The nucleotide sequence of the gene encoding the K88ab protein subunit of procine enterotoxigenic Escherichia coli

Gaastra, W

doi:10.1016/0378-1097(81)90034-3

Cited by 30 publications

(32 citation statements)

References 10 publications

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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). 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).…”

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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).…”

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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%

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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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“…Subsequently, a methyl group is attached to the newly formed amino terminus. Most pili from Escherichia coli appear to be synthesized as true secretory precursors, with the characteristic hydrophobic signal sequences (2,12) being cleaved without any additional posttranslational modification of the mature protein. Thus evolutionarily distinct group of proteins with a conserved primary sequence and specific machinery for modification and assembly.…”

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confidence: 99%

Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli

Strom¹,

Lory²

1986

J Bacteriol

104

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Many strains of Pseudomonas aeruginosa possess pili which have been implicated in the pathogenesis of the organism. This report presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the pili of P. aeruginosa PAK. Total DNA from this strain was partially digested with Sau3A and inserted into the cloning vector pUCI8. Recombinant E. coli clones were screened with oligonucleotide probes prepared from the constant region of the previously published amino acid sequence of the mature pilin subunit. Several positive clones were identified, and restriction maps were generated. Each clone contained an identical 1.1-kilobase HindIII fragment which hybridized to the oligonucleotide probes. Western blot analysis showed that all of the clones expressed small amounts of the P. aeruginosa pilin subunit, which has a molecular mass of ca. 18,000. This expression occurred independently of the orientation'of the inserted DNA fragments in the cloning vector, indicating that synthesis was directed from an internal promoter. However, subclones containing the 1.1-kilobase HindIl fragment in a specific orientation produced an order of magnitude more of the pilin subunit. While the expressed pilin antigen was located in both the cytoplasmic and outer membrane fractions of E. coli, none appeared to be polymerized into a pilus structure.Pili have been observed on a variety of clinical and laboratory strains of Pseudomonas aeruginosa, and recently, they have been implicated in pathogenesis as well. Woods et al. (34), using an in vitro binding assay, demonstrated that there was a direct interaction of purified pili and piliated organisms with mammalian buccal epithelial cells. Pseudomonas pili resemble those from other bacterial genera in their morphological appe'arance and subunit molecular weights. They are flexible structures with a diameter of 6 nm and a length of about 30 nm. The molecular mass of the pilin subunit from several strains is about 18,000 daltons.Amino acid sequencing of pilin from P. aeruginosa PAK revealed the presence of an unusual amino acid, methylphenylalanine, at the amino terminus, followed by a stretch of predominantly hydrophobic amino acids (27). This sequence of about 30 amino acids appears to be a constant component of antigenically related pilin from various P. aeruginosa isolates, while the immunological differences are due to different epitopes in the C terminus. Furthermore, the same constant amino-terminal sequence is found in pilin from a number of rather diverse genera, such as Neisseria gonorrhoeae (14, 29), Moraxella nonliquefaciens (11) evolutionarily distinct group of proteins with a conserved primary sequence and specific machinery for modification and assembly.Despite the great amount of biochemical data available about the pilin of P. aeruginosa and the availability of a well-defined genetic system, relatively little work has been done on the elucidation of the regulation of pilin expression. Selecting for resistance to pilus-specific phages, Bradl...

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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).…”

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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 gene encoding the K88ab protein subunit of procine enterotoxigenic Escherichia coli

Cited by 30 publications

References 10 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

Cloning and expression of the pilin gene of Pseudomonas aeruginosa PAK in Escherichia coli

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

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