Characterization of the Binding Specificity of K88ac and K88ad Fimbriae of Enterotoxigenic Escherichia coli by Constructing K88ac/K88ad Chimeric FaeG Major Subunits

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) fimbriae are the major cause of diarrhea in young pigs. Three antigenic variants of K88 fimbriae (K88ab, K88ac, and K88ad) have been identified among porcine ETEC strains. Each K88 fimbrial variant shows a unique pattern in binding to different receptors on porcine enterocytes. Such variant specificity in fimbrial binding is believed to be controlled by the major subunit (FaeG) of the K88 fimbriae, because the genes coding for the only other fimbrial subunit are identical among the three variants. Uniqueness in binding to host receptors may be responsible for differences in the virulence levels of porcine diarrhea disease caused by K88 ETEC strains. To better understand the relationships between the structure of FaeG proteins and fimbrial binding function, and perhaps virulence in disease, we constructed and expressed various K88ac/K88ad faeG gene chimeras and characterized the binding activity of each K88 chimeric fimbria. After verifying biosynthesis of the chimeric fimbriae, we examined their binding specificities in bacterial adherence assays by using porcine brush border vesicles that are specific to either the K88ac or K88ad fimbria. Results showed that each fimbria switched binding specificity to that of the reciprocal type when a peptide comprising amino acids 125 to 163 was exchanged with that of its counterpart. Substitutions of a single amino acid within this region negatively affected the binding capacity of each fimbria. These data indicate that the peptide including amino acids 125 to 163 of the FaeG subunit is essential for K88 variant-specific binding.

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Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

AMB Express ◽

10.1186/s13568-021-01201-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pengpeng Xia ◽

Yunping Wu ◽

Siqi Lian ◽

Guomei Quan ◽

Yiting Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Clinical Symptoms ◽

Mucosal Damage ◽

Enterotoxigenic Escherichia Coli ◽

Intestinal Epithelial ◽

Antibody Microarray ◽

E Coli ◽

Fimbrial Subunit ◽

Induced Apoptosis ◽

Weaning Piglets

AbstractEnterotoxigenic Escherichia coli (ETEC) F4ac is a major constraint to the development of the pig industry, which is causing newborn and post-weaning piglets diarrhea. Previous studies proved that FaeG is the major fimbrial subunit of F4ac E. coli and efficient for bacterial adherence and receptor recognition. Here we show that the faeG deletion attenuates both the clinical symptoms of F4ac infection and the F4ac-induced intestinal mucosal damage in piglets. Antibody microarray analysis and the detection of mRNA expression using porcine neonatal jejunal IPEC-J2 cells also determined that the absence of FaeG subunit alleviated the F4ac promoted apoptosis in the intestinal epithelial cells. Thus, targeted depletion of FaeG is still beneficial for the prevention or treatment of F4ac infection.

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Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3850-3859.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3850-3859

Author(s):

T A Coleman ◽

C Kunsch ◽

M Maher ◽

S M Ruben ◽

C A Rosen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Fusion Protein ◽

Binding Specificity ◽

Single Amino Acid ◽

Nf Kappa B ◽

Dna Binding Specificity ◽

Dna Motif ◽

Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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Gastrointestinal ecology response of piglets’ diets containing non-starch polysaccharide hydrolysis products and egg yolk antibodies following an oral challenge with Escherichia coli (k88)

Canadian Journal of Animal Science ◽

10.4141/cjas09007 ◽

2009 ◽

Vol 89 (3) ◽

pp. 341-352 ◽

Cited By ~ 7

Author(s):

E Kiarie ◽

B A Slominski ◽

D O Krause ◽

C M Nyachoti

Keyword(s):

