Glycan Biosynthesis Ability of Gut Microbiota Increased in Primary Hypertension Patients Taking Antihypertension Medications and Potentially Promoted by Macrophage-Adenosine Monophosphate-Activated Protein Kinase

Increasing evidences suggest that the gut microbiota have their contributions to the hypertension, but the metagenomic characteristics and potential regulating mechanisms in primary hypertension patients taking antihypertension drugs are not clear yet. We carried out a metagenomic analysis in 30 primary hypertension patients taking antihypertension medications and eight healthy adults without any medication. We found that bacterial strains from species, such as Bacteroides fragilis, Bacteroides vulgatus, Escherichia coli, Klebsiella pneumoniae, and Streptococcus vestibularis, were highly increased in patients; and these strains were reported to generate glycan, short-chain fatty acid (SCFA) and trimethylamine (TMA) or be opportunistic pathogens. Meanwhile, Dorea longicatena, Eubacterium hallii, Clostridium leptum, Faecalibacterium prausnitzii, and some other strains were greatly decreased in the patient group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that ortholog groups and pathways related to glycan biosynthesis and multidrug resistance were significantly increased in the patient group, and some of the hub genes related to N-glycan biosynthesis were increased in the patient group, while those related to TMA precursor metabolism and amino acid metabolism both increased and decreased in the patient group. Metabolites tested by untargeted liquid chromatography–mass spectrometry (LC-MS) proved the decrease of acetic acid, choline, betaine, and several amino acids in patients’ fecal samples. Moreover, meta-analysis of recent studies found that almost all patients were taking at least one kind of drugs that were reported to regulate adenosine monophosphate-activated protein kinase (AMPK) pathway, so we further investigated if AMPK regulated the metagenomic changes by using angiotensin II-induced mouse hypertensive model on wild-type and macrophage-specific AMPK-knockout mice. We found that the changes in E. coli and Dorea and glycan biosynthesis-related orthologs and pathways were similar in our cohort and hypertensive wild-type mice but reversed after AMPK knockout. These results suggest that the gut microbiota-derived glycan, SCFA, TMA, and some other metabolites change in medication-taking primary hypertension patients and that medications might promote gut microbiota glycan biosynthesis through activating macrophage-AMPK.

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YakA, a protein kinase required for the transition from growth to development in Dictyostelium

Development ◽

10.1242/dev.125.12.2291 ◽

1998 ◽

Vol 125 (12) ◽

pp. 2291-2302 ◽

Cited By ~ 7

Author(s):

G.M. Souza ◽

S. Lu ◽

A. Kuspa

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Basic Protein ◽

Mrna Levels ◽

Wild Type ◽

E Coli ◽

Cycle Arrest ◽

Expression Of Genes ◽

Extracellular Signal ◽

Nutrient Rich Medium

When Dictyostelium cells starve they arrest their growth and induce the expression of genes necessary for development. We have identified and characterized a protein kinase, YakA, that is essential for the proper regulation of both events. Amino acid sequence and functional similarities indicate that YakA is a homolog of Yak1p, a growth-regulating protein kinase in S. cerevisiae. Purified YakA expressed in E. coli is able to phosphorylate myelin basic protein. YakA-null cells are smaller and their cell cycle is accelerated relative to wild-type cells. When starved, YakA-null cells fail to decrease the expression of the growth-stage gene cprD, and do not induce the expression of genes required for the earliest stages of development. YakA mRNA levels increase during exponential growth and reach a maximum at the point of starvation, consistent with a role in mediating starvation responses. YakA mRNA also accumulates when cells are grown in medium conditioned by cells grown to high density, suggesting that yakA expression is under the control of an extracellular signal that accumulates during growth. Expression of yakA from a conditional promoter causes cell-cycle arrest in nutrient-rich medium and promotes developmental events, such as the expression of genes required for cAMP signaling. YakA appears to regulate the transition from growth to development in Dictyostelium.

