cyclic-gmp and Escherichia-coli-Infections

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function.

Topics: Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Escherichia coli Infections; Gastrointestinal Microbiome; Humans; Inflammasomes; Inflammation; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Salmonella Infections

Heat-stable enterotoxin, produced by Escherichia coli, binds to particulate guanylate cyclase to increase cyclic GMP in intestinal cells. This in turn stimulates the cyclic-GMP- or cyclic-AMP-dependent protein kinase, activating the same chloride channel that is defective in cystic fibrosis. It is possible that the relatively high prevalence of cystic fibrosis in humans results from its protective effect against diarrhea.

Topics: Animals; Bacterial Toxins; Chloride Channels; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterotoxins; Escherichia coli Infections; Escherichia coli Proteins; Humans; Ion Transport; Membrane Proteins

In several bacterial diseases, the clinical, laboratory, and histologic findings result from the elaboration by the organism of a toxic product that binds to and may enter the host cell to alter its metabolism. In some cases, the intracellular mediators of toxin action are the cyclic nucleotides, cyclic adenosine 5'-monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP), the ubiquitous second messengers through which numerous hormones, neurotransmitters, and drugs exert their effects. Certain toxins act by enhancing the activity of cellular enzymes that synthesize cAMP or cGMP; and others, by themselves catalyzing cAMP synthesis after entering the cell. Studies of the mechanism of action of these toxins have helped in deciphering the enzymatic components within animal cells that are responsible for cyclic nucleotide synthesis, degradation, and function as well as in understanding the pathogenesis of the diseases in which they are involved.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Bacillus anthracis; Bacterial Toxins; Bordetella pertussis; Cell Membrane; Cholera Toxin; Cyclic GMP; Enterotoxins; Enzyme Activation; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Guanylate Cyclase; Humans; Intestinal Diseases; Intestinal Mucosa; Nucleotides, Cyclic; Organ Specificity; Virulence Factors, Bordetella

Topics: Adenylyl Cyclases; Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Guanylate Cyclase; Humans; Mice; Vibrio cholerae

Enteric infections cause more than a billion episodes of diarrhoeal disease in humans each year killing many millions of people, especially young children, in developing countries. Recent progress, reviewed in this article, has enabled that a specific pathogen now can be isolated in the majority of patients with acute diarrhoea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents. Based on this understanding it now seems possible to devise new techniques for the treatment and prevention of diarrhoeal disease to complement those based on fluid replacement therapy and sanitation; prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed.. Enteric infections cause more than 1 billion episodes of diarrheal disease in humans each year killing many millions of people, especially young children, in developing countries. Recent progress, reviewed here, has enabled that a specific pathogen can now be isolated in the majority of patients with acute diarrhea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents. Based on this understanding, it now seems possible to devise new techniques for the treatment and prevention of diarrheal disease to complement those based on fluid replacement therapy and sanitation. Prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed.

Topics: Antidiarrheals; Bacterial Vaccines; Calcium; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Dysentery, Amebic; Escherichia coli Infections; G(M1) Ganglioside; Humans; Reoviridae Infections; Rotavirus; Viral Vaccines

Enterotoxigenic

Topics: Animals; Cell Line; Cyclic GMP; Cytokines; Disease Models, Animal; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Female; Host-Pathogen Interactions; Humans; Immunity, Mucosal; Immunization; Inflammation Mediators; Interleukin-33; Intestinal Mucosa; Mice

Topics: Acute-Phase Reaction; Animals; Cyclic AMP; Cyclic GMP; Escherichia coli; Escherichia coli Infections; Hepatocytes; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic

Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic

Topics: Bacterial Toxins; Cyclic GMP; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Escherichia coli Proteins; Humans; Intestine, Small; Jejunum; Signal Transduction; Stress, Mechanical; Virulence Factors

Topics: Bacterial Toxins; Caco-2 Cells; Cell Line, Tumor; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Diarrhea; Enterotoxigenic Escherichia coli; Enterotoxins; Epithelial Cells; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae Proteins; Humans; Jejunum; Membrane Glycoproteins; Peptide Hydrolases; Propionates; Quinolines; Virulence Factors

