Characterization of a fibronectin‐binding protein from Lactobacillus casei BL23

Aims: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin‐binding protein. Methods and Results: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease‐sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco‐2 or HT‐29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose‐dependent manner. Western blot experiments with FbpA‐specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole‐cell ELISA. Conclusions: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. Significance and Impact of the Study: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.     

Response of Lactobacillus casei BL23 to phenolic compounds

Aims: To determine the inhibitory effect of phenolic compounds on Lactobacillus casei BL23, the role of two component signal transduction systems (TCS) and the response of Lact. casei BL23 to p‐coumaric acid. Methods and Results: Growth of Lact. casei BL23 and 17 derivative strains defective in each TCS harboured by this strain in the presence of p‐coumaric acid, ferulic acid, caffeic acid or methyl gallate was monitored. Furthermore, changes in the protein content of Lact. casei BL23 when exposed to p‐coumaric acid were evaluated by 2D‐SDS‐PAGE. Eleven proteins differentially expressed in the presence of p‐coumaric acid were detected. Six of them could be identified: ClpP and HtrA, involved in protein turnover and folding, acetyl‐CoA carboxylase, involved in lipid metabolism, and an arginyl‐tRNA synthetase were more abundant, whereas PurL and PurN, involved in purine biosynthesis, were less abundant. Conclusions: No significant differences were observed between the parental strain and the TCS‐defective mutants. p‐Coumaric acid elicited a response against membrane and cytoplasmic damages. Significance and Impact of the Study: The inhibitory effect of phenolic compounds on Lact. casei BL23 has been determined. For the first time, cytoplasmic proteins presumably involved in the response of Lact. casei BL23 against p‐coumaric acid have been identified.     

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD⁺ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.     

Functional analysis of the p40 and p75 proteins from Lactobacillus casei BL23

The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria.     

Attenuation of Colitis by Lactobacillus casei BL23 Is Dependent on the Dairy Delivery Matrix

The role of the food delivery matrix in probiotic performance in the intestine is not well understood. Because probiotics are often provided to consumers in dairy products, we investigated the contributions of milk to the health-benefiting performance of Lactobacillus casei BL23 in a dextran sulfate sodium (DSS)-induced murine model of ulcerative colitis. L. casei BL23 protected against the development of colitis when ingested in milk but not in a nutrient-free buffer simulating consumption as a nutritional supplement. Consumption of (acidified) milk alone also provided some protection against weight loss and intestinal inflammation but was not as effective as L. casei and milk in combination. In contrast, L. casei mutants deficient in DltD (lipoteichoic acid d-alanine transfer protein) or RecA (recombinase A) were unable to protect against DSS-induced colitis, even when consumed in the presence of milk. Mice fed either L. casei or milk contained reduced quantities of colonic proinflammatory cytokines, indicating that the L. casei DltD⁻ and RecA⁻ mutants as well as L. casei BL23 in nutrient-free buffer were effective at modulating immune responses. However, there was not a direct correlation between colitis and quantities of these cytokines at the time of sacrifice. Identification of the cecal microbiota by 16S rRNA gene sequencing showed that L. casei in milk enriched for Comamonadaceae and Bifidobacteriaceae; however, the consumption of neither L. casei nor milk resulted in the restoration of the microbiota to resemble that of healthy animals. These findings strongly indicate that probiotic strain efficacy can be influenced by the food/supplement delivery matrix.     

Identification of Surface Proteins from Lactobacillus casei BL23 Able to Bind Fibronectin and Collagen

Strains of lactobacilli show the capacity to attach to extracellular matrix proteins. Cell-wall fractions of Lactobacillus casei BL23 enriched in fibronectin, and collagen-binding proteins were isolated. Mass spectrometry analysis of their protein content revealed the presence of stress-related proteins (GroEL, ClpL), translational elongation factors (EF-Tu, EF-G), oligopeptide solute-binding proteins, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The latter two enzymes were expressed in Escherichia coli and purified as glutathione-S-transferase (GST) fusion proteins, and their in vitro binding activity to fibronectin and collagen was confirmed. These results reinforce the idea that lactobacilli display on their surfaces a variety of moonlighting proteins that can be important in their adaptation to survive at intestinal mucosal sites and in the interaction with host cells.     

