Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

Arabinoxylan oligosaccharides (AXOS) are a promising class of prebiotics that have the potential to stimulate the growth of bifidobacteria and the production of butyrate in the human colon, known as the bifidogenic and butyrogenic effects, respectively. Although these dual effects of AXOS are considered beneficial for human health, their underlying mechanisms are still far from being understood. Therefore, this study investigated the metabolic interactions between Bifidobacterium longum subsp. longum NCC2705 (B. longum NCC2705), an acetate producer and arabinose substituent degrader of AXOS, and Eubacterium rectale ATCC 33656, an acetate-converting butyrate producer. Both strains belong to prevalent species of the human colon microbiota. The strains were grown on AXOS during mono- and coculture fermentations, and their growth, AXOS consumption, metabolite production, and expression of key genes were monitored. The results showed that the growth of both strains and gene expression in both strains were affected by cocultivation and that these effects could be linked to changes in carbohydrate consumption and concomitant metabolite production. The consumption of the arabinose substituents of AXOS by B. longum NCC2705 with the concomitant production of acetate allowed E. rectale ATCC 33656 to produce butyrate (by means of a butyryl coenzyme A [CoA]:acetate CoA-transferase), explaining the butyrogenic effect of AXOS. Eubacterium rectale ATCC 33656 released xylose from the AXOS substrate, which favored the B. longum NCC2705 production of acetate, explaining the bifidogenic effect of AXOS. Hence, those interactions represent mutual cross-feeding mechanisms that favor the coexistence of bifidobacterial strains and butyrate producers in the same ecological niche. In conclusion, this study provides new insights into the bifidogenic and butyrogenic effects of AXOS.     

Resistance to Simulated Gastrointestinal Conditions and Adhesion to Mucus as Probiotic Criteria for Bifidobacterium longum Strains

Eight Bifidobacterium longum strains, including reported probiotic strains (commercial and noncommercial), collection strains, and laboratory isolates, were investigated for their ability to adhere to mucin as well as their ability to tolerate acid and bile. Strains could be discriminated based on their sensitivity at pH values of 2.0 to 2.5 and bile concentrations of 0.5% to 2.0%. B. longum NCC 2705, a strain known for its probiotic properties, showed the highest resistance to gastrointestinal conditions, whereas the commercial probiotic strains B. longum BB 536 and SP 07/3 were the least resistant. In parallel, the human isolate B. longum BIF 53 showed the highest adhesion to mucin, whereas the commercial probiotic strains B. longum W 11, BB 536, and SP 07/3 were the least adhesive. The bacterial adhesion to mucin of strains B. longum NCC 2705 and BIF 53 could be reduced by lysozyme, indicating that cell-wall components are involved in the adhesion process. These results showed that there is no obvious link between adhesion and resistance to gastrointestinal conditions and the probiotic status of the studied strains. This calls for a definition of conditions for in vitro tests that better predict the in vivo functionality of probiotic strains.     

Intrinsic and inducible resistance to hydrogen peroxide in <Emphasis Type="Italic">Bifidobacterium</Emphasis> species

Interest in, and use of, bifidobacteria as a probiotic delivered in functional foods has increased dramatically in recent years. As a result of their anaerobic nature, oxidative stress can pose a major challenge to maintaining viability of bifidobacteria during functional food storage. To better understand the oxidative stress response in two industrially important bifidobacteria species, we examined the response of three strains of B. longum and three strains of B. animalis subsp. lactis to hydrogen peroxide (H₂O₂). Each strain was exposed to a range of H₂O₂ concentrations (0–10 mM) to evaluate and compare intrinsic resistance to H₂O₂. Next, strains were tested for the presence of an inducible oxidative stress response by exposure to a sublethal H₂O₂ concentration for 20 or 60 min followed by challenge at a lethal H₂O₂ concentration. Results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H₂O₂ resistance of all strains tested and B. animalis subsp. lactis BL-04 had the highest resistance among B. lactis strains. Inducible H₂O₂ resistance was detected in four strains, B. longum NCC2705, B. longum D2957, B. lactis RH-1, and B. lactis BL-04. Other strains showed either no difference or increased sensitivity to H₂O₂ after induction treatments. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific in B. longum and B. lactis and suggest that for some strains, sublethal H₂O₂ treatments might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.     

