抗大肠埃希氏菌K88ab,K88ac和K88ad特异单克隆抗体

A panel of twelve hybridoma cell lines, secreting specific antibodies to K88 adhesin antigens of enterotoxigenic Escherichia coli (ETEC) were established from eight separate fusions between mouse myeloma cell line Sp 2/0-Ag-14 and spleen cells from mice immunized with purified K88 antigens. Among the 12 monoclonal antibodies (MCA), K-A, K-35, K-11, and K-15 were K88a specific and reacted with all K88 adhesin bearing Escherichia coli strains tested, whatever K88ab, K88ac or K88ad they might be, as shown either in enzyme-linked immunosorbent assay (ELISA) or in direct agglutination test, whereas K32, K-4, and K-3 were specific for G88ab, K88ac, and K88ad respectively. The antigen patterns of 33 K88 bearing Escherichia coli strains covering 3 serotypes of K88ab, K88ac, and K88ad were analyzed by the use of these MCAs. The preliminary results showed that all Escherichia strains with the same serotype of K88 antigen shared at least one common type-specific antigenic determinant, that K88ad and K88ac strains enjoyed one common antigenic determinant that did not exist on K88ab strains, and that there were a few K88 antigenic determinants that appeared only on limited Escherichia coli strains of the same K88 serotype.     

Episome-carried Surface Antigen K88 of Escherichia coli III. Morphology

Two nonfimbriate strains of Escherichia coli and their K88(+) counterparts, obtained by episomal transfer of this antigen, were studied with the electron microscope. Specimens were prepared with the spray drop method and were studied after shadow-casting. Under these conditions, the alteration in surface structure, due to the acquisition of K88 antigen, appeared as a fur of fine filaments, distinctly more flexible than fimbriae. Purified K88 antigen was also studied and found to have the same structure.     

Cloning of chromosomal DNA encoding the F41 adhesin of enterotoxigenic Escherichia coli and genetic homology between adhesins F41 and K88.

The genetic determinant for production of the adhesive antigen F41 was isolated from a porcine enterotoxigenic Escherichia coli strain by cosmid cloning. The cloned DNA included sequences homologous to those of hybridization probes prepared from the K88 adhesive antigen operon. Transposon insertions which inactivated F41 production mapped to the same region of DNA showing homology with the K88 genes, demonstrating the genetic relatedness of F41 and K88. Hybridization of a K88 gene probe to plasmid and total DNA from the porcine E. coli isolate from which the F41 gene was cloned indicated that F41 is chromosomally encoded by this strain. This observation was extended to other F41-producing animal isolates. A large number of animal E. coli isolates were examined with K88, F41, and K99 gene probes and for mannose-resistant hemagglutination of human group O erythrocytes and K88 and F41 antigen production. All K88 and F41 antigen producers possessed genetic homology with the K88 and F41 gene probes. Most, but not all, F41-producing strains possessed homology to the K99 gene probe, reflecting the previously observed association of F41 and K99 antigen production. In the strains examined, homology with the K99 gene probe was plasmid associated, whereas homology with the F41 gene probe was chromosomal. The K88 antigen-producing strains showed no homology with the K99 probe. A number of strains possessed homology with the K88 and F41 gene probes and were mannose-resistant hemagglutination positive, but did not produce K88 or F41 antigens. This suggests that there are adhesins among animal isolates of E. coli which are genetically related to but antigenically distinct from K88 and F41.     

Genetic Mapping of the Antigenic Determinants of Two Polysaccharide K Antigens, K10 and K54, in Escherichia coli

The genes controlling synthesis of the Escherichia coli acidic polysaccharide capsular antigens K10 and K54 were transferred by conjugation to E. coli strains of other serotypes. The genes concerned with these K antigen determinants showed genetic linkage with the serA locus. We propose to name the K antigen-controlling gene kpsA. The genetic determinants of the two K antigens could also be transferred to enteropathogenic serotypes, even though such strains have never been found in nature with special acidic polysaccharide K antigens. A noncapsulated derivative, K(-), of the K10 strain can transfer the genetic determinant of the K antigen, demonstrating the probable existence of another chromosomal locus involved in the production of such acidic polysaccharide K antigens.     