Topics: Bacterial Proteins; Biofilms; Biological Variation, Population; Cyclic GMP; Escherichia coli; Escherichia coli Infections; Extracellular Matrix; Fluorescent Antibody Technique; Gene Expression Regulation, Bacterial; Models, Biological; Mutation; Phenotype; Signal Transduction

The

Topics: Animals; ATP-Binding Cassette Transporters; Cyclic GMP; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Mice; Multigene Family; Mutation; Operon; Phosphates; Phosphorus-Oxygen Lyases; Recombinases; Regulon; Transcription Factors; Urinary Bladder; Urinary Tract Infections; Uropathogenic Escherichia coli; Virulence

Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease.

Topics: Analysis of Variance; Animals; Bacterial Toxins; Cyclic GMP; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Disease Models, Animal; Enterotoxigenic Escherichia coli; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Escherichia coli Proteins; Humans; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Transgenic; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)--producing the biofilm-promoting second messenger c-di-GMP--that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.

Topics: Biofilms; Cyclic GMP; Disease Outbreaks; Escherichia coli Infections; Escherichia coli Proteins; Female; Germany; Hemolytic-Uremic Syndrome; Humans; Middle Aged; Phosphorus-Oxygen Lyases; Shiga Toxin; Shiga-Toxigenic Escherichia coli

Escherichia coli O157:H7 is an important foodborne pathogen that causes serious illness in humans at low infectious doses. The main source of infections is beef or greens contaminated with E. coli O157:H7 shed by cattle. Here we investigated the role of c-di-GMP-dependent signal transduction in cattle gut colonization of E. coli O157:H7. To manipulate intracellular c-di-GMP levels, we introduced into E. coli O157:H7 a c-di-GMP specific phosphodiesterase (PDE). Liquid chromatography tandem mass spectrometry analysis confirmed that in E. coli O157:H7, over-expression of PDE decreased c-di-GMP level. Consistent with the altered c-di-GMP level, PDE overexpression resulted in decreased biofilm formation in E. coli O157:H7. Furthermore, this diminished c-di-GMP levels reduced adhesion of E. coli O157:H7 to both cultured HT-29 cells and cattle colon explants. Consistently, mRNA levels of genes involved in adhesion were down-regulated including genes encoding E. coli common pili, long polar fimbriae 1, hemorrhagic coli pilus, as well as intimin and tir. We further observed decreased curli fimbriae synthesis in the strain with decreased c-di-GMP levels, which was supported by the reduction in the transcription of curli large subunit gene csgA and the curli expression regulator gene csgD. Genes for enterocyte effacement encoded regulator (Ler) and type III secretion system effectors, EspA and EspB, were also down-regulated. Collectively, data indicated that c-di-GMP signaling positively regulates E. coli O157:H7 intestinal epithelial cell and tissue colonization and expression of associated adhesion factors.

Topics: Animals; Cattle; Cyclic GMP; Enterocytes; Epithelial Cells; Escherichia coli Infections; Escherichia coli O157; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; HT29 Cells; Humans; Intestines; Signal Transduction; Time Factors

During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.

Topics: Animals; Bacterial Proteins; Cellulose; Cyclic GMP; Cytoplasm; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Female; Fimbriae, Bacterial; Gene Deletion; Hydrogen Peroxide; Iron; Mice; Phosphorus-Oxygen Lyases; Urinary Tract; Urinary Tract Infections; Urine; Uropathogenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

Topics: Adhesins, Bacterial; Antigens, Surface; Caco-2 Cells; Cell Line, Tumor; Cyclic GMP; DNA-Binding Proteins; Enterotoxigenic Escherichia coli; Epithelial Cells; Escherichia coli Infections; Escherichia coli Proteins; Gene Expression; Host-Pathogen Interactions; Humans; Intestinal Mucosa; Intestines; Promoter Regions, Genetic; Receptors, Cyclic AMP; Transcription, Genetic; Transcriptome; Virulence

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT(192(R-->G)) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa(11(N-->K)), STa(12(P-->F)), and STa(13(A-->Q))] and used the full-length pLT(192) as an adjuvant to carry the pSTa toxoid for pLT(192):pSTa-toxoid fusion antigens. Rabbits immunized with pLT(192):pSTa(12) or pLT(192):pSTa(13) fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT(192):pSTa(13) fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.

