987P fimbriae from porcine enterotoxigenic Escherichia coli: Characterization, N-terminal amino acid sequence and immunization with purified antigen

Abstract The 987P fimbrial antigen was purified from a spontaneous overproducing variant. The protein was characterized with respect to M _r, amino acid sequence and partial covalent structure. The purified protein was used for vaccination trials, and excellent protection of piglets upon challenge with 987P positive enterotoxigenic strains was seen.     

Hemagglutination by pilus antigen 987P of enterotoxigenic Escherichia coli

The hemagglutination (HA) by pilus antigen 987P of an enterotoxigenic Escherichia coli strain 987 was examined using fresh and glutaraldehyde (GA)-fixed erythrocytes (RBC) of various animals. Only when GA-fixed RBC was employed, a strain 987 exhibited striking HA activities. This was also demonstrated by using latex heads sensitized with the 987P antigen. The 987P-specific antiserum inhibited HA of strain 987 and 987P sensitized latex beads against GA-fixed RBC. We concluded that HA of strain 987 against GA-fixed RBC was specifically associated with the presence of 987P pilus antigen but do not exclude a possibility that adhesin is distinct from pili antigen.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli

The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32-35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P.     

Erythrocyte and porcine intestinal glycosphingolipids recognized by F4 fimbriae of enterotoxigenic Escherichia coli

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO₃-3Galß1Cer), sulf-lactosylceramide (SO₃-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).     

Binding of F41 and K99 fimbriae of enterotoxigenic Escherichia coli to glycoproteins from bovine and porcine colostrum

F41 and K99 fimbriae of enterotoxigenic Escherichia coli were found to bind to periodate-sensitive oligosaccharides of glycoproteins from bovine and porcine colostrum. Only a minor component of casein fractions (kappa-casein) possessed receptors for one type of fimbriae (K99). Both whey and fat globule membranes were rich in glycoproteins with receptor structures. Porcine colostrum seemed to contain a higher quantity of receptors than bovine colostrum.     

Purification and analysis of colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae from enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli fimbriae are immunogenic and play a key role in intestinal colonization. Native colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae were purified by a common method involving shearing, differential centrifugation, gel filtration, and density gradient ultracentrifugation. The compositions and N-terminal sequences were determined. Coli surface antigen 3 possesses two N-terminal isoforms, one of which matches the published DNA sequence, except for the previously proposed signal sequence cleavage point.     

Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors

Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adR^FA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adR^PA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adR^PA receptor is masked by the F4adR^FA. The hypothesis that F4adR^FA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy–Weinberg equilibrium statistics. The F4adR^PA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abR^PA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abR^FA/F4acR⁺/F4adR^FA); A2 (F4abR^FA/F4acR⁺/F4adR^PA); B (F4abR^FA/F4acR⁺/F4adR⁻); C1 (F4abR^PA/F4acR⁻/F4adR^FA); C2 (F4abR^PA/F4acR⁻/F4adR^PA); D1 (F4abR⁻/F4acR⁻/F4adR^FA); D2 (F4abR⁻/F4acR⁻/F4adR^PA); E (F4abR⁻/F4acR⁻/F4adR⁻).     

毒素源性大肠杆菌定居因子抗原CS6基因的克隆

通过构建人源毒素源性大肠杆菌野生株E519/66A大质粒的基因文库,成功地筛选出能表达定居因子抗原CS6菌毛的阳性克隆,初步确定了克隆DNA片段的限制性内切酶图谱。CS6抗原的编码和调控基因集中在一大小为4.6kb的DNA区段中,该片段能产生两种分子量大小不同、但抗原反应交叉的菌毛蛋白。本研究获得的CS6抗原阳性的重组菌株可用于人源ETEC多价疫苗的研制,克隆的基因片段亦可作为研究CS6菌毛蛋白基因表达及调控的基础。     

Permissive linker insertion sites in the outer membrane protein of 987P fimbriae of Escherichia coli.

The FasD protein is essential for the biogenesis of 987P fimbriae of Escherichia coli. In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein. In addition, the accessibility of FasD to proteases established the presence of surface-exposed FasD domains on both sides of the outer membrane. The fasD gene was sequenced, and the deduced amino acid sequence was shown to share homologous domains with a family of outer membrane proteins from various fimbrial systems. Similar to porins, fimbrial outer membrane proteins are relatively polar, lack typical hydrophobic membrane-spanning domains, and posses secondary structures predicted to be rich in turns and amphipathic beta-sheets. On the basis of the experimental data and structural predictions, FasD is postulated to consist essentially of surface-exposed turns and loops and membrane-spanning interacting amphipathic beta-strands. In an attempt to test this prediction, the fasD gene was submitted to random in-frame linker insertion mutagenesis. Preliminary experiments demonstrated that it was possible to produce fasD mutants, whose products remain functional for fimbrial export and assembly. Subsequently, 11 fasD alleles, containing linker inserts encoding beta-turn-inducing residues, were shown to express functional proteins. The insertion sites were designated permissive sites. The inserts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly involved in fimbrial export. In contrast, FasD is not expected to accommodate such residues in its amphipathic beta-strands without being destabilized in the membrane and losing function. All permissive sites were sequenced and shown to be located in or one residue away from predicted turns. In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic beta-strands. These results are consistent with the structural predictions for FasD.     

