Binding sites in the rat brain for Escherichia coli S fimbriae associated with neonatal meningitis.

Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal meningitis, we have studied the presence of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat organs and their binding was assessed by indirect immunofluorescence. In the brain of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and brain ventricles. The binding was completely inhibited by the trisaccharide NeuAc alpha 2-3Gal beta 1-4Glc, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressing S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, whereas the non-fimbriated host strain and a recombinant strain expressing P fimbriae did not adhere to brain tissues. The results suggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatal brain has a pathogenetic role during bacterial invasion from circulation into the cerebrospinal fluid.

Bacterial plasminogen activators and receptors.

Invasive bacterial pathogens intervene at various stages and by various mechanisms with the mammalian plasminogen/plasmin system. A vast number of pathogens express plasmin(ogen) receptors that immobilize plasmin(ogen) on the bacterial surface, an event that enhances activation of plasminogen by mammalian plasminogen activators. Bacteria also influence secretion of plasminogen activators and their inhibitors from mammalian cells. The prokaryotic plasminogen activators streptokinase and staphylokinase form a complex with plasmin(ogen) and thus enhance plasminogen activation. The Pla surface protease of Yersinia pestis resembles mammalian activators in function and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators turn bacteria into proteolytic organisms using a host-derived system. In Gram-negative bacteria, the filamentous surface appendages fimbriae and flagella form a major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules as well as M-protein-related structures have been identified as plasminogen receptors, the former receptor type also occurs on mammalian cells. Plasmin is a broad-spectrum serine protease that degrades fibrin and noncollagenous proteins of extracellular matrices and activates latent procollagenases. Consequently, plasmin generated on or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrices. In a few instances plasminogen activation has been shown to enhance bacterial metastasis in vitro through reconstituted basement membrane or epithelial cell monolayers. In vivo evidence for a role of plasminogen activation in pathogenesis is limited to Y. pestis, Borrelia, and group A streptococci. Bacterial proteases may also directly activate latent procollagenases or inactivate protease inhibitors of human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers.

Fibrinolytic and procoagulant activities of Yersinia pestis and Salmonella enterica.

Pla of the plague bacterium Yersinia pestis and PgtE of the enteropathogen Salmonella enterica are surface-exposed, transmembrane ß-barrel proteases of the omptin family that exhibit a complex array of interactions with the hemostatic systems in vitro, and both proteases are established virulence factors. Pla favors fibrinolysis by direct activation of plasminogen, inactivation of the serpins plasminogen activator inhibitor-1 and α2-antiplasmin, inactivation of the thrombin-activable fibrinolysis inhibitor, and activation of single-chain urokinase. PgtE is structurally very similar but exhibits partially different functions and differ in expression control. PgtE proteolysis targets control aspects of fibrinolysis, and mimicry of matrix metalloproteinases enhances cell migration that should favor the intracellular spread of the bacterium. Enzymatic activity of both proteases is strongly influenced by the environment-induced variations in lipopolysaccharide that binds to the ß-barrel. Both proteases cleave the tissue factor pathway inhibitor and thus also express procoagulant activity.

Organization of genes expressing the blood-group-M-specific hemagglutinin of Escherichia coli: identification and nucleotide sequence of the M-agglutinin subunit gene.

The organization of genes encoding the blood group M-specific hemagglutinin (M-agglutinin) of Escherichia coli strain IH11165 was studied with a cloned 6.5-kb DNA segment. This DNA segment contains at least five genes which code for the polypeptides of 12.5, 30, 80, 18.5 and 21 kDa. The 30-, 80- and 21-kDa polypeptides are synthesized as precursors that are approximately 2 kDa larger. The 21-kDa polypeptide was identified as the M-agglutinin subunit by its reactivity with anti-M-agglutinin serum. Nucleotide sequence analysis of the corresponding gene showed that the M-agglutinin precursor had a 24-amino acid (aa) signal sequence, while the mature protein is 146 aa residues long. Although the organization of the M-agglutinin gene cluster resembles those of other E. coli adhesins, there is no significant sequence homology between the M-agglutinin subunit and the subunits of the other potentially related proteins in E. coli.

Binding of plasminogen to Escherichia coli adhesion proteins.

