Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.     

A Collagen-Binding S-Layer Protein in Lactobacillus crispatus

Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer.     

Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin

In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro. Addition of B. subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested. Since B. subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L. acidophilus JCM 1132. Both catalase and B. subtilis (natto) enhanced the growth of L. reuteri JCM 1112, whereas B. subtilis (natto) but not catalase enhanced the growth of L. acidophilus JCM 1132. In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L. reuteri JCM 1112 was abolished by catalase or B. subtilis (natto). In addition, a serine protease from B. licheniformis, subtilisin, improved the growth and viability of L. reuteri JCM 1112 and L. acidophilus JCM 1132 in the absence of hydrogen peroxide. These results indicate that B. subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin.     

Interferon induction in murine peritoneal macrophage by stimulation with Lactobacillus acidophilus.

Induction of interferon for a kind of dairy lactic acid bacteria, Lactobacillus acidophilus (L. acidophilus), was investigated in murine peritoneal macrophage (M phi) cultures. Lactobacillus acidophilus JCM 1034, 1132T, 1229 and 2125 induced IFN (12-34 I.U./ml) in M phi cultures in vitro. Strain 1132T- and 2125-induced IFNs were characterized as IFN alpha/beta by treatment with anti-IFNs serum. The results indicate that the inducing activity of IFNs may be one of the available biological parameters for designating the dairy products containing L. acidophilus as "physiologically functional foods."     

A new assay using surface plasmon resonance (SPR) to determine binding of the Lactobacillus acidophilus group to human colonic mucin

A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcalpha1-3(Fucalpha1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.     

乳酸菌黏附小鼠派伊尔结及影响因子的研究

目的研究乳酸菌结合小鼠派伊尔结（PP）的性质,确定乳酸菌黏附小鼠PP的影响因子。方法测定10株荧光标记的乳酸菌对PP组织切片黏附性,分析细菌表面疏水性和酵母、红细胞凝集性与黏附性的关系。结果凝集酵母、红细胞能力强的乳酸菌,PP黏附性强。除嗜酸乳杆菌（L.acidophilm）外,其他9株乳酸菌的表面疏水性与凝集酵母、红细胞的能力呈显著的负相关性（r=-0．60和r=-0．53,P〈0．05）。L.acidophilm黏附PP的能力最强。L．acidophilm黏附PP能力和红细胞凝集性均可被D（＋）-甘露糖、D（＋）-半乳糖抑制。结论乳酸菌黏附PP的能力受疏水性和表面特异性凝集素的影响。L.acidophilm表面D（＋）-甘露糖、D（＋）-半乳糖特异性凝集素可能参与黏附PP的过程。     

几种水稻植株中血凝活性物质的分布与变化及其稻胚凝集素的比较

开花前的水稻旗叶提取液中不含有血凝活力,但开花后旗叶提取液中能测得血凝活力,并随着开花后天数增加而逐渐升高。雄蕊和成熟花药中无血凝活力,开花前和开花早期的子房提取液中有血凝活性,开花5d后子房或胚乳中测不到血凝活性,但胚中血凝活性随种子发育而明显增加。分别从“寒丰”和“双丰一号”水稻成熟胚中分离得到凝集素,用电泳、免疫学方法和血凝试验表明两者的分子特性和血凝性质完全相同。     

Screening of Lactobacilli derived from chicken feces and partial characterization of Lactobacillus acidophilus A12 as an animal probiotics

This study was performed to screen and select Lactobacillus strains from chicken feces for probiotic use in animals. Of these strains, strain A12 had the highest immunostimulatory effect. Therefore, strain A12 was characterized as a potential probiotic. Strain A12 was tentatively identified as Lactobacillus acidophilus A12, using the API 50 CHL kit based on a 99.9% homology. L. acidophilus A12 was highly resistant to artificial gastric juice (pH 2.5) and bile acid (oxgall). Based on results from the API ZYM kit, leucine arylamidase, crystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alpha-galactosidase, beta- galactosidase, alpha-glucosidase, beta-glucosidase, and N-acetyl-beta- glucosamidase were produced by strain A12. L. acidophilus A12 showed resistance to several antibiotics (nisin, gentamycin, and erythromycin). The amount of interleukin (IL)-1alpha in 20x concentrated supernatant from L. acidophilus A12 was approximately 156 pg/ml. With regard to antioxidant activity, L. acidophilus A12 supernatant showed 60.6% DPPH radical scavenging activity. These results demonstrate the potential use of L. acidophilus A12 as a health-promoting probiotics.     

