Exopolysaccharide is the potential effector of Lactobacillus fermentum PS150, a hypnotic psychobiotic strain.

Psychobiotics are a class of probiotics that confer beneficial effects on the mental health of the host. We have previously reported hypnotic effects of a psychobiotic strain, Lactobacillus fermentum PS150 (PS150), which significantly shortens sleep latency in experimental mice, and effectively ameliorate sleep disturbances caused by either caffeine consumption or a novel environment. In the present study, we discovered a L. fermentum strain, GR1009, isolated from the same source of PS150, and found that GR1009 is phenotypically distinct but genetically similar to PS150. Compared with PS150, GR1009 have no significant hypnotic effects in the pentobarbital-induced sleep test in mice. In addition, we found that heat-killed PS150 exhibited hypnotic effects and altered the gut microbiota in a manner similar to live bacteria, suggesting that a heat-stable effector, such as exopolysaccharide (EPS), could be responsible for these effects. Our comparative genomics analysis also revealed distinct genetic characteristics in EPS biosynthesis between GR1009 and PS150. Furthermore, scanning electron microscopy imaging showed a sheet-like EPS structure in PS150, while GR1009 displayed no apparent EPS structure. Using the phenol-sulfate assay, we found that the sugar content value of the crude extract containing EPS (C-EPS) from PS150 was approximately five times higher than that of GR1009, indicating that GR1009 has a lower EPS production activity than PS150. Through the pentobarbital-induced sleep test, we confirmed the hypnotic effects of the C-EPS isolated from PS150, as evidenced by a significant reduction in sleep latency and recovery time following oral administration in mice. In summary, we utilized a comparative approach to delineate differences between PS150 and GR1009 and proposed that EPS may serve as a key factor that mediates the observed hypnotic effect.

Neuroprotective Effects of Lactobacillus plantarum PS128 in a Mouse Model of Parkinson's Disease: The Role of Gut Microbiota and MicroRNAs.

Parkinson's disease (PD) is a neurodegenerative disease characterized by motor deficits and marked neuroinflammation in various brain regions. The pathophysiology of PD is complex and mounting evidence has suggested an association with the dysregulation of microRNAs (miRNAs) and gut dysbiosis. Using a rotenone-induced PD mouse model, we observed that administration of Lactobacillus plantarum PS128 (PS128) significantly improved motor deficits in PD-like mice, accompanied by an increased level of dopamine, reduced dopaminergic neuron loss, reduced microglial activation, reduced levels of inflammatory factors, and enhanced expression of neurotrophic factor in the brain. Notably, the inflammation-related expression of miR-155-5p was significantly upregulated in the proximal colon, midbrain, and striatum of PD-like mice. PS128 reduced the level of miR-155-5p, whereas it increased the expression of suppressor of cytokine signaling 1 (SOCS1), a direct target of miR-155-5p and a critical inhibitor of the inflammatory response in the brain. Alteration of the fecal microbiota in PD-like mice was partially restored by PS128 administration. Among them, Bifidobacterium, Ruminiclostridium_6, Bacteroides, and Alistipes were statistically correlated with the improvement of rotenone-induced motor deficits and the expression of miR-155-5p and SOCS1. Our findings suggested that PS128 ameliorates motor deficits and exerts neuroprotective effects by regulating the gut microbiota and miR-155-5p/SOCS1 pathway in rotenone-induced PD-like mice.

Lactobacillus paragasseri BBM171 Ameliorates Allergic Airway Inflammation Induced by Ovalbumin in Mice via Modulating the Th1/Th2 Balance

Supplementation with specific probiotics has been shown to improve allergic airway symptoms. This study aimed to investigate immunomodulatory effects of a potential probiotic strain isolated from breast milk, Lactobacillus paragasseri BBM171 (BBM171), in an ovalbumin (OVA)-induced allergic mouse model. OVA-sensitized and OVA-challenged BALB/c mice were orally administered live or heat-inactivated BBM171 for 48 consecutive days. After the last allergen challenge, serum immunoglobulin (Ig) levels, inflammatory cell levels in the lungs, and cytokine levels in bronchoalveolar lavage fluid (BALF) were assessed. The results showed that oral administration of live or heat-inactivated BBM171 decreased serum levels of total IgE, OVA-specific IgE, and OVA-specific IgG1, while increasing OVA-specific IgG2a and reducing the extent of airway inflammation in OVA-induced allergic mice. In addition, both live and heat-inactivated BBM171 modulated the cytokine profile in BALF to a type 1 T helper (Th1) response. Furthermore, ex vivo experiments using OVA-induced allergic mouse splenocytes showed that both live and heat-inactivated BBM171 could regulate the Th1/Th2 balance, decrease the proinflammatory cytokine interleukin (IL)-17 level, and increase the anti-inflammatory cytokine IL-10 level. Taken together, these results suggest that oral administration of live or heat-inactivated BBM171 improved allergen-induced airway inflammation symptoms by modulating the host immune response toward Th1 dominance.

