Evaluation of the ability of Bifidobacterium longum to metabolize human intestinal mucus

The ability of Bifidobacterium longum to use intestinal mucus as a metabolizable source was characterized. Bifidobacterium longum biotype longum NCIMB8809 was grown in a chemically semi-defined medium supplemented with human intestinal mucus, and the cytoplasmic protein profiles and several glycosyl hydrolase activities were analysed and compared with those obtained from the same bacterium grown in the absence of mucus. We were able to identify 22 different proteins in the cytoplasmic fraction, of which nine displayed a different concentration in the presence of mucus. Among the proteins whose concentrations varied, we found specific enzymes that are involved in the response to different environmental conditions, and also proteins that mediate interaction with mucus in bacteria. Significant changes in some glycoside-hydrolysing activities were also detected. In addition, stable isotope labelling of amino acids in cell culture demonstrated that B. longum incorporates leucine from the glycoprotein matrix of mucin within its proteins. This study provides the first proteomic data regarding the interaction of B. longum with intestinal mucus, and contributes to the understanding of the behaviour of this intestinal species in its natural ecological niche.     

Analysis of host-inducing proteome changes in bifidobacterium longum NCC2705 grown in Vivo

To investigate the molecular mechanisms underlying the adaptation of Bifidobacterium longum to the intestinal tract, we utilized a new model for rabbit intestinal culture of B. longum and reported the changes in proteomic profiles after incubation in the in vivo environment. By 2D-PAGE coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analyses, proteomic profiles of B. longum strain NCC2705 grown in the in vivo and in vitro environments were compared. Confirmed by semiquantitative RT-PCR, which exhibited at least a 3-fold change or greater, 19 up-regulated proteins, 14 down-regulated proteins, and 4 proteins with mobility changes were identified during intestinal growth. These identified proteins include key stress proteins, metabolism-related proteins, and proteins related to translation. Our results indicate that some useful proteins are expressed at higher levels in cells during intestinal growth. These proteins reflected the adaptation of B. longum NCC2705 to the intestine, such as EF-Tu which contributes to the retention or attachment as a Bifidobacterium adhesin-like factor, bile salt hydrolase (BSH) which might play an important role in the molecular mechanisms for the initial interaction of probiotic with the intestinal environment, and stress proteins which defend B. longum against the action of bile salts and other harmful ingredients of the gastrointestinal tract (GIT). The most striking fact of our observation was that four proteins GlnA1, PurC, LuxS, and Pgk exhibit clear post-translational modification. Western blot (WB) analysis and Pro-Q Diamond staining revealed that substances of the GIT trigger Pgk and LuxS phosphorylation at Ser/Thr residues for bacteria grown in vivo. These proteins were identified for the first time as bifidobacterial phosphoproteins. Our data suggest that the phosphorylated autoinducer-2 production protein LuxS of B. longum NCC2705 (LuxS-P) is the active form of LuxS and that LuxS-P may play a key role in the regulation of quorum sensing.     

Molecular characterization of three plasmids from Bifidobacterium longum

The complete nucleotide sequences for pNAC1 (3538bp) from strain RW048 as well as for pNAC2 (3684bp) and pNAC3 (10,224bp) from strain RW041 of Bifidobacterium longum were determined. The largest ORF (repB) of pNAC1 encodes a putative protein similar to those involved in a rolling-circle (RC) replication mechanism, which was confirmed by demonstration of single-strand intermediates in the host cell. The putative RepB gene product of pNAC2 is most similar to the replication protein of pDOJH10L and pKJ36. A second gene (mob) is similar to mobilization proteins involved in conjugation. Plasmid pNAC3 is the largest bifidobacterial plasmid to be sequenced to date. Of the eight putative gene products coded by pNAC3, one is similar to replication proteins (RepB), and another (Orf2) to putative transfer proteins (Tra). Bifidobacterial plasmids were divided into five groups based on Rep amino acid sequence homology and the results suggest a new plasmid family for B. longum.     

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。     

Cloning and expression in Escherichia coli of a gene,hup, encoding the histone-like protein HU of Bifidobacterium longum

A genomic DNA library of Bifidobacterium longum ATCC15707 was transfected into an Escherichia coli strain deficient in both HU and IHF, the growth of which is cold-sensitive because of the deficiency in these proteins. Cold-resistant colonies were selected and the DNA was cloned and sequenced. A polypeptide consisted of 93 amino acids, a predicted molecular mass of 9983 Da with an isoelectric point of 10.35, was deduced from an orf in the middle of the DNA fragment. The amino acid sequence was highly similar to HU family proteins, and 26 aas of N terminal was identical to a histone-like protein, HBI, a HU family protein of B. longum. Incapabilities of Mu phage propagation in an E. coli mutant deficient in HU or IHF could be suppressed by DNA bearing this orf. These results showed that the orf is a gene hup encoding HBI, a histone-like protein HU of B. longum.     

Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.     

