Heinz Hoschützky

A possible new adhesive site in the cell-adhesion molecule uvomorulin

The calcium-dependent cell-adhesion molecule uvomorulin is a member of the cadherin gene family. Recent studies on the homophilic binding of molecules from neighbouring cells have shown that the amino-terminal part of these proteins plays an important role in the adhesive mechanism. We show here that the epitope for monoclonal antibody DECMA-1, capable of blocking uvomorulin function, is located close to the membrane proximal part of the extracellular domain. To test the effect of structural changes in this membrane proximal region on the adhesive function of uvomorulin, we have studied the cluster of cysteine residues located in the vicinity of the DECMA-1 epitope. Treatment of cells with the reducing agent dithiothreitol (DTT) cleaved the di-sulphide bonds in uvomorulin and affected the adhesive properties of cells. Close cell-cell contacts accompanied by cell flattening and changes in cell shape were blocked by DTT; however, cell aggregation was not inhibited. Consistent with this, uvomorulin became more susceptible in its membrane proximal part to trypsin digestion after treatment with DTT, indicating that conformational changes in this region of the molecule affect the adhesive function. These results suggest that the membrane proximal region of uvomorulin is involved in the adhesive mechanism.

Isolation and characterization of the non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7 : K98 : H6

The non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6 mediates the agglutination of human red cells (RBC), notably of blood group MM. The adhesin can be separated from the bacteria by heat extraction and was purified to homogeneity by ammonium sulphate precipitation and anion exchange chromatography in the presence of 8 M urea. NFA-4 consists of non-covalently linked 28 kDa subunits which tend to form aggregates of an apparent molecular weight in excess of 10(6) Da. The first 23 amino-terminal amino acids were sequenced, and no homology of this region was found with that of the blood group M specific non-fimbrial adhesin of an unrelated uropathogenic E. coli. It has, however, an about 70% homology to the corresponding region of the K88 antigen from animal-pathogenic enterotoxic E. coli. Both polyclonal and monoclonal antibodies against NFA-4 were prepared. One of the monoclonal antibodies strongly inhibits the hemagglutinating activity of both whole bacteria and purified NFA-4.

Function and Molecular Architecture of E.Coli Adhesins

The pathogenicity of E.coli has been ascribed to different virulence factors such as toxins, cytolysins, serum resistance, O- and K-antigens or adhesive properties. The adherence of the bacteria to epithelial surfaces — e.g. cell membranes and/or mucosal surfaces — is an important early event in host parasite interactions leading to bacterial colonization and infection (1). The term adhesion as used in this context describes a specific interaction of recognition proteins attached to the bacterial surface with complex carbohydrate moieties of glycoproteins and/or glycolipids on mammalian cells. These recognition proteins (also termed adhesins, hemagglutinins or lectins) may have different appearances in the electron microscope. Structures that can be demonstrated directly by negative staining procedures have been termed fimbriae or pili (rigid, 5–7 nm diameter) and fibrillae (flexible, 2–3 nm diameter). Nonfimbrial adhesive structures can only be visualized after stabilization with specific antibodies and then have a capsule like appearance (2).

Function and Organization of Escherichia Coli Adhesins

The adhesion of pathogenic bacteria to the epithelial surfaces of a host is a prelude to infection, followed by multiplication of the bacteria and their penetration into the host’s cells or their damaging of the epithelium by the action of their toxins. Adhesion is mediated by interaction between bacterial recognition proteins (adhesins, hemagglutinins, lectins) and receptors on the host ce11.1,2 Most receptors are complex carbohydrates but components of the extracellular matrix and other proteins have also been identified as such2,3 (see Korhonen et al., in this volume). Adhesion can be measured by counting the adhering cells under standard conditions; however, since most adhesive bacteria agglutinate erythrocytes, adhesion can also be monitored easily by hemagglutination. Both adhesion and hemagglutination are inhibited by receptor analogs (glycoproteins, glycolipids, oligosaccharides) and this can be used to determine adhesion specificity. Thus, one way of characterizing bacterial adhesins is via the specificity of their receptors.

Chemical Nature and Cellular Location of Adhesins of E. Coli

The term adhesion describes a specific interaction of bacterial recognition proteins (adhesins) with the carbohydrate moiety of glycoproteins or glycolipids on mammalian cells. Adhesion of pathogenic bacteria to epithelial cells initiates the infective process, enabling the bacteria to withstand the rinsing forces of body fluids or to optimize the delivery of toxins to target cells. It can also be considered as a first stage in bacterial invasion. A function hitherto less appreciated may be the acquisition of nutritional advantages on a substratum rich in nutrients.

Characterization and Surface Organization of E. Coli Adhesins

An important initial step in bacterial infections is the adhesion of the pathogenic bacteria to host cells or tissue. This phenomenon has been studied with intestinally pathogenic as well as with extraintestinal and invasive bacteria. Adhesion is mediated by bacterial recognition proteins, which are termed as adhesins. Since adhesive bacteria also induce agglutination of host red blood cells (RBC), the recognition proteins are also called hemagglutinins (1). They are associated with extracellular structures which may have different appearances in the electron microscope. Some of these extracellular appendages are relatively thick and rigid and others are much thinner, flexible and curly. Both can be demonstrated directly by negative staining procedures. A third group of extracellular adhesive structures can only be visualized after stabilization with specific antibodies and has then a capsule-like appearance. These structures, which are schematized in Fig. 1, are termed as fimbriae (rigid, 5–7 nm diameter), fimbrillae (flexible, 2–3 nm diameter) and nonfimbrial (no fine structure demonstrable). Pending their morphological analysis, the term “nonfimbrial” for the latter structures is only tentative.