Restoration of Lactobacillus johnsonii and Enterococcus faecalis Caused the Elimination of Tritrichomonas sp. in a Model of Antibiotic-Induced Dysbiosis.

Inflammatory bowel disease (IBD) is a multifactorial disease involving the interaction of the gut microbiota, genes, host immunity, and environmental factors. Dysbiosis in IBD is associated with pathobiont proliferation, so targeted antibiotic therapy is a rational strategy. When restoring the microbiota with probiotics, it is necessary to take into account the mutual influence of co-cultivated microorganisms, as the microbiota is a dynamic community of species that mediates homeostasis and physiological processes in the intestine. The aim of our study was to investigate the recovery efficacy of two potential probiotic bacteria, L. johnsonii and E. faecalis, in Muc2-/- mice with impaired mucosal layer. Two approaches were used to determine the efficacy of probiotic supplementation in mice with dysbiosis caused by mucin-2 deficiency: bacterial seeding on selective media and real-time PCR analysis. The recovery time and the type of probiotic bacteria relocated affected only the number of E. faecalis. A significant positive correlation was found between colony-forming unit (CFU) and the amount of E. faecalis DNA in the group that was replanted with probiotic E. faecalis. As for L. johnsonii, it could be restored to its original level even without any additional bacteria supplementation after two weeks. Interestingly, the treatment of mice with L. johnsonii caused a decrease in the amount of E. faecalis. Furthermore, either L. johnsonii or E. faecalis treatment eliminated protozoan overgrowth caused by antibiotic administration.

Bile promotes Lactobacillus johnsonii N6.2 extracellular vesicle production with conserved immunomodulatory properties.

Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived ßlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.

Beneficial Effect of Heat-Killed Lactic Acid Bacterium Lactobacillus johnsonii No. 1088 on Temporal Gastroesophageal Reflux-Related Symptoms in Healthy Volunteers: A Randomized, Placebo-Controlled, Double-Blind, Parallel-Group Study.

A randomized, placebo-controlled, double-blind, parallel-group clinical study was conducted to examine the effects of ingesting a heat-killed lactic acid bacterium, Lactobacillus johnsonii No. 1088 (LJ88) on temporal gastroesophageal reflux-related symptoms in healthy volunteers. A total of 120 healthy Japanese volunteers of both sexes, aged between 21 and 63 years, whose Frequency Scale for the Symptoms of Gastroesophageal Reflux Disease (FSSG) total score was 8 or greater, but who were not diagnosed with functional dyspepsia according to the Rome IV classification, were enrolled. They were randomly assigned to either the LJ88 or placebo group and instructed to ingest the test food (1 billion heat-killed LJ88 or placebo) once a day for six weeks. Gastroesophageal reflux-related symptoms were evaluated using FSSG scores as a primary endpoint. The Gastrointestinal Symptoms Rating Scale (GSRS), stomach state questionnaire, and serum gastrin concentration were used as secondary endpoints. In the FSSG evaluation, the heartburn score was significantly improved at 6 weeks in the LJ88 group compared to the placebo group. No severe adverse events related to the test food were observed. In conclusion, daily ingestion of heat-killed LJ88 improved temporal heartburn symptoms in non-diseased individuals.

Chronic Stress Dampens Lactobacillus Johnsonii-Mediated Tumor Suppression to Enhance Colorectal Cancer Progression.

