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PubMed2006

The probiotic bacterium Lactobacillus casei induces activation of the gut mucosal immune system through innate immunity.

The mechanisms by which probiotic bacteria affect the immune system are unknown yet, but many of them are attributed to an increase in the innate or in the acquired immune response. To study the influence of the probiotic bacterium Lactobacillus casei in the expression of receptors involved in the innate immune response, this bacterium was orally administered to BALB/c mice. After, they were sacrificed; the small intestine and intestinal fluids were collected to measure secretory immunoglobulin A (IgA) specific for L. casei. Mononuclear cells from Peyer's patches were isolated to determine the CD-206 and TLR-2 receptors. In histological slices we determined the number of IgA+, CD4+, CD8+, and CD3+ cells and two cytokines (interleulin-5 [IL-5] and IL-6). CD-206 and TLR-2 increased with respect to the untreated control. We did not observe an increase in the T population or in the IL-5-positive cells. IgA+ cells and IL-6-producing cells increased after 7 days of L. casei administration. We did not find specific antibodies against L. casei. The main immune cells activated after oral L. casei administration were those of the innate immune response, with an increase in the specific markers of these cells (CD-206 and TLR-2), with no modification in the number of T cells.

Administration, Oral↗

PubMed1979

Mechanism of folate transport in Lactobacillus casei: evidence for a component shared with the thiamine and biotin transport systems.

Lactobacillus casei cells have been shown previously to utilize two separate binding proteins for the transport of folate and thiamine. Folate transport, however, was found to be strongly inhibited by thiamine in spite of the fact that the folate-binding protein has no measurable affinity for thiamine. This inhibition, which did not fluctuate with intracellular adenosine triphosphate levels, occurred only in cells containing functional transport systems for both vitamins and was noncompetitive with folate but competitive with respect to the level of folate-binding protein. Folate uptake in cells containing optimally induced transport systems for both vitamins was inhibited by thiamine (1 to 10 muM) to a maximum of 45%; the latter value increased to 77% in cells that contained a progressively diminished folate transport system and a normal thiamine system. Cells preloaded with thiamine could transport folate at a normal rate, indicating that the inhibition resulted from the entry of thiamine rather than from its presence in the cell. In a similar fashion, folate (1 to 10 muM) did not interfere with the binding of thiamine to its transport protein, but inhibited thiamine transport (to a maximum of 25%). Competition also extended to biotin, whose transport was strongly inhibited (58% and 73%, respectively) by the simultaneous uptake of either folate or thiamine; biotin, however, had only a minimal effect on either folate or thiamine transport. The nicotinate transport system was unaffected by co-transport with folate, thiamine, or biotin. These results are consistent with the hypothesis that the folate, thiamine, and biotin transport systems of L. casei each function via a specific binding protein, and that they require, in addition, a common component present in limiting amounts per cell. The latter may be a protein required for the coupling of energy to these transport processes.

Biological Transport, Active↗

PubMed1973

An automated method for the microbiological assay of serum pyridoxal.

A fully automated method for the measurement of serum pyridoxal has been developed. Acid phosphatase was used for dephosphorylation and precipitation of the serum proteins was not required. A chloramphenicol-resistant strain of L. casei was used as the test organism and this removed the need for sterilization. The method gives highly reproducible results, and is suitable for population and institutional studies.

Acid Phosphatase↗

PubMed1977

Measurement of serum folate: experience with 75Se-selenofolate radioassay.

Three hundred and eighty-two serum samples were assayed for folate content by a standard Lactobacillus casei microbiological assay and by radioassay using the 75Se-selenofolate radioassay kit from the Radiochemical Centre, Amersham. Results showed encouraging concordance. Explanations were discovered for the few disparities. The radioassay procedure is simple and an assay batch can be completed in one working day.

Biological Assay↗

PubMed1994

A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase.

The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.

Amino Acid Sequence↗

PubMed1975

Transfer of folate to the fetus.

The accumulation of tritium-labelled folate in the product of conception was examined in eight women undergoing hysterotomy for termination of pregnancy. Folic acid containing a small quantity of tracer material (50n Ci/mug.) was injected intravenously 20 minutes before operation in 4 patients but 24 hours beforehand in the remainder. After operation, maternal blood, placenta, fetal blood, and fetal liver were examined for radioactivity as was the daily urine output during the patient's postoperative hospital stay. The results indicated that only 1/2 per cent of the injected material accumulated in the product of conception--uptake was primarily by fetal liver and no large placental pool of folate was involved. Approximately half of the injected load was excreted in the urine--the majority in the first 24 hours--and the remainder was presumed to have been retained by maternal tissues. Bioassay with L. casei and chromatography on TEAE cellulose suggested that extensive metabolism of the injected material had taken place and that fetal uptake might be more efficient than indicated by counting radioactivity alone.

