RESUMO
Bacterial pathogens utilize the factors of their hosts to infect them, but which factors they exploit remain poorly defined. Here, we show that a pathogenic Salmonella enterica serovar Typhimurium (STm) exploits host polyamines for the functional expression of virulence factors. An STm mutant strain lacking principal genes required for polyamine synthesis and transport exhibited impaired infectivity in mice. A polyamine uptake-impaired strain of STm was unable to inject effectors of the type 3 secretion system into host cells due to a failure of needle assembly. STm infection stimulated host polyamine production by increasing arginase expression. The decline in polyamine levels caused by difluoromethylornithine, which inhibits host polyamine production, attenuated STm colonization, whereas polyamine supplementation augmented STm pathogenesis. Our work reveals that host polyamines are a key factor promoting STm infection, and therefore a promising therapeutic target for bacterial infection.
Assuntos
Poliaminas , Salmonella typhimurium , Sistemas de Secreção Tipo III , Fatores de Virulência , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/genética , Animais , Poliaminas/metabolismo , Camundongos , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Humanos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , FemininoRESUMO
Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota, and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota, and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, that is, they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis and suggests a more prominent role for agmatine.
Assuntos
Bactérias , Proteínas de Bactérias , Espermidina Sintase , Espermina Sintase , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Espermidina/metabolismo , Espermidina/análogos & derivados , Espermidina/biossíntese , Espermidina Sintase/metabolismo , Espermidina Sintase/genética , Espermina/metabolismo , Espermina/análogos & derivados , Espermina/biossíntese , Espermina Sintase/metabolismo , Espermina Sintase/genética , Poliaminas/metabolismo , Alquil e Aril Transferases/biossíntese , Alquil e Aril Transferases/genética , Agmatina/química , Agmatina/metabolismoRESUMO
Hyperuricemia (HUA) is a symptom of high blood uric acid (UA) levels, which causes disorders such as gout and renal urinary calculus. Prolonged HUA is often associated with hypertension, atherosclerosis, diabetes mellitus, and chronic kidney disease. Studies have shown that gut microbiota (GM) affect these chronic diseases. This study aimed to determine the relationship between HUA and GM. The microbiome of 224 men and 254 women aged 40 years was analyzed through next-generation sequencing and machine learning. We obtained GM data through 16S rRNA-based sequencing of the fecal samples, finding that alpha-diversity by Shannon index was significantly low in the HUA group. Linear discriminant effect size analysis detected a high abundance of the genera Collinsella and Faecalibacterium in the HUA and non-HUA groups. Based on light gradient boosting machine learning, we propose that HUA can be predicted with high AUC using four clinical characteristics and the relative abundance of nine bacterial genera, including Collinsella and Dorea. In addition, analysis of causal relationships using a direct linear non-Gaussian acyclic model indicated a positive effect of the relative abundance of the genus Collinsella on blood UA levels. Our results suggest abundant Collinsella in the gut can increase blood UA levels.
Assuntos
Microbioma Gastrointestinal , Hiperuricemia , Aprendizado de Máquina , RNA Ribossômico 16S , Ácido Úrico , Humanos , Hiperuricemia/microbiologia , Hiperuricemia/sangue , Masculino , Feminino , Adulto , RNA Ribossômico 16S/genética , Ácido Úrico/sangue , Fezes/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Pessoa de Meia-IdadeRESUMO
In recent years, with the development of non-cultivation approaches, it has become evident that intestinal bacteria have a significant impact on human health. However, because one-third of the genes cannot be annotated, it is difficult to elucidate the function of all intestinal bacteria by in silico analysis, and it is necessary to study the intestinal bacteria by culturing them. In addition, various media recommended for each individual bacterium have been used for culturing intestinal bacteria; however, the preparation of each medium is complex. To simultaneously culture many bacteria and compare bacterial phenotypes under the same conditions, a medium capable of culturing a wide range of bacteria is needed. In this study, we developed GAM + blood medium (GB medium), which consists of Gifu anaerobic medium containing 5% (v/v) horse blood; it is easy to prepare and it allowed the successful cultivation of 85% of the available predominant species in the human intestinal microbiota.