Hemagglutination by Rabies Virus

Goose erythrocytes were agglutinated by five strains of rabies virus grown in monolayer cell cultures at pH 6.4 and at 0 to 4 C. Hemagglutination was not affected by the cell type in which the virus was grown. Prerequisites for occurrence of hemagglutination are absence of hemagglutination inhibitors (such as those contained in bovine serum) and a relatively high virus concentration (> 10(6) plaque-forming units of virus per ml). "Soluble" hemagglutinin was not present in crude preparations of extracellular virus. Treatment of purified preparations of extracellular virus with Tween 80 and ether did not result in release of a "soluble" hemagglutinin. The hemagglutinating property of extracellular virus seemed to be conditioned by the integrity of its coat. Preparations of infectious intracellular virus exhibited about 15 times lower hemagglutinating activity than extracellular virus. This decreased hemagglutinating activity did not seem to be caused by binding of hemagglutination inhibitors to the virus particles. Rabies virus can be quantitatively adsorbed onto and eluted from erythrocytes. Erythrocytes pretreated with rabies virus retained their ability to be agglutinated by the same virus strain. The reaction with rabies virus of erythrocytes treated with the receptor-destroying enzyme or KIO(4) was the same as that of nontreated erythrocytes. The hemagglutinating component of rabies virus, therefore, does not exhibit neuraminidase activity. Treatment of extracellular virus by various agents indicated that the hemagglutinating component consists of protein or lipoprotein. Sulfhydryl groups present in the viral hemagglutinin are essential for hemagglutination.     

Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri LA 6

Lactobacillus reuteri LA 6, isolated from infant faeces, produced an antimicrobial agent active against Lactobacillus acidophilus JCM 2125, Lactobacillus delbrueckii subsp. bulgaricus JCM 1002 and Lactobacillus delbrueckii subsp. lactis JCM 1148 and JCM 1248. The agent was sensitive to proteolytic enzymes and retained activity after heating at 100°C for 20 min. This agent was a bacteriocin and has been designated as reutericin 6. Reutericin 6 exceeds 200 kDa as determined by ultrafiltration studies. Activity against sensitive cells was both bacteriocidal and bacteriolytic.     

Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin.

The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C-6814) forms a large complex composed of 150-kDa neurotoxin (NT), 130-kDa nontoxic-nonhemagglutinin (NTNHA), and hemagglutinin (HA) components. The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26-21 and 17 kDa. We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants. The isolated HA subcomponents, designated as i-HA-33, i-HA-55, i-HA-70 and i-HA-33/17, were nearly homogeneous on SDS/PAGE, but the HA-17 and HA-26-21 components were not purified. Some HA subcomponents, designated as f-HA-33 and f-HA-33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified. Every HA subcomponent so far isolated shows binding activity to erythrocytes. The hemagglutination activities of each HA subcomponent had a titer of 25 for the f-HA-33/17 complex, and below 23 for the other f- and i-HA subcomponents, while the parent progenitor L toxin was 28. The reconstitution of various combinations of f- and i-HA subcomponents was attempted via mixing and tested for hemagglutination activity. When the i-HA-33/17 complex and i-HA-55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin. These data imply that a combination of at least HA-33, -17 and -55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.     

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).     

