Novel LysM motifs for antigen display on lactobacilli for mucosal immunization.

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.

Probiotic Lactobacillus fermentum strain JDFM216 stimulates the longevity and immune response of Caenorhabditis elegans through a nuclear hormone receptor.

Here, we examined the functionality of Lactobacillus fermentum strain JDFM216, a newly isolated probiotic bacterium, using a Caenorhabditis elegans model. We determined bacterial colonization in the intestinal tract of C. elegans by plate counting and transmission electron microscopy and examined the survival of C. elegans using a solid killing assay. In addition, we employed DNA microarray analysis, quantitative real time-polymerase chain reaction, and immunoblotting assays to explore health-promoting pathways induced by probiotic bacteria in C. elegans. Initially, we found that the probiotic bacterium L. fermentum strain JDFM216 was not harmful to the C. elegans host. Conditioning with JDFM216 led to its colonization in the nematode intestine and enhanced resistance in nematodes exposed to food-borne pathogens, including Staphylococcus aureus and Escherichia coli O157:H7. Interestingly, this probiotic strain significantly prolonged the life span of C. elegans. Whole-transcriptome analysis and transgenic worm assays revealed that the health-promoting effects of JDFM216 were mediated by a nuclear hormone receptor (NHR) family and PMK-1 signaling. Taken together, we described a new C. elegans-based system to screen novel probiotic activity and demonstrated that preconditioning with the probiotic L. fermentum strain JDFM216 may positively stimulate the longevity of the C. elegans host via specific pathway.

Differential intestinal anti-inflammatory effects of Lactobacillus fermentum and Lactobacillus salivarius in DSS mouse colitis: impact on microRNAs expression and microbiota composition.

SCOPE: To compare the intestinal anti-inflammatory effects of two probiotics Lactobacillus fermentum and Lactobacillus salivarius in mouse colitis, focusing on their impact on selected miRNAs and microbiota composition. METHODS AND RESULTS: Male C57BL/6J mice were randomly assigned to four groups (n = 10): non-colitic, DSS colitic and two colitic groups treated with probiotics (5 × 108 CFU/mouse/day). Both probiotics ameliorated macroscopic colonic damage. They improved the colonic expression of markers involved in the immune response, and the expression of miR-155 and miR-223. L. fermentum also restored miR-150 and miR-143 expression, also linked to the preservation of the intestinal barrier function. Besides, these beneficial effects were associated with the amelioration of the microbiota dysbiosis and a recovery of the SCFAs- and lactic acid-producing bacterial populations, although only L. fermentum improved Chao richness, Pielou evenness and Shannon diversity. Moreover, L. fermentum also restored the Treg cell population in MLNs and the Th1/Th2 cytokine balance. CONCLUSION: Both probiotics exerted intestinal anti-inflammatory effects in DSS-mouse colitis, maybe due to their ability to restore the intestinal microbiota homeostasis and modulate the immune response. L. fermentum showed a greater beneficial effect compared to L. salivarius, which makes it more interesting for future studies.

Genome Characteristics of Lactobacillus fermentum Strain JDFM216 for Application as Probiotic Bacteria.

Lactobacillus fermentum strain JDFM216, isolated from a Korean infant feces sample, possesses the ability to enhance the longevity and immune response of a Caenorhabditis elegans host. To explore the characteristics of strain JDFM216 at the genetic level, we performed whole-genome sequencing using the PacBio system. The circular draft genome has a total length of 2,076,427 bp and a total of 2,682 encoding sequences were identified. Five phylogenetically featured genes possibly related to the longevity and immune response of the host were identified in L. fermentum strain JDFM216. These genes encode UDP-N-acetylglucosamine 1-carboxyvinyltransferase (E.C. 2.5.1.7), ErfK/YbiS/YcfS/YnhG family protein, site-specific recombinase XerD, homocysteine S-methyltransferase (E.C. 2.1.1.10), and aspartate-ammonia ligase (E.C. 6.3.1.1), which are involved in peptidoglycan synthesis and amino acid metabolism in the gut environment. Our findings on the genetic background of L. fermentum strain JDFM216 and its potential candidate genes for host longevity and immune response provide new insight for the application of this strain in the food industry as newly isolated functional probiotic.

