N-Carbamoylputrescine Amidohydrolase of Bacteroides thetaiotaomicron, a Dominant Species of the Human Gut Microbiota.

Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N-carbamoylputrescine amidohydrolase (NCPAH) that converts N-carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His)6 was analyzed for enzymatic activity and found to be capable of converting N-carbamoylputrescine to putrescine, with a Michaelis constant (Km) and turnover number (kcat) of 730 µM and 0.8 s-1, respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron.

Gut bacterial aromatic amine production: aromatic amino acid decarboxylase and its effects on peripheral serotonin production.

Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.

Structural differences in bacterial lipopolysaccharides determine atherosclerotic plaque progression by regulating the accumulation of neutrophils.

BACKGROUND AND AIMS: Gut microbial lipopolysaccharide (LPS) induces endotoxemia, an independent risk factor for cardiovascular disease (CVD). However, no studies have demonstrated how structural differences in each bacterial LPS contribute to endotoxemia. Here, we investigated the effects of different acyl chains in the lipid A moiety of LPS on endotoxemia and the subsequent immune response and atherosclerotic plaque formation. METHODS: Apoe-/- mice were intraperitoneally administered 2 mg/kg of Escherichia coli-derived LPS (E. LPS, as a representative of hexa-acylated lipid A), Bacteroides-derived LPS (B. LPS, as a representative of penta- or tetra-acylated lipid A), or saline (control) once a week, six times. An immunohistological assessment was performed on plaque sections. RESULTS: E. LPS administration induced endotoxemia, but B. LPS and saline did not. In E. LPS-treated mice, total plaque areas in the aortic root were significantly increased, and neutrophil accumulation and increased formation of neutrophil extracellular traps (NETs) were observed at the plaque lesions, but not in B. LPS-treated mice. A single dose of E. LPS significantly increased the accumulation of neutrophils in plaque lesions on day 3, and NET formation on day 7. E. LPS also increased interleukin-1 beta (IL-1ß) production in plaque lesions on day 7. Furthermore, NET formation and IL-1ß production were also observed in human coronary plaques. CONCLUSIONS: We identified a previously unknown link between structural differences in LPS and atherosclerosis. Lowering microbial LPS activity may reduce NET formation in plaques and prevent CVD progression.

Dietary-protein sources modulate host susceptibility to Clostridioides difficile infection through the gut microbiota.

Clostridioides difficile causes nosocomial antibiotic-associated diarrhea on a global scale. Susceptibility to C. difficile infection (CDI) is influenced by the composition and metabolism of gut microbiota, which in turn are affected by diet. However, the mechanism underlying the interplay between diet and gut microbiota that modulates susceptibility to CDI remains unclear. Here, we show that a soy protein diet increases the mortality of antibiotic-treated, C. difficile-infected mice while also enhancing the intestinal levels of amino acids (aas) and relative abundance of Lactobacillus genus. Indeed, Ligilactobacillus murinus-mediated fermentation of soy protein results in the generation of aas, thereby promoting C. difficile growth, and the process involves the anchored cell wall proteinase PrtP. Thus, mutual interaction between dietary protein and the gut microbiota is a critical factor affecting host susceptibility to CDI, suggesting that dietary protein sources can be an important determinant in controlling the disease.

Putrescine Production by Latilactobacillus curvatus KP 3-4 Isolated from Fermented Foods.

Polyamines are aliphatic hydrocarbons with terminal amino groups and are essential for biological activities. It has been reported that polyamines have health-promoting effects in animals, such as the extension of lifespan by polyamine intake. The identification of a high polyamine-producing bacterium from foods could lead to the development of a novel probiotic candidate. We aimed to identify high polyamine-producing bacteria from food, and isolated and collected bacteria from vegetables and fermented foods produced in Japan. We successfully acquired Latilactobacillus curvatus KP 3-4 isolated from Kabura-zushi as a putrescine producing lactic acid bacteria. Comparing the polyamine synthesis capability of L. curvatus KP 3-4 with that of typical probiotic lactic acid bacteria and L. curvatus strains available from the Japan Collection of Microorganisms, it was found that only L. curvatus KP 3-4 was capable of exporting high levels of putrescine into the culture supernatant. The enhancement of putrescine production by the addition of ornithine, and whole-genome analysis of L. curvatus KP 3-4, suggest that putrescine is synthesized via ornithine decarboxylase. The administration of L. curvatus KP 3-4 to germ-free mice increased the concentration of putrescine in the feces.

A possible beneficial effect of Bacteroides on faecal lipopolysaccharide activity and cardiovascular diseases.