Topics: Animals; Antibodies, Bacterial; Antibodies, Neutralizing; Bacterial Toxins; Bacterial Vaccines; Cyclic GMP; Enterotoxigenic Escherichia coli; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genetic Engineering; Rabbits; Recombinant Proteins; Swine; Swine Diseases

Topics: Acute Kidney Injury; Anti-Bacterial Agents; Biopsy; Cyclic GMP; Escherichia coli Infections; Histiocytes; Humans; Ischemia; Kidney; Kidney Diseases; Malacoplakia; Male; Middle Aged; Periodic Acid-Schiff Reaction; Postoperative Complications; Pyelonephritis; Transplants; Urinary Tract Infections

Acute kidney injury (AKI) in septic patients drastically increases the mortality to 50-80%. Sepsis is characterized by hemodynamic perturbations as well as overwhelming induction of proinflammatory cytokines. Since ghrelin has been shown to have anti-inflammatory properties, we hypothesized that ghrelin may afford renal protection during endotoxemia-induced AKI. Studies were conducted in a normotensive endotoxemia-induced AKI model in mice by intraperitoneal injection of 3.5 mg/kg LPS. Serum ghrelin levels were increased during endotoxemia accompanied by increased ghrelin receptor (GHSR-1a) protein expression in the kidney. Ghrelin administration (1.0 mg/kg sc 6 h and 30 min before and 14 h after LPS) significantly decreased serum cytokine levels (TNF-alpha, IL-1beta, and IL-6) and serum endothelin-1 levels which had been induced by LPS. The elevated serum nitric oxide (NO) levels and renal inducible NO synthase expression were also decreased by ghrelin. Renal TNF-alpha levels were also increased significantly in response to LPS and ghrelin significantly attenuated this increase. When administrated before LPS, ghrelin protected against the fall in glomerular filtration rate at 16 h (172.9 +/- 14.7 vs. 90.6 +/- 15.2 microl/min, P < 0.001) and 24 h (147.2 +/- 20.3 vs. 59.4 +/- 20.7 microl/min, P < 0.05) as well as renal blood flow at 16 h (1.65 +/- 0.07 vs. 1.47 +/- 0.04 ml/min, P < 0.01) and 24 h (1.56 +/- 0.08 vs. 1.22 +/- 0.03 ml/min, P < 0.05) after LPS administration without affecting mean arterial pressure. Ghrelin remained renal protective even when it was given after LPS. In summary, ghrelin offered significant protection against endotoxemia-induced AKI. The renal protective effect of ghrelin was associated with an inhibition of the proinflammatory cytokines. Of particular importance was the suppression of TNF-alpha both in the circulation and kidney tissues. Thus, ghrelin may be a promising peptide in managing endotoxemia-induced AKI.

Topics: Acute Kidney Injury; Animals; Body Weight; Cyclic GMP; Endothelin-1; Endotoxemia; Escherichia coli Infections; Ghrelin; HMGB1 Protein; Kidney; Kidney Function Tests; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type II; Receptors, Ghrelin; Tumor Necrosis Factor-alpha

We have studied the cellular alterations, after in vitro adherence, of purified K88ab fimbriae to membranes of porcine enterocytes. Effects on enzymatic activities as disaccharydases and alkaline phosphatase show low changes. While cAMP levels were decreased (44%), guanylyl cyclase was increased (up to 200%), and levels of cGMP were in consequence significantly affected. This study support the role of cyclic GMP as intracellular mediator for adherence, and suggest their implication in disease, affecting a membrane-mediated mechanisms for guanylate cyclase activation, that is unique in the intestine.