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae

Abstract Porcine Escherichia coli strains isolated from cases fo postweaning diarrhea or edema disease were analysed for the presence of fedA , the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA -specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.     

产肠毒素性大肠杆菌K88菌毛装配

K88菌毛介导产肠毒索性大肠杆菌在小肠上皮细胞的粘附,是引起新生仔猪腹泻的主要致病因子之一.菌毛的合成与装配是由fae操纵子调控的,fae操纵子包含10个基,faeA-fae J,其中有些基因表达菌毛装配所需的各种结构蛋白、分子伴侣和调控因子.菌毛的装配过程是由fae操纵子调控,通过分子伴侣,锚定蛋白的相互协同作用完成,组装成结构蛋白的多聚体.继阐明K88菌毛装配调控机理之后,K88菌毛在非毒素源性大肠杆菌及其它原核生物中装配也取得成功,同时菌毛结构蛋白在真核生物中组装也取得了很大进展.     

Molecular serogrouping of porcine enterotoxigenic Escherichia coli from Australia

Enterotoxigenic Escherichia coli (ETEC) is a common etiological agent of neonatal, pre and post weaning diarrhoea in piglets. One of the most important steps in the diagnosis and epidemiological understanding of this organism is accurate serogrouping. In many instances, however, conventional serogrouping fails to produce accurate identification of serogroups. In this communication we report a modified and simplified molecular serogrouping method (rfb-RFLP) for the accurate identification of the most common porcine ETEC strains that cause neonatal, pre and post weaning diarrhoea in Australia.     

Structural analysis of F18 fimbriae expressed by porcine toxigenic Escherichia coli

The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes. The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA. The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm. Analyzing their helical symmetry showed the axially repeating units to alternate in a "zigzag" manner around the helical axis with an axial rise of 2.2 nm. Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed. Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae. Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF. The presence of the latter two proteins might cause the observed 27-nm axial pattern.     

Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli

Summary The Escherichia coli enterotoxin STII gene is carried by Tn4521. The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2. In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition. The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition. Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition. This protein may be the transposase of Tn4521. A pair of 11 bp repeat sequences flanking the ORF was also found to be important. The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region. The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.     

Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways

Pathogenic enterotoxigenic Escherichia coli (ETEC) are the major bacterial cause of diarrhea in young children in developing countries and in travelers, causing significant mortality in children. Adhesive fimbriae are a prime virulence factor for ETEC, initiating colonization of the small intestinal epithelium. Similar to other Gram‐negative bacteria, ETEC express one or more diverse fimbriae, some assembled by the chaperone‐usher pathway and others by the alternate chaperone pathway. Here, we elucidate structural and biophysical aspects and adaptations of each fimbrial type to its respective host niche. CS20 fimbriae are compared with colonization factor antigen I (CFA/I) fimbriae, which are two ETEC fimbriae assembled via different pathways, and with P‐fimbriae from uropathogenic E. coli. Many fimbriae unwind from their native helical filament to an extended linear conformation under force, thereby sustaining adhesion by reducing load at the point of contact between the bacterium and the target cell. CFA/I fimbriae require the least force to unwind, followed by CS20 fimbriae and then P‐fimbriae, which require the highest unwinding force. We conclude from our electron microscopy reconstructions, modeling and force spectroscopy data that the target niche plays a central role in the biophysical properties of fimbriae that are critical for bacterial pathophysiology.     

Binding to erythrocyte membrane glycoproteins and carbohydrate specificity of F41 fimbriae of enterotoxigenic Escherichia coli

Abstract Haemagglutination of enterotoxigenic Escherichia coli (ETEC) possessing F41 fimbriae was found to be inhibited by N -acetylgalactosamine. Other monosaccharides, such as N -acetylglucosamine, galactose and fucose were also inhibitors, although less effective than N -acetylgalactosamine. Purified F41 fimbriae bound to glycoproteins of human erythrocytes and glycophorin was found to act as an erythrocyte receptor for F41.