Colorectal cancer development and outcome are impacted by modifiable risk factors, including psychologic stress. The gut microbiota has also been shown to be linked to psychologic factors. Here, we found a marked deteriorative effect of chronic stress in multiple colorectal cancer models, including chemically induced (AOM/DSS), genetically engineered (APCmin/+), and xenograft tumor mouse models. RNA sequencing data from colon tissues revealed that expression of stemness-related genes was upregulated in the stressed colorectal cancer group by activated ß-catenin signaling, which was further confirmed by results from ex vivo organoid analyses as well as in vitro and in vivo cell tumorigenicity assays. 16S rRNA sequencing of the gut microbiota showed that chronic stress disrupted gut microbes, and antibiotic treatment and fecal microbiota transplantation abolished the stimulatory effects of chronic stress on colorectal cancer progression. Stressed colorectal cancer mice displayed a significant decrease in Lactobacillus johnsonii (L. johnsonii) abundance, which was inversely correlated with tumor load. Moreover, protocatechuic acid (PCA) was identified as a beneficial metabolite produced by L. johnsonii based on metabolome sequencing and LC/MS-MS analysis. Replenishment of L. johnsonii or PCA blocked chronic stress-induced colorectal cancer progression by decreasing ß-catenin expression. Furthermore, PCA activated the cGMP pathway, and the cGMP agonist sildenafil abolished the effects of chronic stress on colorectal cancer. Altogether, these data identify that stress impacts the gut microbiome to support colorectal cancer progression. SIGNIFICANCE: Chronic stress stimulates cancer stemness by reducing the intestinal abundance of L. johnsonii and its metabolite PCA to enhance ß-catenin signaling, forming a basis for potential strategies to circumvent stress-induced cancer aggressiveness. See related commentary by McCollum and Shah, p. 645.

Lactobacillus johnsonii CPN23 vis-à-vis Lactobacillus acidophilus NCDC15 Improves Gut Health, Intestinal Morphometry, and Histology in Weaned Wistar Rats.

The present investigation was carried out with the aim to establish the comparative efficacy of a canine-sourced probiotic meant for canine feeding and a conventional dairy-sourced probiotic. For this purpose, canine-origin Lactobacillus johnsonii CPN23 and dairy-origin Lactobacillus acidophilus NCDC15 were evaluated for potential probiotics health benefits in the rat model. Forty-eight weaned Wistar rats enrolled in this experiment of 8 weeks were fed a basal diet and divided into three dietary treatments. Rats of group I enrolled as control (CON) were given MRS placebo at 1 mL/head/day, while rats of group II (LAJ) and III (LAC) were administered with overnight MRS broth grown-culture of L. johnsonii CPN23 and L. acidophilus NCDC15, respectively, at 1 mL/head/day (108 cfu/mL). The average daily gain and net gain in body weight were significantly higher (p < 0.05) in LAJ and LAC than in CON. Fecal and digesta biochemical attributes altered (p < 0.05) positively in response to both probiotics. Total fecal and pooled digesta SCFAs were higher (p < 0.05) in both LAJ and LAC than in CON. The microbial population in cecal and colonic digesta responded (p < 0.05) positively to both probiotics. The diameter of intestinal segments was higher (p < 005) in LAJ as compared to CON. The number and height of villi in jejunum tended to be higher in LAJ as compared to CON. The humoral immune response to sheep erythrocytes as well as chicken egg-white lysozyme was higher in LAJ as compared to CON. Overall, the results of the study have demonstrated the effectiveness of the canine-sourced L. johnsonii CPN23 as a potential probiotic, with a comparatively better response than the dairy-sourced L. acidophilus NCDC15. It could thus be recommended for use in feeding dogs to help augment their health.

Fingerprinting and characterization of the polysaccharides from Polygonatum odoratum and the in vitro fermented effects on Lactobacillus johnsonii.