Gnotobiotic Mouse Immune Response Induced by Bifidobacterium sp. Strains Isolated from Infants

Bifidobacterium, which is a dominant genus in infants’ fecal flora and can be used as a probiotic, has shown beneficial effects in various pathologies, including allergic diseases, but its role in immunity has so far been little known. Numerous studies have shown the crucial role of the initial intestinal colonization in the development of the intestinal immune system, and bifidobacteria could play a major role in this process. For a better understanding of the effect of Bifidobacterium on the immune system, we aimed at determining the impact of Bifidobacterium on the T-helper 1 (T_H1)/T_H2 balance by using gnotobiotic mice. Germfree mice were inoculated with Bifidobacterium longum NCC2705, whose genome is sequenced, and with nine Bifidobacterium strains isolated from infants’ fecal flora. Five days after inoculation, mice were killed. Transforming growth factor β1 (TGF-β1), interleukin-4 (IL-4), IL-10, and gamma interferon (IFN-γ) gene expressions in the ileum and IFN-γ, tumor necrosis factor alpha (TNF-α), IL-10, IL-4, and IL-5 secretions by splenocytes cultivated for 48 h with concanavalin A were quantified. Two Bifidobacterium species had no effect (B. adolescentis) or little effect (B. breve) on the immune system. Bifidobacterium bifidum, Bifidobacterium dentium, and one B. longum strain induced T_H1 and T_H2 cytokines at the systemic and intestinal levels. One B. longum strain induced a T_H2 orientation with high levels of IL-4 and IL-10, both secreted by splenocytes, and of TGF-β gene expression in the ileum. The other two strains induced T_H1 orientations with high levels of IFN-γ and TNF-α splenocyte secretions. Bifidobacterium's capacity to stimulate immunity is species specific, but its influence on the orientation of the immune system is strain specific.     

The role of Acanthamoeba castellanii (T4 genotype) antioxidant enzymes in parasite survival under H2O2-induced oxidative stress

Acanthamoeba castellanii (A. castellanii) is an important opportunistic parasite. Induction of oxidative stress by the host immune system is one of the most important defense strategies against parasites. Hence, parasites partly deal with oxidative stress by different mechanisms. Identifying resistance mechanisms of A. castellanii parasites against oxidative stress is important to achieve a new therapeutic approach. Thus, this study aimed to understand the resistance mechanisms of A. castellanii, against oxidative stress. Trophozoites of A. castellanii were treated with different concentrations of H₂O₂. The half maximal inhibitory concentration (IC₅₀) of H₂O₂ was determined using the MTT assay. The induction of oxidative stress was confirmed by flow cytometer. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) were determined. The gene expression levels of CAT and SOD were measured by qRT-PCR. Furthermore, 3-amino-1:2:4-triazole (3-AT) and potassium cyanide (KCN) were used as specific inhibitors of CAT and SOD, respectively. Cell cycle assay and the apoptosis were evaluated by flow cytometer. The activities of SOD, CAT, GR, and GPx, showed an increase in oxidative stress. The cell cycle analysis revealed that most of the cellular population was in G0 and G1 phases. The apoptosis increased in oxidative stress conditions. Moreover, the apoptosis significantly increased after the specific inhibition of CAT and SOD under oxidative stress. The gene expression levels of CAT and SOD significantly increased under oxidative stress. A. castellanii can resist the host immune system through various mechanisms, including evoking its antioxidant enzymes. Therefore, by reducing or inhibiting the activity of the parasite's antioxidant enzymes such as SOD and CAT, it is possible to cope with A. castellanii.     

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

Background  

Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H₂O₂ from 0 to 130 ppm.     

SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion

Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O₂⁻) into oxygen and hydrogen peroxide. It is a Cu²⁺/Zn²⁺ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.     

Prevention of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infection in gnotobiotic mice associated with <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.     

