Enterotoxigenic Escherichia coli is detectable in water samples from an endemic area by real-time PCR

Aims: We aimed to develop an assay for sensitive detection and quantification of enterotoxigenic Escherichia coli (ETEC) in different types of water samples. Methods and Results: Real‐time polymerase chain reaction (PCR) assays with primers against ETEC enterotoxin genes estA (STh) estB (STp) and eltB (LT) were designed and the detection levels were determined to be three bacteria per PCR reaction. Gene copy numbers were estimated to be four (LT), two (STh) and one (STp) per bacteria. Twenty‐six household and 13 environmental water samples from Bangladesh were filtered through 0·22‐μm filters; DNA was extracted from the filters and analysed by real‐time PCR. The results were compared with toxin GM1‐enzyme‐linked immunosorbent assay (ELISA), in which colonies were tested for toxin production after cultivation of the filters. Out of the 39 samples tested, 18 household and 8 environmental samples were positive for ETEC in real‐time PCR, but only 6 positive samples were found with GM1‐ELISA. Conclusions: The method allows for highly sensitive detection and quantification of ETEC based on detection of toxin DNA in water samples. Significance and Impact of the Study: The method facilitates detection and identification of ETEC in water and allows comparison between water contamination and incidence of ETEC diarrhoea in endemic areas.     

Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)

Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.     

Guanylin, uroguanylin, and heat-stable euterotoxin activate guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubule cells

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.     

STa peptide analogs for probing guanylyl cyclase C

Guanylyl cyclase C (GC-C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18-residue heat-stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12-residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC-C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.     

Protection of mice against enterotoxigenic E. coli by immunization with a polyvalent enterotoxin comprising a combination of LTB, STa, and STb

Currently available enterotoxigenic Escherichia coli (ETEC) vaccines are based on colonization factors and/or the heat-labile enterotoxin B subunit (LTB). However, the induction of antitoxic responses against heat-stable enterotoxin a (STa) and b (STb) has merit as these two poorly immunogenic toxins are frequently associated with ETEC strains. In this study, we genetically constructed a trivalent enterotoxin fusion protein (STa–LTB–STb, abbreviated to SLS) in an effort to develop a single toxoid containing these three enterotoxins for vaccination against ETEC. Mutagenesis at one disulfide-bridge-forming cysteine in STa led to a dramatic reduction in the STa toxicity of SLS; however, the fusion peptide retained the STb-associated toxicity. Immunization of mice with SLS protein elicited significant antibody responses to LTB, STa, and STb. Significantly, the mice antisera were able to neutralize the biological activity of both STa and STb. In the experiment to assess the protective effect of SLS immunization, the mortality of mice receiving SLS was significantly lower than their control cohorts (P < 0.01) after intraperitoneal challenge with ETEC. These results show that the trivalent fusion enterotoxin SLS has the potential to serve as a useful toxin-based vaccine against ETEC-induced diarrheal disease via a single immunogen.     

Expression and characterization of the extracellular domain of guanylyl cyclase C from a baculovirus and Sf21 insect cells

Guanylyl cyclase (GC)-C, a single-transmembrane receptor protein for heat-stable enterotoxin, guanylin, and uroguanylin, and its N-terminal extracellular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant GC-C, containing the complete sequence, retained its binding affinity to heat-stable enterotoxin with a KD value (6.2 x 10(-10) M) and cyclase catalytic activity at a level similar to those of GC-C expressed in mammalian cell lines, such as COS-7. The N-terminal extracellular domain was prepared in a form which contained the hexahistidine tail at its C-terminus and was purified as a homogenous protein by Con A and Ni-chelating affinity chromatography from the culture medium of the insect cells. The purified N-terminal extracellular domain of GC-C exhibited the high (KD = 4 x 10(-10) M) and low (KD = 7 x 10(-8) M) affinity sites in binding to heat-stable enterotoxin. These results clearly indicate that the N-terminal extracellular domain of GC-C possesses the same biochemical characteristics as the complete GC-C protein even in the membrane-free form. Moreover, the extracellular domain is able to form an oligomer in a ligand-dependent manner, suggesting that the N-terminal extracellular domains interact with one another in binding to ligands.     

Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

To determine the modes of three disulfide linkages in the heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6-18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138-142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6-18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6-18) are Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys.     

