F K de Graaf

The Fimbrial Adhesins of Escherichia Coli

Publisher Summary This chapter reviews the current knowledge of E. coli adhesions and their receptors. The main emphasis is given on the genetic and physiological aspects of adhesin production, the primary structure of adhesion subunits, their adhesive properties, and the characterization of adhesion receptors. Adhesins are macromolecular structures present on the bacterial cell and mediate the attachment of these cells to a surface. Pathogenic E. coli strains associated with intestinal or urinary tract infections are able to produce one or more different types of fimbrial adhesins that enable the bacteria to colonize the host epithelia. The majority of these fimbriae are composed of a single repeating protein subunit, but some fimbriae may contain other polypeptides as well. It is noted that all fimbriae appear to recognize and bind to relatively simple glycolipids or to glycoproteins present on the host epithelial cell surface or on certain types of erythrocytes. The chapter also discusses the classification of fimbriae according to their affinity for a particular oligosaccharide structure present on these glycoconjugates.

Molecular biology of fimbriae of enterotoxigenic Escherichia coli.

Particular strains of Escherichia coli that produce enterotoxins are an important cause of diarrheal disease in man and domestic animals. The ability of these enterotoxigenic E. coli (ETEC) strains to adhere to the intestinal epithelium is required as an initial step in establishing diarrheal disease. The bacterial cell surface structures that are responsible for adherence have been called adhesins or colonization factors (see also GAASTRA and DE GRAAF 1982, for a recent review). A special class of adhesins is formed by the proteinaceous filamentous surface appendages called fimbriae (DUGUID et al. 1955) or pili (BRINTON 1959). These fimbrial adhesins are host specific and include K88 (ORSKOV et al. 1964) and 987P (NAGY et al. 1976) on porcine strains; K99 (ORSKOV et al. 1975) and F41 (DE GRAAF and ROORDA 1982; MORRIS et al. 1982) on porcine, ovine and bovine strains; and CFA/I and CFA/II (EVANS et al. 1975; EVANS and EVANS 1978) associated with strains of human origin. Some characteristics of these fimbrial adhesins are shown in Table 1. It should be noted that this list is not complete and that the number of discovered fimbrial adhesins is rapidly increasing.

Production and Release of Cloacin DF13 and Related Colicins

Protein export by bacteria has been widely studied in Escherichia coli and in Bacillus species. The mechanism of protein translocation across the cytoplasmic membrane of these organisms appears to be comparable to the translocation of proteins across the membrane of the rough endoplasmic reticulum of eukaryotic cells, and is described in several reviews (Michaelis and Beckwith 1982; Silhavy et al. 1983; Randall and Hardy 1984; Pugsley and Schwartz 1985). In gram-positive bacteria the exported proteins either remain anchored in the bacterial cell envelope, like the lipoproteins, or are released from the cells, like various proteases, amylase, and other proteins. In gram-negative bacteria the exported proteins are released into the periplasm, or are incorporated in the outer membrane of these cells. Apparently, the outer membrane of these organisms, which is both functionally and structurally completely different from the cytoplasmic membrane, forms an extra barrier for protein excretion into the extracellular space. Several gram-negative bacterial species, however, have been found to release extracellular proteins (Pugsley and Schwartz 1985), but very little is known about the mechnisms by which proteins are translocated across the outer membrane and about the possible role of signal or other topogenic sequences in this process.

Age and serotype dependent binding of K88 fimbriae to porcine intestinal receptors

The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.

K88 fimbriae as carriers of heterologous antigenic determinants.

The K88 fimbriae of enterotoxigenic Escherichia coli are strongly immunogenic antigens that can be used to evoke protective immunity. To find out whether these fimbriae can be used as carriers for foreign epitopes, a highly variable region present in the primary structure of the different K88 variants was replaced with five different heterologous epitopes to investigate to what extent these insertions affected the expression, assembly (biogenesis), stability and immunogenic properties of the resulting hybrid fimbriae. Amino acid residues 163-173, were replaced using site-directed in vitro mutagenesis and the hybrid fimbriae were tested for these aspects using ELISA, immunoelectronmicroscopy and immunoblotting. Replacement of this highly variable region did not affect the biosynthesis of fimbriae, although all mutations tested resulted in a reduced expression depending on the epitope inserted. Testing of the different hybrid fimbriae with a panel of monoclonal antibodies raised against the various K88 serotypes K88ab, K88ac and K88ad indicated that replacement of amino acid sequence 163-173 did not affect conserved or K88ab specific epitopes but the K88ac and K88ad specific conformation was lost. Immunization with hybrid fimbriae raises antibodies specific for the inserted heterologous epitopes.