Overexpression of SOD in retina: need for increase in H2O2-detoxifying enzyme in same cellular compartment

In retinitis pigmentosa (RP), various mutations cause rod photoreceptor cell death leading to increased oxygen levels in the outer retina, progressive oxidative damage to cones, and gradual loss of cone cell function. We have been exploring the potential of overexpressing components of the endogenous antioxidant defense system to preserve cone cell function in rd10^+/+ mice, a model of RP. rd10^+/+ mice deficient in superoxide dismutase 1 (SOD1) showed increased levels of superoxide radicals and carbonyl adducts (a marker of oxidative damage) in the retina and more rapid loss of cone function than rd10^+/+ mice with normal levels of SOD1. This suggests that SOD1 is an important component of the antioxidant defense system of cones, but increased expression of SOD1 in rd10^+/+ mice increased oxidative damage and accelerated the loss of cone function. Coexpression of SOD1 with glutathione peroxidase 4 (Gpx4), which like SOD1 is localized in the cytoplasm, but not with catalase targeted to the mitochondria, reduced oxidative damage in the retina and significantly slowed the loss of cone cell function in rd10^+/+ mice. Gene transfer resulting in increased expression of SOD2, but not coexpression of SOD2 and mitochondrial Gpx4, resulted in high levels of H₂O₂ in the retina. These data suggest that to provide benefit in RP, overexpression of an SOD must be combined with expression of a peroxide-detoxifying enzyme in the same cellular compartment.     

长双歧杆菌NCC2705菌株应激蛋白的研究

研究长双歧杆菌NCC2705菌株发酵至稳定期时应激蛋白的表达情况。根据乳酸乳球菌IL1403菌株蛋白质参考图谱及长双歧杆菌NCC2705基因组注释中应激蛋白的分子量与等电点,确定应激蛋白在双向电泳凝胶上的相应蛋白点,并利用MALDI-TOF和/或ESI-MS/MS对相应蛋白质点进行鉴定。每个蛋白质点的肽指纹图谱均在长双歧杆菌NCC2705的蛋白质数据库用Mascot进行检索,共鉴定到44个蛋白点对应8个应激蛋白。这些蛋白为亲水性酸性蛋白,大多具有翻译后修饰现象,它们基因的CAI值除DnaJ外,其余均在0.5以上,在全细胞表达谱中为高丰度蛋白;此外,菌体具有较强的抗脂质过氧化和清除DPPH自由基的能力,而对羟自由基和超氧负离子的清除力较弱,推测鉴定到的具有逆转氧化损害作用的碱性过氧化氢还原酶(ahpC)可能是体内表达的降低氧损伤的主要酶。     

Proteomic comparison of the cytosolic proteins of three <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> human isolates and <Emphasis Type="Italic">B. longum</Emphasis> NCC2705

Background  

Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels.     

Correlation between oxidative stress and G6PD activity in neonatal jaundice

Fetal distress represents a pathophysiological condition in which oxygen is not available to the fetus in sufficient quantities. In cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency, under conditions of oxidative stress, the residual G6PD and complimentary antioxidant mechanisms may become insufficient to neutralize the large amounts of ROS and to prevent severe hemolysis. Alteration in the oxidant–antioxidant profile is also known to occur in neonatal jaundice. The study group included 22 neonates presented with fetal distress during labor and 24 neonates with no evidence of fetal distress (control group). Umbilical cord blood samples were taken immediately after delivery, and the following blood tests were carried out after birth and at discharge from the hospital: erythrocyte count, total bilirubin, G6PD activity, and parameters presenting oxidative status [thiobarbituric acid reactive substances (TBARS), NO, O₂ ^?, H₂O₂, SOD, CAT, O₂ ^?/SOD, and H₂O₂/CAT]. There were no significant differences in TBARS and NO values among neonates with or without fetal distress. However, the values of O₂ ^?, H₂O₂, SOD, O₂ ^?/SOD, and H₂O₂/CAT among neonates born after fetal distress were significantly higher than in neonates without fetal distress (p < 0.01). In neonates with fetal distress, the total number of RBCs at delivery was significantly lower, accompanied with higher bilirubin content. Also neonates with fetal distress had lower activity of G6PD and lower CAT activity. Higher values of oxidative stress parameters in newborns delivered after fetal distress do not indicate strictly what occurred first—oxidative stress or basic lower G6PD activity.     