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Depletion of microbiome-derived molecules in the host using Clostridium genetics

10.1101/401489 ◽

2018 ◽

Cited By ~ 3

Author(s):

Chun-Jun Guo ◽

Breanna M. Allen ◽

Kamir J. Hiam ◽

Dylan Dodd ◽

Will van Treuren ◽

...

Keyword(s):

Fatty Acids ◽

Gut Microbiota ◽

Metabolic Pathways ◽

General System ◽

Short Chain Fatty Acids ◽

Wild Type ◽

Bacterial Strains ◽

High Concentrations ◽

Host Tissues ◽

Molecule Production

ABSTRACTThe gut microbiota produce hundreds of molecules that are present at high concentrations in circulation and whose levels vary widely among humans. In most cases, molecule production has not been linked to specific bacterial strains or metabolic pathways, and unraveling the contribution of each molecule to host biology remains difficult. A general system to ‘toggle’ molecules in this pool on/off in the host would enable interrogation of the mechanisms by which they modulate host biology and disease processes. Such a system has been elusive due to limitations in the genetic manipulability of Clostridium and its relatives, the source of many molecules in this pool. Here, we describe a method for reliably constructing clean deletions in a model commensal Clostridium, C. sporogenes (Cs), including multiply mutated strains. We demonstrate the utility of this method by using it to ‘toggle’ off the production of ten Cs-derived molecules that accumulate in host tissues. By comparing mice colonized by wild-type Cs versus a mutant deficient in the production of branched short-chain fatty acids, we discover a previously unknown IgA-modulatory activity of these abundant microbiome-derived molecules. Our method opens the door to interrogating and sculpting a highly concentrated pool of chemicals from the microbiome.

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Antimicrobial Peptides Display Strong Synergy with Vancomycin Against Vancomycin-Resistant E. faecium, S. aureus, and Wild-Type E. coli

International Journal of Molecular Sciences ◽

10.3390/ijms21134578 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4578 ◽

Cited By ~ 3

Author(s):

Chih-Lung Wu ◽

Ju-Yun Hsueh ◽

Bak-Sau Yip ◽

Ya-Han Chih ◽

Kuang-Li Peng ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Synergistic Effects ◽

Synergistic Activity ◽

Wild Type ◽

Bacterial Strains ◽

Antibiotic Resistant ◽

Minimal Inhibitory Concentrations ◽

E Coli ◽

Large Size ◽

Vancomycin Resistant

There is an urgent and imminent need to develop new antimicrobials to fight against antibiotic-resistant bacterial and fungal strains. In this study, a checkerboard method was used to evaluate the synergistic effects of the antimicrobial peptide P-113 and its bulky non-nature amino acid substituted derivatives with vancomycin against vancomycin-resistant Enterococcus faecium, Staphylococcus aureus, and wild-type Escherichia coli. Boron-dipyrro-methene (BODIPY) labeled vancomycin was used to characterize the interactions between the peptides, vancomycin, and bacterial strains. Moreover, neutralization of antibiotic-induced releasing of lipopolysaccharide (LPS) from E. coli by the peptides was obtained. Among these peptides, Bip-P-113 demonstrated the best minimal inhibitory concentrations (MICs), antibiotics synergism, bacterial membrane permeabilization, and supernatant LPS neutralizing activities against the bacteria studied. These results could help in developing antimicrobial peptides that have synergistic activity with large size glycopeptides such as vancomycin in therapeutic applications.

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P076 Role of gut microbiota in mediating the effects of thiopurines

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjz203.205 ◽

2020 ◽

Vol 14 (Supplement_1) ◽

pp. S173-S174

Author(s):

M Franzin ◽

M Lucafò ◽

C Lagatolla ◽

G Stocco ◽

G Decorti

Keyword(s):