Assuntos
Bactérias , Microbioma Gastrointestinal , Humanos , Animais , Cavalos , Técnicas de Cultura , Bactérias/genética , Microbioma Gastrointestinal/genética , Meios de CulturaRESUMO
Polyamine intake has been reported to help extend the lifespan of animals. Fermented foods contain high concentrations of polyamines, produced by fermenting bacteria. Therefore, the bacteria, isolated from fermented foods that produce large amounts of polyamines, are potentially used as a source of polyamines for humans. In this study, the strain Levilactobacillus brevis FB215, which has the ability to accumulate approximately 200 µM of putrescine in the culture supernatant, was isolated from fermented foods, specifically the Blue Stilton cheese. Furthermore, L. brevis FB215 synthesized putrescine from agmatine and ornithine, which are known polyamine precursors. When cultured in the extract of Sakekasu, a byproduct obtained during the brewing of Japanese rice wine containing high levels of both agmatine and ornithine, L. brevis FB215 grew to OD600 = 1.7 after 83 h of cultivation and accumulated high concentrations (~1 mM) of putrescine in the culture supernatant. The fermentation product also did not contain histamine or tyramine. The Sakekasu-derived ingredient fermented by the food-derived lactic acid bacteria developed in this study could contribute to increasing polyamine intake in humans.
Assuntos
Agmatina , Queijo , Levilactobacillus brevis , Humanos , Putrescina/farmacologia , Lactobacillaceae , Poliaminas/análise , OrnitinaRESUMO
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.
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It has been reported that the intake of polyamines contributes to the extension of healthy life span in animals. Fermented foods contain high concentrations of polyamines thought to be derived from fermentation bacteria. This suggests that bacteria that produce high levels of polyamines could be isolated from fermented foods and utilized as a source of polyamines for human nutrition. In this study, Staphylococcus epidermidis FB146 was isolated from miso, a Japanese fermented bean paste, and found to have a high concentration of putrescine in its culture supernatant (452 µM). We analyzed the presence of polyamines in the culture supernatants and cells of the type strains of 21 representative Staphylococcus species in addition to S. epidermidis FB146, and only S. epidermidis FB146 showed high putrescine productivity. Furthermore, whole-genome sequencing of S. epidermidis FB146 was performed, and the ornithine decarboxylase gene (odc), which is involved in putrescine synthesis, and the putrescine:ornithine antiporter gene (potE), which is thought to contribute to the release of putrescine into the culture supernatant, were present on plasmid DNA harbored by S. epidermidis FB146.
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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.
Assuntos
Carbidopa , Microbioma Gastrointestinal , Animais , Descarboxilases de Aminoácido-L-Aromático/genética , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Benserazida/farmacologia , Humanos , Camundongos , Fenetilaminas , Serotonina/metabolismoRESUMO
Polyamines (putrescine, spermidine, and spermine) are compounds with amino groups at both ends of a hydrocarbon. Polyamines produced by intestinal bacteria suppress chronic inflammation and enhance the intestinal barrier in the colon, and are also transferred into the blood via the colonic epithelium, resulting in significant improvement of host cognitive performance and life extension in mice. Upregulation of polyamine production by gut microbes can help compensate for the aging-associated decrease in polyamine content through the uptake of intestinal luminal polyamine, thereby extending the healthy life expectancy of the host. This review summarizes recent advances in the study of polyamine metabolism and transport in gut microbes, with particular reference to Escherichia coli and the most predominant species of the gut microbiota. Furthermore, we describe polyamine production by a novel hybrid system comprised of multiple gut microbes, as well as from high-polyamine-producing lactic acid bacteria derived from fermented foods.
Assuntos
Microbioma Gastrointestinal , Animais , Transporte Biológico , Escherichia coli/metabolismo , Camundongos , Poliaminas/metabolismo , Putrescina , Espermidina , EsperminaRESUMO
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.