NOD1 in the modulation of host-microbe interactions and inflammatory bone resorption in the periodontal disease model.

Periodontitis is a chronic inflammatory condition characterized by destruction of non-mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram-negative microorganisms. Nucleotide-binding oligomerization domain containing 1 (NOD1) is a member of the Nod-like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram-negative bacteria. This study assessed the role of NOD1 in inflammation-mediated tissue destruction in the context of host-microbe interactions. We used mice with whole-genome deletion of the NOD1 gene in a microbe-induced periodontitis model using direct injections of heat-killed Gram-negative or Gram-negative/Gram-positive bacteria on the gingival tissues. In vitro experiments using primary bone-marrow-derived macrophages from wild-type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram-negative and Gram-negative/Gram-positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram-negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram-positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat-killed Gram-negative bacteria induced the same biological processes in wild-type and NOD1-deficient cells; however, expression of pro-inflammatory mediators was increased in NOD1-deficient cells. These results suggest a bone-sparing role for NOD1 in this model.

Biofilms of Lactobacillus plantarum and Lactobacillus fermentum: Effect on stress responses, antagonistic effects on pathogen growth and immunomodulatory properties.

Few studies have extensively investigated probiotic functions associated with biofilms. Here, we show that strains of Lactobacillus plantarum and Lactobacillus fermentum are able to grow as biofilm on abiotic surfaces, but the biomass density differs between strains. We performed microtiter plate biofilm assays under growth conditions mimicking to the gastrointestinal environment. Osmolarity and low concentrations of bile significantly enhanced Lactobacillus spatial organization. Two L. plantarum strains were able to form biofilms under high concentrations of bile and mucus. We used the agar well-diffusion method to show that supernatants from all Lactobacillus except the NA4 isolate produced food pathogen inhibitory molecules in biofilm. Moreover, TNF-α production by LPS-activated human monocytoid cells was suppressed by supernatants from Lactobacillus cultivated as biofilms but not by planktonic culture supernatants. However, only L. fermentum NA4 showed anti-inflammatory effects in zebrafish embryos fed with probiotic bacteria, as assessed by cytokine transcript level (TNF-α, IL-1ß and IL-10). We conclude that the biofilm mode of life is associated with beneficial probiotic properties of lactobacilli, in a strain dependent manner. Those results suggest that characterization of isolate phenotype in the biofilm state could be additional valuable information for the selection of probiotic strains.

Amelioration of ovalbumin induced allergic symptoms in Balb/c mice by potentially probiotic strains of lactobacilli.

To evaluate the antiallergic effect of newly characterised probiotic strains, Lactobacillus fermentum NWS29, Lactobacillus casei NWP08 and Lactobacillus rhamnosus NWP13, mice were divided into six experimental groups: control, ovalbumin (OVA), NWS29, NWP08, NWP13 and L. rhamnosus GG (LGG). Mice were immunised and probiotics were administered via oral gavage followed by challenge with OVA. After last challenge with OVA, inflammatory cells in bronchoalveolar lavage fluid (BALF), recruitment of inflammatory cells in airways and OVA-specific immunoglobulin E (IgE) in serum were determined by Giemsa, haematoxylin and eosin (HE) staining, and ELISA, respectively. Relative mRNA expression of interleukins (IL-4, IL-5, IL-10, IL-13 and IL-17), transforming growth factor-ß (TGF-ß) and interferon-γ (IFN-γ) in lung and spleen tissue was determined by real time RT-PCR. OVA-specific IgE levels, recruitment of eosinophils and mRNA expressions of inflammatory cytokines were remarkably increased in OVA-exposed mice compared with the control group. Administration of NWS29 and NWP13 suppressed inflammatory cell infiltration in airways and BALF, and level of OVA-specific IgE in serum of OVA-exposed mice. Furthermore, NWS29 and NWP13 also abrogated the mRNA expression of 1L-4, IL-5, IL-13 and TGF-ß in mice immunised and exposed to OVA. Our findings suggest that NWS29 and NWP13 might be good candidates for the prevention of allergic airway inflammation.