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension

Guanylyl cyclase C (GC-C) was found to function as the principal receptor for heat-stable enterotoxins (STa), major causative factors in E. coli-induced secretory diarrhea. GC-C is enriched in intestinal epithelium, but was also detected in other epithelial tissues. The enzyme belongs to the family of receptor guanylyl cyclases, and consists of an extracellular receptor domain, a single transmembrane domain, a kinase homology domain, and a catalytic domain. GC-C is modified by N-linked glycosylation and, at least in the small intestine, by proteolysis, resulting in a STa receptor that is coupled non-covalently to the intracellular domain. So far two endogenous ligands of mammalian GC-C have been identified i.e. the small cysteine-rich peptides guanylin and uroguanylin. The guanylins are released in an auto- or paracrine fashion into the intestinal lumen but may also function as endocrine hormones in gut-kidney communication and as regulators of ion transport in extra-intestinal epithelia. They are thought to activate GC-C by inducing a conformational change in the extracellular portion of the homotrimeric GC-C complex, which allows two of the three intracellular catalytic domains to dimerize and form two active catalytic clefts. In the intestine, activation of GC-C results in a dual action: stimulation of Cl and HCO₃ secretion, through the opening of apical CFTR Cl channels; and inhibition of Na absorption, through blockade of an apical Na/H exchanger. The principal effector of the GC-C effect on ion transport is cGMP dependent protein kinase type II, which together with GC-C and the ion transporters, may form a supramolecular complex at the apical border of epithelial cells.     

Molecular Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Diarrheal Patients in Korea during 2003–2011

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of infectious diarrhea in developing countries. In order to characterize the molecular features of human ETEC isolates from Korea, we investigated the profiles of enterotoxin and colonization factor (CF) genes by polymerase chain reaction (PCR) and performed multilocus sequence typing (MLST) with a total of 291 ETEC strains. The specimens comprised 258 domestic strains isolated from patients who had diarrhea and were from widely separated geographic regions in Korea and 33 inflow strains isolated from travelers visiting other Asian countries. Heat-stable toxin (STh)-possessing ETEC strains were more frequent than heat-labile toxin (LT)-possessing ETEC strains in the domestic isolates, while the detection rates of both enterotoxin genes were similar in the inflow isolates. The profile of CF genes of domestic isolates was similar to that of inflow isolates and the major CF types of the strains were CS3-CS21-CS1/PCF071 and CS2-CS3-CS21. Most of these 2 CF types were detected in ETEC strains that possess both lt and sth genes. The major MLSTST types of domestic isolates were ST171 and ST955. Moreover, the 2 major CF types were usually found concomitantly with the 2 major MLST STs, ST171 and ST955. In conclusion, our genotyping results may provide useful information for guiding the development of geographically specific vaccines against human ETEC isolates.     

Colonocyte basolateral membranes contain Escherichia coli heat-stable enterotoxin receptors

Heat-stable enterotoxin (ST(a)) elaborated by E. coli is a major cause of diarrhea. The transmembrane protein guanylyl cyclase C (GC-C) is the acknowledged receptor for ST(a) and for the mammalian peptides guanylin and uroguanylin. Binding to GC-C results in generation of cGMP, activation of type II cGMP-dependent protein kinase, phosphorylation of CFTR and increased chloride and bicarbonate secretion. We had previously shown that ST(a) receptors (GC-C) are found on the brush border membranes of small intestinal enterocytes and of colonocytes. However, since it has subsequently been shown that the endogenous ligands for these receptors, guanylin and uroguanylin, circulate in blood, we proposed the existence of ST(a) binding sites on the basolateral membranes (BLM) of colonocytes. Specific binding of 125I-ST(a) to rat colonocyte BLM was seen. The kinetics of binding to the BLM were similar to binding to BBM. The nature of the BLM receptor is unknown. This suggests that circulating guanylin and uroguanylin, analogues of ST(a), may also function via the basolateral surface.     

The Heat-Stable Enterotoxin-Guanylin Receptor is Expressed in Rat Hepatocytes and in a Rat Hepatoma (H-35) Cell Line

Abstract

Background/Aims: Guanylyl cyclase C (GC-C) is an intestinal transmembrane receptor which binds both guanylin, an endogenous ligand, and Escherichia coli heat-stable enterotoxin (STa) resulting in 5′-cyclic guanosine monophosphate (cGMP) accumulation and chloride secretion. In the adult rat, there is a high basal level of GC-C expression in the intestine, but not in the liver. Increased expression of GC-C in the rat liver has been demonstrated during the perinatal period as well as with liver regeneration and during an acute phase response. The aim of this study was to identify and utilize cell culture models to further characterize the expression of GC-C in the liver. Methods: STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression by Northern analysis were determined in primary cultures of rat hepatocytes and H-35 cells, a rat hepatoma cell line, following treatment with dexamethasone and/or interleukin-6 (IL-6). Results: In rat hepatocytes treated with the combination of dexamethasone and IL-6, there was an increase in STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression as compared to untreated cells. In H-35 cells treated with dexamethasone alone, there was an increase in STa binding, STa-stimulated cGMP accumulation, and GC-C RNA expression as compared to untreated cells. Conclusion: Primary cultures of rat hepatocytes and H-35 cells can be utilized to further study upregulation of GC-C in the hepatocyte. The expression of this receptor in hepatocytes, combined with the recent demonstration of circulating guanylin, is consistent with a functional role for GC-C in the liver.     

