Blocked conversion of Lactobacillus johnsonii derived acetate to butyrate mediates copper-induced epithelial barrier damage in a pig model.

BACKGROUND: High-copper diets have been widely used to promote growth performance of pigs, but excess copper supplementation can also produce negative effects on ecosystem stability and organism health. High-copper supplementation can damage the intestinal barrier and disturb the gut microbiome community. However, the specific relationship between high-copper-induced intestinal damage and gut microbiota or its metabolites is unclear. OBJECTIVE: Using fecal microbiota transplantation and metagenomic sequencing, responses of colonic microbiota to a high-copper diet was profiled. In addition, via comparison of specific bacteria and its metabolites rescue, we investigated a network of bacteria-metabolite interactions involving conversion of specific metabolites as a key mechanism linked to copper-induced damage of the colon. RESULTS: High copper induced colonic damage, Lactobacillus extinction, and reduction of SCFA (acetate and butyrate) concentrations in pigs. LefSe analysis and q-PCR results confirmed the extinction of L. johnsonii. In addition, transplanting copper-rich fecal microbiota to ABX mice reproduced the gut characteristics of the pig donors. Then, L. johnsonii rescue could restore decreased SCFAs (mainly acetate and butyrate) and colonic barrier damage including thinner mucus layer, reduced colon length, and tight junction protein dysfunction. Given that acetate and butyrate concentrations exhibited a positive correlation with L. johnsonii abundance, we investigated how L. johnsonii exerted its effects by supplementing acetate and butyrate. L. johnsonii and butyrate administration but not acetate could correct the damaged colonic barrier. Acetate administration had no effects on butyrate concentration, indicating blocked conversion from acetate to butyrate. Furthermore, L. johnsonii rescue enriched a series of genera with butyrate-producing ability, mainly Lachnospiraceae NK4A136 group. CONCLUSIONS: For the first time, we reveal the microbiota-mediated mechanism of high-copper-induced colonic damage in piglets. A high-copper diet can induce extinction of L. johnsonii which leads to colonic barrier damage and loss of SCFA production. Re-establishment of L. johnsonii normalizes the SCFA-producing pathway and restores colonic barrier function. Mechanistically, Lachnospiraceae NK4A136 group mediated conversion of acetate produced by L. johnsonii to butyrate is indispensable in the protection of colonic barrier function. Collectively, these findings provide a feasible mitigation strategy for gut damage caused by high-copper diets. Video Abstract.

The PKA-SREBP1c Pathway Plays a Key Role in the Protective Effects of Lactobacillus johnsonii JNU3402 Against Diet-Induced Fatty Liver in Mice.

SCOPE: The present study aims to assess the protective effect of Lactobacillus johnsonii JNU3402 (LJ3402) against diet-induced non-alcoholic fatty liver disease (NAFLD) and determine the mechanism underlying its beneficial effect on the liver in mice. METHODS AND RESULTS: Seven-week-old male mice are fed a high-fat diet (HFD) with or without oral supplementation of LJ3402 for 14 weeks. In mice fed an HFD, LJ3402 administration alleviates liver steatosis, diet-induced obesity, and insulin resistance with a decreased hepatic expression of sterol-regulatory element-binding protein-1c (SREBP-1c), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), and an increased phosphorylation of SREBP-1c. The mechanistic study shows that LJ3402 inhibits SREBP-1c transcriptional activity by enhancing protein kinase A (PKA)-mediated phosphorylation and reduces the expression of its lipogenic target genes in AML12 and HepG2 cells, thereby attenuating hepatic lipid accumulation. Moreover, silencing the PKA α catalytic subunit or the inhibition of PKA activity by H89 abolishes LJ3402 suppression of free fatty acid (FFA)-induced SREBP-1c activity in hepatocytes. In addition, LJ3402 administration elevates the plasma lactate levels in mice fed an HFD; this lactate increases PKA-mediated SREBP-1c phosphorylation in AML12 cells with a decreased expression of its target genes, reducing hepatic lipid accumulation. CONCLUSION: LJ3402 attenuates HFD-induced fatty liver in mice through the lactate-PKA-SREBP-1c pathway.

Lactobacillus johnsonii L531 Ameliorates Salmonella enterica Serovar Typhimurium Diarrhea by Modulating Iron Homeostasis and Oxidative Stress via the IRP2 Pathway.