Heat-killed probiotic bacteria differentially regulate colonic epithelial cell production of human ß-defensin-2: dependence on inflammatory cytokines.

The inducible antimicrobial peptide human ß-defensin-2 (hBD-2) stimulated by pro-inflammatory cytokines and bacterial products is essential to antipathogen responses of gut epithelial cells. Commensal and probiotic bacteria can augment such mucosal defences. Probiotic use in the treatment of inflammatory bowel disease, however, may have adverse effects, boosting inflammatory responses. The aim of this investigation was to determine the effect of selected probiotic strains on hBD-2 production by epithelial cells induced by pathologically relevant pro-inflammatory cytokines and the role of cytokine modulators in controlling hBD-2. Caco-2 colonic intestinal epithelial cells were pre-incubated with heat-killed probiotics, i.e. Lactobacillus casei strain Shirota (LcS) or Lactobacillus fermentum strain MS15 (LF), followed by stimulation of hBD-2 by interleukin (IL)-1ß and tumour necrosis factor alpha (TNF-α) in the absence or presence of exogenous IL-10 or anti-IL-10 neutralising antibody. Cytokines and hBD-2 mRNA and protein were analysed by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. LcS augmented IL-1ß-induced hBD-2, whereas LF enhanced TNF-α- and suppressed IL-1ß-induced hBD-2. LF enhanced TNF-α-induced TNF-α and suppressed IL-10, whereas augmented IL-1ß-induced IL-10. LcS upregulated IL-1ß-induced TNF-α mRNA and suppressed IL-10. Endogenous IL-10 differentially regulated hBD-2; neutralisation of IL-10 augmented TNF-α- and suppressed IL-1ß-induced hBD-2. Exogenous IL-10, however, suppressed both TNF-α- and IL-1ß-induced hBD-2; LcS partially rescued suppression in TNF-α- and IL-1ß-stimulation, whereas LF further suppressed IL-1ß-induced hBD-2. It can be concluded that probiotic strains differentially regulate hBD-2 mRNA expression and protein secretion, modulation being dictated by inflammatory stimulus and resulting cytokine environment.

Lactobacillus fermentum CJL-112 protects mice against influenza virus infection by activating T-helper 1 and eliciting a protective immune response.

We have previously reported that nasally administered Lactobacillus fermentum CJL-112 (CJL-112) efficiently improves resistance against lethal influenza infection in both mice and chicken. The aim of the present study was to understand the underlying mechanisms of the significant anti-influenza activity of this lactobacilli strain. In vitro, co-culturing of the chicken macrophage cell line HD-11 with CJL-112 significantly increased nitric oxide (NO) production. In vivo, CJL-112 was nasally administered to BALB/c mice for 21 days prior to influenza A/NWS/33 (H1N1) virus (IFV) infection. Significant up-regulation of T-helper 1 (Th1) cytokines (IL-2, IFN-γ) was observed, while the levels of T-helper 2 (Th2) cytokines (IL-4, IL-5, IL-10) was either reduced or unchanged than that in control mice were. Furthermore, IgA and specific anti-influenza IgA levels increased significantly in the treated mice than those in untreated mice. Therefore, CJL-112 likely protects the mice against lethal IFV infection via stimulation of macrophages, activation of Th1 and augmentation of IgA production, when directly delivered into the respiratory tract.

Strain dependent protection conferred by Lactobacillus spp. administered orally with a Salmonella Typhimurium vaccine in a murine challenge model.