PCR assay for detection and differentiation of K88ab1, K88ab2, K88ac, and K88ad fimbrial adhesins inE. coli strains isolated from diarrheic piglets

Primers were designed and prepared and conditions were determined for PCR detection and differentiation of enterotoxigenic E. coli bacterial strains isolated from diarrheic pigs. Primers K88/1 and K88/2 are 25 bp oligomers that correspond to a region of genes encoding one of serological variants of the K88 antigen (K88ab(1), K88ab(2), K88ac or K88ad). A positive result of PCR is an amplificate of 792 bp in size for K88ab and K88ad variant or 786 bp for K88ac variant. The individual serological variants of genes of the K88 antigen could be differentiated by cutting the obtained PCR amplificates by restriction endonucleases. The PCR analysis of 674 E. coli strains isolated from diarrheic pigs showed that 184 strains were K88 positive. By using restriction endonucleases the K88-positive strains were in 4 cases classified as K88ab variant, 180 as K88ac variant and none contained gene for the K88ad variant. Ninety-five % coincidence with serological examination using K88ab, K88ac and K88ad specific antibodies was shown.     

Identification and cloning of the genetic determinant that encodes for the K88ac adherence antigen.

Strains of Escherichia coli capable of causing diarrhea in young pigs are often able to proliferate in the upper small intestine of the infected animal due to the presence of a specific surface antigen, K88. The genetic determinants for K88 antigen production and the ability to utilize the trisaccharide raffinose (Raf) are carried on a 50-megadalton plasmid. Recombinant deoxyribonucleic acid techniques were used to insert an 8.2-megadalton HindIII fragment carrying the K88ac gene(s) from the K88/Raf plasmid pPS100 into the vector pBR322. At lease six polypeptides encoded by this fragment were expressed in minicells. These polypeptides ranged in size from 18,000 to 70,000 daltons. The K88ac antigenic subunit, which has an apparent molecular weight of 23,500, was identified by immunoprecipitation with staphylococcal protein A as the coprecipitant.     

Construction and characterization of mutants impaired in the biosynthesis of the K88ab antigen

Plasmid pFM205 contains the genetic determinant for the K88ab antigen and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 NA of pFM205 contains five genes, which code for polypeptides with apparent molecular weights of 17,000, 26,000 (the K88ab subunit), 27,000 27,500, and 81,000. All five polypeptides were synthesized as precursors approximately 2,000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in one of the five genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. However, these strains did not adhere to brushborders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.     

Comparative study of inhibition of mannose-resistant haemagglutination caused by CFA/I, CFA/II, K88 and K99-positive Escherichia coli strains

Abstract We have studied the inhibition of mannose-resistant haemagglutination (MRHA) caused by Escherichia coli strains with CFA/I, CFA/II, K88, K99 and by other faecal E. coli lacking these colonisation antigens, by means of 30 sugar compounds and by enzymatic treatment of erythrocytes with neuraminidase, α-mannosidase, β-galactosidase, trypsin and pronase, and with formaldehyde. Inhibition of MRHA by sugars was effective only in K88-positive strains with d (+)glucosamine, mucic acid and bovine submaxillary mucin. Enzymatic treatment and the formolisation of erythrocytes gave different results on MRHA activity in strains possessing each colonisation antigen type. Results suggest that the erythrocyte receptor for CFA/I and CFA/II may possibly be sialoglycoprotein in which N -acetylneuraminic acid (NANA) plays an important role, because MRHA activity in these strains was inhibited by treatment of erythrocytes with neuraminidase and pronase. On the other hand, erythrocyte receptors for K88 and K99, like receptors for haemagglutinins of faecal E. coli lacking these colonization antigens, may have other glycoconjugate structures in which proteins and NANA are not essential. Our observations also suggest that the nature (or structure) of the receptor for a specific colonisation antigen on diverse erythrocyte types may be different.     

