The nucleotide sequence of the K88ad protein subunit of porcine enterotoxigenic Escherichia coli

Gaastra, Wim

doi:10.1016/0378-1097(83)90323-3

Cited by 17 publications

(20 citation statements)

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“…Comparison of a part of the primary structure of six lectins: K88ab, K88ac, and K88ad adhesin (5,6,12), gonococcal MS11 and R10 fimbrial subunits (20), and fragment B of diphtheria toxin (3). Identical or functionally identical residues are boxed.…”

Section: Methodsmentioning

confidence: 99%

“…Three serological variants of K88 fibrillae have been described and designated K88ab, K88ac, and K88ad (7). The primary structure of all three proteins has been determined, showing both conserved and variable regions (5,6,12 Purification of fibrillae. K88ab, K88ac, K88ad, F41, and K99 fibrillae were isolated and purified as described previously (10).…”

mentioning

confidence: 99%

“…Three serological variants of K88 fibrillae have been described and designated K88ab, K88ac, and K88ad (7). The primary structure of all three proteins has been determined, showing both conserved and variable regions (5,6,12). The conserved regions are supposed to be involved in common features such as subunit-subunit interaction and receptor binding, while the variable regions might have evolved to evade the immune response of the host (16).…”

mentioning

confidence: 99%

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Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin

Jacobs¹,

Venema²,

Leeven³

et al. 1987

J Bacteriol

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A cyanogen bromide fragment derived from the K88ab adhesin inhibited the hemagglutinating activity of K88 fibrillae. Smaller fragments which inhibited the adherence of K88 fibrillae to erythrocytes or to intestinal epithelial cells were obtained by digestion of K88ab fibrillae with oa-chymotrypsin. Active peptides were isolated from the digestion mixture and identified as Ser-Leu-Phe and Ala-Ile-Phe. Both tripeptides correspond to the peptide stretches Ser-148-Leu-Phe-150 and Ala-156-Ile-Phe-158, respectively, which are part of conserved regions in the primary structure of the K88 variants ab, ac, and ad. The isolated tripeptides inhibited the hemagglutinating activity of purified K88 fibrillae in the 1 to 5 ,uM range, while adherence of the fibrillae to intestinal epithelial cell brush borders was inhibited in the 10 to 50 ,uM range. Furthermore, the tripeptides were capable of eluting attached bacteria from agglutinated erythrocytes. The inhibitory activity of the isolated peptides was confirmed by testing various synthetic peptides for their ability to inhibit the interaction of the different K88 variants with various species of erythrocytes. The significance of these findings for the localization of the receptor-binding domain is discussed.K88 fibrillae are nonflagellar, filamentous adhesins found on many enterotoxigenic Escherichia coli strains that cause neonatal diarrhea in pigs (4). They enable the bacteria to colonize the small intestinal epithelium, which is considered to be a prerequisite for the establishment of diarrheal disease. The K88 fibrillae consist of multimers of the K88 adhesin subunit with a molecular weight of 27,500 (4). Three serological variants of K88 fibrillae have been described and designated K88ab, K88ac, and K88ad (7). The primary structure of all three proteins has been determined, showing both conserved and variable regions (5,6,12 Purification of fibrillae. K88ab, K88ac, K88ad, F41, and K99 fibrillae were isolated and purified as described previously (10).Isolation of brush borders. Brush borders were prepared from the pig intestine as described by Middeldorp and Witholt (15) and stored in 50% glycerol at -20°C.Hemagglutination inhibition test. Suspensions of washed erythrocytes (2%) in PBSM (50 mM sodium phosphate [pH 7.3] containing 0.9% NaCl and 0.5% mannose) were mixed 1:1 with various amounts of peptide fractions in the same buffer. Subsequently, 50-pl portions of these suspensions were added to serial twofold dilutions (50 ,ul) of purified fibrillae in PBSM, using polystyrene microtiter trays with V-shaped cups. After 2 h of incubation at 4°C, the trays were examined. The initial concentrations of K88ab, -ac, and -ad fibrillae used were 50, 500, and 25 [Lg/ml, respectively

