Ppx1 putative exopolyphosphatase is essential for polyphosphate accumulation in Lacticaseibacillus paracasei.

The linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In many Lactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1 and ppx2) each having different domain compositions, with the gene order ppx1-ppk-ppx2. However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans. Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis in Lc. paracasei and have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis.IMPORTANCEPoly-P is a pivotal molecular player in bacteria, participating in a diverse array of processes ranging from stress resilience to pathogenesis while also serving as a functional component in probiotic bacteria. The synthesis of poly-P is tightly regulated, but the underlying mechanisms remain incompletely elucidated. Our study sheds light on the distinctive role played by the two exopolyphosphatases (Ppx) found in the Lactobacillaceae bacterial group, of relevance in food and health. This particular group is noteworthy for possessing two Ppx enzymes, supposedly involved in poly-P degradation. Remarkably, our investigation uncovers an unprecedented function of Ppx1 in Lacticaseibacillus paracasei, where its absence leads to the total cessation of poly-P synthesis, paralleling the impact observed upon eliminating the poly-P forming enzyme, poly-P kinase. Unlike the anticipated role as a conventional exopolyphosphatase, Ppx1 demonstrates an unexpected function. Our results added a layer of complexity to our understanding of poly-P dynamics in bacteria.

Phosphate Uptake and Its Relation to Arsenic Toxicity in Lactobacilli.

The use of probiotic lactobacilli has been proposed as a strategy to mitigate damage associated with exposure to toxic metals. Their protective effect against cationic metal ions, such as those of mercury or lead, is believed to stem from their chelating and accumulating potential. However, their retention of anionic toxic metalloids, such as inorganic arsenic, is generally low. Through the construction of mutants in phosphate transporter genes (pst) in Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains, coupled with arsenate [As(V)] uptake and toxicity assays, we determined that the incorporation of As(V), which structurally resembles phosphate, is likely facilitated by phosphate transporters. Surprisingly, inactivation in Lc. paracasei of PhoP, the transcriptional regulator of the two-component system PhoPR, a signal transducer involved in phosphate sensing, led to an increased resistance to arsenite [As(III)]. In comparison to the wild type, the phoP strain exhibited no differences in the ability to retain As(III), and there were no observed changes in the oxidation of As(III) to the less toxic As(V). These results reinforce the idea that specific transport, and not unspecific cell retention, plays a role in As(V) biosorption by lactobacilli, while they reveal an unexpected phenotype for the lack of the pleiotropic regulator PhoP.

Impact of Chronic Exposure to Arsenate through Drinking Water on the Intestinal Barrier.

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 noncarcinogenic pathologies. Although the oral pathway is the main source of exposure, in vivo studies conducted to verify the intestinal toxicity of this metalloid are scarce and are mainly focused on evaluating the toxicity of As(III). The aim of this study was to evaluate the effect of chronic exposure (6 months) of BALB/c mice to As(V) (15-60 mg/L) via drinking water on the different components of the intestinal barrier and to determine the possible mechanisms involved. The results show that chronic exposure to As(V) generates a situation of oxidative stress (increased lipid peroxidation and reactive species) and inflammation (increased contents of several proinflammatory cytokines and neutrophil infiltrations) in the intestinal tissues. There is also evidence of an altered expression of constituent proteins of the intercellular junctions (Cldn1, Cldn3, and Ocln) and the mucus layer (Muc2) and changes in the composition of the gut microbiota and the metabolism of short-chain fatty acids. All of these toxic effects eventually may lead to the disruption of the intestinal barrier, which shows an increased paracellular permeability. Moreover, signs of endotoxemia are observed in the serum of As(V)-treated animals (increases in lipopolysaccharide-binding protein LBP and the proinflammatory cytokine IL-1ß). The data obtained suggest that chronic exposure to As(V) via drinking water affects the intestinal environment.

Culture of Human Rotaviruses in Relevant Models Shows Differences in Culture-Adapted and Nonculture-Adapted Strains.

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo-blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models.

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion.

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1-3 months) or an auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators.

Complex Oligosaccharide Utilization Pathways in Lactobacillus.

Lactobacillus is the bacterial genus that contains the highest number of characterized probiotics. Lactobacilli in general can utilize a great variety of carbohydrates. This characteristic is an essential trait for their survival in highly competitive environments such as the gastrointestinal tract of animals. In particular, the ability of some strains to utilize complex carbohydrates such as milk oligosaccharides as well as their precursor monosaccharides, confer upon lactobacilli a competitive advantage. For this reason, many of these carbohydrates are considered as prebiotics. Genome sequencing of many lactobacilli strains has revealed a great variety of genes involved in the metabolism of carbohydrates and some of them have already been characterized. In this review, the current knowledge at biochemical and genetic levels on the catabolic pathways of complex carbohydrates utilized by lactobacilli will be summarized.

