Impact of the Pla Protease Substrate α2-Antiplasmin on the Progression of Primary Pneumonic Plague

Abstract

Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor ␣2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection. The activation of coagulation, resulting in fibrin deposition and fibrin cross-linking, is a host mechanism to stop bleeding and to physically contain invading microorganisms (1, 2). Subsequently, mechanisms to induce fibrinolysis are activated via tightly regulated proteolytic cascades (3). During this phase, tissue-bound and secreted activators mediate the conversion of the circulating zymogen plasminogen into its active serine protease form, plasmin. In addition, the host innate immune system is tightly linked to the hemostatic system. By coordinating host platelet activation and aggregation, coagulation, fibrinolysis, and inflammation, the innate immune response can synchronize the sensing of invading pathogens, immune cell recruitment, and pathogen elimination, as well as subsequent tissue repair (3-5).As a part of the fibrinolytic system, plasmin degrades fibrin clots with additional biological activities related to tissue remodeling, cell migration, and inflammation (6). In addition, there exist endogenous inhibitors of both active plasmin and plasmin generation to maintain homeostasis. To control the generation of plasmin, plasminogen activator inhibitor 1 (PAI-1) inactivates the plasminogen activators urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) (7,8). On the other hand, ␣2-antiplasmin (A2AP) inhibits plasmin directly. A2AP is synthesized in the liver and maintained in the circulation to rapidly inhibit freely circulating active plasmin (9). Currently, the only known biological function of A2AP is to inhibit active plasmin; this occurs via a set of interaction steps, which culminates in the formation of an enzymatically inactive 1:1 stoichiometric complex of p...

Depletion of Glucose Activates Catabolite Repression during Pneumonic Plague

“…Measurement of plasmin activity. Plasmin activity was determined as described previously (10). BALF (100 l) was incubated in the presence of 50 M D-AFK-ANSNH-iC4H9-2Hbr fluorogenic substrate (SN5; Hematologic Technologies), and fluorescence was recorded over time at 460 nm in 96-well plates with a Molecular Devices SpectraMax M5 fluorescence microplate reader.…”

“…plague (7,8), manipulates programmed cell death pathways and deregulates the host fibrinolysis pathways (9). Not only does Pla activate host plasminogen into plasmin, but it also inactivates inhibitors of plasmin activation, ␣ 2 -antiplasmin and plasminogen activation inhibitor 1 (PAI-1) (10,11). In the absence of catalytic activity of Pla, Y. pestis fails to proliferate in the lungs and poorly disseminates into the bloodstream (7).…”

Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis

“…Based upon the data presented here and collected by our group and others previously, we propose classifying substrates of Pla into three categories: group I substrates that are cleaved both in vitro and in vivo and contribute to bacterial virulence and the host response (these substrates would be represented by the apoptotic molecule FasL [43]); group II substrates such as Prdx6 that are cleaved in vitro and in vivo but do not play a detectable role in bacterial virulence or the host response; and finally, group III substrates that are cleaved in vitro but not in vivo and thus have a limited role during disease progression; A2AP would be a representative example of this class of substrate (50). Therefore, while the identification of new host substrates of Pla in vitro can provide new insights into the mechanisms by which Y. pestis causes disease in mammals, it is important to consider that any individual host substrate may not necessarily play a role in vivo, at least at our current level of investigation.…”

“…As Pla is required for the full virulence of Y. pestis during pneumonic plague (34,35), we set out to determine if (i) the total levels of Prdx6 are altered in the lungs of mice during respiratory infection with Y. pestis and (ii) if Pla cleaves Prdx6 in vivo. As the ⌬pla mutant of Y. pestis is attenuated during respiratory infections (35), to compare the effects of Pla on Prdx6 levels when bacterial loads are normalized between the wild-type and ⌬pla strains, we can increase the inoculum of the ⌬pla mutant ("input CFU") so that the CFU in the lungs at 48 h ("output CFU") is equivalent between the wild-type and mutant strains, as we have done previously (43,50). To do so, we infected C57BL/6 mice via the i.n.…”

Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence

“…pestis to cause primary pneumonic plague [17]. Also, it was shown that Pla can directly inactivate major plasmin inhibitor α 2 -antiplasmin [18, 19] and mediate adhesion to eukaryotic cells (extracellular matrices and basement membranes) which invasive bacteria must penetrate in order to reach the circulation [20, 21]. In addition to its role in adhesion, invasion and tissue damage, Pla has been reported to cleave complement component C3 [11, 22], to possess weak coagulase activity [15], and to mediate the proteolysis of Yersinia virulence factors Yops [23].…”