Gut Microbiota ◽

Growth Phase ◽

Statistical Significance ◽

Jurkat Cells ◽

Uv Spectrophotometry ◽

Bacterial Strains ◽

Cytotoxic Effects ◽

E Coli

Abstract Background A general consensus exists that patients with inflammatory bowel disease (IBD) present compositional changes in the gut microbiota (dysbiosis), including an increase in the abundance of Enterobacteriaceae. Thiopurine drugs are commonly used in the maintenance of remission in IBD. In this context, the purpose of the project is to explore the role of candidate bacterial strains in mediating the effects of thiopurines in vitro. Methods Azathioprine (AZA), mercaptopurine (MP) and thioguanine (TG) (400 µM) were incubated in minimal salts medium (M9) in presence or not of E. coli, S. enterica and K. pneumoniae and of their growth phase broths (GPB) for 4 h at 37°C. The viability of NALM6 (B cells) and JURKAT (T cells) exposed to serial dilution of drugs (ranging from 0.2 to 15 μM of AZA, from 0.3 to 20 μM of MP, from 0.08 to 5 μM of TG) previously incubated or not with bacteria and with their GPB was determined by the MTT assay. Absorbance peaks of thiopurines were analysed by UV spectrophotometry. Statistical significance was assessed by two-way ANOVA and Bonferroni’s post-test for MTT tests and by one-way ANOVA for UV spectra. Results In NALM6 cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 45 ± 2.9%; 34 ± 2.5%% and 21 ± 0.6%) and its GPB (respectively 41 ± 7.7%; 41 ± 5.1% and 27 ± 3.5%) compared with the drugs not previously exposed (respectively 76 ± 2.3%; 69 ± 1.7% and 43 ± 3.8%). In JURKAT cells, the cytotoxic effects of 15 μM of AZA, 2.5 μM of MP and 1.25 μM of TG decreased significantly (p < 0.001) after incubation with K. pneumoniae (respectively 46 ± 2.8%; 38 ± 1.29% and 19 ± 3.3%) and its GPB (respectively 49 ± 9.4%; 38 ± 1.5% and 26 ± 1.5%) in comparison with the drugs not exposed (respectively 75 ± 4.0%; 50 ± 3.5% and 54 ± 4.0%). E. coli and S. enterica did not affect the cytotoxicity of the thiopurines. UV analysis evidenced a reduction of absorbance peaks of AZA (21 ± 0.05%), MP (32 ± 0.015%) and TG (30 ± 0.03%) after incubation with K. pneumoniae but not with its growth phase broth (GPB). Conclusion The activity of thiopurines decreased after incubation with both K. pneumoniae and its GPB. UV analysis suggested that the lower cytotoxicity of thiopurines exposed to the bacterial strain is due to the reduction of the concentration of the drugs exposed to K. pneumoniae. Moreover, the reduction of drug availability after the exposure to GPB could be explained with a possible interaction between thiopurines and capsular polysaccharides released by the bacteria.

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Chemical Composition and Antimicrobial Activities of Essential Oils of Four Lines of Origanum Vulgare Subsp. Hirtum (Link) Ietswaart Grown in Hungary

Natural Product Communications ◽

10.1177/1934578x0700201122 ◽

2007 ◽

Vol 2 (11) ◽

pp. 1934578X0700201 ◽

Cited By ~ 6

Author(s):

Katalin Veres ◽

Erzsébet Varga ◽

Zsuzsanna Schelz ◽

József Molnár ◽

Jenő Bernáth ◽

...

Keyword(s):

Essential Oils ◽

Staphylococcus Epidermidis ◽

Antimicrobial Activities ◽

Deficient Mutant ◽

Wild Type ◽

Bacterial Strains ◽

Origanum Vulgare ◽

Gram Negative ◽

E Coli ◽

Yeast Strains

The essential oils of four lines of Origanum vulgare L. subsp. hirtum (Link) Ietswaart cultivated in Hungary were analysed by GC and GC-MS methods. These oils were found to contain carvacrol, γ-terpinene and p-cymene as main constituents. The antimicrobial activities of the various oils and their authentic individual components were tested on Gram-positive and Gram-negative bacterial strains, two Saccharomyces cerevisiae strains and two Candida albicans strains. No difference in sensitivity was found between Escherichia coli, Staphylococcus epidermidis and the yeast strains tested, but there were marked differences in sensitivity between the proton pump-deficient mutant of E. coli and its wild type as regards the growth inhibition and MIC values.