Influence of two-component signal transduction systems of Lactobacillus casei BL23 on tolerance to stress conditions

Lactobacillus casei BL23 carries 17 two-component signal transduction systems. Insertional mutations were introduced into each gene encoding the cognate response regulators, and their effects on growth under different conditions were assayed. Inactivation of systems TC01, TC06, and TC12 (LCABL_02080-LCABL_02090, LCABL_12050-LCABL_12060, and LCABL_19600-LCABL_19610, respectively) led to major growth defects under the conditions assayed.     

myo-Inositol dehydrogenase and scyllo-inositol dehydrogenase from Lactobacillus casei BL23 bind their substrates in very different orientations

Many bacteria can use myo-inositol as the sole carbon source using enzymes encoded in the iol operon. The first step is catalyzed by the well-characterized myo-inositol dehydrogenase (mIDH), which oxidizes the axial hydroxyl group of the substrate to form scyllo-inosose. Some bacteria, including Lactobacillus casei, contain more than one apparent mIDH-encoding gene in the iol operon, but such redundant enzymes have not been investigated. scyllo-Inositol, a stereoisomer of myo-inositol, is not a substrate for mIDH, but scyllo-inositol dehydrogenase (sIDH) enzymes have been reported, though never observed to be encoded within the iol operon. Sequences indicate these enzymes are related, but the structural basis by which they distinguish their substrates has not been determined. Here we report the substrate selectivity, kinetics, and high-resolution crystal structures of the proteins encoded by iolG1 and iolG2 from L. casei BL23, which we show encode a mIDH and sIDH, respectively. Comparison of the ternary complex of each enzyme with its preferred substrate reveals the key variations allowing for oxidation of an equatorial versus an axial hydroxyl group. Despite the overall similarity of the active site residues, scyllo-inositol is bound in an inverted, tilted orientation by sIDH relative to the orientation of myo-inositol by mIDH.     

Characterization of a membrane-regulated sugar phosphate phosphohydrolase from Lactobacillus casei.

One of the key components of the futile xylitol cycle of Lactobacillus casei Cl-16 is a phosphatase which dephosphorylates xylitol 5-phosphate to xylitol prior to the expulsion of the pentitol from cells. This enzyme has been partially purified and characterized. The phosphatase is active against a variety of four-, five-, and six-carbon sugars and sugar alcohols phosphorylated at the terminal 4, 5, and 6 positions, respectively, but exhibits little or no affinity for substrates phosphorylated at the C-1 position. The enzyme has an apparent molecular weight of 62,000 and a pH optimum between 5.5 and 6, and it requires a divalent cation (Mg2+) for maximal activity. A single protein band, exhibiting phosphatase activity, was excised from polyacrylamide gels and used to prepare antiphosphatase sera in rabbits. The antiserum was used to detect the enzyme on polyacrylamide gels and to determine the molecular weight of the monomer on sodium dodecyl sulfate-polyacrylamide gels. With a subunit molecular weight of 32,000, the native enzyme appears to be a dimer. Phosphatase activity and substrate specificity are regulated by some component associated with the cytoplasmic membrane.     

干酪乳杆菌JH-23中单胺氧化酶抑制剂的初步分离

采用丙酮抽提法得到了干酪乳杆菌JH-23中具有抑制单胺氧化酶作用的无细胞提取物,并利用硅胶薄层色谱从中分离出4组活性片段,其中片段Ⅰ对单胺氧化酶的抑制效果最佳,反应浓度在120μg/mL时抑制率为55.4%。该活性片段经国家新药筛选中心确证有抑制单胺氧化酶的效果,具有潜在的抗衰老作用。     

Characterization of antagonistic and acid formation properties of Lactobacillus casei

Cultural, morphologic, and biochemical characteristics of Lactobacillus casei were studied as well as their acid-forming and antagonistic activity and resistance to antibiotics. 60 cultures identified as L. caseiwere isolated from 250 samples of sour-milk products and clinical specimens. All isolated strains had respectively high antagonistic activity regardless of their source. Dependence between antagonistic activity and acid formation was not detected. Conclusion about promise for using these lactobacilli for manufacturing of probiotics has been done.     

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.     

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca²⁺-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes β-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.     

Characterization of a pAM beta 1 deletion derivative isolated from Lactobacillus casei after conjugation

Streptococcal plasmid pAM beta 1 was conjugally transferred from Streptococcus lactis KB953 (a transformant of pAM beta 1) into Lactobacillus casei 239. A unique transconjugant, L. casei C2, was found to contain a small (11.1 kilobase pair) plasmid, pLY201, which was derived by a deletion event from pAM beta 1. Restriction analysis revealed that pLY201 was missing approximately 58% of the original pAM beta 1 genome, and contained 5 single restriction sites for AvaI, EcoRI, PvuII, HpaI and KpnI. Physical analyses revealed that the stability and copy number of pLY201 were elevated compared with those of pAM beta 1 in L. casei. In addition, pLY201 was no longer transferable by conjugation.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.