Abortion, Induced↗

PubMed2001

Lactobacillus casei acquires the binding activity to fibronectin by the expression of the fibronectin binding domain of Streptococcus pyogenes on the cell surface.

Fibronectin binding domain was expressed on the cell surface of Lactobacillus casei strain Shirota which hardly adheres to fibronectin. DNA for the fibronectin binding domain of the sfbl gene, which encodes a fibronectin binding protein of Streptococcus pyogenes ATCC 21059, was amplified with polymerase chain reaction, cloned into a surface display vector pSAK332, and introduced into L. casei. The fibronectin binding domain was expressed as a fusion protein consisting of staphylokinase of Staphylococcus aureus and the anchor sequence of cell wall-associated 763 proteinase of Lactococcus lactis NCDO 763. The fibronectin binding ability of the resulting L. casei was confirmed with Western blot analysis, immunoelectron microscopic analysis, and adherence to fibroblast cells. These results indicate that L. casei has acquired a new phenotype to bind fibronectin upon the expression of the fibronectin binding domain on the cell surface. This L. casei also shows binding affinity to fibrinogen, indicating that fibronectin binding domain is involved in the binding to fibrinogen as well.

Adhesins, Bacterial↗

PubMed1987

The role of humoral immunity in Lactobacillus casei cell wall induced arthritis.

Although both cellular and humoral immunity have been shown to play important roles in the development of collagen induced arthritis, their roles in the pathogenesis of bacterial cell wall induced arthritis remain unclear. Previous studies of humoral immunity in bacterial cell wall induced arthritis were carried out in outbred animals and measured the humoral response to only the eliciting bacteria. We compared the humoral immune responses of arthritis susceptible Lewis and arthritis resistant Fisher rats after the injection of arthritogenic group B Lactobacillus casei cell wall fragments. Both strains developed significant IgG and IgM responses to the Lactobacillus casei cell wall fragments. Neither strain developed a significant immune response to types I, II, IX or 1a2a3a collagens. These data suggest that humoral immune responses to the Lactobacillus casei cell wall fragments and types I, II, IX, or 1a2a3a collagens do not play a significant role in determining the differing arthritis susceptibilities of Lewis and Fisher rats.

Animals↗

PubMed2006

Lactobacillus casei administration reduces lung injuries in a Streptococcus pneumoniae infection in mice.

The effect of the oral administration of Lactobacillus casei on the prevention of a Streptococcus pneumoniae lung infection in a mouse experimental model was studied, analyzing the innate and specific immune response. Adult Swiss albino mice were treated with L. casei (10(9)CFU/day) for 2, 5 and 7 d. Mice were infected intranasally with S. pneumoniae (10(6)CFU/mouse) after each treatment and the microbiological, histopathological and host responses were determined for 15 d after infection. Feeding L. casei for 2 d induced a faster clearance of S. pneumoniae, with a lower number of pneumococci in lung and a shorter period of septicemia than in the control group. L. casei administration induced activation of phagocytes as evidenced by the strong myeloperoxidase activity and the nitro blue tetrazolium assay in lung. Mice given L. casei for 2 d showed higher levels of anti-pneumococcal serum IgG and bronchoalveolar lavage IgA than the control mice. The group fed L. casei for 2 d could beneficially regulate the balance between tumor necrosis factor alpha and interleukin 10, allowing a more effective immune response against infection and modulating the inflammatory response, with less damage to the lung.

Animals↗

PubMed2004

Selection of single-chain antibodies against the VP8* subunit of rotavirus VP4 outer capsid protein and their expression in Lactobacillus casei.

Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.

Amino Acid Sequence↗

PubMed1996

The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA.

S-proteins are proteins which form a regular structure (S-layer) on the outside of the cell walls of many bacteria. Two S-protein-encoding genes are located in opposite directions on a 6.0-kb segment of the chromosome of Lactobacillus acidophilus ATCC 4356 bacteria. Inversion of this chromosomal segment occurs through recombination between two regions with identical sequences, thereby interchanging the expressed and the silent genes. In this study, we show that the region involved in recombination also has a function in efficient S-protein production. Two promoter sequences are present in the S-protein gene expression site, although only the most downstream promoter (P-1) is used to direct mRNA synthesis. S-protein mRNA directed by this promoter has a half-life of 15 min. Its untranslated leader can form a stable secondary structure in which the 5' end is base paired, whereas the ribosome-binding site is exposed. Truncation of this leader sequence results in a reduction in protein production, as shown by reporter gene analysis of Lactobacillus casei. The results obtained indicate that the untranslated leader sequence of S-protein mRNA is involved in efficient S-protein production.