Escherichia Coli ◽

Egg Yolk ◽

Intestinal Morphology ◽

Oral Challenge ◽

Enterotoxigenic Escherichia Coli ◽

Adaptation Period ◽

Egg Yolk Antibodies ◽

Hydrolysis Products ◽

K88 Fimbriae ◽

Polysaccharide Hydrolysis

The gastrointestinal ecology (GE) of piglets fed diets containing non-starch polysaccharide hydrolysis products (HP) and egg yolk antibodies against K88 fimbriae (EYA) following oral challenge with enterotoxigenic Escherichia coli K88 (ETEC) was investigated. The HP were products of incubating feedstuffs with a blend of carbohydrase enzymes. Forty, 21-d-old pigs (two pigs/pen) were assigned to four diets to give five pens per diet. The diets were: a control fed without or with 5 g kg-1 of HP and EYA either singly or in combination forming a 2 × 2 factorial arrangement. Following a 9-d adaptation period, all pigs were orally challenged with ETEC and killed at 24 and 48 h post-challenge (one pig/pen on each occasion). Feeding HP increased pre-challenge average daily gain (252 vs. 207 g d-1; P = 0.01). An interaction (P < 0.10) between EYA and HP was observed such that when fed in combination they resulted in higher ileal digesta lactic acid and cecal DM contents and lower ileal digesta ammonia. The main effects (P < 0.05) were such that pigs fed EYA-diets had shorter intestinal crypt whilst pigs fed HP-diets showed low gastric pH and high ileal mucosal adherent lactobacilli counts. In conclusion, HP and EYA influenced indices of fermentative characteristics and intestinal morphology in the gastrointestinal ecology of piglets orally challenged with enterotoxigenic E. coli (k88).Key words: Egg yolk antibodies, ETEC, gastrointestinal ecology, non-starch polysaccharides hydrolysis products, piglet

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Genetic Characterization and Immunogenicity of Coli Surface Antigen 4 from Enterotoxigenic Escherichia coli when It Is Expressed in a Shigella Live-Vector Strain

Infection and Immunity ◽

10.1128/iai.71.3.1352-1360.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1352-1360 ◽

Cited By ~ 19

Author(s):

Zeev Altboum ◽

Myron M. Levine ◽

James E. Galen ◽

Eileen M. Barry

Keyword(s):

Escherichia Coli ◽

Shigella Flexneri ◽

Regulatory Protein ◽

Electron Microscopic Examination ◽

Amino Acid Sequences ◽

Enterotoxigenic Escherichia Coli ◽

Electron Microscopic ◽

Bacterial Surface ◽

Fimbrial Subunit ◽

Mucosal Antibody

ABSTRACT The genes that encode the enterotoxigenic Escherichia coli (ETEC) CS4 fimbriae, csaA, -B, -C, -E, and -D′, were isolated from strain E11881A. The csa operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa tip-associated protein (CsaE), a 27-kDa chaperone-like protein (CsaA), a 97-kDa usher-like protein (CsaC), and a deleted regulatory protein (CsaD′). The predicted amino acid sequences of the CS4 proteins are highly homologous to structural and assembly proteins of other ETEC fimbriae, including CS1 and CS2, and to CFA/I in particular. The csaA, -B, -C, -E operon was cloned on a stabilized plasmid downstream from an osomotically regulated ompC promoter. pGA2-CS4 directs production of CS4 fimbriae in both E. coli DH5α and Shigella flexneri 2a vaccine strain CVD 1204, as detected by Western blot analysis and bacterial agglutination with anti-CS4 immune sera. Electron-microscopic examination of Shigella expressing CS4 confirmed the presence of fimbriae on the bacterial surface. Guinea pigs immunized with CVD 1204(pGA2-CS4) showed serum and mucosal antibody responses to both the Shigella vector and the ETEC fimbria CS4. Among the seven most prevalent fimbrial antigens of human ETEC, CS4 is the last to be cloned and sequenced. These findings pave the way for CS4 to be included in multivalent ETEC vaccines, including an attenuated Shigella live-vector-based ETEC vaccine.