Polygonatum odoratum (Yu-Zhu) can be utilized to treat the digestive and respiratory illness. Previous studies have revealed that the underlying therapeutic mechanism of P. odoratum polysaccharides (POPs) is associated with remodeling the gut microbiota. However, POPs in terms of the chemical composition and fermentation activities have been understudied. Here we developed the three-level fingerprinting approaches to characterize the structures of POPs and probed into the beneficial effects on promoting the growth and fermentation of Lactobacillus johnsonii. POPs were prepared by water decoction followed by alcohol sedimentation, while trifluoroacetic acid under different conditions to prepare the hydrolyzed oligosaccharides and monosaccharides. POPs exhibited three main molecular distribution of 601-620 kDa, 4.12-6.09 kDa, and 3.57-6.02 kDa. Hydrolyzed oligosaccharides with degree of polymerization (DP) 2-13 got primarily characterized by analyzing the rich fragmentation information obtained by hydrophilic interaction chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (HILIC/IM-QTOF-MS). Amongst them, the DP5 oligosaccharide was characterized as 1,6,6-kestopentaose. The molecular ratio of Fru: Ara: Glc: Gal: Xyl was 87.72: 0.30: 11.56: 0.19: 0.23. In vitro fermentation demonstrated that 4.5 mg/mL of POPs could significantly promote the growth of L. johnsonii. Co-cultivated with 4.5 mg/mL of POPs, L. johnsonii exhibited stronger antimicrobial activity against Klebsiella pneumoniae. The concentrations of short-chain fatty acids in the POPs-lactobacilli fermented products, including acetic acid, isobutyric acid, and isovaleric acid, were increased. Conclusively, POPs represent the promising prebiotic candidate to facilitate lactobacilli, which is associated with exerting the health benefits.

Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection.

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein-protein docking modelling suggests the helveticin-J homolog may be able to block VP28-PmRab7 interactions and interrupt WSSV infection.

Propionic Acid Driven by the Lactobacillus johnsonii Culture Supernatant Alleviates Colitis by Inhibiting M1 Macrophage Polarization by Modulating the MAPK Pathway in Mice.

In this study, we investigated the effects of Lactobacillus johnsonii on the mouse colitis model. The results showed that the supernatant of the L. johnsonii culture alleviated colitis and remodeled gut microbiota, represented by an increased abundance of bacteria producing short-chain fatty acids, leading to an increased concentration of propionic acid in the intestine. Further studies revealed that propionic acid inhibited activation of the MAPK signaling pathway and polarization of M1 macrophages. Macrophage clearance assays confirmed that macrophages are indispensable for alleviating colitis through propionic acid. In vitro experiments showed that propionic acid directly inhibited the MAPK signaling pathway in macrophages and reduced M1 macrophage polarization, thereby inhibiting the secretion of pro-inflammatory cytokines. These findings improve our understanding of how L. johnsonii attenuates inflammatory bowel disease (IBD) and provide valuable insights for identifying molecular targets for IBD treatment in the future.

Lactobacillus johnsonii N6.2 phospholipids induce immature-like dendritic cells with a migratory-regulatory-like transcriptional signature.

Shifts in the gut microbiota composition, called dysbiosis, have been directly associated with acute and chronic diseases. However, the underlying biological systems connecting gut dysbiosis to systemic inflammatory pathologies are not well understood. Phospholipids (PLs) act as precursors of both, bioactive inflammatory and resolving mediators. Their dysregulation is associated with chronic diseases including cancer. Gut microbial-derived lipids are structurally unique and capable of modulating host's immunity. Lactobacillus johnsonii N6.2 is a Gram-positive gut symbiont with probiotic characteristics. L. johnsonii N6.2 reduces the incidence of autoimmunity in animal models of Type 1 Diabetes and improves general wellness in healthy volunteers by promoting, in part, local and systemic anti-inflammatory responses. By utilizing bioassay-guided fractionation methods with bone marrow-derived dendritic cells (BMDCs), we report here that L. johnsonii N6.2 purified lipids induce a transcriptional signature that resembles that of migratory (mig) DCs. RNAseq-based analysis showed that BMDCs stimulated with L. johnsonii N6.2 total lipids upregulate maturation-mig related genes Cd86, Cd40, Ccr7, Icam1 along with immunoregulatory genes including Itgb8, Nfkbiz, Jag1, Adora2a, IL2ra, Arg1, and Cd274. Quantitative reverse transcription (qRT)-PCR analysis indicated that PLs are the bioactive lipids triggering the BMDCs response. Antibody-blocking of surface Toll-like receptor (TLR)2 resulted in boosted PL-mediated upregulation of pro-inflammatory Il6. Chemical inhibition of the IKKα kinase from the non-canonical NF-κB pathway specifically restricted upregulation of Il6 and Tnf. Phenotypically, PL-stimulated BMDCs displayed an immature like-phenotype with significantly increased surface ICAM-1. This study provides insight into the immunoregulatory capacity of Gram-positive, gut microbial-derived phospholipids on innate immune responses.