Topics: Animals; Antigens, Bacterial; Bacterial Adhesion; Cyclic AMP; Cyclic GMP; Enterocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae Proteins; Fimbriae, Bacterial; Guanylate Cyclase; Intestinal Mucosa; Microvilli; Swine; Swine Diseases

Various invasive pathogens cause diarrhea, but the mechanism(s) are poorly understood. We hypothesized that nitric oxide and prostaglandins might modulate chloride secretory and barrier properties of the infected intestinal epithelium and that diarrhea is caused, in part, by altered expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2).. Studies were conducted in human intestinal epithelial cell lines (HT29/cl.19A, Caco-2, and T84). Cells were infected with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]). Infected cells and controls were tested for transepithelial resistance, chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate, and protein expression.. Cells infected with EIEC or SD, but not uninfected controls or ST/LA-exposed monolayers, showed a progressive reduction in transepithelial resistance starting at 6-12 hours. Infected HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an increase in total nitrite. Expression of iNOS, and consequently COX-2, was also increased, followed by increased production of prostaglandins and cyclic nucleotides. Furthermore, basal and stimulated chloride secretory responses to various agonists were enhanced in HT29/cl.19A and Caco-2 cells after infection with enteroinvasive bacteria, and this effect was reversed for some agonists by iNOS or COX-2 inhibitors. Increased expression of cystic fibrosis transmembrane conductance regulator and NKCC1 was also observed in EIEC or SD-infected cells vs. controls, secondary to NO synthase activity.. Up-regulation of iNOS and COX-2 by enteroinvasive bacteria can modulate chloride secretion and barrier function in intestinal epithelial cells. Thus, these enzymes represent possible therapeutic targets in infectious diarrhea.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Biological Transport; Caco-2 Cells; Chlorides; Cyclic AMP; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Dinoprostone; Electric Impedance; Enzyme Inhibitors; Epithelial Cells; Escherichia coli Infections; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; HT29 Cells; Humans; Intestinal Mucosa; Isoenzymes; Membrane Proteins; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrobenzenes; Peroxynitrous Acid; Prostaglandin-Endoperoxide Synthases; Salmonella Infections; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Sulfonamides; Up-Regulation; Water-Electrolyte Balance

Nitric oxide (NO) is a vasodilator agent that is cytotoxic and negatively inotropic in the heart. More recently, it has been shown that during sepsis there is a high amount of NO production by a NO synthase (NOS) that is inducible by cytokines. The aim of this study was to investigate the role of NO in the genesis of diaphragmatic dysfunction during sepsis. Rats were inoculated i.p. injection with 10 mg/kg of Escherichia coil endotoxin (E animals) or saline (C animals). Six hours after endotoxin or saline inoculation, diaphragmatic force and muscularc GMP (Cyclic guanosine monophosphate) were assessed by in vitro force frequency curves and ELISA method, respectively. As compared to C animals, E animals showed a significant decrease in diaphragmatic force for all the frequencies of stimulation (p < 0.01). This reduction was associated with a significant increase in muscular cGMP. Inhibition of NO synthesis in E animals with either dexamethasone (4 mg/kg IV, 45 min before endotoxin or saline) or NG-monomethyl-L-arginine (L-NMMA, 8 mg/kg IV, 90 min after endotoxin or saline) prevented the effects of endotoxin. However, no modification was seen with NG-monomethyl-D-arginine (D-NMMA), a molecule which does not inhibit NO synthesis. Administration of dexamethasone or L-NMMA in C animals did not induce any significant change in diaphragmatic force, and cGMP ratio. We conclude that NO has a contributive role in diaphragmatic dysfunction during Escherichia coli induced sepsis in rats.

Topics: Animals; Anti-Inflammatory Agents; Biopsy; Cyclic GMP; Dexamethasone; Disease Models, Animal; Endotoxemia; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Male; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Random Allocation; Rats; Rats, Sprague-Dawley; Respiratory Mechanics; Respiratory Paralysis

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [125I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a Kd of 0.5 x 10(-10) M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [125I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.