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SCOPE: Isoliquiritigenin (ILG) has been reported to attenuate adipose tissue inflammation and metabolic disorder; however, the underlying mechanisms remain to be elucidated. The aim of this study is to elucidate whether ILG shows the anti-inflammatory and antimetabolic syndrome effects through gut microbiota modification. METHODS AND RESULTS: Mice are fed a high-fat diet (HFD) with or without ILG for up to 12 weeks. The effect of ILG on body weight, blood glucose level, adipose tissue inflammation, gut barrier function, and gut microbiota composition are investigated. ILG supplementation alleviates HFD-induced obesity, glucose tolerance, and insulin resistance and suppresses inflammatory gene expression in epididymal white adipose tissue (eWAT). Moreover, ILG supplementation modifies gut bacterial composition by increasing the abundance of antimetabolic disease-associated species (e.g., Parabacteroides goldsteinii and Akkemansia muciniphila) and up-regulated genes associated with gut barrier function. Fecal microbiome transplantation (FMT) from ILG-fed donors counteract HFD-induced body and eWAT weight changes, inflammation-related gene expression, glucose tolerance, and insulin resistance, thereby suggesting that ILG-responsive gut bacteria exerts anti-inflammatory and antimetabolic syndrome effects. CONCLUSION: Alterations in gut bacteria underly the beneficial effects of ILG against adipose tissue inflammation and metabolic disorders. ILG may be a promising prebiotic for the prevention and treatment of metabolic syndrome.
Assuntos
Resistência à Insulina , Síndrome Metabólica , Tecido Adiposo/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Bactérias , Chalconas , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Inflamação/metabolismo , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Certain existing prebiotics meant to facilitate the growth of beneficial bacteria in the intestine also promote the growth of other prominent bacteria. Therefore, the growth-promoting effects of ß-galactosides on intestinal bacteria were analyzed. Galactosyl-ß1,4-l-rhamnose (Gal-ß1,4-Rha) selectively promoted the growth of Bifidobacterium. Bifidobacterium longum subsp. longum 105-A (JCM 31944) has multiple solute-binding proteins belonging to ATP-binding cassette transporters for sugars. Each strain in the library of 11 B. longum subsp. longum mutants, in which each gene of the solute-binding protein was disrupted, was cultured in a medium containing Gal-ß1,4-Rha as the sole carbon source, and only the BL105A_0502 gene-disruption mutant showed delayed and reduced growth compared to the wild-type strain. BL105A_0502 homolog is highly conserved in bifidobacteria. In a Gal-ß1,4-Rha-containing medium, Bifidobacterium longum subsp. infantis JCM 1222T, which possesses BLIJ_2090, a homologous protein to BL105A_0502, suppressed the growth of enteric pathogen Clostridioides difficile, whereas the BLIJ_2090 gene-disrupted mutant did not. In vivo, administration of B. infantis and Gal-ß1,4-Rha alleviated C. difficile infection-related weight loss in mice. We have successfully screened Gal-ß1,4-Rha as a next-generation prebiotic candidate that specifically promotes the growth of beneficial bacteria without promoting the growth of prominent bacteria and pathogens.
Assuntos
Bifidobacterium longum subspecies infantis/crescimento & desenvolvimento , Bifidobacterium/crescimento & desenvolvimento , Clostridioides difficile/crescimento & desenvolvimento , Dissacarídeos/farmacologia , Prebióticos/análise , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bifidobacterium/genética , Bifidobacterium longum subspecies infantis/genética , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
This study aims to understand the mechanistic basis underlying the response of Bifidobacterium to lactulose ingestion in guts of healthy Japanese subjects, with specific focus on a lactulose transporter. An in vitro assay using mutant strains of Bifidobacterium longum subsp. longum 105-A shows that a solute-binding protein with locus tag number BL105A_0502 (termed LT-SBP) is primarily involved in lactulose uptake. By quantifying faecal abundance of LT-SBP orthologues, which is defined by phylogenetic analysis, we find that subjects with 107 to 109 copies of the genes per gram of faeces before lactulose ingestion show a marked increase in Bifidobacterium after ingestion, suggesting the presence of thresholds between responders and non-responders to lactulose. These results help predict the prebiotics-responder and non-responder status and provide an insight into clinical interventions that test the efficacy of prebiotics.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium/crescimento & desenvolvimento , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Lactulose/administração & dosagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/metabolismo , Estudos Transversais , Feminino , Fármacos Gastrointestinais/administração & dosagem , Trato Gastrointestinal/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.