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PSVII-16 Galactosylated chitosan-oligosaccharides have anti-adhesive effect against enterotoxigenic Escherichia coli in piglets

Journal of Animal Science ◽

10.1093/jas/skz122.393 ◽

2019 ◽

Vol 97 (Supplement_2) ◽

pp. 224-224

Author(s):

Charlotte Maria Elisabeth Heyer ◽

Weilan Wang ◽

Yalu Yan ◽

Michael G Gänzle ◽

Ruurd T Zijlstra

Keyword(s):

Escherichia Coli ◽

Enterotoxigenic Escherichia Coli ◽

Rrna Genes ◽

Saline Control ◽

Fluid Loss ◽

Bacterial Gene ◽

Test Product ◽

Chitosan Oligosaccharides ◽

Galactosylated Chitosan ◽

K88 Fimbriae

Abstract Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets. In vitro, high molecular weight β-galactosylated chitosan-oligosaccharides (Gal-COS) had strong anti-adhesive activity against ETEC-expressing K88 fimbriae (ETEC K88) binding to porcine erythrocytes. This study assessed the effects of Gal-COS differing in structure on anti-adhesive properties against ETEC in a small intestinal segment perfusion (SISP) model in 8 piglets (BW 10 kg; 5-wk old). With 10 jejunal segments in each pig, 5 segments were infected with ETEC K88, and the other 5 segments were infused with saline (non-ETEC). Every 2 paired segments (ETEC or non-ETEC) from the same pig were treated for 8 h with 64 ml of 10 g L-1 of one of the following test products: 1) α-Gal-COS; 2) β-Gal-COS; 3) exopolysaccharides produced by Lactobacillus reuteri; and 4) raffinose in a double 4 × 4 Latin square with a saline control. Infection by ETEC K88 was verified by quantitative PCR. Net fluid loss was calculated as difference of fluid loss between ETEC segment and its paired non-ETEC segment. Data were analyzed using the mixed model with segment and test product as fixed effects, and pig as random effect. Number of eubacterial rRNA genes was 10-fold greater (P < 0.001) in ETEC segments than non-ETEC segments, indicating that ETEC K88 accounted for > 90% of bacterial gene counts. Test product did not affect (P > 0.10) the number of ETEC bacteria in the outflow fluid. Furthermore, net fluid loss caused by ETEC tended (P = 0.08) to be decreased by β-Gal-COS compared to all other treatments. In conclusion, the in vivo SISP model confirmed that Gal-COS had anti-diarrheal effects, indicating that β-Gal-COS is a potential feed additive to reduce the ETEC-induced diarrhea in piglets.

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Increasing dietary tryptophan in conjunction with decreasing other large neutral amino acids increases weight gain and feed intake in weaner pigs regardless of experimental infection with enterotoxigenic Escherichia coli

Journal of Animal Science ◽

10.1093/jas/skaa190 ◽

2020 ◽

Vol 98 (8) ◽

Author(s):