Faecal lipopolysaccharides (LPS) have attracted attention as potent elements to explain a correlation between the gut microbiota and cardiovascular disease (CVD) progression. However, the underlying mechanism of how specific gut bacteria contribute to faecal LPS levels remains unclear. We retrospectively analysed the data of 92 patients and found that the abundance of the genus Bacteroides was significantly and negatively correlated with faecal LPS levels. The controls showed a higher abundance of Bacteroides than that in the patients with CVD. The endotoxin units of the Bacteroides LPS, as determined by the limulus amoebocyte lysate (LAL) tests, were drastically lower than those of the Escherichia coli LPS; similarly, the Bacteroides LPS induced relatively low levels of pro-inflammatory cytokine production and did not induce sepsis in mice. Fermenting patient faecal samples in a single-batch fermentation system with Bacteroides probiotics led to a significant increase in the Bacteroides abundance, suggesting that the human gut microbiota could be manipulated toward decreasing the faecal LPS levels. In the clinical perspective, Bacteroides decrease faecal LPS levels because of their reduced LAL activity; therefore, increasing Bacteroides abundance might serve as a novel therapeutic approach to prevent CVD via reducing faecal LPS levels and suppressing immune responses.

Development of a new chromogenic method for putrescine quantification using coupling reactions involving putrescine oxidase.

Quantification of polyamines, including putrescine, is generally performed using high-performance liquid chromatography (HPLC) or gas chromatography. However, these methods are time-consuming because of sample derivatization and analytical reagent preparation. In this study, we developed a simple and high-throughput putrescine quantification method on a 96-well microtiter plate using putrescine oxidase from Rhodococcus erythropolis NCIMB 11540, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt. The developed method (named as PuO-POD-4AA-TOPS method) was applicable to bacterial culture supernatants. Furthermore, putrescine concentrations determined by the developed method roughly corresponded to the concentrations determined by HPLC.

Measuring the Antimicrobial Activity of Lauric Acid against Various Bacteria in Human Gut Microbiota Using a New Method.

Lauric acid (LA) has a broad spectrum of anti-microbiological activities against enveloped viruses and various bacteria, and might be useful to protect against microbial infection and control the balance and distribution of bacteria in human gut microbiota. It is not necessarily more difficult to measure antimicrobial activity the traditional way, but it is, however, more laborious. In the present study, we developed a new method to measure the antimicrobial activity of LA in multiple samples with a microplate reader. A "test complex" (TC) was produced consisting of 100 µL of agar medium with LA in the bottom layer and 300 µL of broth in the top layer in 96-well deep-well microplates. Afterward, analysis of the broth in the top layer showed that the antimicrobial activity was the same as that of the "control complex," (CC) which consisted of 100 µL of agar medium in the bottom layer and 300 µL of broth with LA in the top layer. Furthermore, evaluation of the antimicrobial effect of the TC when using a microplate reader was the same as that with the use of the colony counting method. The colony counting method has confirmed that the antimicrobial activity of LA when bacteria are inoculated into the broth was equivalent between CC and TC, and we validated this by correlating the number of bacteria with absorbance. In addition, the broth itself in TC was transparent enough that the turbidity of broth can be used as an index of the number of bacteria, which enabled the use of a microplate reader for multiple samples. For human gut microbes, LA was shown to have low antimicrobial activity against commensal lactic acid bacteria, but high antimicrobial activity against pathogenic Bacteroides and Clostridium, suggesting that LA might modulate intestinal health, as confirmed by the proposed method.

Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis.

The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.

5-Aminolevulinic acid regulates the immune response in LPS-stimulated RAW 264.7 macrophages.

BACKGROUND: Macrophages are crucial players in a variety of inflammatory responses to environmental cues. However, it has been widely reported that macrophages cause chronic inflammation and are involved in a variety of diseases, such as obesity, diabetes, metabolic syndrome, and cancer. In this study, we report the suppressive effect of 5-aminolevulinic acid (ALA), via the HO-1-related system, on the immune response of the LPS-stimulated mouse macrophage cell line RAW264.7. RESULTS: RAW264.7 cells were treated with LPS with or without ALA, and proinflammatory mediator expression levels and phagocytic ability were assessed. ALA treatment resulted in the attenuation of iNOS and NO expression and the downregulation of proinflammatory cytokines (TNF-α, cyclooxygenase2, IL-1ß, IL-6). In addition, ALA treatment did not affect the phagocytic ability of macrophages. To our knowledge, this study is the first to investigate the effect of ALA on macrophage function. Our findings suggest that ALA may have high potential as a novel anti-inflammatory agent. CONCLUSIONS: In the present study, we showed that exogenous addition of ALA induces HO-1 and leads to the downregulation of NO and some proinflammatory cytokines. These findings support ALA as a promising anti-inflammatory agent.

Analysis of polyamine biosynthetic- and transport ability of human indigenous Bifidobacterium.

Bifidobacteria are members of the human intestinal microbiota, being numerically dominant in the colon of infants, and also being prevalent in the large intestine of adults. In this study, we measured the concentrations of major polyamines (putrescine, spermidine, and spermine) in cells and culture supernatant of 13 species of human indigenous Bifidobacterium at growing and stationary phase. Except for Bifidobacterium bifidum and Bifidobacterium gallicum, 11 species contained spermidine and/or spermine when grown in Gifu-anaerobic medium (GAM). However, Bifidobacterium scardovii and Bifidobacterium longum subsp. infantis, which contain spermidine when grown in GAM, did not contain spermidine when grown in polyamine-free 199 medium. Of the tested 13 Bifidobacterium species, 10 species showed polyamine transport ability. Combining polyamine concentration analysis in culture supernatant and in cells, with basic local alignment search tool analysis suggested that novel polyamine transporters are present in human indigenous Bifidobacterium. ABBREVIATIONS: Put: putrescine; Spd: spermidine; Spm: spermine; GAM: Gifu anaerobic medium; BHI: brain-heart infusion.