Bifidobacterium longum CECT 7347 modulates immune responses in a gliadin-induced enteropathy animal model

Coeliac disease (CD) is an autoimmune disorder triggered by gluten proteins (gliadin) that involves innate and adaptive immunity. In this study, we hypothesise that the administration of Bifidobacterium longum CECT 7347, previously selected for reducing gliadin immunotoxic effects in vitro, could exert protective effects in an animal model of gliadin-induced enteropathy. The effects of this bacterium were evaluated in newborn rats fed gliadin alone or sensitised with interferon (IFN)-γ and fed gliadin. Jejunal tissue sections were collected for histological, NFκB mRNA expression and cytokine production analyses. Leukocyte populations and T-cell subsets were analysed in peripheral blood samples. The possible translocation of the bacterium to different organs was determined by plate counting and the composition of the colonic microbiota was quantified by real-time PCR. Feeding gliadin alone reduced enterocyte height and peripheral CD4+ cells, but increased CD4+/Foxp3+ T and CD8+ cells, while the simultaneous administration of B. longum CECT 7347 exerted opposite effects. Animals sensitised with IFN-γ and fed gliadin showed high cellular infiltration, reduced villi width and enterocyte height. Sensitised animals also exhibited increased NFκB mRNA expression and TNF-α production in tissue sections. B. longum CECT 7347 administration increased NFκB expression and IL-10, but reduced TNF-α, production in the enteropathy model. In sensitised gliadin-fed animals, CD4+, CD4+/Foxp3+ and CD8+ T cells increased, whereas the administration of B. longum CECT 7347 reduced CD4+ and CD4+/Foxp3+ cell populations and increased CD8+ T cell populations. The bifidobacterial strain administered represented between 75-95% of the total bifidobacteria isolated from all treated groups, and translocation to organs was not detected. These findings indicate that B. longum attenuates the production of inflammatory cytokines and the CD4+ T-cell mediated immune response in an animal model of gliadin-induced enteropathy.     

The antimutagenic properties of a polysaccharide produced by Bifidobacterium longum and its cultured milk against some heterocyclic amines

The antimutagenicity and fermentation pattern of three Bifidobacterium longum strains (B. longum, B. longum PS+, and B. longum PS-) in skim milk were studied. The increase in fermentation time significantly increased antimutagenicity with all strains tested against the mutagenicity of both 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in an Ames-like test using streptomycin-dependent strain SD510 of Salmonella typhimurium TA98. Bifidobacterium longum PS+, a polysaccharide-producing strain, had a longer lag phase but showed the highest inhibition percentage against both mutagens tested. The viability of B. longum PS+ cells was not affected by the low pH of 4.1, probably owing to the protection offered by the polysaccharide produced. The antimutagenicity of the fermented milk against Trp-P-1 was dose dependent. The strains were also able to bind with different amino acid pyrolysates, and B. longum showed the highest binding. Acetone extracts of fermented skim milk dissolved in water showed less antimutagenicity than extracts dissolved in dimethylsulfoxide. The isolated crude polysaccharide from B. longum PS+ showed a dose-dependent inhibition of the mutagenicity of Trp-P-1. Thus, we conclude that the polysaccharide of B. longum PS+ can be used as an antimutagen.     

Complete genome sequence of Bifidobacterium longum subsp. longum BBMN68, a new strain from a healthy chinese centenarian

Bifidobacterium longum subsp. longum BBMN68 was isolated from the feces of a healthy centenarian living in an area of BaMa, Guangxi, China, known for longevity. Here we report the main genome features of B. longum strain BBMN68 and the identification of several predicted proteins associated with the ecological niche of longevity.     

Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.     

Cross-feeding between Bifidobacterium longum BB536 and acetate-converting, butyrate-producing colon bacteria during growth on oligofructose

In vitro coculture fermentations of Bifidobacterium longum BB536 and two acetate-converting, butyrate-producing colon bacteria, Anaerostipes caccae DSM 14662 and Roseburia intestinalis DSM 14610, with oligofructose as the sole energy source, were performed to study interspecies interactions. Two clearly distinct types of cross-feeding were identified. A. caccae DSM 14662 was not able to degrade oligofructose but could grow on the fructose released by B. longum BB536 during oligofructose breakdown. R. intestinalis DSM 14610 could degrade oligofructose, but only after acetate was added to the medium. Detailed kinetic analyses of oligofructose breakdown by the last strain revealed simultaneous degradation of the different chain length fractions, in contrast with the preferential degradation of shorter fractions by B. longum BB536. In a coculture of both strains, initial oligofructose degradation and acetate production by B. longum BB536 took place, which in turn also allowed oligofructose breakdown by R. intestinalis DSM 14610. These and similar cross-feeding mechanisms could play a role in the colon ecosystem and contribute to the combined bifidogenic/butyrogenic effect observed after addition of inulin-type fructans to the diet.     

Isolation and characterization of two plasmids from Bifidobacterium longum

In order to develop a cloning vector system which can be used in Bifidobacterium sp., we screened about 100 bifidobacteria from the faeces of adults and children. Among them, only one strain, identified as B. longum KJ, was shown to contain extrachromosomal DNAs. Bifidobacterium longum KJ showed multiple plasmid DNA bands which were resolved to be multimers of two plasmids designated pKJ36 and pKJ50. These plasmids were cloned into the Escherichia coli vector pUC19 as pMS36 and pMS50, respectively, and restriction-mapped.     