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New Oxadiazole Derivatives: Synthesis and Appraisal of Their Potential as Antimicrobial Agents

Letters in Drug Design & Discovery ◽

10.2174/1570180814666170425160545 ◽

2018 ◽

Vol 15 (1) ◽

pp. 21-30

Author(s):

Deboleena Dhara ◽

Dhanya Sunil ◽

Pooja R. Kamath ◽

K. Ananda ◽

S. Shrilakshmi ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Agents ◽

Drug Resistant ◽

Bacterial Strains ◽

Spectral Techniques ◽

Docking Simulations ◽

E Coli ◽

Oxadiazole Derivatives ◽

Bacteria And Fungi ◽

Almost All

Introduction: The escalating threat due to dwindling effect of antibiotics and challenge of tackling rising drug-resistant infections has gathered high focus in current medicinal research. Methods: In an attempt to find new molecules that can defeat microbial resistance, two new series of 2-[2-substituted ethenyl]-5-(substituted methoxy)-1,3,4-oxadiazole derivatives were synthesized. Various aromatic hydrazides were allowed to undergo cyclization to substituted oxadiazole-2- amines in the presence of cyanogen bromide and further condensed with different heterocyclic aldehydes to give new oxadiazole derivatives. The synthesized molecules were fully characterized by various spectral techniques and tested for antimicrobial activity. Results: Almost all the newly synthesized compounds especially (5g-5l) displayed remarkable growth inhibition against three bacterial strains: M. smegmatis, S. aureus, E. coli and fungi C. albicans. The antimicrobial activity was further confirmed by MIC assay against the same microorganisms. Oxadiazole 5g displayed promising activity with a MIC value of 0.025 mM for two bacteria and fungi, whereas MIC of this compound for E. coli was 0.1 mM. Other active compounds (5h-5l) also exhibited good MIC ranging between 0.313 to 5.0 mM against the selected microorganisms. Docking simulations were generated to explore the potential binding approaches of ligand 5g at the D-alanine:d-alanine ligase (Ddl) protein of E. coli and S. aureus. Conclusion: Molecule 5g was active even at a lower concentration and could probably act as a prospective lead molecule for targeting the drug resistant microorganisms.

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Dysregulated Microbiota-Driven Gasdermin D Activation Promotes Colitis Development by Mediating IL-18 Release

Frontiers in Immunology ◽

10.3389/fimmu.2021.750841 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hanchao Gao ◽

Mengtao Cao ◽

Yikun Yao ◽

Wenjun Hu ◽

Huimin Sun ◽

...

Keyword(s):

Gut Microbiota ◽

Molecular Mechanism ◽

Inflammatory Diseases ◽

Cell Loss ◽

Intestinal Epithelial ◽

Wild Type ◽

E Coli ◽

Host Health ◽

Deficient Mice ◽

Colitis Model

The balance between gut microbiota and host is critical for maintaining host health. Although dysregulation of the gut microbiota triggers the development of various inflammatory diseases, including colitis, the molecular mechanism of microbiota-driven colitis development is largely unknown. Here, we found that gasdermin D (GSDMD) was activated during acute colitis. In the dextran sulfate sodium (DSS)-induced colitis model, compared to wild-type mice, Gsdmd-deficient mice had less colitis severity. Mechanistically, GSDMD expression in intestinal epithelial cells (IECs), but not infiltrating immune cells, was critical for GSDMD-mediated colitis progression. Moreover, commensal Escherichia coli (E. coli) largely overgrew during colitis, and then the dysregulated commensal E. coli mediated GSDMD activation. Furthermore, the activated GSDMD promoted the release of interleukin-18 (IL-18), but not the transcript or maturation level of IL-18, which in turn mediated goblet cell loss to induce colitis development. Thus, GSDMD promotes colitis development by mediating IL-18 release, and the microbiota can mediate colitis pathogenesis through regulation of GSDMD activation. Our results provide a potential molecular mechanism by which the microbiota-driven GSDMD activation contributes to colitis pathogenesis.