Samantha O Sterndale ◽

David W Miller ◽

Josie P Mansfield ◽

Jae C Kim ◽

John R Pluske

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Feed Intake ◽

Stress Responses ◽

Plasma Cortisol ◽

Average Daily Gain ◽

Enterotoxigenic Escherichia Coli ◽

Large Neutral Amino Acids ◽

Neutral Amino Acids ◽

Serum Serotonin

Abstract Dietary tryptophan (Trp) is a precursor for serotonin, a neuromediator involved in stress responses. Tryptophan competes with other large neutral amino acids (LNAA: tyrosine, isoleucine, leucine, valine, and phenylalanine) to cross the blood–brain barrier; therefore, the regulation of circulating LNAA can influence Trp availability in the cortex and serotonin biosynthesis. The hypothesis examined in this study was that increased supplementation of dietary Trp and a reduction in LNAA for weaned pigs experimentally infected with enterotoxigenic Escherichia coli (ETEC; F4) will increase Trp availability in plasma and reduce indices of the stress response, which will translate to reduced production losses. At 21 ± 3 d of age (mean ± SEM), 96 male pigs (Large White × Landrace) weighing 6.3 ± 0.98 kg (mean ± SEM) were individually penned and allocated to a 4 × 2 factorial arrangement of treatments, with respective factors being 1) four dietary standardized ileal digestible (SID) Trp and LNAA contents, being HTrpHLNAA (Low Trp-High LNAA; 0.24% SID Trp: 5.4% SID LNAA), HTrpHLNAA (Low Trp-Low LNAA; 0.24% SID Trp: 4.6% SID LNAA), HTrpHLNAA (High Trp-High LNAA; 0.34% SID Trp: 5.4% SID LNAA), and HTrpHLNAA (High Trp-Low LNAA; 0.34% SID Trp: 4.6% SID LNAA), and 2) without/with ETEC infection. Pigs were orally infected with 0.8 mL (3.6 × 109 CFU/mL) ETEC at days 7 and 8 after weaning. Pigs fed diets high in Trp irrespective of the level of LNAA (HTrpHLNAA and HTrpLLNAA) had higher plasma Trp concentrations (P < 0.001) and a Trp:LNAA ratio (P < 0.001) before infection and 6 d after infection. Following infection, noninfected pigs had higher plasma Trp (P = 0.03) and a Trp:LNAA ratio (P = 0.004) compared with pigs infected with ETEC. Plasma cortisol levels after infection were higher in ETEC-infected pigs (P = 0.05) and altering dietary Trp and LNAA concentrations did not influence (P > 0.05) plasma cortisol. Pigs fed diet HTrpLLNAA had higher serum serotonin levels 24 h after infection (P = 0.02) compared with pigs fed diets LTrpLLNAA and HTrpHLNAA. Similarly, pigs fed diet HTrpLLNAA had a higher (P = 0.02) average daily gain during the 3-wk study. Overall, average daily feed intake tended to be higher in pigs fed an HTrpLLNAA diet compared with the other diets (P = 0.08). These results suggest that the increased supplementation of dietary Trp with reduced LNAA increased circulating Trp levels that, in turn, likely caused higher serum serotonin levels, irrespective of infection with ETEC, and improved aspects of post-weaning performance.

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SPECIFIC BINDING OF PROLACTIN TO SEMINAL VESICLE, PROSTATE AND TESTICULAR HOMOGENATES OF IMMATURE, MATURE AND AGED RATS

Journal of Endocrinology ◽

10.1677/joe.0.0740163 ◽

1977 ◽

Vol 74 (2) ◽

pp. 163-173 ◽

Cited By ~ 48

Author(s):

R. J. BARKEY ◽

J. SHANI ◽

T. AMIT ◽

D. BARZILAI

Keyword(s):

Seminal Vesicle ◽

Binding Capacity ◽

Specific Binding ◽

Age Groups ◽

Liver Homogenate ◽

Binding Specificity ◽

Scatchard Analysis ◽

Total Binding ◽

Ovine Prolactin ◽

Rat Prostate

Ovine prolactin was iodinated by the lactoperoxidase method and purified by gel filtration on Sephadex G-100. The binding ability of the labelled hormone was determined, by incubation with liver homogenate from rabbits in late pregnancy, to be 8·8% total binding/ mg protein, of which 86% was specific. The fraction of 125I-labelled ovine prolactin which bound most strongly was subsequently used to study its binding to rat seminal vesicle, prostate and testicular homogenates. The total binding to the seminal vesicle homogenate taken from mature (80-day-old) rats was the highest (11·69%/mg protein), but the greatest degree of binding specificity (82·6%) was to immature (30-day-old) rat prostate. Both total and specific binding to rat testicular homogenate were consistently very low. The binding specificity was demonstrated by displacement studies: while ovine prolactin caused displacement of specific binding, human chorionic gonadotrophin, rat thyrotrophin and human follicle-stimulating hormone did not cause any significant displacement of bound 125I-labelled ovine prolactin. Affinity constants (Ka) and binding capacities for the seminal vesicle and prostate homogenates were determined by Scatchard analysis and the effect of age on these parameters was studied. There was no difference in Ka between the aged (220-day-old), immature and mature rat tissue homogenates; however, a significant fall in binding capacity was observed in the mature rat prostate, and a further fall in the aged rat prostate. No such change was observed in the binding capacity of the seminal vesicle, as estimated by Scatchard analysis, although total and specific binding to the mature homogenates was higher than that of the other age groups.