Safety assessment of potential lactic acid bacteria Bifidobacterium longum SPM1205 isolated from healthy Koreans

The safety assessment of Bifidobacterium longum SPM1205 isolated from healthy Koreans and this strain's inhibitory effects on fecal harmful enzymes of intestinal microflora were investigated. The overall safety of this strain was investigated during a feeding trial. Groups of SD rats were orally administered a test strain or commercial reference strain B. longum 1 x 10(9) CFU/kg body weight/day for four weeks. Throughout this time, their feed intake, water intake and live body weight were monitored. Fecal samples were periodically collected to test harmful enzyme activities of intestinal microflora. At the end of the four-week observation period, samples of blood, liver, spleen, kidney, and gut tissues were collected to determine for hematological parameters and histological differences. The results obtained in this experiment demonstrated that four weeks of consumption of this Bifidobacterium strain had no adverse effects on rat's general health status, blood biochemical parameters or histology. Therefore, it is likely to be safe for human use. Fecal harmful enzymes such as beta-glucosidase, beta-glucuronidase, tryptophanase and urease, were effectively inhibited during the administration of the B. longum SPM1205. These results suggested that this B. longum SPM 1205 could be used for humans as a probiotic strain.     

Acid stress in the food pathogen Bacillus cereus

AIMS: The pathogen Bacillus cereus, which is associated with a number of foods including dairy products, was studied for its response to acid stress during the exponential phase. METHODS AND RESULTS: Bacillus cereus was found to adapt to acid stress (pH 4.6) when pre-exposed to a non-lethal, inducing pH of 6.3 or to inducing concentrations of heat, ethanol, salt or hydrogen peroxide. Cells were found to maintain their internal pH at a higher level than the external acid pH and adapted cells had a higher internal pH than unadapted cells. A constitutive acid-sensitive mutant that was also heat- and ethanol-sensitive was found to be capable of high levels of adaptation despite its lack of induction of proteins induced in the wild type by exposure to moderate pH (6.3) values. CONCLUSIONS: A number of proteins were found to be underexpressed in the mutant compared with the wild type at pH 6.3, including some with homology to ribosomal proteins and to the sporulation regulator RapK, while one differentially expressed band contained two proteins, one of which was homologous to the competence regulator CodY. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the processing of B. cereus-associated foods by acidification. The linked developmental processes of stationary phase, sporulation and possibly competence appear to be involved in the response to acid stress.     

An immunostimulatory DNA sequence from a probiotic strain of Bifidobacterium longum inhibits IgE production in vitro

The immunostimulatory oligodeoxynucleotide (ODN) BL07 (5'-GCGTCGGTTTCGGTGCTCAC-3') was identified from the genomic DNA of the probiotic strain Bifidobacterium longum BB536. ODN BL07 stimulated B-lymphocyte proliferation and induced interleukin-12 (IL-12) production in macrophage-like J774.1 cells. ODNs BL07 and BL07S (modified with phosphorothioate backbone) significantly inhibited immunoglobulin E (IgE) production and stimulated interferon-gamma (IFN-gamma) and IL-12 production, but did not affect IL-4 secretion in murine splenic cells of ovalbumin-primed BALB/c mice. These ODNs also significantly inhibited production of IgE in purified murine B cells in the presence of IL-4 and anti-CD40. The results suggest the potential of ODNs BL07 and BL07S in preventing IgE-related immune responses and the possible involvement of ODN BL07 in the antiallergic efficacy of B. longum BB536.     

大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

长双歧杆菌可特异地定植于实体瘤低氧区,可用做肿瘤靶向性基因治疗的载体,而构建大肠杆菌-长双歧杆菌穿梭质粒则被证明是外源基因在长双歧杆菌中稳定表达的有效途径。为了构建能在长双歧杆菌中稳定表达外源基因的穿梭质粒并检测携带抑癌基因的工程菌对小鼠实体瘤的抑制效果,利用软件设计并合成了48条部分序列相互重叠的引物,通过PCR合成了长双歧杆菌质粒pMB1序列及长双歧杆菌HU启动子区序列,插入克隆载体pMD18-T,构建穿梭载体pMB-HU,该载体可在大肠杆菌DH5α及长双歧杆菌L17中稳定复制。PTEN基因编码具有蛋白质和酯类双重特异性磷酸酶活性的抑癌因子。将PTEN基因cDNA序列插入载体pMB-HU中HU启动子下游,构建重组质粒pMB-HU-PTEN,电击转化长双歧杆菌后,Western blot检测表明,表达产物中存在55kDa的PTEN蛋白特异条带。抑癌试验表明:与对照组相比,携带PTEN基因的长双歧杆菌可显著抑制小鼠实体瘤的生长。上述结果为以长双歧杆菌为载体的实体瘤靶向性基因治疗研究奠定了基础。