Maini Kukkonen

Lack of O‐antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica

The O‐antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express β‐barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O‐antigen repeats on wild‐type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O‐antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6‐Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla‐mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O‐antigen prevented PgtE‐mediated bacterial adhesion to basement membrane. Substitution of Arg‐138 and Arg‐171 of the motif for protein binding to lipid A 4′‐phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O‐antigen sterically prevents recognition of large‐molecular‐weight substrates. Loss of O‐antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.

Protein regions important for plasminogen activation and inactivation of α2‐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, α2‐antiplasmin, and the bacteria were found to inactivate α2‐antiplasmin. In contrast, only poor plasminogen activation and no cleavage of α2‐antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A β‐barrel topology model for Pla and OmpT predicted 10 transmembrane β‐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 α2‐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 α‐Pla of 292 amino acids was processed into β‐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 α2‐antiplasmin. Cleavage of α2‐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 α2‐antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.

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.

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(plSF101) 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(plSF101) 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 oMgomannoside chains of the lamjnjn network in basement membranes.

Using Every Trick in the Book: The Pla Surface Protease of Yersinia pestis

The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion into human endothelial cell lines; the adhesive and invasive charateristics of Pla can be genetically dissected from its proteolytic activity. Pla is a 10-stranded antiparallel beta-barrel with five surface-exposed short loops, where the catalytic residues are oriented inwards at the top of the beta-barrel. The sequence of Pla contains a three-dimensional motif for protein binding to lipid A of the lipopolysaccharide. Indeed, the proteolytic activity of Pla requires rough lipopolysaccharide but is sterically inhibited by the O antigen in smooth LPS, which may be the selective advantage of the loss of O antigen in Y. pestis. Members of the omptin family are highly similar in structure but differ in functions and virulence association. The catalytic residues of omptins are conserved, but the variable substrate specificities in proteolysis by Pla and other omptins are dictated by the amino acid sequences near or at the surface loops, and hence reflect differences in substrate binding. The closest orthologs of Pla are PgtE of Salmonella and Epo of Erwinia, which functionally differ from Pla. Pla gives a model of how a horizontally transferred protein fold can diverge into a powerful virulence factor through adaptive mutations.

The Single Substitution I259T, Conserved in the Plasminogen Activator Pla of Pandemic Yersinia pestis Branches, Enhances Fibrinolytic Activity

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla beta-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the beta-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in alpha(2)-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution (259)IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution (259)TIDKN/IIDKT in Pla altered proteolysis in favor of alpha(2)-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.

Phylogenetic group‐associated differences in regulation of the common colonization factor Mat fimbria in Escherichia coli

Heterogeneity of cell population is a key component behind the evolutionary success of Escherichia coli. The heterogeneity supports species adaptation and mainly results from lateral gene transfer. Adaptation may also involve genomic alterations that affect regulation of conserved genes. Here we analysed regulation of the mat (or ecp) genes that encode a conserved fimbrial adhesin of E. coli. We found that the differential and temperature‐sensitive expression control of the mat operon is dependent on mat promoter polymorphism and closely linked to phylogenetic grouping of E. coli. In the mat promoter lineage favouring fimbriae expression, the mat operon‐encoded regulator MatA forms a positive feedback loop that overcomes the repression by H‐NS and stabilizes the fimbrillin mRNA under low growth temperature, acidic pH or elevated levels of acetate. The study exemplifies phylogenetic group‐associated expression of a highly common surface organelle in E. coli.

Yersinia pestis Pla Has Multiple Virulence-associated Functions

Our work indicates that Y. pestis Pla is a multifunctional surface protein. It is an efficient protease and also an adhesin and an invasin. The Salmonella homolog of Pla, PgtE, also has proteolytic and adhesive properties, whereas E. coli OmpT is only weakly adhesive, and its substrate specificity differs from that of Pla and PgtE. PgtE and OmpT do not mediate bacterial invasion into any of the human epithelial and endothelial cell lines tested. We are currently characterizing the mechanism and relevance of Pla-mediated invasion as well as the structural requirements of the other functions of Pla.

Functional Variability of Type 1 Fimbriae of Escherichia coli

Type 1 fimbriae and the FimH-adhesins of E. coli differ in adhesive functions: those of a meningitis isolate IHE3034 mediate adhesion to collagens whereas those of an avirulent K-12 strain PC31 do not. Amino acid residue Alanine-62 in the FimH is critical for the collagen binding ability: substitution of Ala62 of IHE3034 FimH into serine abolishes collagen adhrence whereas substitution of Ser62 of PC31 FimH into alanine restores the binding.

Multifunctional Nature of Enterobacterial Fimbriae

Bacterial adhesion at the site of infection is widely accepted as an important early phase of numerous infectious diseases., Adhesion is often mediated by bacterial surface appendages called fimbriae (see Jann et al., in this volume). The fimbrial filaments consist of numerous copies of a major structural subunit, which confers serological properties on the fimbriae, and a few copies of minor subunits, one of which is an adhesin with lectin-like binding properties. A number of fimbrial types occur in Escherichia coli strains associated with human extraintestinal infections, they differ in molecular binding specificity and show distinct reactivity with tissue compartments.1 Normally, the invading bacteria first encounter epithelial surfaces, and it is not suprising that the virulence-associated fimbrial types of E. coli exhibit binding to epithelial surfaces at the relevant tissue sites (see Figure lA for an example). Analysis of potential inhibitors of fimbrial binding in normal human urine showed that the pathogenic function of fimbriae in the ascent of urinary tract infection is dependent on two factors, the presence of receptors on uroepithelia and the lack of soluble inhibitors of binding in urine.1–3