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Hydrophilic Domain II of Escherichia coli Dr Fimbriae Facilitates Cell Invasion

Infection and Immunity ◽

10.1128/iai.73.9.6119-6126.2005 ◽

2005 ◽

Vol 73 (9) ◽

pp. 6119-6126 ◽

Cited By ~ 20

Author(s):

Margaret Das ◽

Audrey Hart-Van Tassell ◽

Petri T. Urvil ◽

Susan Lea ◽

David Pettigrew ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Cell Invasion ◽

Cell Entry ◽

Wild Type ◽

Alanine Scanning Mutagenesis ◽

Decay Accelerating Factor ◽

Fimbrial Subunit ◽

Hydrophilic Domain ◽

Intracellular Compartmentalization

ABSTRACT Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decay-accelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

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Fimbrial Subunit Protein FaeG Expressed in Transgenic Tobacco Inhibits the Binding of F4ac Enterotoxigenic Escherichia coli to Porcine Enterocytes

Transgenic Research ◽

10.1023/b:trag.0000034621.55404.70 ◽

2004 ◽

Vol 13 (3) ◽

pp. 295-298 ◽

Cited By ~ 17

Author(s):

Jussi J. Joensuu ◽

Mirkka Kotiaho ◽

Tero Riipi ◽

Veerle Snoeck ◽

E. Tapio Palva ◽

...

Keyword(s):

Escherichia Coli ◽

Transgenic Tobacco ◽

Enterotoxigenic Escherichia Coli ◽

Subunit Protein ◽

Fimbrial Subunit

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Inactivation of the β-adrenergic receptor in cardiac muscle by dithiols

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-154 ◽

1985 ◽

Vol 63 (8) ◽

pp. 932-936 ◽

Cited By ~ 5

Author(s):

Trevor I. Prior ◽

Vandana Patel ◽

G. I. Drummond

Keyword(s):

Adrenergic Receptor ◽

Reduced Glutathione ◽

Binding Capacity ◽

Specific Binding ◽

Binding Activity ◽

Ventricular Muscle ◽

Adrenergic Antagonist ◽

Equilibrium Dissociation ◽

Rabbit Ventricular Muscle ◽

Membrane Binding Sites

The effect of sulfhydryl reagents on binding of the β-adrenergic antagonist (−)-[3H]dihydroalprenolol hydrochloride ((−)-[3H]DHA) to a microsomal fraction of rabbit ventricular muscle was studied. Incubation with the disulfide reducing agents dithiothreitol (DTT), 2-mercaptocthanol, and reduced glutathione resulted in loss of (−)-[3H]DHA binding. At 500 μM DTT, less than 50% of specific binding activity remained; at 100 mM, binding was completely eliminated. 2-Mercaptoethanol and reduced glutathione were less effective than DTT at inhibiting binding activity. The total binding capacity (Bmax) decreased from 155.4 fmol mg−1 of protein, in the absence of DTT, to 92.4 and 77.5 fmol mg−1 at 0.25 and 0.7 mM DTT, respectively. The equilibrium dissociation constant (KD) increased from 7.6 nM, in the absence of DTT, to 10.3 nM at 0.25 mM DTT and to 20.8 nM at 0.7 mM DTT. Thus, DTT-induced decline in (−)-[3H]DHA binding results from a decrease in both the number and affinity of membrane binding sites for the tracer. Receptors could be protected from DTT inactivation by preincubation with β-adrenergic ligands. Oxidants could not reverse inactivation, with the exception of o-iodosobenzoate which was only partially effective. Thus, the β-adrenergic receptor of rabbit ventricular muscle contains essential disulfide moietie(s) which can be inactivated by reducing thiols.

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