A role for a Hevea latex lectin-like protein in mediating rubber particle aggregation and latex coagulation

An in vitro aggregation of washed lutoid membrane and rubber particles, respectively, prepared from the bottom (lutoid) fraction and rubber layer of centrifuged fresh latex, leading to the formation of rubber coagulum necessary for a latex coagulation was demonstrated. A Triton X-100 extract of washed lutoid membrane proteins, isolated and prepared from the bottom fraction of centrifuged fresh latex was examined for its role in the latex coagulation process. It induced agglutination of rabbit erythrocytes, indicating the presence of a lectin-like protein. Hevea latex lectin-like protein (HLL) was purified to homogeneity by active chitin binding separation, followed by DEAE-Sepharose chromatography. Its M(r) analyzed by SDS-PAGE was 17 kDa, whereas that determined by gel filtration was 267 kDa. The HLL had a pI value of 7.2. Several glycoproteins were shown to inhibit the HLL-induced hemagglutination. The hemagglutinin activity of HLL was enhanced by Ca(2+). Of most interest was the finding that HLL strongly induced aggregation of the Hevea latex rubber particles (RP). This strong RP aggregation leads to latex coagulation, indicating the possibility that it is involved in the formation of the coagulum that plugs the latex vessel ends and stops the flow of latex upon tapping. In addition, the purified HLL also induced aggregation of RP taken from several other non-Hevea latex producing plants. This might indicate either a common or universal role of this lectin-like protein in RP aggregation and hence latex coagulation. This paper, for the first time, provides clear and unequivocal evidence for either a key biological role or physiological function of an endogenous latex lectin-like protein in the sequential process of latex coagulation.     

A N-acetyllactosamine-specific cell-binding activity in a plant pathogen, Erwinia rhapontici

A strain of the phytopathogenic bacterial species, Erwinia rhapontici, was found to cause hemagglutination of human erythrocytes that was specifically inhibited by beta-galactosides. Of the monosaccharides tested, N-acetyl galactosamine and galactose efficiently inhibited the hemagglutination. The most potent inhibitor identified was Ga1 beta 1-4GlcNAc that was 30-100-fold more potent than Ga1 beta 1-3GlcNac or Ga1 beta 1-3GalNAc. Fetuin had no effect on the hemagglutination whereas asialofetuin was inhibitory. No blood group specificity was found for the hemagglutinin. These findings indicate that the E. rhapontici strain possesses a novel bacterial cell-binding activity with specificity for terminal N-acetyllactosamine residues.     

The primary screening of bifidobacteria and lactobacilli strains to develop effective probiotic preparations based on them

10 Bifidobacterium strains and 10 Lactobacillus strains were studied for their antagonistic activity with respect to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and for their sensitivity to antibiotics, widely used in clinical practice. L. acidophilus strain 5/4, L. acidophilus strain 18/4, B. adolescentis strain UX, B. longum strain 44 exhibited the highest antagonistic activity and the highest degree of antibiotic resistance. The restriction analysis of the chromosomal DNA of these strains was then made and their plasmid content was studied, making it possible to recognize these strains in future in the course of in vivo experiments.     

Role of C-terminal region of HA-33 component of botulinum toxin in hemagglutination.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.     

Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in mumps virus hamster encephalitis: analysis of mutants selected with monoclonal antibodies.

With the aid of monoclonal antibodies directed against a specific site on the hemagglutinin-neuraminidase surface glycoprotein, four mutants of the Kilham neurotropic strain of mumps virus were isolated. All four mutants had increased neuraminidase activity. Two mutants (M10 and M12) lost their hemagglutination capacity with human O erythrocytes but retained their ability to agglutinate guinea pig erythrocytes at 4 degrees C. A third mutant (M11) showed a change in the molecular weight of the hemagglutinin-neuraminidase glycoprotein. These three mutants (M10, M11, and M12) showed unaltered capacity to infect tissue cultures and to cause encephalitis in newborn hamsters. A fourth mutant (M13) retained its hemagglutination activity and capacity to infect Vero cell cultures but showed significantly lower neurovirulence in the suckling hamster brain than did the parental Kilham strain and the other three mutants. Both the number of infected neurons and the amount of infectious virus in the brain was reduced. On the other hand, there were no apparent differences in the occurrence of viral antigen in ependymal cells, indicating a selective change in affinity for neurons in the brain. These results suggest that certain changes in the hemagglutinin-neuraminidase glycoprotein may lead to an alteration of the neuropathogenicity of the Kilham strain of mumps virus.     