Psychobiotic supplementation of HK-PS23 improves anxiety in highly stressed clinical nurses: a double-blind randomized placebo-controlled study.

Nurses often experience adverse health effects associated with increasing levels of work-related stress. Stress may induce systemic effects through the HPA axis, glucocorticoid responses, and inflammatory cascades. Psychobiotics may help alleviate stress through associations of the microbiota, anti-inflammation factors, and the gut-brain axis. We aimed to investigate whether interventions with a psychobiotic, heat-killed (HK)-PS23 cells, may help improve perceived stress, anxiety, and related biological markers among highly stressed clinical nurses. This double-blind, randomized, placebo-controlled study included seventy clinical nurses from a medical center in Northern Taiwan who scored 27 or higher on the 10-item version of the Perceived Stress Scale (PSS), and participants were randomized into either taking HK-PS23 or a placebo for 8 weeks. Baseline and endpoint results of the PSS, Job Stress Scale, State and Trait Anxiety Index (STAI), emotional questionnaires, gastrointestinal severity questionnaires, Trails Marking Tests, blood biological markers, and sleep data were analyzed. While both groups demonstrated improvements in most measures over time, only the blood cortisol measure demonstrated significant group differences after the 8-week trial. Further analyses of the subgroup with higher anxiety (nurses with STAI ≥ 103) revealed that anxiety states had improved significantly in the HK-PS23 group but not in the placebo group. In summary, this placebo-controlled trial found significant reduction in the level of blood cortisol after 8 weeks of HK-PS23 use. The distinctive anxiolytic effects of HK-PS23 may be beneficial in improving perceived anxiety and stress hormone levels in female nurses under pressure. Clinical trial registration: https://clinicaltrials.gov/, identifier: NCT04452253-sub-project 1.

Continuous Lactate Monitoring System Based on Percutaneous Microneedle Array.

Lactate measurement is important in the fields of sports and medicine. Lactate accumulation can seriously affect an athlete's performance. The most common problem caused by lactate accumulation in athletes is muscle soreness due to excessive exercise. Moreover, from a medical viewpoint, lactate is one of the main prognostic factors of sepsis. Currently, blood sampling is the most common approach to lactate measurement for lactate sensing, and continuous measurement is not available. In this study, a low-cost continuous lactate monitoring system (CLMS) is developed based on a percutaneous microneedle array that uses a three-electrode lactate sensor. The working electrode has an area of 10 mm × 6 mm, including a 3 × 3 array of stainless-steel microneedles. The length, width, and thickness of each needle are 1 mm, 0.44 mm, and 0.03 mm, respectively. The working electrode is then plated with gold, polyaniline, lactate enzyme, Nafion, and Poly(2-hydroxyethyl methacrylate) (poly HEMA). The reference electrode is a 2 × 1 array covered with AgCl, and the counter electrode is a 2 × 1 array plated with gold. The sensor is incorporated into the CLMS and connected to a smartphone application and the cloud. The CLMS was tested on 40 human subjects who rode indoor bicycles, starting at 100 W and increasing in steps of 25 W at intervals of 5 min until exhaustion. The data acquired from the app connected to the CLMS were analyzed to determine the subjects' lactate response to exercise and the feasibility of assessing exercise performance and training exercise intensity by using the proposed system.

