present the current knowledge on the physiological role of TCSs in species of the families Lactobacillaceae and Leuconostocaceae of the group of lactic acid bacteria (LAB). LAB are microorganisms of great relevance for health and food production as the group spans from starter organisms to pathogens. Whereas the role of TCSs in pathogenic LAB (most of them belonging to the family Streptococcaceae) has focused the attention, the roles of TCSs in commensal LAB, such as most species of Lactobacillaceae and Leuconostocaceae, have been somewhat neglected. However, evidence available indicates that TCSs are key players in the regulation of the physiology of these bacteria. The first studies in food-associated LAB showed the involvement of some TCSs in quorum sensing and production of bacteriocins, but subsequent studies have shown that TCSs participate in other physiological processes, such as stress response, regulation of nitrogen metabolism, regulation of malate metabolism, and resistance to antimicrobial peptides, among others.

Host genetic factors, such as histo-blood group antigens (HBGAs), are associated with susceptibility to norovirus (NoV) and rotavirus (RV) infections. Recent advances point to the gut microbiome as a key player necessary for a viral pathogen to cause infection. In vitro NoV attachment to host cells and resulting infections have been linked to interactions with certain bacterial types in the gut microbiota. We investigated the relationship between host genotype, gut microbiota, and viral infections. Saliva and fecal samples from 35 adult volunteers were analysed for secretor status genotype, the gut microbiota composition by 16S rRNA gene sequencing, and salivary IgA titers to NoV and RV. Higher levels of IgA against NoV and RV were related to secretor-positive status. No significant differences were found between the FUT2 genotype groups, although the multivariate analysis showed a significant impact of host genotype on specific viral susceptibilities in the microbiome composition. A specific link was found between the abundance of certain bacterial groups, such as Faecalibacterium and Ruminococcus spp., and lower IgA titers against NoV and RV. As a conclusion, we can state that there is a link between host genetics, gut microbiota, and susceptibility to viral infections in humans., This work was funded by Ministerio de Economía y Competitividad (MINECO) grants RYC-2013-12442 and AGL2014-52996-C2-2-R to JRD. SVV was recipient of a PhD scholarship from Generalitat Valenciana (Vali+d program, ACIF/2016/437).

Two-component systems (TCSs) are widespread signal transduction pathways mainly found in bacteria where they play a major role in adaptation to changing environmental conditions. TCSs generally consist of sensor histidine kinases that autophosphorylate in response to a specific stimulus and subsequently transfer the phosphate group to their cognate response regulators thus modulating their activity, usually as transcriptional regulators. In this review we present the current knowledge on the physiological role of TCSs in species of the families Lactobacillaceae and Leuconostocaceae of the group of lactic acid bacteria (LAB). LAB are microorganisms of great relevance for health and food production as the group spans from starter organisms to pathogens. Whereas the role of TCSs in pathogenic LAB (most of them belonging to the family Streptococcaceae) has focused the attention, the roles of TCSs in commensal LAB, such as most species of Lactobacillaceae and Leuconostocaceae, have been somewhat neglected. However, evidence available indicates that TCSs are key players in the regulation of the physiology of these bacteria. The first studies in food-associated LAB showed the involvement of some TCSs in quorum sensing and production of bacteriocins, but subsequent studies have shown that TCSs participate in other physiological processes, such as stress response, regulation of nitrogen metabolism, regulation of malate metabolism, and resistance to antimicrobial peptides, among others.

Summary The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

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 PTS(Gnb) . 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.

Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus , a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus , 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase ( ppk ) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk -disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.

Metal sequestration by bacteria has been proposed as a strategy to counteract metal contamination in foodstuffs. Lactobacilli can interact with metals, although studies with important foodborne metals such as inorganic [Hg(II)] or organic (CHLactobacillus strains efficiently bound Hg(II) and CHThis is the first example of the biosorption of Hg(II) and CH