Lactobacillus rhamnosus Strain GG Modulates Intestinal Absorption, Fecal Excretion, and Toxicity of Aflatoxin B1 in Rats

In this study, the modulation of aflatoxin B₁ (AFB₁) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB₁ excretion in GG-treated rats was increased via bacterial AFB₁ binding. Furthermore, AFB₁-associated growth faltering and liver injury were alleviated with GG treatment.     

Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B1 (AFB1) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB1 availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB1 levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB1 transport from the apical to the basolateral chamber was reduced from 11.1%+/-1.9% to 6.4%+/-2.5% (P=0.019) and to 3.3%+/-1.8% (P=0.002) within the first hour in monolayers coincubated with GG (1x10(10) and 5x10(10) CFU/ml, respectively). GG (1x10(10) and 5x10(10) CFU/ml) bound 40.1%+/-8.3% and 61.0%+/-6.0% of added AFB1 after 1 h, respectively. AFB1 caused significant reductions of 30.1% (P=0.01), 49.4% (P=0.004), and 64.4% (P<0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1x10(10) CFU/ml GG after 24 h protected against AFB1-induced reductions in transepithelial resistance at both 24 h (P=0.002) and 48 h (P=0.04). DNA fragmentation was apparent in cells treated only with AFB1 cells but not in cells coincubated with either 1x10(10) or 5x10(10) CFU/ml GG. GG reduced AFB1 uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.     

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.     

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.     

Lactobacillus rhamnosus strain GG modulates intestinal absorption, fecal excretion, and toxicity of aflatoxin B(1) in rats

In this study, the modulation of aflatoxin B(1) (AFB(1)) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB(1) excretion in GG-treated rats was increased via bacterial AFB(1) binding. Furthermore, AFB(1)-associated growth faltering and liver injury were alleviated with GG treatment.     

Comparative Genomic and Functional Analysis of 100 Lactobacillus rhamnosus Strains and Their Comparison with Strain GG

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.     

Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover, L. rhamnosus GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that L. rhamnosus GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the Lactobacillus casei group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by L. rhamnosus GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (wzb), lipoteichoic acid (dltD), and central metabolism (luxS) showed their relative importance in biofilm formation by L. rhamnosus GG.     

Persistence of Colonization of Human Colonic Mucosa by a Probiotic Strain, Lactobacillus rhamnosus GG, after Oral Consumption

Lactobacillus rhamnosus GG is one of the most thoroughly studied probiotic strains. Its advantages in the treatment of gastrointestinal disorders are well documented. The aim of the present study was to demonstrate with colonic biopsies the attachment of strain GG to human intestinal mucosae and the persistence of the attachment after discontinuation of GG administration. A whey drink fermented with strain GG was fed to human volunteers for 12 days. Fecal samples were collected before, during, and after consumption. L. rhamnosus GG-like colonies were detected in both fecal and colonic biopsy samples. Strain GG was identified by its characteristic colony morphology, a lactose fermentation test, and PCR. This study showed that strain GG was able to attach in vivo to colonic mucosae and, although the attachment was temporary, to remain for more than a week after discontinuation of GG administration. The results demonstrate that the study of fecal samples alone is not sufficient in evaluating colonization by a probiotic strain.     

Piliation of Lactobacillus rhamnosus GG Promotes Adhesion,Phagocytosis, and Cytokine Modulation in Macrophages

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.     

Lactobacillus rhamnosus BFE 5264 and Lactobacillus plantarum NR74 Promote Cholesterol Excretion Through the Up-Regulation of ABCG5/8 in Caco-2 Cells

The effect of two putative probiotic strains, Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74, on the control of cholesterol efflux in enterocytes was assessed by focusing on the promotion of ATP-binding cassette sub-family G members 5 and 8 (ABCG5 and ABCG8). Differentiated Caco-2 enterocytes were treated with live bacteria, heat-killed bacteria, a bacterial cell wall fraction, and metabolites and were subjected to cholesterol uptake assay, mRNA analysis, and protein analyses. Following LXR-transfection by incubation with CHO-K1 cells in DNA-lipofectin added media, the luciferase assay was conducted for LXR analysis. Treatment of Caco-2 cells with L. rhamnosus BFE5264 (isolated from traditional fermented Maasai milk) and L. plantarum NR74 (isolated from Korean kimchi) resulted in the up-regulation of LXR, concomitantly with the elevated expression of ABCG5 and ABCG8. This was associated with the promotion of cholesterol efflux at significantly higher levels compared to the positive control strain L. rhamnosus GG (LGG). The experiment with CHO-K1 cells confirmed up-regulation of LXR-beta by the test strains, and treatment with the live L. rhamnosus BFE5264 and L. plantarum NR74 strains significantly increased cholesterol efflux. Heat-killed cells and cell wall fractions of both LAB strains induced the upregulation of ABCG5/8 through LXR activation. By contrast, LAB metabolites did not show any effect on ABCG5/8 and LXR expression. Data from this study suggest that LAB strains, such as L. rhamnosus BFE5264 and L. plantarum NR74, may promote cholesterol efflux in enterocytes, and thus potentially contribute to the prevention of hypercholesterolemia and atherosclerosis.     