Survival and gene expression of enterotoxigenic Escherichia coli during long‐term incubation in sea water and freshwater

Aims: In this study, the main objective was to verify the hypothesis of induction of ‘viable but non‐culturable’ (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. Methods and Results: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat‐labile (LT) or heat‐stable (ST) enterotoxins was observed using GM1‐ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real‐time RT‐PCR on cDNA derived from the bacteria. Conclusions: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. Significance and Impact of the Study: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long‐term incubation in water.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Phosphorylation and activation of the intestinal guanylyl cyclase receptor for Escherichia coli heat-stable toxin by protein kinase C

The heat-stable enterotoxin STa of E. coli causes diarrhea by binding to and stimulating intestinal membrane-bound guanylyl cyclase, triggering production of cyclic GMP. Agents which stimulate protein kinase C (PKC), including phorbol esters, synergistically enhance STa effects on cGMP and secretion. We investigated whether PKC causes phosphorylation of the STa receptor in vivo and in vitro.Immunoprecipitation of the STa receptor-guanylyl cyclase was carried out from extracts of T84 colon cells metabolically labelled with [³²P]-phosphate using polyclonal anti-STa receptor antibody. The STa receptor was phosphorylated in its basal state, and ³²P content in the 150 kDa holoreceptor band increased 2-fold in cells exposed to phorbol ester for 1 h. In vitro, immunopurified STa receptor was readily phosphorylated by purified rat brain PKC. Phosphorylation was inhibited 40% by 5 M of a synthetic peptide corresponding to the sequence around Ser¹⁰²⁹ of the STa receptor, a site previously proposed as a potential PKC phosphorylation site. Treatment of the immunopurified STaR/GC with purified PKC increased STa-stimulated guanylyl cyclase activity 2-fold. We conclude that PKC phosphorylates and activates the STa receptor/guanylyl cyclase in vitro and in vivo; Ser¹⁰²⁹ of the STaR/GC remains a candidate phosphorylation site by PKC.Abbreviations STa the heat-stable enterotoxin of E. coli, which has also been called ST-I and STp. The 18 amino acid variant was used throughout - PBS phosphate-buffered saline - PDB 4--12, 13-phorbol dibutyrate - ANP atrial natriuretic peptide - STaR/GC STa receptor/guanylyl cyclase, also called GC-C - PKC protein kinase C     

Recognition and signal transduction mechanism of Escherichia coli heat-stable enterotoxin and its receptor, guanylate cyclase C.

Guanylate cyclase C (GC-C), a member of the membrane-bound GC family, consists of an extracellular domain (ECD) and an intracellular domain, which are connected by a single-transmembrane region. GC-C is a receptor protein, i.e. specifically stimulated by the endogenous peptides guanylin, uroguanylin, lymphoguanylin, and the exogenous peptide heat-stable enterotoxin (ST(a)), secreted by pathogenic Escherichia coli and acting on the intestinal brush border membranes. The binding of these peptide ligands to the ECD of GC-C results in the synthesis of cyclic GMP in cells, which, in turn, regulates a variety of intracellular physiologic processes. As the cloning of GC-C, its physiologic functions of each domain have been vigorously investigated. The structural characterization of the ligand-binding domain of the receptor promises to provide important clues for better understanding of the mechanisms of receptor recognition and activation. Recently, structural data for each domain of membrane-bound GCs and related proteins has become available. Coupling information obtained from such work and validation of structure-function relationships of GC-C and its ligands should allow for three-dimensional mapping of their interaction site in detail. Our approach to this issue involved designing photoaffinity-labeling ST(a) analogs, capable of binding covalently to the ligand-binding region of the ECD of GC-C. The photoaffinity-labeling ligand was used to covalently label a soluble form of the recombinant ECD protein. Mass spectrometric analyses of an endoproteinase digest of the ECD revealed that the ligand specifically bound to a narrow region contained in the membrane-proximal subdomain of the ECD of GC-C. These results will enable us to identify the possible binding motifs within the ligand-binding domain by computer modeling. In this review, we summarize the available data on the recognition mechanism between ST(a) and GC-C at the molecular level.     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.     

Expression and immunogenicity of an <Emphasis Type="Italic">Escherichia coli</Emphasis> K99 fimbriae subunit antigen in soybean

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T₀ events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T₀ lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T₁ and T₂ generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4⁺ T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.     

Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli

Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H₂O₂. Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.