Consumption of Lactobacillus spp. has been shown to enhance immune responses in mice. This study examined the immuno-adjuvant capacity of two strains: Lactobacillus acidophilus L10 and Lactobacillus fermentum PC2, in the induction of protective humoral immunity in a Salmonella Typhimurium vaccine challenge model. Briefly, BALB/c mice were divided into four groups. Three groups of mice received S. Typhimurium vaccine (10(8) colony forming units (CFU) per dose) on days 0 and 14. In addition to the vaccine, five doses (10(8) CFU per dose) of either L. acidophilus L10 or L. fermentum PC2 were also administered to a group. All mice were challenged with viable S. Typhimurium on day 28. On day 10 post challenge, the study was terminated and microbial and immunological parameters were assessed. Mice dosed with L. fermentum PC2 in addition to the vaccine had a significantly enhanced S. Typhimurium humoral response. The mice in this group had high levels of lactobacilli in the feces and in association with the Peyer's patches, no detectable levels of either lactobacilli or S. Typhimurium in the spleen, and no detectable weight loss. Mice given L. acidophilus L10 with the vaccine were unable to exhibit elevated S. Typhimurium specific humoral responses. However, there was no detectable S. Typhimurium in the spleens of this group. Interestingly, translocation of lactobacilli into the spleen was observed as well as a slight weight loss was noted in mice that received the L. acidophilus L10 with the vaccine. This study shows that, the L. fermentum PC2 had a greater capacity than the L. acidophilus L10 to act as an oral adjuvant in a S. Typhimurium oral vaccine program and afforded greater protection against a live S. Typhimurium challenge.

Adherent properties and macrophage activation ability of 3 strains of lactic acid bacteria.

Lactic acid bacteria (LAB) must possess probiotic properties in order to be beneficial to humans and animals. The adherent properties, the acid and bile tolerance as well as the macrophage activation ability of isolated LAB strains were investigated in this study. The adhesion was analyzed following heat, acid, trypsin, and sodium periodate treatments. Production of the cytokine tumor necrosis factor-α (TNF-α) by RAW 264.7 macrophages was also measured after stimulation with heat-killed LAB strains. The viable strains of Lactobacillus fermentum AF7, L. acidophilus GG5, and L. plantarum BB9 were able to tightly adhere to the intestinal Caco-2 cells. In addition, the GG5 strain was not affected by heating, acid, trypsin, or sodium periodate treatment. However, the adhesion of strains AF7 and BB9 was reduced significantly by heating and trypsin treatment. This result suggested the GG5 and AF7 or BB9 strains had different cell-surface adherent factors. TNF-α production by the RAW 264.7 macrophages was induced significantly following stimulation with heat-killed LAB at 10(8) CFU/mL in a dose-dependent fashion. In addition, macrophage activity was similar whether the treatment consisted of live probiotics, or probiotics treated with heat, acid, or trypsin. However, the activity was reduced after treating with sonication. These in vitro results showed that the LAB studied possess probiotic characteristics, such as acid or bile tolerance, adherent capability, and immune activation, and may suggest that these LAB strains retain their probiotic activity as they pass through the gastrointestinal tract.

Integrative expression system for delivery of antibody fragments by lactobacilli.

A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection.

Oral intake of Lactobacillus fermentum CECT5716 enhances the effects of influenza vaccination.

OBJECTIVE: We studied the coadjuvant capability of oral consumption of the breast-milk-isolated strain Lactobacillus fermentum (CECT5716) for an anti-influenza vaccine. METHODS: A randomized, double-blinded, placebo-controlled human clinical trial including 50 volunteers (31 male and 19 female) was performed to address the immunologic effects of an intramuscular anti-influenza vaccine in adults (33.0 +/- 7.7 y old). Fifty percent of volunteers received an oral daily dose of methylcellulose (placebo) or probiotic bacteria (1 x 10(10) colony-forming units/d) 2 wk before vaccination and 2 wk after vaccination. RESULTS: Two weeks after vaccination there was an increase in the proportion of natural killer cells in the probiotic group but not in the placebo group. The vaccination induced an increase in T-helper type 1 cytokine concentrations and in T-helper and T-cytotoxic proportions in both groups; however, the probiotic group showed a significant higher induction in some of these parameters. Regarding the humoral effects, induction of antibody response in the placebo group could not be detected. In the case of the probiotic group, a significant increase in antigen specific immunoglobulin A was detected. Although an increase in total immunoglobulin M was observed, changes in anti-influenza antigen specific immunoglobulin M were not observed. The incidence of an influenza-like illness during 5 mo after vaccination (October to February) was lower in the group consuming the probiotic bacteria. CONCLUSION: Oral administration of the strain L. fermentum CECT5716 potentates the immunologic response of an anti-influenza vaccine and may provide enhanced systemic protection from infection by increasing the T-helper type 1 response and virus-neutralizing antibodies.