Unraveling the role of the secretor antigen in human rotavirus attachment to histo-blood group antigens.

Rotavirus is the leading agent causing acute gastroenteritis in young children, with the P[8] genotype accounting for more than 80% of infections in humans. The molecular bases for binding of the VP8* domain from P[8] VP4 spike protein to its cellular receptor, the secretory H type-1 antigen (Fuc-α1,2-Gal-ß1,3-GlcNAc; H1), and to its precursor lacto-N-biose (Gal-ß1,3-GlcNAc; LNB) have been determined. The resolution of P[8] VP8* crystal structures in complex with H1 antigen and LNB and site-directed mutagenesis experiments revealed that both glycans bind to the P[8] VP8* protein through a binding pocket shared with other members of the P[II] genogroup (i.e.: P[4], P[6] and P[19]). Our results show that the L-fucose moiety from H1 only displays indirect contacts with P[8] VP8*. However, the induced conformational changes in the LNB moiety increase the ligand affinity by two-fold, as measured by surface plasmon resonance (SPR), providing a molecular explanation for the different susceptibility to rotavirus infection between secretor and non-secretor individuals. The unexpected interaction of P[8] VP8* with LNB, a building block of type-1 human milk oligosaccharides, resulted in inhibition of rotavirus infection, highlighting the role and possible application of this disaccharide as an antiviral. While key amino acids in the H1/LNB binding pocket were highly conserved in members of the P[II] genogroup, differences were found in ligand affinities among distinct P[8] genetic lineages. The variation in affinities were explained by subtle structural differences induced by amino acid changes in the vicinity of the binding pocket, providing a fine-tuning mechanism for glycan binding in P[8] rotavirus.

The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update.

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.

Interaction of Intestinal Bacteria with Human Rotavirus during Infection in Children.

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.

Use of lactic acid bacteria and yeasts to reduce exposure to chemical food contaminants and toxicity.

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.

In vivo evaluation of the effect of arsenite on the intestinal epithelium and associated microbiota in mice.

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-1ß, 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.

Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei.

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.

Physiological Role of Two-Component Signal Transduction Systems in Food-Associated Lactic Acid Bacteria.

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.

Human milk and mucosal lacto- and galacto-N-biose synthesis by transgalactosylation and their prebiotic potential in Lactobacillus species.

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-ß-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-ß-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 ß1,3-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-ß-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.

Characterization of the binding capacity of mercurial species in Lactobacillus strains.

BACKGROUND: 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.

A unique gene cluster for the utilization of the mucosal and human milk-associated glycans galacto-N-biose and lacto-N-biose in Lactobacillus casei.

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-ß-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.

An L-Fucose Operon in the Probiotic Lactobacillus rhamnosus GG Is Involved in Adaptation to Gastrointestinal Conditions.

L-Fucose is a sugar present in human secretions as part of human milk oligosaccharides, mucins, and other glycoconjugates in the intestinal epithelium. The genome of the probiotic Lactobacillus rhamnosus GG (LGG) carries a gene cluster encoding a putative L-fucose permease (fucP), L-fucose catabolic pathway (fucI, fucK, fucU, and fucA), and a transcriptional regulator (fucR). The metabolism of L-fucose in LGG results in 1,2-propanediol production, and their fucI and fucP mutants displayed a severe and mild growth defect on L-fucose, respectively. Transcriptional analysis revealed that the fuc genes are induced by L-fucose and subject to a strong carbon catabolite repression effect. This induction was triggered by FucR, which acted as a transcriptional activator necessary for growth on L-fucose. LGG utilized fucosyl-α1,3-N-acetylglucosamine and contrarily to other lactobacilli, the presence of fuc genes allowed this strain to use the L-fucose moiety. In fucI and fucR mutants, but not in fucP mutant, L-fucose was not metabolized and it was excreted to the medium during growth on fucosyl-α1,3-N-acetylglucosamine. The fuc genes were induced by this fucosyl-disaccharide in the wild type and the fucP mutant but not in a fucI mutant, showing that FucP does not participate in the regulation of fuc genes and that L-fucose metabolism is needed for FucR activation. The l-fucose operon characterized here constitutes a new example of the many factors found in LGG that allow this strain to adapt to the gastrointestinal conditions.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

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.

Preparative scale purification of fucosyl-N-acetylglucosamine disaccharides and their evaluation as potential prebiotics and antiadhesins.

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.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.