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

“…Three serological variants of K88 fibrillae have been described and designated K88ab, K88ac, and K88ad (7). The primary structure of all three proteins has been determined, showing both conserved and variable regions (5,6,12). The conserved regions are supposed to be involved in common features such as subunit-subunit interaction and receptor binding, while the variable regions might have evolved to evade the immune response of the host (16).…”

mentioning

confidence: 99%

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Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin

Jacobs¹,

Venema²,

Leeven³

et al. 1987

J Bacteriol

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“…4B), with identical amino acids occurring at 9 of 26 positions. The sequences of the three main serotypes of K88 (ab, ac, and ad) are identical for this region of the sequence (10,13,14,17). CIE analysis.…”

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confidence: 99%

Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli

Caffrey

Owen

1989

J Bacteriol

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Antigen 43 has been identified as a unique protein complex in the outer membrane of Escherichia coli. The complex contains two different polypeptides, a (Mrs 60,000) and ,( (Mrs 53,000), in equal stoichiometry (P. Owen, P. Caffrey, and L.-G. Josefsson, J. Bacteriol. 169:3770-3777, 1987). The a subunit was released in a water-soluble form upon heating of outer membranes to 60°C and was purified to apparent homogeneity by gel filtration and ion-exchange chromatography. The purified protein was acidic (pl 4.6) and had a polarity of 49.2%. The N-terminal sequence showed homology with the N termini of certain enterobacterial fimbrial subunits. In addition, antigen 43 underwent a reversible phase variation similar to that of type 1 fimbriae. By use of subunit-specific antisera, it was shown that the purified a subunit was capable of reassociating with the 0i polypeptide. However, electron microscopic examination indicated that antigen 43 does not form a recognizable surface structure. The available evidence supports the view that antigen 43 is a complex consisting of a peripheral membrane protein (at) anchored to a subunit (Ii) that is integral to the outer membrane.Extensive immunochemical studies have established a reference profile for Escherichia coli ML308-225 envelope components resolved by crossed immunoelectrophoresis (CIE; 37-40, 43). Antigen 43 is often prominent in the array of precipitin arcs generated when outer membranes are analyzed by this technique. In a previous paper (41), we described the initial characterization of this antigen as a unique protein complex in the outer membrane. The antigen complex contains two chemically and immunologically different polypeptides, ot and 1, with Mrs of 60,000 and 53,000, respectively. These occur in equal stoichiometry. The polypeptide is heat modifiable and shows an apparent Mr of 37,000 when solubilized at temperatures lower than 70°C before analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Analysis by CIE performed in conjunction with zymogram staining and radiolabeling failed to detect the presence of enzyme activity, fatty acyl groups, or other cofactors for the complex (41).Antigen 43 provokes interest for a number of reasons. The complex differs from the well-characterized proteins of the outer membrane of E. coli in that it consists of two different subunits (31, 41). In addition, the antigen is surface exposed, and its yield varies in a manner which appears to be independent of external growth conditions. In this communication, we report on the purification and properties of the a subunit of this unique antigen complex.(Preliminary accounts of this work have been presented

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“…These three variants share the component a, whereas components, b, c, and d are due to variations in amino acid composition. The primary structures of the subunits for each variant have been determined from their nucleotide sequences (8,9,19) and by amino acid sequencing (20). It was recently established that K88 antigens bind to glycolipids and glycoproteins present in the mucus and brush borders of the small bowel of piglets (2,27).…”

mentioning

confidence: 99%

Isolation of K88 Antigen Variants (ab, ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

González

Vázquez

Garabal

et al. 1995

Microbiology and Immunology

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Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes 08:K87, 032, 045, 0138:K81, 0141:K85, 0147:K89, 0149:K91, and 0157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the 0149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.

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The nucleotide sequence of the K88ad protein subunit of porcine enterotoxigenic Escherichia coli

Cited by 17 publications

References 7 publications

Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin

Inhibition of adhesive activity of K88 fibrillae by peptides derived from the K88 adhesin

Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli

Isolation of K88 Antigen Variants (ab, ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

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