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.

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.

Protein regions important for plasminogen activation and inactivation of alpha2-antiplasmin in the surface protease Pla of Yersinia pestis.

The plasminogen activator, surface protease Pla, of the plague bacterium Yersinia pestis is an important virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla-expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, alpha2-antiplasmin, and the bacteria were found to inactivate alpha2-antiplasmin. In contrast, only poor plasminogen activation and no cleavage of alpha2-antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A beta-barrel topology model for Pla and OmpT predicted 10 transmembrane beta-strands and five surface-exposed loops L1-L5. Hybrid Pla-OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards plasminogen and alpha2-antiplasmin. Substitution analysis at 25 surface-located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla-expressing recombinant E. coli. The mature alpha-Pla of 292 amino acids was processed into beta-Pla by an autoprocessing cleavage at residue K262, and residues important for the self-recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on plasminogen activation or cleavage of alpha2-antiplasmin. Cleavage of alpha2-antiplasmin and plasminogen activation were influenced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla-like protease by deleting residues D214 and P215, by substituting residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation of alpha2-antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.

Wire-guided excision of non-palpable breast cancer: determinants and correlations between radiologic and histologic margins and residual disease in re-excisions.

In cases of wire-guided excision of non-palpable breast cancer (WGE), data concerning the determinants and correlations between radiologic and histologic margins and residual cancer in re-excisions are sparse. A total of 21 variables in 66 WGE followed by 49 re-excisions were prospectively analyzed. In multivariate analysis, only large mammographic lesions were clearly related to positive margins in specimen radiography (P<0.05). Multifocality (P<0.001), large pathologic size (P<0.05) and superficial excision (P<0.05) were related to positive histologic margins and multifocality (P=0.001) to residual disease in re-excisions. The sensitivity, specificity and positive predictive values of specimen radiography for predicting histologic margins were 33%, 79% and 53%, and those for predicting residual disease 30%, 80% and 38%, respectively. The ability of histologic margins to predict residual disease was 91%, 58% and 38%, respectively. In WGE, large mammographic lesions carry a significant risk for radiologically incomplete excision, while pathologically large and multifocal tumors may be histologically incompletely excised, especially if the excision does not extend down to the pectoral fascia. The excision sites of multifocal tumors should be re-excised because of the considerable risk of residual disease. The radiologic and histologic margins of the specimen may be misleading.

Plasminogen activation in degradation and penetration of extracellular matrices and basement membranes by invasive bacteria.

Methods to assess in vitro the role of plasminogen activation in enterobacterial degradation of extracellular matrices and their protein components as well as in penetration through basement membrane are described. Development of these methods was initiated after the findings that enterobacterial surface structures (fimbriae and the Pla surface protease) function in plasminogen activation as well as in laminin- and/or fibronectin-specific adhesion. Enterobacteria with these properties degrade radiolabeled laminin as well as metabolically labeled extracellular matrix from cultured endothelial or epithelial cells. Plasmin-coated bacteria also penetrate through the reconstituted basement membrane preparation Matrigel. The processes are dependent on plasminogen activation by the invasive bacteria. The results suggest a pathogenic similarity between enterobacteria and tumor cells in cellular metastasis through tissue barriers.

Apoptosis during breast carcinoma progression.

The purpose of this study was to investigate apoptosis, proliferation, and the expression of apoptosis-influencing proteins bcl-2 and bax and estrogen and progesterone receptors during breast carcinoma progression. The material consisted of 53 paired breast carcinoma samples representing primary and recurrent tumors and 24 control samples. The recurrent sample was located either in the breast scar tissue or at a distant metastatic site. Apoptosis was detected both morphologically and by 3' end labeling of fragmented DNA. Cell proliferation was evaluated immunohistochemically by the MIB index. The expressions of bcl-2, bax, and estrogen and progesterone receptors were studied immunohistochemically. There was a significant increase in the extent of apoptosis and proliferation in recurrent tumors compared to the primary lesions (P = 0.015 and P = 0.038, respectively). In primary tumors with an apoptotic index of >0.50%, the survival of the patients was significantly shorter (P = 0.015). In cases with a significant increase in apoptosis or proliferation in the recurrent tumor, the survival of the patients was significantly shorter (P = 0.009 and P = 0.003, respectively). Of the variables analyzed, bcl-2 expression and a positive estrogen receptor status were significantly associated with a low extent of apoptosis (P = 0.010 and P = 0.042, respectively). Their changes were parallel to the changes in apoptosis during tumor progression, although the associations did not reach statistical significance. The results show that increased apoptosis is associated with a worse prognosis in breast carcinoma. A significant increase in apoptosis in recurrent breast carcinoma lesions predicts a worse clinical outcome.

