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

Abstract

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

Localization of a domain in the FimH adhesin of Escherichia coli type 1 fimbriae capable of receptor recognition and use of a domain-specific antibody to confer protection against experimental urinary tract infection.

“…Our studies are also consistent with earlier transposon insertion analyses of E. coli fimH that indicated mutations localized to the 5Ј region of the fimH gene were more likely to abrogate adhesive functions of the fimbriae than mutations at other sites in the gene (18). However, it is noteworthy that several other fimbrial adhesins appear to mediate binding via their carboxy terminus region (45)(46)(47)(48)(49).…”

Sequence analysis of the clpG gene, which codes for surface antigen CS31A subunit: evidence of an evolutionary relationship between CS31A, K88, and F41 subunit genes

“…Extensive differences in the V2 region result in the CS31A subunit primary sequence losing the two tripeptides Ser-148-Leu-Phe-150 and Ala-156-Ile-Phe-156 which are involved in the K88 receptor binding domains (22). The possibly low structural constraints on the central region V2 may have contributed to the observed diversity of the K88-related subunits and to the apparent absence of an adhesin function in CS31A.…”

“…Although the nucleotide sequence of this octapeptide was highly conserved (90%) in the fimbrial subunit gene of CS31A and K88, it was not possible to know whether this correspondence is fortuitous or not. Interestingly, the two tripeptides Ser-Leu-Phe and Ala-Ile-Phe involved in the K88 binding activity (22) were not found in the primary structure of CS31A.…”

“…Variable regions are probably involved in the antigenic specificity of the three variants, and conserved regions are supposed to be involved in such common features as folding, processing, and functioning of the polypeptide (30). Jacobs et al (22) demonstrated that two conserved tripeptides are involved in the K88 receptor binding domain and suggested that the receptor binding site could be a hydrophobic cleft which encompasses the two conserved amino acid sequences and which interacts with the ligand molecules.…”

Desenvolvimento e avaliação de novas estratégias de imunização contra colibacilose suína

“…No entanto, estes autores não descartaram a possibilidade da proteína FaeC também ter um papel importante pois esta está localizada na extremidade da fímbria. Em E. coli enterotoxigênica que causa diarréia em humanos, Papl, a menor subunidade é responsável pela adesão da bactéria (Jacobs et al 1987). A organização gênica deste operon é parecida com a do K88, desta forma FaeC também poderia participar na adesão da fímbria K88 ao epitélio intestinal.…”