Kaarina Lähteenmäki

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.

pH-Dependent Association of Enolase and Glyceraldehyde-3-Phosphate Dehydrogenase of Lactobacillus crispatus with the Cell Wall and Lipoteichoic Acids

The plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus crispatus were localized on the cell surface at pH 5 but released into the medium at an alkaline pH. These proteins bound to lipoteichoic acids at a pH below their isoelectric point. The results indicate that lactobacilli rapidly modify their surface properties in response to changes in pH.

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.

Invited review: Breaking barriers — attack on innate immune defences by omptin surface proteases of enterobacterial pathogens

The omptin family of Gram-negative bacterial transmembrane aspartic proteases comprises surface proteins with a highly conserved β-barrel fold but differing biological functions. The omptins OmpT of Escherichia coli, PgtE of Salmonella enterica, and Pla of Yersinia pestis differ in their substrate specificity as well as in control of their expression. Their functional differences are in accordance with the differing pathogenesis of the infections caused by E. coli, Salmonella, and Y. pestis, which suggests that the omptins have adapted to the life-styles of their host species. The omptins Pla and PgtE attack on innate immunity by affecting the plasminogen/plasmin, complement, coagulation, fibrinolysis, and matrix metalloproteinase systems, by inactivating antimicrobial peptides, and by enhancing bacterial adhesiveness and invasiveness. Although the mechanistic details of the functions of Pla and PgtE differ, the outcome is the same: enhanced spread and multiplication of Y. pestis and S. enterica in the host. The omptin OmpT is basically a housekeeping protease but it also degrades cationic antimicrobial peptides and may enhance colonization of E. coli at uroepithelia. The catalytic residues in the omptin molecules are spatially conserved, and the differing polypeptide substrate specificities are dictated by minor sequence variations at regions surrounding the catalytic cleft. For enzymatic activity, omptins require association with lipopolysaccharide on the outer membrane. Modification of lipopolysaccharide by in vivo conditions or by bacterial gene loss has an impact on omptin function. Creation of bacterial surface proteolysis is thus a coordinated function involving several surface structures.

Activation, cytokine production, and intracellular survival of bacteria in Salmonella-infected human monocyte-derived macrophages and dendritic cells

Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen causing localized gastroenteritis in humans. Macrophages (Mφs) and dendritic cells (DCs) play an important role in innate immunity against Salmonella. In this report, we have compared the consequences of infection of human Mφs and DCs with wild‐type S. typhimurium and an isogenic PgtE‐defective strain. PgtE is an outer membrane protein hypothesized to have a role in intracellular survival of Salmonella. We observed that DCs undergo full maturation in response to Salmonella infection, as indicated by up‐regulation of cell‐surface marker proteins CD80, CD83, CD86, and human leukocyte antigen class II. CC chemokine ligand 5 (CCL5), CXC chemokine ligand 10, tumor necrosis factor α, interleukin (IL)‐12, and IL‐18 gene expression and protein production were readily induced by Salmonella‐infected Mφs and DCs. CCL20 was preferentially produced by Mφs, whereas DCs secreted higher levels of CCL19 as compared with Mφs. DCs and Mφs infected with S. typhimurium also produced high levels of interferon‐γ (IFN‐γ). Cytokine neutralization and stimulation experiments suggest that the production was partly regulated by Salmonella‐induced type I IFNs, IL‐12, and IL‐18. DC cytokine production induced by Salmonella was much higher as compared with the responses induced by Salmonella lipopolysaccharide or flagellin. Mφs and DCs were capable of internalizing and harboring Salmonella for several days. S. enterica PgtE provided no survival advantage for the bacteria in human Mφs or DCs. Our results demonstrate that although Mφs and DCs share similar functions, they may have different roles during Salmonella infection as a result of differential production of certain chemokines and cytokines.

The surface protease PgtE of Salmonella enterica affects complement activity by proteolytically cleaving C3b, C4b and C5

Complement activity in mammalian serum is fundamentally based on three homologous components C3b, C4b and C5. During systemic infection, the gastrointestinal pathogen Salmonella enterica disseminates within host phagocytic cells but also extracellularly. Consequently, systemic Salmonella transiently confronts the complement system. We show here that the surface protease PgtE of S. enterica proteolytically cleaves C3b, C4b and C5 and that the expression of PgtE enhances bacterial resistance to human serum. Degradation of C3b was further enhanced by PgtE‐mediated plasminogen activation.

Antiprotease inactivation by Salmonella enterica released from infected macrophages

The mammalian serine protease plasmin, which has an important role in extracellular matrix degradation during cell migration, is regulated by the plasma antiprotease α2‐antiplasmin (α2AP). The surface protease PgtE of Salmonella enterica serovar Typhimurium proteolytically inactivated α2AP. PgtE also activates the plasma zymogen plasminogen to plasmin, and bacteria expressing PgtE promoted degradation of extracellular matrix laminin in the presence of plasminogen and α2AP. α2AP inactivation was detected with the rough derivative of S. enterica 14028, but not with the smooth wild‐type strain, suggesting that the O‐antigen of lipopolysaccharide prevented contact of PgtE with the substrate molecule. After growth of S. enterica 14028 in murine J774A.1 macrophage‐like cells, the infected cell lysate as well as bacteria from isolated Salmonella‐containing vacuoles (SCVs) cleaved α2AP. Bacteria from SCVs produced an elevated level of PgtE and had a reduced O‐antigen chain length. The lysate from S. enterica 14028‐infected macrophages promoted formation of plasmin in the presence of α2AP, wheras plasmin formation by lysates from uninfected macrophages, or from macrophages infected with the pgtE‐negative derivative of 14028, was inhibited by α2AP. Salmonella disseminates in the host within macrophages, which utilize plasmin for migration through tissue barriers. The results suggest that intracellular enhancement of PgtE activity in Salmonella may promote macrophage‐associated proteolysis and cellular migration by altering the balance between host plasmin and α2AP.

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.