Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

Abstract

The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. (Blood. 2009;113: 1139-1148) IntroductionAside from its role in minimizing blood loss, the clotting system is also an effector arm of the immune system that modulates release of inflammatory mediators and prevents the spread of invasive microorganisms through extravascular fibrin deposition. This "quarantine through coagulation" strategy is evolutionarily primitive, as evidenced by the sensitivity of the horseshoe crab clotting system to activation by bacterial endotoxin. 1 Indeed, fibrin deposition is one of the hallmarks of inflammation in humans, and the induration accompanying delayed-type hypersensitivity skin reactions is caused by extravascular fibrin deposition. 2 Inflammatory responses to infection include expression of tissue factor on activated monocytes as well as increased acute phase proteins like fibrinogen and PAI-I, which contribute to hypercoagulable states. 3 Some bacteria modulate the host coagulation system to evade immune responses or facilitate dissemination through extravascular tissues. For example, staphylocoagulase and streptokinase form complexes with prothrombin or plasminogen, converting them into prothrombotic (thrombin) or profibrinolytic (plasmin) enzymes. 4 Likewise, plasminogen activator (Pla), a member of the omptin family of bacterial proteases, promotes dissemination of the plague agent, Yersinia pestis, from subcutaneous sites to the lymphatic and circulatory systems. 5 Proteolytic activation of plasminogen by Pla is thought to facilitate Y pestis migration through tissue barriers, because plasmin degrades fibrin clots and extracellular matrix components, activates procollagenases, and inactivates collagenase inhibitors. [6...

C-terminal Peptides of Tissue Factor Pathway Inhibitor Are Novel Host Defense Molecules

“…The finding that the peptides were particularly active in human plasma against E. coli, and to a lesser extent against P. aeruginosa, is particularly relevant and interesting, in the light of the ubiquitous occurrence of P. aeruginosa in chronic leg ulcers, contrasting to the very sparse E. coli colonization in these wounds (35,47). Furthermore, based on the demonstration that bacterial omptins (such as OmpT from E. coli) may release GGL27 fragments from TFPI, it was recently proposed that TFPI has evolved sensitivity to omptin-mediated proteolytic inactivation to potentiate procoagulant responses to E. coli infection in certain conditions (48). Our data further substantiate this hypothesis by adding another host-response mechanism to E. coli infection: OmpT-mediated generation of the host defense peptide GGL27.…”

The Omptins ofYersinia pestisandSalmonella entericaCleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1

“…We observed here that Y. pseudotuberculosis cells and the Yco omptins do not cleave PAI-1, and they neither cleave nor activate plasminogen (J. Haiko, unpublished). The Pla-mediated, wide attack on control of an important hemostatic process in humans, i.e., the fibrinolysis/coagulation balance (32,60,71), represents a considerable difference in pathogenetic potential between the two bacterial species. The omptin Epo also cleaved human PAI-1; this omptin associates with the plant pathogen E. pyrifoliae, which causes pear blight (30), and epo is carried on a mosaic plasmid, pEP36, that is closely related to the virulence plasmid pEA29 of Erwinia amylovora (40).…”

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

“…Many inhibitors are exceptionally stable to proteolysis (6); however, some inhibitors can be gradually inactivated either by the very proteases that they inhibit (7) or by alternative endogenous proteases that they do not inhibit (8 -12). Infectious microorganisms can also produce proteases that perturb the protease-inhibitor balance in human tissue by selective cleavage and inactivation of human protease inhibitors (13)(14)(15). An understanding of the fundamental features that render protein protease inhibitors stable inhibitors versus vulnerable substrates of the proteases that they encounter is critical for understanding the function of these molecules in tissue homeostasis.…”