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Contribution of Escherichia coliAlpha-Hemolysin to Bacterial Virulence and to Intraperitoneal Alterations in Peritonitis

Infection and Immunity ◽

10.1128/iai.68.1.176-183.2000 ◽

2000 ◽

Vol 68 (1) ◽

pp. 176-183 ◽

Cited By ~ 24

Author(s):

Addison K. May ◽

Thomas G. Gleason ◽

Robert G. Sawyer ◽

Timothy L. Pruett

Keyword(s):

Marked Decrease ◽

Bacterial Virulence ◽

Wild Type ◽

Bacterial Strains ◽

E Coli ◽

Alpha Hemolysin ◽

Erythrocyte Lysis ◽

Leukocyte Response ◽

Peritonitis Model ◽

Specific Contribution

ABSTRACT Alpha-hemolysin (Hly) is a common exotoxin produced byEscherichia coli that enhances virulence in a number of clinical infections. The addition of hemolysin production to laboratory bacterial strains is known to increase the lethality of E. coli peritonitis. However, the mechanisms involved have not been determined and the contribution of hemolysin to the alterations in the host intraperitoneal environment and the leukocyte response is not known. Utilizing a rat peritonitis model, we show that wild-type hemolytic E. coli strains have a significant competitive advantage over nonhemolytic strains within the peritoneum. To examine the specific contribution of Hly to E. coli-induced virulence and alterations within the peritoneum, a mixed peritonitis model of E. coli, Bacteroides fragilis, and sterile fecal adjuvant was used. Three transformed E. colistrains were utilized: one strongly secretes active hemolysin (WAF 270), a second secretes active hemolysin but a reduced amount (WAF 260), and the third does not produce hemolysin (WAF 108). After an equal inoculum of each of the three strains, WAF 270 produced a markedly increased lethality and an increased recovery of both E. coli and B. fragilis from the host relative to the other strains. Changes in the intraperitoneal pH, degree of erythrocyte lysis, and recruitment and viability of leukocytes within the peritoneum following the induction of peritonitis differed significantly between the strongly hemolytic and nonhemolytic strains. Induction of peritonitis with WAF 270 caused a pronounced decrease in intraperitoneal pH, lysis of most of the intraperitoneal erythrocytes, and a marked decrease in recoverable viable leukocytes compared to WAF 108. Thus, hemolysin production by E. coli within the peritoneum may alter not only the host's ability to control the hemolytic strain itself but also other organisms.

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Regulation of the Erythroid Transcription Factor NF-E2 by Cyclic Adenosine Monophosphate–Dependent Protein Kinase

Blood ◽

10.1182/blood.v91.9.3193 ◽

1998 ◽

Vol 91 (9) ◽

pp. 3193-3201 ◽

Cited By ~ 18

Author(s):

Darren Casteel ◽

Modem Suhasini ◽

Tanima Gudi ◽

Reza Naima ◽

Renate B. Pilz

Keyword(s):

Transcription Factor ◽

Protein Kinase ◽

Phosphorylation Site ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Transactivation Domain ◽

Wild Type ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Murine Erythroleukemia

Abstract Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase–deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser169; Ala substitution for Ser169 resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore β-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser169 → Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.

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An assay of human tyrosine protein kinase ABL activity using an Escherichia coli protein expression system

BioTechniques ◽

10.2144/btn-2020-0154 ◽

2021 ◽

Author(s):

Emiko Kinoshita-Kikuta ◽

Momoka Yoshimoto ◽

Marina Yano ◽

Eiji Kinoshita ◽

Tohru Koike

Keyword(s):

Escherichia Coli ◽

Protein Kinase ◽

Expression System ◽

Kinase Domain ◽

Wild Type ◽

E Coli ◽

Abl Kinase ◽

Sds Page ◽

Tyrosine Protein Kinase ◽

Comparative Results

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in Escherichia coli. Tyrosine phosphorylation of the substrate in E. coli was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure–activity relationships.

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