Frits R. Mooi

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.

Structure and function of peripiasmic chaperone‐like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli

The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunobiotting of subcellular fractions with an anti‐serum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone‐like protein, Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilizes this polypeptide and prevents its degradation by the ceil‐envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.

Characterization of fimbriae from Bacteroides fragilis

Fimbriae derived from Bacteroides fragilis strain BE1 (BE1 fimbriae) appeared to be composed of subunits with a molecular weight of 40,000. Under the electron microscope the fimbriae could be visualized as straight filaments with a diameter of 4 nm. It appeared that production of the BE1 fimbriae is repressed under conditions of iron limitation, and at a growth temperature of 20 degrees C. Antibodies raised against the 40,000 dalton polypeptide, purified by means of preparative SDS-polyacrylamide gelelectrophoresis, recognized the native fimbriae, as was shown by immunogold labelling of intact bacterial cells, and by immunoprecipitation. Immunoblot experiments showed that other strains of B fragilis tested produced polypeptides, ranging in molecular weight from 40,000 to 42,000, that are antigenically related to the BE1 fimbrial subunit. No haemagglutination activity could be associated with the BE1 fimbriae.

Effect of limited proteolysis on bacteriocin activity in vivo and in vitro.

Cells of Enterobacter cloacae harbouring the bacteriocinogenic factor Clo DF13 produce several plasmid-specific gene products [ 11. Two of these products, the cloacin DF13 and its immunity protein form a very tight and stoichiometric complex which protects the producing cells against the lethal effects of their own bacteriocin [2] . After being released from the bacteriocinogenic cells the complex possesses a very effective killing activity towards sensitive cells. This lethal action can be divided into three major phases: (a) adsorption onto specific receptor sites located in the outer membrane; (b) interaction with the cytoplasmic membrane resulting in the leakage of potassium ions: (c) inactivation of the ribosomes by cleavage of the 16 S ribosomal RNA [3-51. The final step in the interaction with sensitive cells appears to involve a penetration of at least a part of the cloacin molecules into the cytoplasm. Because the cloacin molecules possess no activity towards ribosomes as long as they are bound to the immunity protein, the latter is supposed to be released from the complex at one of the preceeding steps. At the present time it is not known whether the inactivation of the ribosomes requires a penetration of the intact cloacin molecules or possibly only a fragment of these molecules. A possible approach to this question is the examination of the effect of proteolytic enzymes upon the activity of cloacin DF 13. In this communication we describe that limited trypsinolysis of the cloacin-immunity protein complex generates a cloacin fragment-immunity protein complex without loss of in vitro activity. Trypsinolysis of the free cloacin results in a complete digestion of the bacteriocin.

Virulence factors of Bordetella pertussis

Clearly, B. pertussis has evolved very elaborate mechanisms to maintain itself in the human host. Three different proteins (FHA, pertussis toxin and fimbriae) have been implicated in adherence. Furthermore, a number of toxins are produced (pertussis toxin, adenylate cyclase, dermonecrotic toxin, and tracheal cytotoxin) which destroy the clearance mechanisms of the respiratory tract, or suppress the immune response. There is evidence that B. pertussis may survive intracellularly, and the possibility that it is a facultative intracellular parasite should certainly be explored. The availability of a large number of cloned virulence genes, and a system to construct well defined mutants by allelic exchange (Stibbitz et al. 1986) will greatly facilitate the study of Bordetella virulence factors at the molecular level. It opens the possibility to construct avirulent strains, which are still able to colonize and stimulate the local immune response. Such strains may be used as live, oral vaccines, to present (heterologous) antigens to the mucosal immune system of the respiratory tract.

Excretion of proteins by gram-negative bacteria: export of bacteriocins and fimbrial proteins byEscherichia coli

In gram-negative bacteria only few proteins are exported across both the cytoplasmic membrane and the outer membrane which forms an extra barrier for protein excretion.In this review we describe the mechanisms of production and export of two types of plasmid-encoded proteins inEscherichia coli. These proteins are the bacteriocin cloacin DF13 and the K88ab and K99 fimbrial subunits. Specific so-called helper proteins located at different positions in the cell envelope play an essential role in the export of these proteins. The genetic organization, subcellular location and functions of these helper proteins, as well as the effects of mutations and culture conditions on the export of the proteins are described. Models for the export mechanisms are presented and future application possibilities for engineering foreign protein excretion inE. coli with these export systems are discussed.

THE USE OF MINICELLS AND MAXICELLS TO DETECT THE EXPRESSION OF CLONED GENES

An analysis of cloned DNA fragments involves the characterisation of the products of gene expression and the regulation of their synthesis. When it is known that a DNA fragment codes for a particular protein or RNA species, suitable tests can be devised to determine if the cloned DNA is expressed in a particular host and the subcellular localisation of the products can be investigated. Thus, proteins can be detected by sensitive immunoassays, RNA by hybridisation to suitable probes and enzymes by appropriate enzyme assays. But how does one detect expression of genes on cloned DNA fragments when the function of the gene products is not known or if no suitable detection assays are available? The answer is to look for the synthesis of novel polypeptides or RNA species in cells carrying the cloned DNA fragments. However in wild-type cells the detection of new proteins and RNA species is almost impossible because of the concurrent expression of the host genome. Three approaches have been devised to circumvent this problem and to detect expression of cloned DNA (plasmid DNA) borne gene products: DNA-driven in vitro protein and RNA synthesis; minicells and maxicells.

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.

The Use of Restriction Endonucleases and T4 DNA Ligase

Bacteria possess restriction-modification systems, each system of which consists of a modification methylase and a restriction endonuclease. The modification methylase recognises a specific sequence and methylates certain adenines or cytosines within this sequence. The corresponding restriction endonuclease recognises the same sequence and in most cases cleaves the DNA when the sequence has not been methylated by the modification methylase.