Effects of gallic acid on capsular polysaccharide biosynthesis in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a gram-negative opportunistic pathogen that causes diseases mostly in immunocompromised individuals. Recently, hypervirulent K. pneumoniae strains also cause severe disease in healthy individuals. Capsular polysaccharide (CPS) is the major virulence determinant in hypervirulent K. pneumoniae and protects the cell against the bactericidal activity of the immune system. Gallic acid (GA), a natural phenolic compound, is known to exhibit wide spectrum antibacterial activity; however, its effect on hypervirulent K. pneumoniae remains largely unresolved. We aimed to identify the effects of GA on CPS biosynthesis in hypervirulent K. pneumoniae. METHODS: Antibacterial activity of GA was evaluated by counting colonies. CPS amount was determined by glucuronic acid content. The transcriptions of cps gene cluster were measured by quantitative real time PCR (qRT-PCR) and the ß-galactosidase activity. The effect of GA on the resistance of K. pneumoniae to streptonigrin (SNG), an iron-activated antibiotic, was evaluated. The effect of GA on the resistance of K. pneumoniae to serum killing and phagocytosis by macrophages was observed. RESULTS: GA inhibited the growth and CPS biosynthesis in K. pneumoniae. GA may affect the iron availability in K. pneumoniae, thus possibly repressing the cps transcription. In addition, GA reduced the resistance of K. pneumoniae to serum killing and enhanced its susceptibility to phagocytosis. CONCLUSION: GA possesses bactericidal activity and inhibits the CPS biosynthesis in hypervirulent K. pneumoniae, thereby facilitating pathogen clearance by the host immune system. Therefore, GA may represent a promising strategy for the prevention or treatment of patients with hypervirulent K. pneumoniae infections.

Lactobacillus plantarum PS128 Promotes Intestinal Motility, Mucin Production, and Serotonin Signaling in Mice.

Lactobacillus plantarum PS128 has been reported as a psychobiotic to improve mental health through the gut-brain axis in experimental animal models. To explore its mechanism of action in the gut, this study aimed to analyze the effects of L. plantarum PS128 ingestion on naïve and loperamide (Lop)-induced constipation mice. We found that, in the two mouse models, the weight, number, and water content of feces in the L. plantarum PS128 group were higher than those in the vehicle control group. Histological observation revealed that L. plantarum PS128 increased the level of colonic mucins including the major mucin MUC2. In addition, the charcoal meal test showed that L. plantarum PS128 significantly increased the small intestine transit in naïve mice, but not in the Lop-treated mice. Since intestinal serotonin has been found to modulate motility, we further analyzed the expression of genes related to serotonin signal transduction in the small intestine of naïve mice. The results showed that L. plantarum PS128 significantly altered the expression levels of Tph1, Chga, Slc6a4, and Htr4, but did not affect the expression levels of Tph2, Htr3a, and Maoa. Furthermore, immunohistochemistry revealed that L. plantarum PS128 significantly increased the number of serotonin-containing intestinal cells in mice. Taken together, our results suggest that L. plantarum PS128 could promote intestinal motility, mucin production, and serotonin signal transduction, leading to a laxative effect in mice.

Safety Evaluation and Anti-Inflammatory Efficacy of Lacticaseibacillus paracasei PS23.

Lacticaseibacillus paracasei strain PS23 (PS23) exhibits some probiotic properties. In this study, a genomic analysis of PS23 revealed no genes related to virulence or antibiotic resistance. Moreover, ornithine decarboxylase activity was not detected in vitro. In addition, PS23 was sensitive to the tested antibiotics. Genotoxicity tests for PS23 including the Ames test and chromosomal aberrations in vitro using Chinese hamster ovary cells and micronuclei in immature erythrocytes of ICR mice were all negative. Moreover, following a 28-day study involving repeated oral dose toxicity tests (40, 400, and 4000 mg/kg equal 1.28 × 1010, 1.28 × 1011, and 1.28 × 1012 CFU/kg body weight, respectively) using an ICR mouse model, no adverse effects were observed from any doses. In addition, supplementation with live or heat-killed PS23 ameliorates DSS-induced colonic inflammation in mice. Our findings suggest that PS23 is safe and has anti-inflammatory effects and may therefore have therapeutic implications.

Lactobacillus fermentum PS150 promotes non-rapid eye movement sleep in the first night effect of mice.