Topics: Bacterial Toxins; Binding Sites; Carrier Proteins; Cell Membrane; Colon; Colonic Neoplasms; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Guanylate Cyclase; Humans; Kinetics; Neoplasm Proteins; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Tumor Cells, Cultured

Nitric oxide and vasoactive intestinal peptide (VIP) are potent vasodilators and postulated as inducers of hypotension. These mediators activate guanylate cyclase and adenylate cyclase, respectively, with subsequent biosynthesis of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) producing vascular smooth muscle relaxation and vasodilatation. Cyclic nucleotides and VIP were evaluated during Escherichia coli septicemia in two groups of rabbits; 1) sepsis alone and 2) sepsis and a competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine. Arterial blood was obtained for determination of bacteremia, lactic acidemia, nucleotides, nitrites, and VIP levels. Significant bacteremia, endotoxemia, tachycardia, lactic acidosis, and hypotension occurred in all animals (P < 0.005). Circulating blood levels of cGMP, nitrites, cAMP, and VIP (P < 0.005) increased with development of shock. The NG-monomethyl-L-arginine treated animals had less cGMP, nitrites, cAMP, and VIP produced (P < 0.01). Plasma cGMP levels remained stable, suggesting that stimulated phagocytes in whole blood were responsible for increased cGMP levels. Infusion of VIP produced profound hypotension and lactic acidemia. Results of these experiments provide definitive evidence that nitric oxide and VIP are mediators during septic shock and their messengers are cGMP and cAMP, respectively. In addition, phagocytic stimulation with increased production of cGMP may initiate shock, with these mediators acting synergistically to prolong hypotension.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Bacteremia; Blood Pressure; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Escherichia coli Infections; Heart Rate; Lactates; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Rabbits; Reference Values; Vasoactive Intestinal Peptide

Endotoxin and other bacterial products induce the release of mediators which alter the circulation and cellular metabolism. Recent evidence suggests nitric oxide (NO) is one such mediator. The proposed mechanism by which NO produces hypotension is the activation of guanylate cyclase with subsequent biosynthesis of 3':5' cyclic guanosine monophosphate (cGMP). We studied the production of cGMP during Escherichia coli-induced septic shock in two experiments; the first with sepsis alone and the second using NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase. Animals in both experiments experienced significant bacteremia (P < 0.05), endotoxemia (P < 0.05), and lactic acidosis (P < 0.03). Mean arterial blood pressure decreased (P < 0.03) and heart rate increased (P < 0.05) within both groups but did not differ between groups. A significant increase in the production of circulating whole blood cGMP occurred at 3-5 h (P < 0.03). There was significantly less cGMP produced by the L-NMMA-treated animals (P < 0.01). These results demonstrate an elevation in cGMP during septic shock which is attenuated by the addition of L-NMMA. This suggests that NO may be present during gram-negative septic shock and its effects mediated through cGMP.

Topics: Animals; Arginine; Bacteremia; Carbon Dioxide; Cyclic GMP; Escherichia coli Infections; Lactates; Lactic Acid; Male; Nitric Oxide; omega-N-Methylarginine; Rabbits; Shock, Septic

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Base Sequence; Child; Cloning, Molecular; Conjugation, Genetic; Cyclic GMP; Diarrhea; DNA, Bacterial; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Genes, Bacterial; Humans; Ileum; In Vitro Techniques; Intestinal Mucosa; Male; Molecular Sequence Data; Multigene Family; Oligodeoxyribonucleotides; Open Reading Frames; Rabbits; Restriction Mapping; Sequence Homology, Amino Acid

Evidence exists that cyclic nucleotides play an important role in the stabilisation of developing synapses during the early postnatal period, and later on participate in the generation of the slow excitatory and inhibitory postsynaptic potentials. It is postulated that enterotoxins of Escherichia coli, due to their long-lasting and specific action on cyclic nucleotide-dependent processes, provide a unique mechanism for selective impairment of synaptogenesis in the abscence of cell loss. Therefore it seems possible that early enteritis, when caused by certain pathogenic strains of Escherichia coli, might be an aetiological factor in the genesis of idiopathic epilepsy.

Topics: Cyclic AMP; Cyclic GMP; Enterotoxins; Epilepsy; Escherichia coli; Escherichia coli Infections; Membrane Potentials; Synapses

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Adult; Animals; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Diarrhea, Infantile; Enterotoxins; Epithelial Cells; Epithelium; Escherichia coli Infections; Fluorides; Guanosine Triphosphate; Guanylate Cyclase; Humans; Infant; Intestinal Mucosa; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes; Prostaglandins; Temperature; Tritium