Expression of plasminogen activator pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix.

The effect of the plasminogen activator Pla of Yersinia pestis on the adhesiveness of bacteria to the mammalian extracellular matrix was determined. Y. pestis KIM D27 harbors the 9.5-kb plasmid pPCP1, encoding Pla and pesticin; the strain efficiently adhered to the reconstituted basement membrane preparation Matrigel, to the extracellular matrix prepared from human lung NCI-H292 epithelial cells, as well as to immobilized laminin. The isogenic strain Y. pestis KIM D34 lacking pPCP1 exhibited lower adhesiveness to both matrix preparations and to laminin. Both strains showed weak adherence to type I, IV, and V collagens as well as to human plasma and cellular fibronectin. The Pla-expressing recombinant Escherichia coli LE392(pC4006) exhibited specific adhesiveness to both extracellular matrix preparations as well as to laminin. The Pla-expressing strains showed a low-affinity adherence to another basement membrane component, heparan sulfate proteoglycan, but not to chondroitin sulfate proteoglycan. The degradation of radiolabeled laminin, heparan sulfate proteoglycan, or human lung extracellular matrix by the Pla-expressing recombinant E. coli required the presence of plasminogen, and degradation was inhibited by the plasmin inhibitors aprotinin and alpha2-antiplasmin. Our results indicate a function of Pla in enhancing bacterial adhesion to extracellular matrices. Y. pestis also exhibits a low level of Pla-independent adhesiveness to extracellular matrices.

Identification of two laminin-binding fimbriae, the type 1 fimbria of Salmonella enterica serovar typhimurium and the G fimbria of Escherichia coli, as plasminogen receptors.

Escherichia coli strains carrying recombinant plasmids encoding either the type 1 fimbria of Salmonella enterica serovar Typhimurium or the G fimbria of E. coli exhibited binding of human 125I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog epsilon-aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229-237, 1993; S. Saarela et al., Infect. Immun. 64:2857-2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes.

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.

Interaction of Haemophilus influenzae with the mammalian extracellular matrix.

The adhesiveness of 2 unencapsulated nonfimbriated strains of Haemophilus influenzae, 23459 and 23330, and the encapsulated fimbriated strain 770235 to extracellular matrix (ECM) and to its isolated components was studied, as was the potential of H. influenzae plasminogen receptors to enhance degradation of ECM and bacterial penetration through basement membrane. All strains exhibited efficient adhesiveness to reconstituted basement membrane and to ECM from cultured human endothelial cells. Strains 23459 and 23330 efficiently adhered to immobilized laminin, fibronectin, and various collagens. Strain 770235 adhered efficiently to fibronectin and type I and III collagens and with low efficiency to laminin. With all 3 strains, plasmin generated on H. influenzae plasminogen receptors degraded laminin and fibronectin as well as ECM from human endothelial cells. Plasmin bound on H. influenzae cells also potentiated penetration of bacteria through a basement membrane preparation reconstituted on membrane filters. These results give evidence for a role of ECM adherence and plasminogen activation in the spread of H. influenzae through tissue barriers.

Bacterial plasminogen receptors: in vitro evidence for a role in degradation of the mammalian extracellular matrix.

The potential of bacterium-bound plasmin to degrade mammalian extracellular matrix and to enhance bacterial penetration through basement membrane was assessed with the adherent strain SH401-1 of Salmonella enterica serovar Typhimurium. Typhimurium SH401-1 was able to bind plasminogen and to enhance the tissue-type plasminogen activator-mediated activation of the single-chain plasminogen to the two-chain plasmin. The end product, the enzymatically active, bacterium-bound plasmin activity, was also formed in a normal human plasma milieu in the presence of exogenous tissue-type plasminogen activator, indicating that plasmin was protected from the plasminogen activator inhibitors and plasmin inhibitors of plasma. Plasmin bound on Typhimurium cells degraded 125I-labeled laminin as well as 3H-labeled extracellular matrix prepared from the human endothelial cell line EA.hy926. The degradations were not seen with Typhimurium cells without plasminogen and were inhibited by the low-molecular-weight plasmin inhibitor aprotinin. Plasmin bound on Typhimurium cells also potentiated penetration of bacterial cells through the basement membrane preparation Matrigel reconstituted on membrane filters. The results give in vitro evidence for degradation of the mammalian extracellular matrix by bacterium-bound plasmin and for a pathogenetic role for bacterial plasminogen receptors.

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.

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin.

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(pISF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1-fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(pISF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oligomannoside chains of the laminin network in basement membranes.

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.