Enhanced tight junction in Caco-2 cells by the pretreatment with Lactobacillus johnsonii strain MG.

We investigated roles of Lactobacillus johnsonii MG (MG) isolated from mice with interaction with tight junction on gut barrier function with Caco-2 cell model. Pretreatment with MG enhanced barrier function and showed protective effect against Enterococcus faecium provided damage. MG treatment increased the gene expressions of transcriptional regulator NFKB and major tight junction protein, ZO-1.

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.

Intranasal administration of Lactobacillus johnsonii attenuates hyperoxia-induced lung injury by modulating gut microbiota in neonatal mice.

BACKGROUND: Supplemental oxygen impairs lung development in newborn infants with respiratory distress. Lactobacillus johnsonii supplementation attenuates respiratory viral infection in mice and exhibits anti-inflammatory effects. This study investigated the protective effects of intranasal administration of L. johnsonii on lung development in hyperoxia-exposed neonatal mice. METHODS: Neonatal C57BL/6N mice were reared in either room air (RA) or hyperoxia condition (85% O2). From postnatal days 0 to 6, they were administered intranasal 10 µL L. johnsonii at a dose of 1 × 105 colony-forming units. Control mice received an equal volume of normal saline (NS). We evaluated the following four study groups: RA + NS, RA + probiotic, O2 + NS, and O2 + probiotic. On postnatal day 7, lung and intestinal microbiota were sampled from the left lung and lower gastrointestinal tract, respectively. The right lung of each mouse was harvested for Western blot, cytokine, and histology analyses. RESULTS: The O2 + NS group exhibited significantly lower body weight and vascular density and significantly higher mean linear intercept (MLI) and lung cytokine levels compared with the RA + NS and RA + probiotic groups. At the genus level of the gut microbiota, the O2 + NS group exhibited significantly higher Staphylococcus and Enterobacter abundance and significantly lower Lactobacillus abundance compared with the RA + NS and RA + probiotic groups. Intranasal L. johnsonii treatment increased the vascular density, decreased the MLI and cytokine levels, and restored the gut microbiota in hyperoxia-exposed neonatal mice. CONCLUSIONS: Intranasal administration of L. johnsonii protects against hyperoxia-induced lung injury and modulates the gut microbiota.

Membrane vesicles from Lactobacillus johnsonii delay osteoarthritis progression via modulating macrophage glutamine synthetase/mTORC1 axis.

AIMS: The manipulation of macrophage recruitment and their shift in the M1/M2 ratio is a promising approach to mitigate osteoarthritis (OA). Nevertheless, the current clinical medication available for OA is only palliative and may result in undesirable outcomes. Hence, it is urgent to explore alternative disease-modifying drug supplement that are both safer and more effective in OA treatment, like probiotic and probiotic-derived membrane vesicles. METHODS: The synovial inflammation and cartilage damage in collagenase-induced OA (CIOA) mice were observed using haematoxylin and eosin, saffron O-solid green and immunohistochemical staining. Bipedal balance test and open field test were conducted to determine the effectiveness of L. johnsonii-derived membrane vesicles (LJ-MVs) in reducing joint pain of CIOA mice. Additionally, Transwell, western blot, and immunological testing were used to examine the effect of LJ-MVs on macrophage migration and reprogramming. Furthermore, a 4D label-free proteomic analysis of LJ-MVs and their parent bacterium was performed, and the glutamine synthetase (GS)/mTORC1 axis in macrophage was verified by western blot. RESULTS: L. johnsonii and its membrane vesicles, LJ-MVs, exhibit a novel ability to mitigate inflammation, cartilage damage, and pain associated with OA. This is achieved by their ability to impede macrophage migration, M1-like polarization, and inflammatory mediators secretion, while simultaneously promoting the M2/M1 ratio in synovial macrophages. The mechanism underlying this effect involves the modulation of macrophage GS/mTORC1 pathway, at least partially. SIGNIFICANCE: Owing to their probiotic derivation, LJ-MVs will be a more dependable and potent disease-modifying drugs for the prevention and therapy of OA in the long run.