Effects of immune colostrum on the expression of a K88 plasmid encoded determinant: role of plasmid stability and influence of phenotypic expression of K88 fimbriae

Passaging of the K88-positive Escherichia coli strain CN6913 through synthetic medium containing immune colostrum gave rise to large numbers of K88-negative CN6913 variants. These K88-negative variants had all lost a single large plasmid known to encode the K88 genetic determinant. Four other large plasmids harboured by this strain were unaffected. Viable K88-positive and K88-negative variants of CN6913 accumulated at a similar rate in synthetic medium and in medium containing non-immune colostrum. In the presence of immune colostrum, viable cells of the K88-negative variant accumulated faster and to a greater extent in cultures than the K88-positive variant if incubated at 37 degrees C, which favours the phenotypic expression of K88. However, when similar cultures were incubated at 18 degrees C, a temperature known to inhibit phenotypic expression of K88, the accumulation of viable cells of the two variants was strictly comparable in all media and no loss of plasmid or increase in K88-negative variants was observed. Cells containing a pBR322-based K88-encoding recombinant plasmid were also eliminated by immune colostrum whereas cells containing pBR322 were not. Plasmids encoding the K99 antigen were not readily eliminated from strains passaged through medium containing immune colostrum. K99-negative variants that were detected still harboured the K99-encoding plasmid.     

Episome-carried Surface Antigen K88 of Escherichia coli II. Isolation and Chemical Analysis

The K88 antigen was carried by episomal transfer to D282, a nonmotile Escherichia coli strain without K antigen. D520, obtained by this episomal transfer, was used for the extraction of K88 antigen. It was shown by the agar gel precipitation technique that some K88 antigen was released from D520 into suspending aqueous medium. The amount of liberated material was increased by gentle heating (60 C) or treatment in a Waring Blendor. The antigen was obtained from the extracts in a purified form by making use of its insolubility between pH 3.5 and 5.5 and of its high sedimentation rate (S(0) (20,w) = 36.7S). The homogeneity of the material was demonstrated by agar gel precipitation with D520 antiserum, by analytical ultracentrifugation, and by moving-boundary electrophoresis. Chemical analysis revealed that K88 is a pure protein containing all the common amino acids with the exception of cysteine-cystine. Purified K88 selectively precipitated the K88 antibodies from D520 antiserum and was shown to be immunogenic in rabbits.     

Transfer systems of K88 and K99 plasmids

The conjugation systems of three K88-mobilizing plasmids were characterized for the morphology of their pili and type of mating system (surface only or surface + liquid). pREI had a typical IncI1 transfer system with both thick and thin pili. pVIDO determined aggregating thick flexible pili and pPLS nonaggregating thick flexible pili. All three transferred equally well in broth and on plates. pPLS alone was naturally transfer-depressed. pREI and pVIDO were tested for K88 mobilization efficiency, which was greater from their wild-type host strains to Escherichia coli K-12 than between E. coli K-12 strains. The K99 conjugative plasmid from strain B41 was repressed for transfer and determined thick flexible pili that were receptors for the filamentous phage fd.     

The porcine intestinal receptor for Escherichia coli K88ab,K88ac: regional localization on chromosome 13 and influence of IgG response to the K88 antigen

The loci encoding the porcine intestinal receptors for Escherichia coli K88ab and K88ac (K88abR and K88acR) were firmly assigned to chromosome 13 by linkage analysis using a three-generation pedigree. The linear order of these loci and seven other markers on chromosome 13 was determined by multipoint analyses. The K88abR and K88acR loci were tightly linked with the K88abR locus localized 7·4 cM (sex average) proximal to the transferrin locus. The results, together with previous reports from two other groups, provide an unequivocal assignment of the K88 receptor loci to chromosome 13, and reject a previous assignment to chromosome 4. Pigs possessing the receptor had a slightly higher specific IgG response to the K88 antigen after an intramuscular immunization with an E. coli vaccine.     

Cloning, mapping and expression of the genetic determinant that encodes for the K88ab antigen.

The K88 antigen, a plasmid-specified virulence factor of E. coli involved in porcine neonatal diarrhoea, is often found to be associated with the ability to metabolize raffinose (Raf). Plasmid pRI8801 (51 megadalton) was used to clone the determinants of K88 and Raf with the vector pBR322. K88 was found to be encoded by a 7.7 megadalton HindIII fragment. The expression was highly dependent on the orientation of the HindIII fragment within pBR322. By in vitro generation of deletions, the HindIII fragment was reduced in size to 4.3 megadalton. The expression of K88 by pRI8801 and the recombinant plasmids was studied using an enzyme-linked immunosorbent assay. Raf was found to be located on a 4.0 megadalton SalI fragment. A physical map of pRI8801 was constructed. The K88 antigen and Raf genes are not closely linked but separated by a stretch of DNA of about 20 megadalton.     

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)     

Escherichia coli F41 adhesin: genetic organization, nucleotide sequence, and homology with the K88 determinant.

The genetic organization of the polypeptides required for the biosynthesis of the F41 adhesin of enterotoxigenic Escherichia coli strains was investigated. Maxicell analysis demonstrated that a recombinant plasmid which mediated mannose-resistant hemagglutination and F41 antigen production encoded four polypeptides of 29, 30, 32, and 86 kilodaltons. The 29-kilodalton protein was identified as the F41 antigen, and the nucleotide sequence of the gene was determined. Extensive homology was observed between the region encoding the putative signal sequences of the F41 and K88 antigens and in the region immediately upstream of the antigen genes. The nucleotide sequence homology between F41 and K88 determinants was further investigated by Southern blot hybridization. A K88 probe hybridized at high stringency to all fragments shown to be essential for F41 production except for fragments internal to the F41 antigen gene.     

K88ab gene of Escherichia coli encodes a fimbria-like protein distinct from the K88ab fimbrial adhesin.

The K88ab adhesin operon of Escherichia coli encodes for a fimbrial protein (the K88ab adhesin) which is involved in colonization of the porcine intestine. We characterized a structural gene (gene A) which is part of the K88ab adhesin operon and codes for an as yet unidentified polypeptide (pA). A mutation in gene A resulted in accumulation of K88ab adhesin subunits inside the cell. The nucleotide sequence of gene A was determined, and the deduced amino acid sequence suggested that pA is synthesized as a precursor containing a typical N-terminal signal peptide. The molecular weight of pA was calculated to be ca. 17,600. Gene A is preceded by a sequence showing homology with the consensus promoter. Fimbrial subunits from a number of E. coli strains have significant homology at their N- and C-termini. pA also contained some of these conserved sequences and showed a number of other similarities with fimbrial subunits. Therefore, it seems likely that the K88ab adhesin operon codes for a fimbrial subunit (pA) distinct from the K88ab adhesin subunit.     

The interaction of the K88 antigen with porcine intestinal epithelial cell brush borders

The interaction of 125I-labelled K88 antigen with brush borders of the epithelial cells of the pig small intestine has been studied. The iodinated antigen bound avidly to the brush borders prepared from adhesive (receptor-positive) pigs even after pretreatment of the brush borders with formaldehyde, whereas the brush borders from non-adhesive (receptor-negative) pigs failed to bind the antigen under these conditions. Treatment with glutaraldehyde rapidly destroyed the ability of both types of brush border to bind the K88 antigen. Studies on the binding of antigen to brush borders revealed the presence of high affinity receptors, but the non-linearity of the Scatchard plot could be explained by cooperative-like interactions, which view was supported by dissociation experiments. Rapid dissociation only in the presence of unlabelled K88 antigen suggested the existence of receptor site interactions of the negatively cooperative type. Attempts to inhibit the binding of 125I-labelled K88 with simple monosaccharides and oligosaccharides suggested that the binding of antigen to brush borders involves complex interactions and that galactosyl residues may be important.     

猪肠毒素源性大肠杆菌K88ac粘附抗原的核苷酸序列分析

 本文对国内流行的猪肠毒素源性大肠杆菌K88ac抗原的结构基因的核苷酸序列进行了测定。该基因由849对核苷酸组成,编码了283个氨基酸的蛋白亚单位及21个氨基酸的信号肽。与国外报道的K88ac序列的不同是我们发现一个碱基的点突变,导致在抗原决定簇内一个氨基酸的改变。从核苷酸序列推导出的氨基酸序列与另两种亚型进行了比较。     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.