Functional analysis of arginine decarboxylase gene speA of Bacteroides dorei by markerless gene deletion.

Polyamine concentrations in the intestine are regulated by their biosynthesis by hundreds of gut microbial species and these polyamines are involved in host health and disease. However, polyamine biosynthesis has not been sufficiently analyzed in major members of the human gut microbiota, possibly owing to a lack of gene manipulation systems. In this study, we successfully performed markerless gene deletion in Bacteroides dorei, one of the major members of the human gut microbiota. The combination of a thymidine kinase gene (tdk) deletion mutant and a counter-selection marker tdk, which has been applied in other Bacteroides species, was used for the markerless gene deletion. Deletion of tdk in B. dorei caused 5-fluoro-2΄-deoxyuridine resistance, suggesting the utility of B. dorei Δtdk as the host for future markerless gene deletions. Compared to parental strains, an arginine decarboxylase gene (speA) deletion mutant generated in this system showed a severe growth defect and decreased concentration of spermidine in the cells and culture supernatant. Collectively, our results indicate the accessibility of gene deletion and the important role of speA in polyamine biosynthesis in B. dorei.

Comprehensive analysis of polyamine transport and biosynthesis in the dominant human gut bacteria: Potential presence of novel polyamine metabolism and transport genes.

Recent studies have reported that polyamines in the colonic lumen might affect animal health and these polyamines are thought to be produced by gut bacteria. In the present study, we measured the concentrations of three polyamines (putrescine, spermidine, and spermine) in cells and culture supernatants of 32 dominant human gut bacterial species in their growing and stationary phases. Combining polyamine concentration analysis in culture supernatant and cells with available genomic information showed that novel polyamine biosynthetic proteins and transporters were present in dominant human gut bacteria. Based on these findings, we suggested strategies for optimizing polyamine concentrations in the human colonic lumen via regulation of genes responsible for polyamine biosynthesis and transport in the dominant human gut bacteria.

Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles.

Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.

Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation.

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.

Application study of 1,2-α-l-fucosynthase: introduction of Fucα1-2Gal disaccharide structures on N-glycan, ganglioside, and xyloglucan oligosaccharide.

We have recently generated a highly efficient 1,2-α-l-fucosynthase (BbAfcA N423H mutant) by protein engineering of 1,2-α-l-fucosidase from Bifidobacterium bifidum JCM 1254. This synthase could specifically introduce H-antigens (Fucα1-2Gal) into the non-reducing ends of oligosaccharides and in O-linked glycans in mucin glycoprotein. In the present study, we show an extended application of the engineered 1,2-α-l-fucosynthase by demonstrating its ability to insert Fuc residues into N- and O-glycans in fetuin glycoproteins, GM1 ganglioside, and a plant-derived xyloglucan nonasaccharide. This application study broadens the feasibility of this novel H-antigen synthesis technique in functional glycomics.

A Novel Putrescine Exporter SapBCDF of Escherichia coli.

Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 µm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.

Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-α-l-fucosynthase.

Fucα1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via α-(1→2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-α-l-fucosidase (AfcA) into a highly efficient 1,2-α-l-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chain-containing oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced H-antigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving α-1,2-fucosyltransferases and retaining α-fucosidase.

Carboxyspermidine decarboxylase of the prominent intestinal microbiota species Bacteroides thetaiotaomicron is required for spermidine biosynthesis and contributes to normal growth.

Recent studies have indicated that polyamines produced by gut microbes significantly influence host health; however, little is known about the microbial polyamine biosynthetic pathway except for that in Escherichia coli, a minor component of the gastrointestinal microbiota. Here, we investigated the polyamine biosynthetic ability of Bacteroides thetaiotaomicron, a predominant gastrointestinal bacterial species in humans. High-performance liquid chromatography analysis revealed that B. thetaiotaomicron cultured in polyamine-free minimal medium accumulated spermidine intracellularly at least during the mid-log and stationary phases. Deletion of the gene encoding a putative carboxyspermidine decarboxylase (casdc), which converts carboxyspermidine to spermidine, resulted in the depletion of spermidine and loss of decarboxylase activity in B. thetaiotaomicron. The Δcasdc strain also showed growth defects in polyamine-free growth medium. The complemented Δcasdc strain restored the spermidine biosynthetic ability, decarboxylase activity, and growth. These results indicate that carboxyspermidine decarboxylase is essential for synthesizing spermidine in B. thetaiotaomicron and contributes to the growth of this species.

Novel substrate specificities of two lacto-N-biosidases towards ß-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1.

We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galß1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of ß-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.