The first night effect (FNE) is a type of sleep disturbance caused by an unfamiliar environment, which leads to difficulty falling asleep and reduced sleep duration. Previously, we reported that Lactobacillus fermentum PS150 (PS150) improves sleep conditions in a pentobarbital-induced sleep mouse model. In this study, we aimed to evaluate the effect of PS150 on the FNE in mice. Briefly, mice were implanted with electrodes and orally administered PS150 for four weeks, and then the FNE was induced by cage changing. Analysis of polysomnographic signals revealed that intervention with PS150 restored non-rapid eye movement (NREM) sleep length under the FNE. Compared to diphenhydramine, a commonly used sleep aid, PS150 had no unwanted side effects, such as rapid eye movement (REM) sleep deprivation and fragmented sleep. Moreover, temporal analysis revealed that PS150 efficiently reduced both sleep latency and time spent restoring normal levels of REM sleep. Taken together, these results suggest that PS150 efficiently ameliorates sleep disturbance caused by the FNE. Additionally, V3-V4 16S rRNA sequencing revealed significant increases in Erysipelotrichia, Actinobacteria, and Coriobacteriia in fecal specimens of the PS150-treated group, indicating that PS150 induces gut microbiota remodeling.

Psychobiotic Supplementation of PS128TM Improves Stress, Anxiety, and Insomnia in Highly Stressed Information Technology Specialists: A Pilot Study.

Background: Information technology (IT) is an industry related to the production of computers, information processing, and telecommunications. Such industries heavily rely on the knowledge and solutions provided by IT specialists. Previous reports found that the subjective stress scores were higher in IT specialists who developed diabetes, hypertension, and depression. Specific probiotics, known as psychobiotics, may alleviate stress and mood symptoms. This study aimed to examine whether an 8-week intervention of a novel psychobiotic, Lactobacillus plantarum PS128TM (PS128TM), improved self-perceived stress and mood symptoms among high-stress IT specialists. Methods: This open-label, single-arm, baseline-controlled study included IT specialists from a large IT company in Northern Taiwan. Participants with a Perceived Stress Scale (PSS) 10-item version score of 27 or higher were included. Participants were asked to take two capsules containing PS128TM powder, equivalent to 20 billion colony-forming units, daily. Self-report measures, such as the Job Stress Scale, Visual Analog Scale of Stress, the Insomnia Severity Index, the State and Trait Anxiety Index, the Questionnaire for Emotional Trait and State, the Patient Health Questionnaire, the Quality of Life Enjoyment and Satisfaction Questionnaire, and Gastrointestinal Severity Index were compared at baseline and at the end of the trial period. The primary outcome was a 20% reduction in the PSS score at endpoint. Objective measures included salivary levels of stress biomarkers, including cortisol, α-amylase, immunoglobulin A, lactoferrin, and lysozymes, as well as results of the Test of Attentional Performance. Results: Of the 90 eligible IT specialists, 36 met the inclusion criteria. After the 8-week trial period, significant improvements in self-perceived stress, overall job stress, job burden, cortisol level, general or psychological health, anxiety, depression, sleep disturbances, quality of life, and both positive and negative emotions were found. Conclusion: Our results suggest that PS128TM has the distinct advantage of providing stress relief and can improve mental health for people with a high-stress job. Future placebo-controlled studies are warranted to explore the effect and underlying mechanisms of action of PS128TM. Clinical Trial Registration: https://clinicaltrials.gov/ (identifier: NCT04452253-sub-project 2).

Toxicity Studies of Lactobacillus plantarum PS128TM Isolated from Spontaneously Fermented Mustard Greens.

: Probiotics are extensively available to consumers; however, the use of probiotics may not always be safe, and there are few reports on their side effects, including those of Lactobacillus. Lactobacillus plantarum strain PS128TM isolated from spontaneously fermented mustard greens in Taiwan was recently reported to exhibit probiotic properties. In this study, we aimed to assess the safety of strain PS128TM for use in humans via examining genotoxic and oral toxic effects using in vitro and in vivo testing. Five strains of Salmonella typhimurium were evaluated by the Ames test; no signs of increased reverse mutation were observed following exposure to PS128TM. Additional testing of Chinese hamster ovary (CHO) cells exposed to PS128TM revealed that the incidence of chromosomal aberrations in CHO cells had not increased. PS128TM treatment also did not affect the proportion of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Moreover, following a 28 day study involving repeated oral dose toxicity tests (2400, 400, and 40 mg/kg body weight) utilizing an ICR mouse model, no observable adverse level (NOAEL) was found at any of the doses. PS128TM was sensitive to antibiotics; however, genes related to the production of biogenic amines were absent. While further research is required, these toxicological assessments suggest that PS128TM could be safe for human consumption.

