Benita Westerlund

Bacterial proteins binding to the mammalian extracellular matrix.

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best‐characterized bacterial proteins active in these interactions are the mycobacterial fibronectin‐binding proteins, the fibronectin‐ and the collagen‐binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A‐protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein‐protein or on a protein‐carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.

The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein

Interaction of the basement‐membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]‐labelled type IV collagen to 075X‐positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N‐terminal 7S domain of type IV collagen, and the binding of [125I]‐labelled type IV collagen to O75X‐positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase‐H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein‐protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue‐binding specificity of the type‐3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild‐type Kleb‐siella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type‐3 fimbriae, as well as the purified type‐3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowmans capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type‐3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type‐3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type‐3 fimbrial gene cluster in association with the pap‐encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type‐3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type‐3 fimbriae strains of Yeisinia and Salmonella, showing that the ability to use type collagen as tissue target is widespread among enteric bacteria.

Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and feoF influence fimbrial binding to renal tubuli and immobilized fibronectin

P fimbriae of the F71 serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Galα(1–4)Gal‐specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Galα(1–4)Gal‐containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin‐coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F72, F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue‐binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.

A novel lectin‐independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Binding of P fimbriae of uropathogenic Escherichia coli to purified human plasma fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30‐kDa fragment and the 120–140‐kDa carboxyterminal fragments of fibronectin. Binding to the gelatin‐binding 40‐kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by α‐D‐Gal‐(1‐4)‐β‐D‐Gal‐1‐O‐Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I‐labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.

Tissue interactions of Escherichia coli adhesins

ConclusionsThe E. coli adhesions show a remarkable tissue tropism in the human urinary tract. This obviously relates to the known compartmentation of glycoconjugates in the kidney. To function as a virulence factor in human urinary tract infections, an adhesin must evidently recognize such receptors at uroepithelia that are not excreted in soluble form in urine. This prerequisite is filled by P fimbriae but not by type-1 or S fimbriae. Most of the tissue interactions of E.coli adhesins involve binding to carbohydrate receptors, whereas the binding of the 075X adhesin to type IV collagen appears to rely on protein-protein interactions. Binding of P fimbriae to immobilized fibronectin is independent of the lectin activity of the fimbriae and suggests of an additional function for the fimbrillin in mediating interaction with matrix and basement membrane proteins. Such interaction might be useful after colonization and disruption of epithelial surfaces, when the lectin activity of the fimbriae is not any more important.

The O75X adhesin of uropathogenic Escherichia coli: receptor-active domains in the canine urinary tract and in-vitro interaction with laminin.

The complementary binding sites for the purified O75X adhesin of uropathogenic Escherichia coli in canine urinary tract were determined by indirect immunofluorescence. The adhesin bound to the vascular basement membranes in the canine kidney, ureter, bladder and urethra. In the kidney, the adhesin bound also to the tubular basement membranes and to the Bowmans capsule and mesangium of the glomeruli. In the bladder and ureter, but not in the urethra, the O75X adhesin bound to the basal lamina of smooth muscle cells. Connective tissue was negative for the adhesin. Interestingly, considerable intraepithelial heterogeneity was revealed as the O75X adhesin bound to epithelium of the ureter but not to that of the kidney, bladder or urethra. The purified O75X adhesin bound specifically to laminin, a basement membrane glycoprotein, immobilized on nitrocellulose or polystyrene, suggesting that laminin is involved in the binding of the O75X adhesin to basement membranes.

P Fimbriae of Uropathogenic Escherichia coli as Multifunctional Adherence Organelles

P fimbriae are the major single virulence factor of uropathogenic Escherichia coli strains. Recent analyses have shown that P fimbriae possess two distinct binding specificities mediated by different fimbrial subunits. P fimbriae bind to Gal alpha (1-4)Gal-containing globoseries of glycolipids of epithelial cells; this binding is mediated by the lectin-like minor protein G of the filament. In vitro mapping of the human urinary tract for binding sites of P fimbriae has revealed that they bind in a Gal alpha (1-4)Gal-inhibitable manner to epithelia of kidney and bladder. On the other hand, P fimbriae bind to immobilized fibronectin and its amino- and carboxyterminal fragments; this binding is dependent on the E and the F minor proteins of the P-fimbrial filament and seems to be based on a protein-protein interaction. The P fimbriae-fibronectin interaction has been demonstrated also on frozen sections of kidney. P fimbriae thus possess two tissue-adherence properties: one specific for epithelial glycoconjugates and the other for fibronectin of subepithelial extracellular matrices. P-fimbrial binding to epithelial glycoconjugates seems to be important in determining the host tropism and enabling the ascent of E. coli urinary tract infections. Binding to fibronectin may be important in secondary phases of the infection, e.g. after epithelial injury.

Fluorescent microparticles as a rapid tool in bacterial adherence studies

Fluorescent microparticles (FMPs) coated with the O75X adhesin of uropathogenic Escherichia coli were evaluated for use in adhesion studies. The purified o75X adhesin coated efficiently onto the microparticles, and the O75X-FMPs retained the agglutination properties of the intact O75X adhesin molecules. Binding tests on frozen sections of human kidney and with immobilized target proteins showed that the tissue- and molecular-binding specificities of the O75X-FMPs were closely similar to those of the intact O75X adhesin. The results show that adhesin-coated FMPs are rapid and stable reagents for functional study of bacterial adhesion proteins.

Heterogeneity of Renal Glycoconjugates: Biochemical and Bacteriological Aspects

Glycoconjugates (glycoproteins and glycolipids) are abundant functional and structural elements of all cell types (5,32,42). Intracellularly they can be found in various organelles, mostly in the Golgi apparatus, where the construction and trimming of the glycan side chain of glycoconjugates takes place. Thereafter the glycans are transported to their intra- or extracellular destinations according to specific target codes (6,41). At the plasma membranes the glycan side chains of glycoconjugates project outwards from the cell surface, constituting the bulk of the cellular glycocalyx (27,32,42). Interest towards the structure and function of glycoconjugates was greatly enhanced after findings showing an association between malignant transformation and altered glycoconjugate pattern of cell surfaces (4,32). The structure and integrity of the glycan side chain seem to be crucial for some glycoconjugates, whereas for others they are of marginal importance (2,3). For instance, desialylation of lymphocyte sure glycans and of circulating glycoproteins leads to their capture by specific receptors in the liver (38).