Immobilization of plasminogen via its lysine-binding sites is regarded as a prerequisite for its activation and function in fibrinolysis and pericellular proteolysis. In the present study, the interaction of plasminogen with fimbriae found on Escherichia coli strains causing invasive human infections was studied. Plasminogen displayed concentration-dependent and saturable binding to immobilized type 1 fimbriae and, several fold lower binding to P and S fimbriae. The binding to fimbriae was effectively inhibited by epsilon-aminocaproic acid indicating that it was mediated by the lysine-binding sites of plasminogen. Binding studies with mutated fimbriae and inhibition tests indicated that the interaction was not dependent on the lectin subunit of the fimbriae. These results indicate the existence of a novel type of host-microbe interaction which may be important in the invasion by bacteria of host tissues.

Novel cell-binding activity specific for N-acetyl-D-glucosamine in an Escherichia coli strain.

Escherichia coli strains isolated from patients with different levels of urinary tract infection and from healthy persons were tested for their ability to haemagglutinate endo-beta-galactosidase-treated human erythrocytes. Among the 104 strains studied one revealed a strong agglutination reaction with the enzyme-treated erythrocytes. From the monosaccharides tested N-acetyl-D-glucosamine inhibited agglutination most effectively. Orosomucoid and asialo-orosomucoid had no effect on the haemagglutination whereas beta-galactosidase treated asialo-orosomucoid was inhibitory. These findings indicate that the E. coli strain studied contains a novel cell-binding activity with specificity for terminal N-acetyl-D-glucosamine residues.

The Pla surface protease/adhesin of Yersinia pestis mediates bacterial invasion into human endothelial cells.

The plasminogen activator Pla of Yersinia pestis belongs to the omptin family of enterobacterial surface proteases and is responsible for the highly efficient invasion of the plague bacterium from the subcutaneous infection site into the circulation. Y. pestis has been reported to invade human epithelial cells. Here, we investigated the role of Pla in bacterial invasion into human endothelial cells. Expression of Pla in recombinant Escherichia coli XL1(pMRK1) enhanced bacterial invasion into ECV304 cells. The invasiveness was not affected by substitution mutation at the residues S99 or D206 that are needed for the proteolytic activity of Pla. Pla-expressing bacteria adhered to the extracellular matrix of ECV304 cells. Only weak adhesion and poor invasion were seen with the recombinant E. coli XL1(pMRK2), which expresses the omptin homolog from E. coli. The results identify Pla as an invasion protein of Y. pestis and show that the invasive function does not involve the proteolytic activity of Pla.

A N-acetyllactosamine-specific cell-binding activity in a plant pathogen, Erwinia rhapontici.

A strain of the phytopathogenic bacterial species, Erwinia rhapontici, was found to cause hemagglutination of human erythrocytes that was specifically inhibited by beta-galactosides. Of the monosaccharides tested, N-acetyl galactosamine and galactose efficiently inhibited the hemagglutination. The most potent inhibitor identified was Ga1 beta 1-4GlcNAc that was 30-100-fold more potent than Ga1 beta 1-3GlcNac or Ga1 beta 1-3GalNAc. Fetuin had no effect on the hemagglutination whereas asialofetuin was inhibitory. No blood group specificity was found for the hemagglutinin. These findings indicate that the E. rhapontici strain possesses a novel bacterial cell-binding activity with specificity for terminal N-acetyllactosamine residues.

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 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 alpha-D-Gal-(1-4)-beta-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.

Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis.

The effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by epsilon-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PA-catalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that lack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.

Determination of taxonomic resolution capacity of conventional one-dimensional SDS-polyacrylamide gel electrophoresis of whole-cell proteins using Enterobacteriaceae.

The capacity of one-dimensional SDS-PAGE of whole bacterial cells to both separate and cluster taxonomic units is studied using members of Enterobacteriaceae as test material. The results show that intraspecies variation can be detected and on the other hand the degree of taxonomic divergence which still can be grouped together is determined. In addition the system has high tolerance to changes in cell culture conditions making the usage of SDS-PAGE suitable for applications where rapid and reliable bacterial identification is needed.

Immobilization of plasminogen on Escherichia coli flagella.