Genetic organization and biogenesis of adhesive fimbriae of Escherichia coli

The genetic organization of the determinants of type 1, K88ab, K99 fimbriae and P(pap)pili of Escherichia coli is presented. The functions of the various gene products are described and a model for the process of fimbriae biogenesis is presented and discussed.

Localization and function of FanH and FanG, minor components of K99 fimbriae of enterotoxigenic Escherichia coli

Specific antisera against FanG and against FanH were prepared by immunization with hybrid Cro-LacZ-FanG and Cro-LacZ-FanH proteins, respectively. Immunoblotting with these antisera revealed the presence of FanG and FanH as minor components in purified K99 fimbriae. Mutations were constructed in fanG and fanH and cells defective in FanG or FanH were characterized by ELISA, immunoblotting, adhesion assays and electron microscopy. A minicell experiment showed that the mutations in fanG or fanH had no effect on the expression of the other K99-specific proteins. Cells defective in FanG produced no fimbriae and did not agglutinate horse erythrocytes, but cell-free heat-shock preparations of these cells still bound the K99 glycolipid receptor. Cells defective in FanH produced 1-2% of the K99 fimbriae as compared with wild-type K99 producing cells. These mutant fimbriae appeared to be shorter but were still capable of binding the K99 glycolipid receptor. Apparently, FanG and FanH are not required for binding the K99 receptor. These results and analysis of K99 mutants by immunoblotting using a specific antiserum against another K99 minor component, FanF, indicated that the combinations FanF/FanG and FanF/FanH are required for the initiation and elongation (length determination) of K99 fimbriae formation, respectively.

Properties and Synthesis of K88 Fimbriae

Neonatal and immediate post-weaning diarrhoea in piglets are frequently caused by enterotoxigenic Escherichia coli strains of particular serotypes. Besides producing and excreting enterotoxins these strains possess adhesive antigens which enable the bacteria to adhere to the mucosal surface and colonize the small intestine.1 Well-known adhesive antigens of porcine enterotoxigenic E. coli are the K88, K99 and 987P fimbriae, also designated as fimbrial serotypes F4, F5 and F6, respectively.2

Characterization and expression of the cloacin DF13/aerobactin uptake system in Enterobacteriaceae

Since in natural habitats the free iron concentration is generally low, the effect of iron limitation on the sensitivity of Klebsietla aerogenes to penicillin G (pen G) was studied. Also, the influence of this growth condition on permeability and binding capacity for pen G of the cell envelope was investigated. The organism was cultured at a growth rate of 0.5 h -1 in a glass/Teflon chemostat. Iron (Fe) limitation was obtained by application of medium components with a low iron content. The sensitivity to pen G was tested by following the time course of the viable count after addition of the antibiotic. The application of sublethal pen G concentrations resulted in the lengthening of cells and in a concomitant slow decrease in the viable count. A lethal effect, i.e., a rate of decrease in the viable count at least equal to the culture wash-out rate, was found after addition of 1024 gg pen G. m.1-1. With excess of iron, e.g., under ammonia-limited conditions, the lethal concentration Was 128 ggml1 In the outer membrane (OM) of Fe-limited cells, a protein with molecular weight 78000 was present in higher amounts than under conditions with excess of iron, and additional protein bands in the 70 000-85 000 molecular weight range were found. In order to measure the effect of the altered OM composition on its permeability for pen G, a strain harbouring the plasmid pBR322 was grown under Fe limitation. The plasmid-coded [Mactamase activity was measured in intact cells and cellfree extracts, and from the difference between these enzyme activities the OM permeability for pen G was calculated. It appeared to be comparable to the value found under ammonia limitation. Cell envelopes from Fe-limited cells were treated with 14C-pen G, and the penicillin-binding proteins (PBPs) were analysed. In these cells, as well as in ammonia-limited cells, 8 PBPs were detected. In the relative amounts of PBPs only minor differences were found between the enveiopes of cells, grown under both conditions. Under Fe-limited conditions no derepression of synthesis of 13-1actamase was found in the wiIdtype strain. Furthermore, since the altered protein composition of the OM had no effect on its permeability for pen G, and no major alterations in.the PBP-pattern were found, it remains unclear which factor(s) determined the low sensitivity to pen G of Fe-limited K. aerogenes.

A comparison of two operons involved in export and assembly of fimbrial subunits

NIKAIDO, H. and Wu, H. C. P. 1984. Amino acid sequence homology among the major outer membrane proteins of Escherichia coil Proc. Natl Acad. Sci. USA 81:1048-1052. OUDEGA, B., OLDENZIEL-WERNER, W. J. M., KLAASEN-BOOR, P., REZEE, A., GLAS, J. and DE GRAAF, F. K. 1979. Purification and characterization of cloacin DF13 receptor from Enterobacter cloacae and its interaction with cloacin DF13 in vitro. J , Bacteriol. 138: 7-16.