Bacterial Outer Membrane Proteins↗

PubMed1963

NATURE OF THE LIPIDS OF SOME LACTIC ACID BACTERIA.

Ikawa, Miyoshi (University of California, Berkeley). Nature of the lipids of some lactic acid bacteria. J. Bacteriol. 85:772-781. 1963.-Lactobacillus casei, L. plantarum, Leuconostoc mesenteroides, Pediococcus cerevisiae, and Streptococcus faecalis were grown on a lipid-free medium, and the amounts of acetone-soluble lipids, acetone-insoluble phospholipids, and unextracted or bound lipid in the cells were estimated. Neutral glycerides appeared to be absent, or present only in traces, in L. casei and S. faecalis. L. plantarum, L. mesenteroides, and P. cerevisiae, in addition to containing glycerides, appeared to contain glycolipids of glucose and galactose. Glycerol was present in the phospholipid fraction of all the organisms, and, in addition, galactose was present in L. mesenteroides. Paper chromatography of hydrolysates of the phospholipid fractions showed the absence of the usual phospholipid nitrogen bases, serine, ethanolamine, and choline, in these organisms. Microbiological assays of lyophilized unextracted cells for myo-inositol and choline also showed that lipids containing these components occurred in very small amounts at most. The principal ninhydrin-reacting substance in the phospholipid fraction of L. casei, L. plantarum, P. cerevisiae, and S. faecalis was lysine, which seemed to be exclusively of the l configuration. The principal ninhydrin-reacting substance in the L. mesenteroides phospholipid fraction was d-alanine. These amino acids appeared to be bound in components of the phospholipid fraction and not free.

Enterococcus faecalis↗

PubMed1985

Nucleic acids of species of Lactobacillus.

Nucleic acid composition of 17 cultures representing the type or neotype strains of 15 named Lactobacillus species was studied. Nucleic acid characterization of these isolates was accompanied by a comparative study of conventional phenotypic reactions. The overall guanine plus cytosine mean deoxyribonucleic acid base composition ranged from 33 to 50% and genome sizes varied between 700 and 1500 X 10(6) daltons. Sporolactobacillus inulinus contained 2500 X 10(6) daltons of chromosomal deoxyribonucleic acid and was therefore similar in size to members of the genus Bacillus. Deoxyribonucleic acid hybridization at a temperature 15 degrees C below the thermal melting temperature confirmed the extreme molecular heterogeneity of many species. The genus can be divided into three major groups, each containing four or more species based on a combination of nucleic acid characteristics and conventional phenotypic reactions. Group I (33 to 39% guanine plus cytosine) contains Lactobacillus jensenii, Lactobacillus helveticus, Lactobacillus helveticus nov. ssp. jugurt, Lactobacillus acidophilus, Lactobacillus salivarius, and Lactobacillus sanfrancisco. Group II (42 to 48% guanine plus cytosine contains Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus brevis, and Group III (48 to 50% guanine plus cytosine) contains Lactobacillus delbrueckii, Lactobacillus delbrueckii nov. ssp. bulgaricus, Lactobacillus delbrueckii nov. ssp. lactis, Lactobacillus delbrueckii nov. ssp. leichmanii, Lactobacillus fermentum, and Lactobacillus fermentum nov. ssp. cellobiosis.

Base Composition↗

PubMed1995

Involvement of host cell energy in the transfection of Lactobacillus casei protoplasts with phage PL-1 DNA.

Transfection of Lactobacillus casei ATCC 27092 protoplasts with phage PL-1 DNA was studied under various conditions. The process of transfection was dependent on the incubation temperature, and the apparent activation energy was calculated to be about 11 kcal/mol. Transfection was inhibited by treating the cells before protoplasting either with monoiodoacetate, N,N'-dicyclohexylcarbodiimide (DCCD), or NaN3, without affecting both the viability of uninfected cells and protoplasting. The addition of DCCD after mixing protoplasts and DNA had no influence on transfection efficiencies. The transfection of L. casei protoplasts with phage PL-1 DNA was considered to require cell energy such as proton-motive force, probably in the initial stages, although the direct involvement of cell energy in the transfer of DNA across the cell membrane is still unclear.

Adenosine Triphosphate↗

PubMed1978

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. II. Structures of glycerophosphoglycolipids.