红藻条斑紫菜凝集素的纯化及其色氨酸化学修饰对其活性的抑制作用

为了探索条斑紫菜凝集素(Porphyra yezoensis Ueda lectin, PYL)的作用机理,对其进行了分离和纯化．条斑紫菜经磷酸盐缓冲液浸泡、20%～75%硫酸铵分级、DEAE 纤维素52离子交换层析和Sephadex G-200凝胶过滤层析,得到PYL纯品. Sephadex G-200分子筛层析测得其分子量为63.2 kD,在非还原SDS-PAGE上显示1条蛋白染色带,分子量为63.1 kD,还原SDS-PAGE显示1条蛋白染色带,亚基分子量为15.8 kD．PYL在对兔、大鼠、鸡、羊、狗血细胞的凝集作用中,对大鼠红细胞的凝集活性最高.PYL在pH 6.50～10.53范围内均有活性,在pH 8.40～8.91活性最高．经42 ℃热处理10 min后,仍然对大鼠红细胞血凝活性保留12.5%,其活性最大温度范围为4 ℃～20 ℃, 48 ℃加热10 min后,其活性完全丧失．EDTA对PYL的凝集活性有抑制作用,最小抑制浓度为156 mmol/L,而 Ca2+和Mg2+未发生凝集抑制现象．PYL凝集大鼠红细胞的作用不被D 果糖、葡萄糖、D-半乳糖、D-甘露糖、菊粉、γ球蛋白、牛甲状腺球蛋白等所抑制,但可被蔗糖和麦芽糖抑制,最小抑制浓度蔗糖为20 mmol/L,麦芽糖为40 mmol/L．用N 溴代丁二酰亚胺(NBS) 对PYL分子中的Trp残基进行化学修饰,有2.1个Trp残基被修饰,修饰后PYL活性丧失, 表明Trp残基是PYL凝集活性所必需的基团．     

Hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

The hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin (LTc-B) produced by chicken enterotoxigenic Escherichia coli was studied by hemagglutination and hemagglutination inhibition. No or weak hemagglutination of intact human erythrocytes was found by the LTc-B at the highest concentration used, whereas strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was found. Enhancement in hemagglutination of treated human erythrocytes induced by the LTc-B was over 2 to 120-fold for type A and B erythrocytes and over 8-fold for type O erythrocytes, respectively. With intact and treated sheep erythrocytes, on the other hand, no hemagglutination was found by the LTc-B at the highest concentration used. Hemagglutination of pronase-treated human type B erythrocytes by the LTc-B was inhibited by methyl-alpha-D-galactopyranoside, galactose, melibiose, hog A + H, asialo-bovine salivary mucin and asialo-thyroglobulin among mono-, di- and polysaccharides and glycoproteins used as inhibitors. These results suggest that the LTc-B is a galactose-specific bacterial lectin.     

Herpes simplex virus type 1-induced hemagglutination: glycoprotein C mediates virus binding to erythrocyte surface heparan sulfate.

We recently reported that herpes simplex virus type 1 (HSV-1) can cause agglutination of murine erythrocytes (E. Trybala, Z. Larski, and J. Wisniewski, Arch. Virol. 113:89-94, 1990). We now demonstrate that the mechanism of this hemagglutination is glycoprotein C-mediated binding of virus to heparan sulfate moieties at the surface of erythrocytes. Hemagglutination was found to be a common property of all gC-expressing laboratory strains and clinical isolates of HSV-1 tested. Mutants of HSV-1 deficient in glycoprotein C caused no specific hemagglutination, whereas their derivatives transfected with a functional gC-1 gene, thus reconstituting gC expression, regained full hemagglutinating activity. Hemagglutination activity was inhibited by antibodies against gC-1 but not by antibodies with specificity for glycoproteins gB, gD, or gE or by murine antiserum raised against the MP strain of HSV-1, which is gC deficient. Finally, purified gC-1 protein, like whole HSV-1 virions, showed high hemagglutinating activity which was inhibited by heparan sulfate and/or heparin and was completely prevented by pretreatment of erythrocytes with heparitinase, providing evidence that gC-1 mediates hemagglutination by binding to heparan sulfate at the cell surface. Thus, HSV-1-induced hemagglutination is gC-1 dependent and resembles the recently proposed mechanism by which HSV-1 attaches to surface heparans on susceptible cells, providing a simple model for initial events in the virus-cell interaction.