The interaction of plasminogen with flagella of Escherichia coli was investigated. Plasminogen bound to flagella purified from E. coli LE392, a commonly used cloning host, and E. coli IH3069, an O25H1 strain isolated from a case of newborn bacteremia. The binding was inhibited by the lysine analog epsilon-aminocaproic acid, suggesting involvement of the lysine-binding Kringle domains of plasminogen in the binding. Purified flagella enhanced the formation of plasmin activity in the presence of tissue-type plasminogen activator; a similar enhancement was observed with flagella-expressing LE392 cells.

Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans.

Candida albicans infections in severely immunocompromized patients are not confined to mucosal surfaces; instead the fungus can invade through epithelial and endothelial layers into the bloodstream and spread to other organs, causing disseminated infections with often fatal outcome. We investigated whether secretion of the C. albicans acid proteinase facilitates invasion into deeper tissues by degrading the subendothelial basement membrane. After cultivation under conditions that induce the secretion of the acid proteinase, C. albicans degraded radioactively metabolically labeled extracellular matrix proteins from a human endothelial cell line. The degradation was inhibited in the presence of pepstatin A, an inhibitor of acid proteinases. Pepstatin A-sensitive degradation of the soluble and immobilized extracellular matrix proteins fibronectin and laminin by proteinase-producing C. albicans was also detected, whereas no degradation was observed when the expression of the acid proteinase was repressed. Our results demonstrate that the C. albicans acid proteinase degrades human subendothelial extracellular matrix; this may be of importance in the penetration of C. albicans into circulation and deep organs.

Penetration of fimbriate enteric bacteria through basement membranes: a hypothesis.

A mechanism for penetration of basement membranes by Escherichia coli is presented. The mechanism is based on the ability of the S fimbriae of meningitis-associated E. coli to bind to vascular endothelium and choroid plexuses in brain and to basement membranes. On the other hand, the S and the type 1 fimbriae of E. coli immobilize plasminogen and tissue-type plasminogen activator; this process generates proteolytic plasmin activity on the surface of fimbriate cells. Our hypothesis is that bacterium-bound plasma activity, directed to basement membranes through fimbrial binding, promotes bacterial penetration through basement membranes.

Interaction of clinical isolates of nonencapsulated Haemophilus influenzae with mammalian extracellular matrix proteins.

The adherence of clinical isolates of nonencapsulated Haemophilus influenzae strains from patients with chronic bronchitis to distinct immobilized extracellular matrix components was determined. With selected strains the induction of plasmin formation by these isolates was studied. The strains could be divided into two groups: strains that showed a very high level of adherence to laminin and type I collagen, as well as adhesion to fibronectin and strains that showed only a moderate level of adhesion to laminin and a low level of adhesion to fibronectin. Plasmin formation was demonstrated for three out of eight isolates. Persisting and nonpersisting strains did not differ quantitatively or qualitatively with respect to the level of adhesiveness to the distinct matrix proteins and in their ability to induce plasmin formation.

Plasminogen receptors. Turning Salmonella and Escherichia coli into proteolytic organisms.

We evaluated in vitro the hypothesis that bacterial adhesiveness to the mammalian extracellular matrix and the activation of plasminogen on bacterial plasminogen receptors promote bacterial penetration through basement membranes. We used the strain SH401 of Salmonella enterica serovar Typhimurium, which adheres to the high-mannose chains of laminin, a major glycoprotein of basement membranes, and expresses plasminogen receptors. Bacterium-bound plasmin was able to degrade laminin and extracellular matrix preparations as well as to potentiate the penetration of bacteria through a reconstituted basement membrane. The results suggest that metastatic tumour cells and bacterial pathogens use similar mechanisms to penetrate through tissue barriers.

Characterisation of Escherichia coli strains associated with canine urinary tract infections.

Of 33 Escherichia coli strains isolated from canine urinary tract infections, 22 were haemolytic and 27 were classified into O serogroups, the most common being O4, O6, O2 and O83. P-fimbriated strains were haemolytic and belonged mainly to serogroups O4 and O6. Twenty-nine strains possessed type-1 fimbriae but only small numbers possessed S fimbriae, type-1C fimbriae, X adhesins or the aerobactin system. It is postulated that P fimbriae and haemolysin production contribute to bacterial virulence in canine pyelonephritis and cystitis.

Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis.

BACKGROUND: Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation. RESULTS: Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI by the omptins. CONCLUSIONS: Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection.