Preliminary characterization of cell-free K99 antigen isolated from Escherichia coli B41.

The K99 antigen of Escherichia coli B41 was isolated by isoelectric precipitation from heated bacterial suspensions. Chromatography and immunoabsorption experiments suggested that the mannose-resistant haemagglutinating activity of partially purified preparations of antigen was K99. The antigen was partially susceptible to bacterial proteases and was inactivated by periodate oxidation. Haemagglutination inhibition experiments with sugars and absorption of K99 with antisera to human blood groups A and B substances suggested that K99 contains a terminal alpha-linked N-acetylgalactosamine moiety, which is involved in the haemagglutination reaction, and an adjacent terminal alpha-linked galactose moiety, which plays no part in the reaction.     

Morphological description of surface structures on strain B41 of bovine enterotoxigenic Escherichia coli bearing both K99 and F41 antigens

In order to describe morphologically the structures on the cell surface of bovine enterotoxigenic Escherichia coli, variants of reference strain B41 (K99+F41+) either negative for K99 and positive for F41 antigens (variants B41A, B41*C), or phenotypically negative for both antigens (variants B41B1, B41B2, B41*CB), and a transconjugant harbouring the K99 plasmid and expressing the K99 adhesin [transconjugant B41 x H510a:H510(2)] were examined by transmission electron microscopy using negative staining. Several negative staining procedures were tested for strain B41 and variant B41A: direct harvesting of strains into ammonium molybdate (2%, w/v), with bacitracin (50 micrograms ml-1) as wetting agent, gave the best results. Three morphologically distinct structures on the cell surface could be identified in cultures grown on Minca medium. Firstly, thin, filamentous, flexible fibrillar structures, presenting a helical structure and a mean diameter of approximately 3 nm, were recognized as K99 fimbriae, since they were present on strain B41 and on transconjugant H510(2), but not on K99-negative variants nor on the recipient strain H510a. Secondly, coil-like structures with a diameter of about 17-20 nm were observed on strain B41 and on variants B41A and B41*C. These structures appeared to consist of two or more curled filaments (diameter 3 nm) joined to coil on themselves into dense spirals. They were very rare in variants B41B1 and B41B2 and were absent on variant B41*CB and on a transconjugant B41* x B41*CB, which had re-acquired the K99 plasmid and which again exhibited K99 fimbriae. Strains B41 and variant B41A gown at 37 degrees C for 24 h on sheep-blood agar exhibited coiled structures like those seen on Minca medium. In contrast, after growth at 18 degrees C for 48 h (which inhibits the synthesis of F41 antigen), coiled structures were no longer expressed on the cell surface of strain B41 and variants B41A and B41*C. Thus the presence of coiled structures correlated with the expression of F41 antigen in strains and variants, which suggests that F41 had a coiled morphology. Finally, straight fimbriae (diameter 6.5-7 nm) were observed on the cell surface of every strain and variant. Their expression on the cell surface was enhanced by several subcultures in th e static broth, and it was inhibited by subculture on agar, but not by culture at 18 degrees C after serial subcultures in static broth. These facts indicated that the straight fimbriae could be common fimbriae, and excluded their being F41 structures.     

Partial purification and characterization of two non K99 mannose-resistant haemagglutinins of Escherichia coli B41

A K99-variant of Escherichia coli B41 was produced by growing the parent strain in the presence of antiserum to E. coli K12K99. Two mannose-resistant and eluting (MRE) haemagglutinins with molecular weights greater than 20 x 10(6) were extracted from the cell surface of the variant. One was an anionic antigen, partially purified by ammonium sulphate and isoelectric point precipitation, which adhered to calf intestinal brush borders; it was a protein composed of subunits with mol. wt 34000. Electron microscopy showed that this material did not have a regular fimbrial appearance, but contained some fine fibrillar structures. A second MRE haemagglutinin which was also partially purified by ammonium sulphate precipitation, had a definite fimbrial structure, being a protein composed of two subunits of mol. wt 49500 and 48000. This antigen was probably responsible for the fimbrial appearance of the K99-variant, but it was antigenically distinct from the anionic adhesin and did not adhere to calf intestinal brush borders.     

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.     

Effect of glucose and amino acids on expression of K99 antigen in Escherichia coli

K99 antigen production by enterotoxigenic Escherichia coli strains of bovine origin was investigated by slide agglutination and in vitro attachment to intestinal villi. Work with two strains (B41 and B44) showed that on minimal medium M2, K99 antigen was not repressed by a high concentration of glucose (2%, w/v). Growth on synthetic or complex medium did not affect K99 antigen detection, which was independent of capsular antigens, and its synthesis was not repressed by Casamino acids or glucose. A survey of 12 strains revealed two groups: in one group K99 antigen production was constitutive on basal medium without glucose, and in the second group K99 antigen was produced only in the presence of glucose. Immunoelectrophoresis patterns, and the results of slide agglutination and attachment tests, were dependent upon K99 type, whereas haemagglutination patterns were not.     

Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/ F41) and the genetic relationship to other O101 rfb loci

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.     

Structural and genetic characterization of Escherichia coli O99 antigen

The influence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota was monitored in 26 infants including five antibiotic-treated (AT) subjects orally administered a broad-spectrum antibiotic for the first 4 days of life and three caesarean-delivered (CD) subjects whose mothers were intravenously injected by the similar type of antibiotics in the same period. The faecal bacterial composition was analysed daily for the first 5 days and monthly for the first 2 months. Terminal restriction fragment length polymor-phisms in the AT subjects showed less diversity with the attenuation of the colonization of some bacterial groups, especially in Bifidobacterium and unusual colonization of Enterococcus in the first week than the control antibiotic-free infants (AF, n =18). Quantitative real-time PCR showed overgrowth of enterococci (day 3, P =0.01; day 5, P =0.003; month 1, P =0.01) and arrested growth of Bifidobacterium (day 3, P =0.03) in the AT group. Furthermore, after 1 month, the Enterobacteriaceae population was markedly higher in the AT group than in the AF group (month 1, P =0.02; month 2, P =0.02). CD infants sustained similar, although relatively weaker, alteration in the developing microbiota. These results indicate that antibiotic exposure at the beginning of life greatly influences the development of neonatal intestinal microbiota.     

Episome-carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers

?rskov, Ida (Statens Seruminstitut, Copenhagen, Denmark), and Frits ?rskov. Episome-carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers. J. Bacteriol. 91:69-75. 1966.-The transmission of the determinant of the Escherichia coli K88 antigen in mixed cultures of E. coli strains is described. The K88 factor could not be transferred by filtrates, nor could responsible phages or colicines be detected. Acriflavine was shown to "cure" the bacteria for the K88 antigen. Generally, the strains having acquired the K88 antigen also acquired the ability to transfer chromosomal markers, but this ability was in some cases retained by segregants which had lost the K88 antigen. Introduction into an F(+) strain caused reduction of the recombination frequency and disappearance of the f(+) antigen. Not all wild-type strains with the K88 antigen are genetic donors of this antigen, at least not to a discernible degree. It was concluded that the K88 antigen determinant is carried by an episome.     

Structural studies on the Escherichia coli O101 lipopolysaccharide found in association with F41 and K99 fimbriae

Abstract Pseudomonas aeruginosa PAO1 used a broad range of alkanesulfonic acids as sole sulfur source for growth, with molar growth yields of 2.2 to 2.9 kg protein per mol sulfur. 4-Phenylbutane-1-sulfonate was desulfonated in vivo to yield 4-pheny 1-1-butyric acid quantitatively as the sole product, suggesting that the desulfonation mechanism is the same as when alkanesulfonates serve as a carbon source for growth. This contrasts with aromatic sulfonate utilization in other organisms, where different desulfonation reactions are used to provide carbon and sulfur. Desulfonation of alkanesulfonates to provide sulfur was repressed by sulfate or thiocyanate, and derepressed in their absence. The alkanesulfonatase system is hence controlled as part of the sulfate starvation-induced stimulon.     

Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae

Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae.     

A chromatographic method for the purification of K99 pili from enterotoxigenic Escherichia coli

Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (w/v) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0.02 M-Tris/HCl, pH 8.6 containing 0.05 M-NaCl and DEAE-Sephadex A-50 equilibrated with 0.05 M-phosphate buffer, pH 7.2. The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. MgCl2 precipitation and chromatofocusing were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously the N-terminal amino acid was apparently blocked.     

Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli

A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor. The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R. The distance between the Shine-Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp. The expression level of HBsAg by E. coli 294[pTRP P31-R] was significantly elevated, in contrast to that of HBsAg by E. coli 294[pTRP SS-6]. Western blotting analysis has shown that E. coli[pTRP P31-R] synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody. The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin.     

Application of the peroxidase-antiperoxidase technique for the detection of enterotoxigenic Escherichia coli K99 pilus antigen in paraffin-embedded tissues

The peroxidase-antiperoxidase (PAP) technique was evaluated as a method for detecting enterotoxigenic Escherichia coli (ETEC) K99 pilus antigen in paraffin-embedded tissues of swine. Antigenic reactivity was optimal when tissues were processed in cold methanol according to a modified method of Sainte-Marie. This method of processing adequately preserved tissue morphology. The PAP technique was applicable to tissues where endogenous peroxidase activity was minimal.     

A single-step isolation of K99 pili from B-44 strain of Escherichia coli

A single-step procedure has been developed to isolate pili from enterotoxigenic Escherichia coli strain B44 of calf origin. Pili, removed from the bacteria by heat shock treatment, were allowed to aggregate at 4 degrees C for 16 h. The precipitated pili, isolated by centrifugation, had typical pili morphology as shown by electron microscopy; ability to bind pig brush border; molecular weight greater than 6 million; and predominance of hydrophobic amino acids. On sodium dodecyl sulfate gel electrophoresis, the subunit pilin migrated as a single polypeptide of molecular weight 17,000.     

Homoserine kinase from Escherichia coli K12.

Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.     

大肠杆菌K99菌毛fan操纵子的克隆、表达及活性

[目的]在体外克隆和表达猪产肠毒素大肠杆菌(ETEC) K99菌毛操纵子fan结构基因,并检测重组菌毛的相关生物学活性.[方法]以猪源分离的表达K99菌毛ETEC C83907株制取模板,成功PCR扩增出编码K99菌毛的fan操纵子,约5.7 kb.将fan操纵子克隆人表达质粒载体pBR322,筛选出含正确阳性质粒的重组菌.进一步将上述的重组质粒DNA转化至不含任何菌毛的大肠杆菌SE5000株,同时将空载体pBR322质粒转化入SE5000构建阴性对照菌株.[结果]该重组菌能与鼠抗K99菌毛单克隆抗体发生明显的凝集反应,与新生仔猪小肠上皮细胞刷状缘BBV分子有强烈凝集反应.电镜观察到上述重组菌表面大量表达K99菌毛,用热抽提法提纯其表达的K99菌毛,并经SDS-PAGE电泳和考马斯亮蓝染色,可以得到分子量约为18.5kDa的主要蛋白条带.纯化菌毛免疫小鼠后制备出高效价的鼠抗血清,能与携带K99菌毛的C83907、C83914、C83260野生株发生强烈的凝集反应,而与携带其他菌毛的ETEC不反应.玻板凝集试验和Western blot结果表明:体外表达的K99菌毛具有和野生K99菌毛相同的抗原性.用表达K99菌毛的重组菌进行HeLa细胞体外黏附试验和黏附抑制实验,结果表明:重组菌和野生菌株一样具有较强的粘附性,而且用重组菌毛制备的鼠抗血清能有效地抑制上述重组菌或野生菌株对细胞系的黏附结合.[讨论]本研究为进一步研究K99菌毛生物学作用建立了良好的实验平台.