Assuntos
Colo/metabolismo , Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Putrescina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana/toxicidade , Células Epiteliais/metabolismo , Feminino , Microbioma Gastrointestinal/fisiologia , Homeostase , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
The purpose of this paper is to summarize the latest information on the various aspects of polyamines and their health benefits. In recent years, attempts to treat cancer by reducing elevated polyamines levels in cancer cells have been made, with some advancing to clinical trials. However, it has been reported since 2009 that polyamines extend the healthy life span of animals by inducing autophagy, protecting the kidneys and liver, improving cognitive function, and inhibiting the progression of heart diseases. As such, there is conflicting information regarding the relationship between polyamines and health. However, attempts to treat cancer by decreasing intracellular polyamines levels are a coping strategy to suppress the proliferation-promoting effects of polyamines, and a consensus is being reached that polyamine intake does not induce cancer in healthy individuals. To provide further scientific evidence for the health-promoting effects of polyamines, large-scale clinical studies involving multiple groups are expected in the future. It is also important to promote basic research on polyamine intake in animals, including elucidation of the polyamine balance between food, intestinal bacteria, and biosynthesis.
Assuntos
Poliaminas/farmacologia , Animais , Dieta , Humanos , NeoplasiasRESUMO
Comparisons of the changes in the gut microbiota and transcriptomes as a result of changes in diet have demonstrated that the regulation of the gene functions of intestinal bacteria is fundamental for the regulation of the intestinal environment. However, the functions of only about half of the genes can be predicted using nucleotide sequences obtained from the metagenomic data of the human gut microbiota. Therefore, the regulation of gut bacterial gene functions is hindered. To resolve this issue, the functions of the genes of intestinal bacteria must be identified. In our previous study, a high-throughput cultivation system was established for the dominant species of indigenous human intestinal microbiota. Using this system, we analyzed the synthesis and transport of polyamines by intestinal bacteria. Comparison of the results with those obtained by in silico analysis indicated the existence of novel polyamine synthetic enzymes and transport proteins. Next, strains with gene deletions and complementation for the polyamine synthetic system of the genus Bacteroides were analyzed. Furthermore, we co-cultured genetically engineered Escherichia coli and Enterococcus faecalis strains to demonstrate the presence of a polyamine synthetic pathway spanning multiple bacterial species. Here, we outline the trends of research using genetically engineered intestinal bacteria and the ripple effects of studies in which intestinal bacteria have been analyzed genetically. Moreover, because studies on intestinal bacteria at the gene level are indispensable for improving our understanding of their regulation, the importance of this research will continue to increase in the future.
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Improving the physicochemical properties of okara for various applications in foods is of great importance. Here, okara and microcrystalline cellulose (MCC) were atomized using a water jet (WJ) system. The WJ-treated okara and MCC dispersed homogeneously in water, and their median sizes in particle size distribution were 6.6 µm and 9.5 µm, respectively. The dispersions of WJ-treated okara and MCC showed high apparent viscosity and shear thinning behavior. Moreover, the inhibition of α-amylase activities by WJ-treated okara was more effective than that by untreated MCC and cellulose. Furthermore, the production of short-chain fatty acids by 32 dominant species of human gut microbes was determined. An increase in butyrate production by Roseburia intestinalis was observed in the presence of WJ-treated okara, but not in untreated okara or WJ-treated MCC. These results demonstrate that WJ system can be used on okara to increase inhibited α-amylase activities and butyrate production by gut microbiota.
Assuntos
Butiratos/metabolismo , Clostridiales/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Água/química , alfa-Amilases/antagonistas & inibidores , Celulose/química , Celulose/farmacologia , Clostridiales/efeitos dos fármacos , Ácidos Graxos Voláteis/biossíntese , Microbioma Gastrointestinal , Humanos , Nebulizadores e Vaporizadores , Tamanho da Partícula , Alimentos de Soja , Glycine max/química , ViscosidadeRESUMO
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.
Assuntos
Proteus mirabilis/metabolismo , Putrescina/análise , Ampirona/química , Compostos Cromogênicos/química , Colorimetria/métodos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Rhodococcus/enzimologiaRESUMO
Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcß1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcß1-Asn-peptides/proteins generated by the action of endo-ß- N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibody-dependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.
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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.