Evidence of host specificity in Lactobacillus johnsonii genomes and its influence on probiotic potential in poultry.

To date, the selection of candidate strains for probiotic development in production animals has been largely based upon screens for desired phenotypic traits. However, increasing evidence indicates that the use of host-specific strains may be important, because coevolution with the animal host better prepares a bacterial strain to colonize and succeed in its respective host animal species. This concept was applied to Lactobacillus johnsonii in commercial poultry production because of its previous correlation with enhanced bird performance. Using 204 naturally isolated chicken- and turkey-source L. johnsonii, we demonstrate that there is a strong phylogenetic signal for coevolution with the animal host. These isolates differ phenotypically, even within host source, and these differences can be correlated with certain L. johnsonii phylogenetic clades. In commercial turkey poults, turkey-specific strains with strong in vitro phenotypes performed better early in life than strains lacking those phenotypes. A follow-up performance trial in broiler chickens demonstrated that chicken-specific strains result in better overall bird performance than nonchicken-specific strains. Collectively, this work provides evidence for the impact of host adaptation on a probiotic strain's potential. Furthermore, this top-down approach is useful for screening larger numbers of isolates for probiotic candidates.

Intravaginal injection of Lactobacillus johnsonii may modulates oviductal microbiota and mucosal barrier function of laying hens.

The avian oviduct connects to the gastrointestinal tract through cloaca, where it is exposed to pathogenic bacteria from intestinal contents. Therefore, improvement of mucosal barrier function in the oviduct is important for safe poultry production. Lactic acid bacteria are known to contribute to strengthening the mucosal barrier function in the intestinal tract, and a similar effect is expected in the oviduct mucosa of chickens. This study aimed to clarify the effects of vaginal administration of lactic acid bacteria on the mucosal barrier function of the oviduct. White Leghorn laying hens (500-days old) were intravaginally administered 1 mL of Lactobacillus johnsonii suspension (1 × 105 and 1 × 108 cfu/mL: low concentration of Lactobacillus (LL) and high concentration of Lactobacillus (HL) groups, respectively) or without bacteria (control: C group) for 7 d (n = 6). The oviductal magnum, uterus, and vagina were collected for histological observations and mucosal barrier function-related gene expression analysis. Amplicon sequence analysis of oviductal mucus bacteria was also performed. Eggs were collected during the experimental period and their weight was measured. Vaginally administering L. johnsonii for 7 d caused 1) an increase in α-diversity of vaginal mucosa microbiota with an increase in the abundance ratio of beneficial bacteria and a decrease in pathogenic bacteria, 2) enhanced claudin (CLA) 1 and 3 gene expression in the magnum and vaginal mucosa, and 3) a decrease in avian ß-defensin (AvBD) 10, 11, and 12 gene expression in the magnum, uterus, and vaginal mucosa. These results suggest that transvaginal administration of L. johnsonii contributes to protection against infection in the oviduct by improving the microflora of the oviductal mucosa and strengthening the mechanical barrier function of the tight junctions. In contrast, transvaginal administration of lactic acid bacteria does not enhance the production of AvBD10, 11, and 12 in the oviduct.

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 BFE6154 Ameliorates Diet-Induced Hypercholesterolemia.