1. Eight glycerophosphoglycolipids were isolated from six Gram-positive bacteria. Besides sn-glycero-1-phospho-beta-gentiobiosyldiacylglycerol (i) and sn-glycero-1-phospho-alpha-kojibiosyldiacylglycerol (ii), three novel structures have been established: 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (iii), 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl]glycerol (iv), and 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (v). 2. Compound i was isolated from Bacillus licheniformis, Bacillus subtilis and Staphylococcus aureus, compound ii from a group B Streptococcus, compounds ii and iii from Streptococcus lactis, compounds iv and v from Lactobacillus casei. Lactobacillus plantarum contained besides compounds iv and v a glycerophosphate derivative of 1,2-di-O-acyl-3-O-[alpha-D-galactopyranosyl (1 leads to 2)-alpha-D-glucopyranosyl]glycerol. 3. Identical structural features of the described glycerophosphoglycolipids and the corresponding lipoteichoic acids are discussed.

Bacillus↗

PubMed1990

Visualization of folate transport proteins by covalent labeling with fluorescein methotrexate.

Fluorescein-methotrexate (F-MTX) has been synthesized by an improved procedure and separated via chromatography on DEAE-Trisacryl into the alpha- and gamma-isomers. Purity of each isomer was verified by TLC, HPLC, and absorbance spectra. Identity of the alpha- and gamma-isomers was established by the following biological criteria: the gamma-isomer inhibited dihydrofolate reductase and was hydrolyzed by carboxypeptidase G2 (at the pteroate-glutamate linkage). The alpha-isomer, conversely, was unreactive in both systems, which is consistent with the specificity of these enzymes. Based upon these results, the gamma-isomer was selected for covalent labeling of proteins. Fluorescent bands were observed when the 22 kDa human dihydrofolate reductase and the 18 kDa folate transporter from Lactobacillus casei were treated with gamma-F-MTX (activated by N-hydroxysuccinimide) and subjected to SDS-PAGE. The probe was also useful for visualizing in situ the micromolar folate transport protein in L1210 cells.

Animals↗

PubMed2005

Modulation of natural killer cell activity by supplementation of fermented milk containing Lactobacillus casei in habitual smokers.

BACKGROUND: Habitual smoking significantly reduces natural killer (NK) cell activity. To clarify whether the intake of fermented milk containing lactic acid bacteria restores NK cell activity in habitual smokers, we conducted a placebo-controlled double-blind test. METHODS: Ninety-nine subjects with smoking habits were randomly divided into two groups and daily for 3 weeks were given fermented milk containing Lactobacillus casei or placebo. NK cell activity in the peripheral blood mononuclear cells (PBMNC) was determined before and after the intake. Average number of cigarettes smoked and number of cigarettes that the subjects smoked before giving blood after getting up on the inspection day were asked, and these data were used to adjust the influence of smoking on NK cell activity. RESULTS: NK cell activity in individuals was inversely correlated to numbers of cigarettes smoked. Averaged NK cell activity adjusted by the numbers of cigarettes was significantly higher in individuals drinking fermented milk containing L. casei than those drinking a placebo. However, the proportion of NK cells was not different between individuals drinking either fermented milk containing L. casei or the placebo. CONCLUSIONS: Intake of fermented milk containing lactic acid bacteria was considered effective for restoring the NK cell activity of habitual smokers.

Adult↗

PubMed1996

Suppression of lymphocyte proliferation in vitro by bovine caseins hydrolyzed with Lactobacillus casei GG-derived enzymes.

BACKGROUND: Processing of proteins in the gut and activation of T-cell suppression leads to systemic hyporesponsiveness to ingested protein antigens. OBJECTIVE: The study was designed to determine whether lactobacilli, a major part of human intestinal microflora, can contribute to degradation of food antigens in the gut and modify their immunoactivities. METHODS: Lymphocyte transformation tests were carried out in healthy adults to determine the mitogen-induced lymphocyte proliferative responses to bovine caseins hydrolyzed with pepsin and trypsin and to bovine caseins additionally hydrolyzed with enzymes derived from Lactobacillus casei strain GG (ATCC 53103). RESULTS: In experiments done with caseins hydrolyzed with pepsin and trypsin, beta- and alpha(s1)-caseins significantly suppressed the proliferation of lymphocytes at 0.1 and 10 micrograms/ml, respectively, when compared with corresponding control cultures without these hydrolysates. In contrast, kappa-casein significantly stimulated the proliferation of lymphocytes at 10 micrograms/ml. In experiments done with caseins additionally hydrolyzed with L. casei GG-derived enzymes, there was one consistent effect on lymphocyte proliferation: suppression by alpha(sl)-, beta-, and kappa-caseins at 0.1, 10, and 1000 micrograms/ml, respectively. CONCLUSIONS: Hydrolysis of caseins with L. casei GG-derived enzymes generates molecules with suppressive effects on lymphocyte proliferation. In addition, intestinal bacteria can be beneficial in the downregulation of hypersensitivity reactions to ingested proteins in patients with food allergy.

Adult↗