FNR-Dependent RmpA and RmpA2 Regulation of Capsule Polysaccharide Biosynthesis in Klebsiella pneumoniae.

Fumarate nitrate reduction regulator (FNR) is a direct oxygen-responsive transcriptional regulator containing an iron-sulfur (Fe-S) cluster. During anaerobic growth, the [4Fe-4S] cluster in FNR (holo-FNR) binds specifically to DNA, whereas exposure to oxygen results in the loss of its DNA-binding activity via oxidation of the [4Fe-4S] cluster. In this study, we aimed to investigate the role of FNR in regulation of capsular polysaccharide (CPS) biosynthesis, serum resistance, and anti-phagocytosis of K. pneumoniae. We found that the CPS amount in K. pneumoniae increased in anaerobic conditions, compared to that in aerobic conditions. An fnr deletion mutant and a site-directed mutant (fnr 3 CA), with the three cysteines (C20, C23, and C29) replaced with alanines to mimic an FNR lacking the [4Fe-4S] cluster, showed marked increase in CPS amount under anaerobic conditions. A promoter-reporter assay and qRT-PCR confirmed that the transcription of the cps genes was repressed by holo-FNR. In addition, we found that holo-FNR could repress the transcription of rmpA and rmpA2, encoding cps transcriptional activators. Deletion of rmpA or rmpA2 in the Δfnr strain reduced CPS biosynthesis, suggesting that RmpA and RmpA2 participated in the holo-FNR-mediated repression of cps transcription, thereby regulating the CPS amount, serum resistance, and anti-phagocytosis. Taken together, our results provided evidence that RmpA and RmpA2 participated in the holo-FNR-mediated repression of CPS biosynthesis, and resistance to the host defense in response to oxygen availability.

Hypnotic Effects of Lactobacillus fermentum PS150TM on Pentobarbital-Induced Sleep in Mice.

The bidirectional communication between the gastrointestinal tract and the central nervous system appears to be functionally linked to the intestinal microbiome, namely the microbiome-gut-brain axis (MGBA). Probiotics with health benefits on psychiatric or neurological illnesses are generally called psychobiotics, and some of them may also be able to improve sleep by targeting the MGBA. This study aimed to investigate the effects of a psychobiotic strain, Lactobacillus fermentum PS150TM (PS150TM), on sleep improvement by using a pentobarbital-induced sleep mouse model. Compared with the vehicle control group, the oral administration of PS150TM, but not the other L. fermentum strains, significantly decreased the sleep latency and increased the sleep duration of mice, suggesting strain-specific sleep-improving effects of PS150TM. Moreover, the ingestion of diphenhydramine, an antihistamine used to treat insomnia, as a drug control group, only increased the sleep duration of mice. We also found that the sleep-improving effects of PS150TM are time- and dose-dependent. Furthermore, the oral administration of PS150TM could attenuate a caffeine-induced sleep disturbance in mice, and PS150TM appeared to increase the expression of the gene encoding the adenosine 1 receptor in the hypothalamus of mice, as assessed by quantitative real-time polymerase chain reaction. Taken together, our results present a potential application of PS150TM as a dietary supplement for sleep improvement.

Psychobiotics in mental health, neurodegenerative and neurodevelopmental disorders.

Psychobiotics are a group of probiotics that affect the central nervous system (CNS) related functions and behaviors mediated by the gut-brain-axis (GBA) via immune, humoral, neural, and metabolic pathways to improve not only the gastrointestinal (GI) function but also the antidepressant and anxiolytic capacity. As a novel class of probiotics, the application of psychobiotics has led researchers to focus on a new area in neuroscience. In the past five years, some psychobiotics strains were reported to inhibit inflammation and decreased cortisol levels, resulting in an amelioration of the symptoms of anxiety and depression. Psychobiotics are efficacious in improving neurodegenerative and neurodevelopmental disorders, including autism spectrum disorder (ASD), Parkinson's disease (PD) and Alzheimer's disease (AD). Use of psychobiotics can improve GI function, ASD symptoms, motor functions of patients with PD and cognition in patients with AD. However, the evidence for the effects of psychobiotics on mental and neurological conditions/disorders remains limited. Further studies of psychobiotics are needed in order to determine into their effectiveness and mechanism as treatments for various psychiatric disorders in the future.