The functional characteristics of Lactobacillus johnsonii BFE6154, first isolated from Maasai traditional fermented milk, were previously identified in vitro, but its cholesterol-lowering properties have not been verified yet. In this study, we investigated the effect of L. johnsonii BFE6154 on cholesterol regulation and the mode of action. Stimulation of Caco-2 intestinal epithelial cells with L. johnsonii BFE6154 downregulated the gene expression of Niemann-Pick C1-like 1 (NPC1L1) through the activation of liver X receptor (LXR). Also, stimulation of HepG2 cells with the metabolites produced by L. johnsonii BFE6154 revealed an increase in the gene expression of low-density lipoprotein receptor (LDLR). Oral administration of L. johnsonii BFE6154 in mice receiving a high-fat and high-cholesterol diet (HFHCD), reduced total cholesterol and low-density lipoprotein-cholesterol (LDL) and increased high-density lipoprotein-cholesterol (HDL) in the blood, compared to the control. Diet-induced hypercholesterolemic mice receiving L. johnsonii BFE6154 showed a suppression of cholesterol absorption under the control of NPC1L1 in the intestine. Furthermore, L. johnsonii BFE6154 consumption ameliorated the hepatic cholesterol level and LDLR expression, which was reduced by HFHCD. These molecular modulations led to the increase of cholesterol excretion and the decrease of cholesterol levels in the feces and liver, respectively. Taken together, these results suggest that L. johnsonii BFE6154 may protect against diet-induced hypercholesterolemia through the regulation of cholesterol metabolism in the intestine and liver.

In Vitro Assessment of Lactobacillus crispatus UBLCp01, Lactobacillus gasseri UBLG36, and Lactobacillus johnsonii UBLJ01 as a Potential Vaginal Probiotic Candidate.

In this study, Lactobacillus crispatus UBLCp01, Lactobacillus gasseri UBLG36, and Lactobacillus johnsonii UBLJ01 isolated from the vagina of healthy reproductive age Indian women were screened for beneficial probiotic properties. These strains showed the ability to survive acidic and simulated vaginal fluid conditions and could adhere to mucin. Lact. gasseri UBLG36, and Lact. johnsonii UBLJ01 produced D- and L-lactic acid, whereas Lact. crispatus UBLCp01 produced hydrogen peroxide and D- and L-lactic acid. All strains inhibited the growth of pathogens (Escherichia coli, Gardnerella vaginalis, Proteus mirabilis, and Candida albicans) and were capable of co-aggregating with them with varying degrees. Strains secreted exopolysaccharides and formed biofilms under in vitro conditions. Safety assessment showed that these strains had a usual antibiotic susceptibility profile, did not produce hemolysins, gelatinases, and mucin degrading enzymes. Based on strain characteristics and beneficial properties, we believe that these strains are promising candidates for human trials to confirm their ability to prevent/treat vaginal dysbiosis and maintain a healthy vaginal eco-system.

Poly-ß-hydroxybutyrate alleviated diarrhea and colitis via Lactobacillus johnsonii biofilm-mediated maturation of sulfomucin.

Maintainance of sulfomucin is a key end point in the treatment of diarrhea and inflammatory bowel disease (IBD). However, the mechanisms underlying the microbial sense to sulfomucin are poorly understood, and to date, there are no therapies targeting the secretion and maturation of sulfomucin in IBD. Herein, we biosynthesized poly-ß-hydroxybutyrate (PHB) and found that PHB could alleviate inflammation caused by diarrhea and colitis by enhancing the differentiation of sulfomucin. Microbiota transplantation and clearance together demonstrate that PHB promoting sulfomucin is mediated by Lactobacillus johnsonii (L. johnsonii). Further studies revealed that PHB provides a favorable niche for L. johnsonii biofilm formation to resist disturbance and support its growth. L. johnsonii-biofilm alleviates colitis by regulating fucose residues to promote goblet cell differentiation and subsequent sulfomucin maturation. Importantly, PHB alleviates colitis by enhancing sulfomucin secretion and maturation in a L. johnsonii-dependent manner. PHB represents a class of guardians, acting as a safe probiotic-biofilm delivery system that significantly promotes probiotic proliferation. Altogether, this study adds weight to the possible role of probiotics and functional materials in the treatment of intestinal inflammation. The application of PHB and biofilm self-coating L. johnsonii carries high translational potential and may be of clinical relevance.