Genometrics as an essential tool for the assembly of whole genome sequences: the example of the chromosome of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> NCC2705

Background  

Analysis of the first reported complete genome sequence ofBifidobacterium longumNCC2705, an actinobacterium colonizing the gastrointestinal tract, uncovered its proteomic relatedness toStreptomyces coelicolorandMycobacterium tuberculosis. However, a rapid scrutiny by genometric methods revealed a genome organization totally different from all so far sequenced high-GC Gram-positive chromosomes.     

Complete Genome Sequence of Bifidobacterium longum JDM301

Bifidobacteria, known as probiotic bacteria, are high-G+C Gram-positive bacteria which naturally inhabit the human gastrointestinal tract and vagina. Recently, we completely sequenced Bifidobacterium longum JDM301, which is a widely used Chinese commercial strain with several probiotic properties.Bifidobacterium spp., which are considered model probiotic bacteria like Lactobacillus spp., play an important role in the stability of the intestinal microflora, the modulation of the immune response, and so on (6, 8). We determined the complete genome sequence of B. longum JDM301, which is commercially used in China as a probiotic strain, using the GS 20 system (454 Life Science Corporation) (7). A total of 192,888 reads with an average length of 210 bp were assembled into 112 contigs by the 454 assembly tool. Among these, 92 large contigs were larger than 500 bp. We determined the order of the largest contigs through BLAST analysis with the reference strain B. longum ATCC 15697 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP001095","term_id":"213522389","term_text":"CP001095"}}CP001095) (10) and arranged the others by multiplex PCR. Gaps were closed by sequencing gap-spanning PCR products or clones using ABI 3730 xl DNA sequencers. Primer design and sequence assembly were performed with the Phred/Phrap/Consed software package (2, 3).The complete genome of B. longum JDM301 is composed of a 59.8% G+C circular chromosome of 2,477,838 bp without any plasmid. The genome of JDM301 (2.48 Mb) is smaller than that of B. longum ATCC 15697 (2.83 Mb) and slightly larger than the complete genomes of B. longum NCC2705 (2.26 Mb; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AE014295","term_id":"58012118","term_text":"AE014295"}}AE014295) and DJO10A (2.38 Mb; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000605","term_id":"189427298","term_text":"CP000605"}}CP000605). The JDM301 genome contains 1,959 protein-coding genes, three rRNA operons, and 55 tRNA genes. There are four rRNA operons with two cascaded in the three reference B. longum strains, while there are three in JDM301. We resequenced the three regions containing rRNA operons in the genome of JDM301 and found no cascaded rRNA operon. However, the locations of the three rRNA operons in JDM301 are the same as in the other three B. longum strains. No complete prophages were found in the genome sequence, but 12 phage-related fragments were identified. The genome also contains 15 pseudogenes, which is evidence of the recent and ongoing genome reduction of lactic acid bacilli (9). In addition, 15 complete or disrupted insertion sequence (IS) elements were found in the entire genome, which were identified as 5 derivatives of ISBlo5, 3 complete copies of ISBlo3, and 7 other ISs or derivatives belonging to the IS256, IS3, IS21, and IS30 family elements, respectively. One of them, named ISBad1, was 98.08% identical to that of B. adolescentis, and the others have been reported in the B. longum NCC2705 genome (8).Genome analysis revealed 14 response regulators and 14 sensor histidine kinases throughout the JDM301 chromosome, which may suggest a less complex regulatory network in JDM301 than that in ATCC 15697 (10). JDM301 has been grown as a commercial strain in stable and rich nutritional medium for such a long time that the regulatory networks in the genome may degenerate slowly, which is required for responses to dynamic environmental cues in the gastrointestinal tract.A gene (for BLJ_1359) encoding a serpin (serine protease inhibitor) with 92.69% identity to that of NCC2705 (4) was found in the genome sequence. The serpin is a potential probiotic effector molecule, and it may contribute to the immunomodulation of this B. longum strain. As we know, modulation of the immune system is one of probiotic functions of lactic acid bacteria (5).As model probiotic bacteria, Bifidobacterium spp. attract more and more interest since the molecular mechanisms of their probiotic activities remain unclear to a large degree (11). On the other hand, the biosafety of probiotics has attracted more attention with their enlarged and wide applications in food and drug products (1). We believe that this work will bring us deeper insight into the probiotic activities and safety of the strain widely used in China.     

Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.