Lactobacillus plantarum PS128 ameliorates 2,5-Dimethoxy-4-iodoamphetamine-induced tic-like behaviors via its influences on the microbiota-gut-brain-axis.

We previously reported a novel psychobiotic strain of Lactobacillus plantarum PS128 (PS128) which could ameliorate anxiety-like& depression-like behaviors and modulate cerebral dopamine (DA) and serotonin (5-HT) in mice. Here, we examine the possibility of using PS128 administration to improve tic-like behaviors by using a 5-HT2A and 5-HT2C receptor agonist 2,5-Dimethoxy-4-iodoamphetamine (DOI). PS128 was orally administered to male Wistar rat for 2 weeks before two daily DOI injections. We recorded the behaviors immediately after the second DOI injection and compared the results with control and haloperidol treatment groups. PS128 significantly reduced tic-like behaviors and pre-pulse inhibition deficit in a threshold-dose of 109 CFU per day. Brain tissue analysis showed that DOI induced abnormal DA efflux in the striatum and prefrontal cortex, while PS128 ingestion improved DA metabolism and increased norepinephrine (NE) levels in these two regions. In addition, PS128 ingestion increased DA transporter and ß-arrestin expressions and decreased DOI-induced phosphorylation of DA and cAMP regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) at Thr34 and extracellular regulated protein kinases (ERK). PS128 ingestion also modulated peripheral 5-HT levels and shaped the cecal microbiota composition, which helps to alleviate DOI-induced dysbiosis. These results suggested that PS128 ameliorated DOI-induced tic-like hyper-active behaviors via stabilizing cerebral dopaminergic pathways through its modulation of host's microbiota-gut-brain axis. Thus, we believe there are potentials for utilizing psychobiotics to improve syndromes caused by DA dysregulation in DA-related neurological disorders and movement disorders such as Tourette syndrome.

Antidepressant-like activities of live and heat-killed Lactobacillus paracasei PS23 in chronic corticosterone-treated mice and possible mechanisms.

Emerging evidence indicates that ingestion of specific probiotics, known as "psychobiotics", confer beneficial effects on mental health. This study investigated antidepressant-like effects and possible underlying mechanisms of Lactobacillus paracasei PS23 (PS23), live or heat-killed, in a mouse model of corticosterone-induced depression using fluoxetine as standard drug. PS23 were orally gavaged to mice from day 1 to 41 or fluoxetine from day 17 to 41 and injected with corticosterone from day 17 to 37. After the last corticosterone treatment, anxiety- and depression-like behaviors were tested within 4 days. On day 42, serum and brain tissue were collected 24 min after forced swim stress. Abnormal behavioral changes induced by corticosterone were ameliorated by treatment with live PS23 in open field and sucrose preference tests, with heat-killed PS23 in open field, forced swim and sucrose preference tests, and with fluoxetine in open field and forced swim tests. Furthermore, both live and heat-killed PS23 and fluoxetine reversed corticosterone-reduced protein levels of brain-derived neurotropic factor, mineralocorticoid, and glucocorticoid receptors in the hippocampus. In addition, live PS23 also reverses corticosterone-reduced serotonin levels in hippocampus, prefrontal cortex and striatum; whereas heat-killed PS23 reverses corticosterone-reduced dopamine levels in hippocampus and prefrontal cortex. And fluoxetine normalized reduced corticosterone level in serum. These studies showed that both live and heat-killed PS23 can reverse chronic corticosterone-induced anxiety- and depression-like behaviors and that may provide insights into the mechanism and a potential psychobiotic for depression management.

Phosphorylated OmpR Is Required for Type 3 Fimbriae Expression in Klebsiella pneumoniae Under Hypertonic Conditions.