Characterization of Biosynthesized Silver Nanoparticles Using Lactobacillus rhamnosus GG and its In Vitro Assessment Against Colorectal Cancer Cells

Silver nanoparticles are the most desirable nanoparticles broadly used in diverse fields. This study intends to investigate the anticancer properties of synthesized silver/Lactobacillus rhamnosus GG nanoparticles (Ag-LNPs) as a reducing and stabilizing agent in the synthesis process. To prepare silver/Lactobacillus rhamnosus GG nanoparticles, 1 mg/ml cell lysate of Lactobacillus rhamnosus GG and 1 mM silver nitrate solution were mixed and incubated for 72 h. XRD, FTIR, and TEM methods were used for nanoparticle characterization. MTT assay and annexin/PI staining were employed to analyze the toxicity and apoptotic cells levels of Ag-LNPs, respectively. TEM showed that these nanoparticles are spherical shaped about 233 nm in size. FTIR spectroscopy demonstrated that Ag-LNPs were functionalized with biomolecules. XRD pattern showed high purity and face-centered crystal structure of Ag-LNPs. MTT assay revealed that the percentages of HT-29 live cells significantly reduced in the high concentration of Ag-LNPs. Annexin/PI staining showed that these nanoparticles could lead HT-29 cells to apoptosis. This study showed the new Ag-LNP-synthesizing method using Lactobacillus rhamnosus GG as a cost-effective and efficient approach. Also, it showed that these nanoparticles can be considered as a potential active agent for biomedical applications and drug delivery due to their anticancer activities.

     

Genome Sequence and Characteristics of Lrm1, a Prophage from Industrial Lactobacillus rhamnosus Strain M1

Prophage Lrm1 was induced with mitomycin C from an industrial Lactobacillus rhamnosus starter culture, M1. Electron microscopy of the lysate revealed relatively few intact bacteriophage particles among empty heads and disassociated tails. The defective Siphoviridae phage had an isometric head of approximately 55 nm and noncontractile tail of about 275 nm with a small baseplate. In repeated attempts, the prophage could not be cured from L. rhamnosus M1, nor could a sensitive host be identified. Sequencing of the phage Lrm1 DNA revealed a genome of 39,989 bp and a G+C content of 45.5%. A similar genomic organization and mosaic pattern of identities align Lrm1 among the closely related Lactobacillus casei temperate phages A2, ΦAT3, and LcaI and with L. rhamnosus virulent phage Lu-Nu. Of the 54 open reading frames (ORFs) identified, all but 8 shared homology with other phages of this group. Five unknown ORFs were identified that had no homologies in the databases nor predicted functions. Notably, Lrm1 encodes a putative endonuclease and a putative DNA methylase with homology to a methylase in Lactococcus lactis phage Tuc2009. Possibly, the DNA methylase, endonuclease, or other Lrm1 genes provide a function crucial to L. rhamnosus M1 survival, resulting in the stability of the defective prophage in its lysogenic state. The presence of a defective prophage in an industrial strain could provide superinfection immunity to the host but could also contribute DNA in recombination events to produce new phages potentially infective for the host strain in a large-scale fermentation environment.     

A Lactobacillus rhamnosus Strain Induces a Heme Oxygenase Dependent Increase in Foxp3+ Regulatory T Cells

We investigated the consequences of feeding with a Lactobacillus species on the immune environment in GALT, and the role of dendritic cells and heme oxygenase-1 in mediating these responses. Feeding with a specific strain of Lactobacillus rhamnosus induced a significant increase in CD4+CD25+Foxp3+ functional regulatory T cells in GALT. This increase was greatest in the mesenteric lymph nodes and associated with a marked decrease in TNF and IFNγ production. Dendritic cell regulatory function and HO-1 expression was also increased. The increase in Foxp3+ T cells could be prevented by treatment with a heme oxygenase inhibitor. However, neither inhibition of heme oxygenase nor blockade of IL-10 and TGFβ prevented the inhibition of inflammatory cytokine production. In conclusion Lactobacillus feeding induced a tolerogenic environment in GALT. HO-1 was critical to the enhancement of Foxp3+ regulatory T cells while additional, as yet unknown, pathways were involved in the down-regulation of inflammatory cytokine production by T cells.     

Aflatoxin B1: Toxicity,bioactivation and detoxification in the polyphagous caterpillar Trichoplusia ni

Trichoplusia ni caterpillars are polyphagous foliage‐feeders and rarely likely to encounter aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how T. ni copes with AFB1, we evaluated the toxicity of AFB1 to T. ni caterpillars at different developmental stages and found that AFB1 tolerance significantly increases with larval development. Diet incorporation of AFB1 at 1 μg/g completely inhibited larval growth and pupation of newly hatched larvae, but 3 μg/g AFB1 did not have apparent toxic effects on larval growth and pupation of caterpillars that first consume this compound 10 days after hatching. Piperonyl butoxide, a general inhibitor of cytochrome P450 monooxygenases (P450s), reduced the toxicity of AFB1, suggesting that AFB1 is bioactivated in T. ni and this bioactivation is mediated by P450s. Some plant allelochemicals, including flavonoids such as flavones, furanocoumarins such as xanthotoxin and imperatorin, and furanochromones such as visnagin, that induce P450s in other lepidopteran larvae ameliorated AFB1 toxicity, suggesting that P450s are also involved in AFB1 detoxification in T. ni.