Effects of commensal bacteria on intestinal morphology and expression of proinflammatory cytokines in the gnotobiotic pig.

A germ-free neonatal pig model was established to determine the effects of bacterial colonization by different species on small intestinal morphology and proinflammatory cytokine gene expression. Two experimental groups of 16 pigs were aseptically delivered by cesarian section and allocated into 4 gnotobiotic isolators. Pigs were either maintained germ-free (GF), or were orally inoculated with either a single strain of nonpathogenic Escherichia coli (EC) or Lactobacillus fermentum (LF) or conventionalized with adult porcine feces (CV). After 13 days tissue samples were collected at 5 regions corresponding to 5%, 25%, 50%, 75%, and 95% of the small intestine (SI) length. In Experiment 2, the GF isolator became contaminated with Staphylococcus epidermidis (SE). In general, intestinal responses to bacterial colonization were similar among GF, LF, and SE pigs, and intestinal responses in EC pigs were more similar to CV pigs. Responses to bacterial colonization were most pronounced in the distal SI regions (50%-95%), suggesting that nonmicrobial factors may be more important in the proximal SI. Relative to CV pigs, the distal intestines of GF, LF, and SE pigs were characterized by long villi, shallow crypts, increased relative intestinal mass, and decreased lamina propria cellularity, whereas SI morphology was intermediate in EC pigs. Relative expression of proinflammatory cytokines interleukin-1beta (IL-1beta ) and IL-6 generally increased distally in the SI and was highest in EC and CV pigs. We observed regional variation in SI morphology and proinflammatory cytokine expression, which differed with bacterial species. This study demonstrates that bacterial species differentially affect intestinal morphology and expression of proinflammatory cytokines and suggests that neonatal bacterial colonization patterns may have long-term effects on intestinal health and development.

Importance of the host specificity in the selection of probiotic bacteria.

The gastrointestinal tract is a complex and dynamic ecosystem. Commensal microorganisms (C), which proliferate in the intestine from birth, are crucial for gut homeostasis while non commensal (NC) microorganisms are transient and enter the organism from the environment and foods. We studied comparatively the influence of oral administration of C and NC Lactobacillus fermentum and Lactobacilus acidophilus on the gut-associated lymphoid tissue (GALT) of conventional mice. To determine the importance of the selection of probiotic host-specificity bacteria with immunomodulating capacity, we examined the interaction with the gut by transmission electron microscopy and FITC-labelled bacteria. We compared the immunomodulation capacities of C and NC strains by studying the number of IgA secreting cells and cytokine profile. No differences were found in the number of IgA+ cells; however, the pattern of cytokine response to C and NC bacteria was different. With regard to proinflammatory cytokine (IFNgamma and TNFalpha), we found that TNFalpha was mainly produced by NC bacteria, while C bacteria were able to elicit mainly IFNgamma. The regulatory cytokines (IL-10 and IL-4) were induced with different patterns for both C and NC strains. No differences in the pathway of internalization to the gut between C and NC were found. In summary, we determined that C and NC bacteria interact with the intestine in the same way; both C and NC bacteria were able to reinforce the surveillance of the gut mucosal immune system. The cytokine profile showed that C bacteria would be involved in the regulation of intestinal homeostasis rather than in the immune activation as the NC bacteria.