OmpR/EnvZ is a two-component system that senses osmotic signals and controls downstream gene expression in many species of Enterobacteriaceae. However, the role of OmpR/EnvZ in Klebsiella pneumoniae remains unknown. In this study, we found that production of MrkA, the major subunit of type 3 fimbriae, was decreased under hypertonic conditions. A deletion mutant of ompR and a site-directed mutant with a single amino acid substitution of aspartate 55 to alanine (D55A), which mimics the unphosphorylated form of OmpR, markedly reduced MrkA production under hypertonic conditions. These results indicate that K. pneumoniae type 3 fimbriae expression is activated by the phosphorylated form of OmpR (OmpR∼P). Although no typical OmpR∼P binding site was found in the P mrkA sequence, mrkA mRNA levels and P mrkA activity were decreased in the ΔompR and ompR D55A strains compared with the wild type (WT) strain, indicating that OmpR∼P mediates type 3 fimbriae expression at the transcriptional level. Previous reports have demonstrated that a cyclic-di-GMP (c-di-GMP) related gene cluster, mrkHIJ, regulates the expression of type 3 fimbriae. We found that both the ompR and ompR D55A mutants exhibited decreased mrkHIJ mRNA levels, intracellular c-di-GMP concentration, and bacterial biofilm amount, but increased total intracellular phosphodiesterase activity in response to hypertonic conditions. These results indicate that OmpR∼P regulates type 3 fimbriae expression to influence K. pneumoniae biofilm formation via MrkHIJ and modulation of intracellular c-di-GMP levels. Taken together, we herein provide evidence that OmpR∼P acts as a critical factor in the regulation of the c-di-GMP signaling pathway, type 3 fimbriae expression, and biofilm amount in K. pneumoniae in response to osmotic stresses.

IscR Regulation of Type 3 Fimbriae Expression in Klebsiella pneumoniae CG43.

In Klebsiella pneumoniae, we have previously shown that IscR, an Fe-S cluster-containing transcriptional factor, plays a dual role in controlling capsular polysaccharide biosynthesis and iron-acquisition systems by switching between its holo and apo forms. In this study, the effect of IscR on type 3 fimbriae expression and biofilm formation was investigated. We found that production of the major subunit of type 3 fimbriae, MrkA, was increased in the ΔiscR and iscR3CA strains, a strain expressing a mutant IscR that mimics apo-IscR, at both the translational and transcriptional levels. Based on the fact that type 3 fimbriae expression is the major factor affecting biofilm formation, increased biofilm formation was also found in ΔiscR or iscR3CA, suggesting that holo-IscR represses biofilm formation. However, the repression of type 3 fimbriae expression by IscR is indirect. To further understand the regulatory mechanism of IscR, the effect of IscR on the expression of mrkHIJ, which encodes cyclic di-GMP (c-di-GMP)-related regulatory proteins that control type 3 fimbriae expression, was studied. We found that holo-IscR could directly repress mrkHI transcription, indicating that MrkHI is required for IscR regulation of type 3 fimbriae expression. Finally, deletion of iscR attenuated K. pneumoniae virulence in a peritonitis model of mouse infection, while the absence of the [2Fe-2S] cluster of IscR had no effect on K. pneumoniae virulence during infection. Taken together, our results demonstrate the underlying mechanism of the [2Fe-2S] cluster of IscR in controlling type 3 fimbriae expression and its effect on K. pneumoniae pathogenesis.

CRP-Cyclic AMP Regulates the Expression of Type 3 Fimbriae via Cyclic di-GMP in Klebsiella pneumoniae.

Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP) signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP)-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP and c-di-GMP signaling pathways in K. pneumoniae.

Alteration of behavior and monoamine levels attributable to Lactobacillus plantarum PS128 in germ-free mice.

Probiotics, defined as live bacteria or bacterial products, confer a significant health benefit to the host, including amelioration of anxiety-like behavior and psychiatric illnesses. Here we administered Lactobacillus plantarum PS128 (PS128) to a germ-free (GF) mouse model to investigate the impact of the gut-brain axis on emotional behaviors. First, we demonstrated that chronic administration of live PS128 showed no adverse effects on physical health. Then, we found that administration of live PS128 significantly increased the total distance traveled in the open field test and decreased the time spent in the closed arm in the elevated plus maze test, whereas the administration of PS128 had no significant effects in the depression-like behaviors of GF mice. Also, chronic live PS128 ingestion significantly increased the levels of both serotonin and dopamine in the striatum, but not in the prefrontal cortex or hippocampus. These results suggest that the chronic administration of PS128 is safe and could induce changes in emotional behaviors. The behavioral changes are correlated with the increase in the monoamine neurotransmitters in the striatum. These findings suggest that daily intake of the L. plantarum strain PS128 could improve anxiety-like behaviors and may be helpful in ameliorating neuropsychiatric disorders.