Salmonella enterica serovar Typhimurium (S. Typhimurium) has evolved mechanisms to evade the host's nutritional immunity and thus promote bacterial growth by using the iron in the host. However, the detailed mechanisms of S. Typhimurium induce dysregulation of iron homeostasis and whether Lactobacillus johnsonii L531 can alleviate the iron metabolism disorder caused by S. Typhimurium has not been fully elucidated. Here, we show that S. Typhimurium activated the expression of iron regulatory protein 2 (IRP2), transferrin receptor 1, and divalent metal transporter protein 1 and suppressed the expression of iron exporter ferroportin, which resulted in iron overload and oxidative stress, inhibiting the key antioxidant proteins NF-E2-related factor 2, Heme Oxygenase-1, and Superoxide Dismutase in vitro and in vivo. L. johnsonii L531 pretreatment effectively reversed these phenomena. IRP2 knockdown inhibited iron overload and oxidative damage induced by S. Typhimurium in IPEC-J2 cells, while IRP2 overexpression promoted iron overload and oxidative damage caused by S. Typhimurium. Interestingly, the protective effect of L. johnsonii L531 on iron homeostasis and antioxidant function was blocked following IRP2 overexpression in Hela cells, demonstrating that L. johnsonii L531 attenuates disruption of iron homeostasis and consequent oxidative damage caused by S. Typhimurium via the IRP2 pathway, which contributes to the prevention of S. Typhimurium diarrhea in mice.

Lactobacillus johnsonii enhances the gut barrier integrity via the interaction between GAPDH and the mouse tight junction protein JAM-2.

Commensal intestinal microbiota interacts with gut epithelial cells in the host by binding to specific host receptors. Several pattern recognition receptors on the gut that sense conserved microbial-associated molecular patterns have been reported; however, many of the gut receptor molecules involved in bacterial binding have not yet been identified. In this study, commensal intestinal bacteria interacting with mouse gut surface proteins were screened from fecal bacterial samples, to identify novel receptors on the epithelial cells in the mouse gut. Among the screened intestinal lactic acid bacteria, the frequently isolated Lactobacillus johnsonii MG was used for the purification of gut receptor proteins. An approximately 30 kDa protein was purified using affinity resin coupled surface layer proteins isolated from L. johnsonii MG. The purified gut protein was identified as a member of the tight junction protein family, junctional adhesion molecule-2 (JAM-2). As expected, the tight junctions of Caco-2 cells damaged by H2O2 were repaired by incubation with L. johnsonii MG. RNA sequence analysis showed significant upregulation of the expression of genes for tight junctions, anti-inflammatory effects, transcriptional regulation, and apoptosis in Caco-2 cells, following L. johnsonii MG treatment. In L. johnsonii MG, the surface layer 40 kDa protein was purified with gut protein-coupled affinity resin and identified as the moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These results suggest that L. johnsonii MG promotes the barrier function integrity in Caco-2 cells via GAPDH-JAM-2 binding. Here, we propose a promising approach to identify novel gut receptor molecules based on commensal bacterial interactions and understand host-bacterial communication in a mouse model.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

Lactobacillus johnsonii 3-1 and Lactobacillus crispatus 7-4 promote the growth performance and ileum development and participate in lipid metabolism of broilers.

Long-term use of antibiotic growth promoter (AGP) in animal production is the main cause of antimicrobial resistance of pathogenic bacteria. Therefore, seeking alternatives to AGP is crucial for animal husbandry. Among all AGP alternatives, probiotics are promising candidates. In this study, two strains of lactic acid bacteria, L. johnsonii 3-1 and L. crispatus 7-4, were isolated from the feces of wild Gallus gallus, which exhibited obvious anti-pathogenic activity and improved the growth performance of broilers. Furthermore, we found that these two strains participated in the lipid metabolism of broilers by reducing the content of TC and TG in ileal epithelial cells and up-regulating the liver AMPKα/PPARα/CPT-1 pathway, which affects abdominal fat deposition. In summary, L. johnsonii 3-1 and L. crispatus 7-4 have the potential to be used as AGP substitutes and participate in the lipid metabolism of broilers to reduce abdominal fat deposition. Importantly, our study reveals for the first time that L. crispatus participates in liver lipid metabolism to reduce abdominal fat deposition in broilers.

Effect of the administration of Lactobacillus spp. strains on neonatal diarrhoea, immune parameters and pathogen abundance in pre-weaned calves.

Neonatal calf diarrhoea is one of the challenges faced by intensive farming, and probiotics are considered a promising approach to improve calves' health. The objective of this study was to evaluate the effect of potential probiotic lactobacilli on new-born dairy calves' growth, diarrhoea incidence, faecal score, cytokine expression in blood cells, immunoglobulin A (IgA) levels in plasma and faeces, and pathogen abundance in faeces. Two in vivo assays were conducted at the same farm in two annual calving seasons. Treated calves received one daily dose of the selected lactobacilli (Lactobacillus reuteri TP1.3B or Lactobacillus johnsonii TP1.6) for 10 consecutive days. A faecal score was recorded daily, average daily gain (ADG) was calculated, and blood and faeces samples were collected. Pathogen abundance was analysed by absolute qPCR in faeces using primers directed at Salmonella enterica, rotavirus, coronavirus, Cryptosporidium parvum and three Escherichia coli virulence genes (eae, clpG and Stx1). The faecal score was positively affected by the administration of both lactobacilli strains, and diarrhoea incidence was significantly lower in treated calves. No differences were found regarding ADG, cytokine expression, IgA levels and pathogen abundance. Our findings showed that oral administration of these strains could improve gastrointestinal health, but results could vary depending on the calving season, which may be related to pathogen seasonality and other environmental effects.

Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.

Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species-Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal B. pseudolongum modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A2A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies.

Bacteroides thetaiotaomicron and Lactobacillus johnsonii modulate intestinal inflammation and eliminate fungi via enzymatic hydrolysis of the fungal cell wall.

Alterations to the gut microbiota can cause an amplification of the inflammatory response to intestinal pathogens. We assessed the effect of Bacteroides thetaiotaomicron and Lactobacillus johnsonii on the elimination of Candida species and whether restoration of these two anaerobic bacteria could attenuate the development of colitis in mice. In this study, L. johnsonii and B. thetaiotaomicron interacted directly with Candida species and induced a degradation of the fungal cell wall, mediated via chitinase-like and mannosidase-like activities, which promoted the inhibition of Candida species growth. In the DSS-induced colitis model, oral administration of L. johnsonii and B. thetaiotaomicron to mice reduced the overgrowth of Escherichia coli, Enterococcus faecalis and Candida glabrata populations and resulted in a significant reduction in inflammatory parameters. L. johnsonii and B. thetaiotaomicron decreased pro-inflammatory mediators and enhanced the anti-inflammatory cytokine response with high TLR9 expression and chitinase-like protein-1 activation, which promoted the elimination of C. glabrata from the gut. Overall, these findings provide evidence that L. johnsonii and B. thetaiotaomicron decrease the development of colitis mediated by TLR9 and promote the elimination of C. glabrata from the gut via chitinase-like and mannosidase-like activities.

Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism.

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsoniiIMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

Transformation kinetics of fermented milk using Lactobacillus casei (Lc1) and Streptococcus thermophilus: comparison of results with other Inocula.

Probiotic-based starter cultures are generally used to produce fermented milks with improved characteristics in the final product. In this study, Lactobacillus casei and Streptococcus thermophilus (Lc1-St) were used as the starter inoculum. The transformation kinetics and properties of the final product were compared with systems produced with other inocula. The Lc1-St inoculum delayed the production of lactic acid from 40 to 70 min (depending on temperature and concentration) when compared to Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus (Lb-St) and Lactobacillus johnsonii and Streptococcus thermophilus (La1-St). The Lc1-St inoculum reached the aggregation system faster (30-80 min) than Lb-St (120-210 min) and La1-St (160-220 min), however, the production of exopolysaccharides and organic phosphates was delayed as a consequence of the lack of synergy between Lc1 and St.

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked ßGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.

Structural robustness of the gut mucosal microbiota is associated with Crohn's disease remission after surgery.

OBJECTIVES: Preventing postoperative recurrence after ileocolonic resection (ICR) for Crohn's disease (CD) is challenging. Defining the disturbances of the microbial composition and community structure after ICR and their link with early disease recurrence is crucial. DESIGN: Microbiota composition (fingerprinting and 16S rDNA sequencing) and community structure (correlation networks of bacterial species) were assessed from ileal mucosa sampled in 20 patients undergoing ICR and 6 months later during endoscopy from above (neoterminal ileum) and below (subanastomotic colon) the surgical anastomosis. RESULTS: ICR had a dramatic effect on gut microbial ecosystem. At surgery, CD mucosa harboured a dysbiotic microbiota with high proportions of α/ß Proteobacteria and Bacilli. Six months later, half of the patients had recurrent lesions at ileocolonoscopy and presented higher numbers of Lachnospiraceae. Recurrence of endoscopic lesions was associated with enrichment in Enterococcus durans while patients in remission had increased proportions of Dorea longicatena and Bacteroides plebeius. Structural differences were striking between recurrence and remission microbiota; while the microbiota of patients with CD recurrence exhibited a loose community structure, the microbiota of patients in remission displayed communities that were robustly correlated to each other. Microbiota colonising the neoterminal ileum and subanastomotic colon 6 months after ICR only differed in patients with recurrence. CONCLUSIONS: ICR modifies the gut microbiome. Remission after 6 months was associated with homogenous bacterial distribution around the anastomosis. Community structure and bacterial networks highlight target species, including Faecalibacterium prausnitzii and Ruminococcus gnavus, which may allow precise modulations of the overall microbial ecosystem towards remission pattern.