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Title: A Key Role for the Urokinase Plasminogen Activator (uPA) in Invasive Group A Streptococcal Infection}
Article:
Abstract:
Recruitment of the serine protease plasmin is central to the pathogenesis of many bacterial species, including Group A streptococcus( GAS), a leading cause of morbidity and mortality globally. A key process in invasive GAS disease is the ability to accumulate plasmin at the cell surface, however the role of host activators of plasminogen in this process is poorly understood. Here, we demonstrate for the first time that the urokinase-type plasminogen activator( uPA) contributes to plasmin recruitment and subsequent invasive disease initiation in vivo. In the absence of a source of host plasminogen activators, streptokinase( Ska) was required to facilitate cell surface plasmin acquisition by GAS. However, in the absence of Ska, host activators were sufficient to promote cell surface plasmin acquisition by GAS strain 5448 during incubation with plasminogen or human plasma. Furthermore, GAS were able mediate a significant increase in the activation of zymogen pro-uPA in human plasma. In order to assess the contribution of uPA to invasive GAS disease, a previously undescribed transgenic mouse model of infection was employed. Both C57/black 6J, and AlbPLG1 mice expressing the human plasminogen transgene, were significantly more susceptible to invasive GAS disease than uPA−/− mice. The observed decrease in virulence in uPA−/−mice was found to correlate directly with a decrease in bacterial dissemination and reduced cell surface plasmin accumulation by GAS. These findings have significant implications for our understanding of GAS pathogenesis, and research aimed at therapeutic targeting of plasminogen activation in invasive bacterial infections.
Introduction:
An emerging theme in bacterial pathogenesis is sequestration of host plasminogen during disease initiation[1]. This has inspired research to develop therapeutic inhibitors of bacterial plasminogen activation and recruitment[2],[3],[4],[5]. To be successful, such strategies require a comprehensive understanding of how bacteria interact with the host fibrinolytic system. Group A streptococcus( GAS) is a globally significant human pathogen, responsible for 600, 000 cases of invasive infection each year, approximately 25% of which are fatal[6]. The ability of GAS to accumulate cell surface plasmin activity is an essential step in the initiation of invasive disease[7],[8], but the mechanistic basis of this virulence property has yet to be fully elucidated. While in vitro analyses suggest a role for host plasminogen activators in GAS disease[9],[10],[11], this hypothesis has yet to be conclusively demonstrated in vivo. The glycoprotein plasminogen is found in plasma and extracellular fluids at concentrations of approximately 2 µM. Activation of plasminogen leads to the generation of the serine protease plasmin. Plasmin is able to degrade fibrin clots, connective tissue, extracellular matrix( ECM) and adhesion proteins. Additionally, activation of pro-metalloproteases by plasmin results in degradation of the collagen structural components of the ECM, leading to widespread tissue destruction[12]. Conversion of plasminogen to plasmin can be facilitated by both host and bacterial activators. The major circulating inhibitor of plasmin is α2-antiplasmin. However, surface bound plasmin is less susceptible to inactivation by α2-antiplasmin[13]. GAS secrete the plasminogen activator streptokinase( Ska), which is highly specific for human plasminogen[14]. The contribution of Ska to GAS virulence is well established, however previous studies have shown that deletion of ska from the GAS chromosome significantly, but not completely, attenuates GAS virulence[9],[10],[11]. This implies a role for host activators. There are two distinct eukaryotic activators of plasminogen, urokinase-type plasminogen activator( uPA) and tissue plasminogen activator( tPA). uPA is primarily involved in cell-associated plasminogen activation. The zymogen pro-uPA can be activated by a variety of proteases, including plasmin[15],[16],[17]. Cleavage of the inactive form of the urokinase plasminogen activator pro-uPA by cell bound plasmin generates the active two-chain uPA. This feedback activation results in a significant increase in plasmin activation within biological systems[18]. uPA is localized on the surface of human cells that contribute to epithelial and innate immune defense against bacterial infection, including keratinocytes, neutrophils and macrophages[19]. Furthermore, uPA is upregulated in response to bacterial sepsis, and elevated uPA levels can be correlated to poor patient outcome[20],[21]. Here we utilise a series of isogenic GAS mutants, in conjunction with a newly developed mouse model of infection, to assess the role of uPA in invasive GAS disease.
Discussion:
Despite increased research efforts over the past 20 years, GAS remains a significant human pathogen, with continued outbreaks of serious GAS infection globally[6]. Interaction with the host protease plasmin is central to the onset of invasive GAS disease[1]. A major consequence of plasmin acquisition in GAS infection is to facilitate bacterial escape from fibrin networks. uPA can facilitate efficient fibrin clearance in the absence of both uPAR and tPA[27], and the results of the current study provide for the first time, evidence that uPA mediated plasmin acquisition facilitates widespread systemic infection by GAS. GAS were able to establish a subcutaneous infection at the site of inoculation, but showed a decreased propensity for dissemination from the site of local infection and systemic disease initiation in the absence of uPA. This reduced virulence could be directly correlated to reduced bacterial plasmin acquisition in plasma from uPA−/− mice. Previously, uPA deficiency has been linked to impaired wound healing[27],[28],[29]. No significant difference was seen 3 days post infection in lesion sizes for AlbPLG1 and AlbPLG1/uPA−/− mice. This may reflect the contribution of numerous secreted GAS virulence factors to localised inflammation and lesion development. Comparison of lesion size between AlbPLG1 and AlbPLG1/uPA−/− mice was not performed at later time points due to poor survival of AlbPLG1 mice following GAS infection, however, it appears that in this model, lesion size is not indicative of the propensity of GAS to initiate systemic disease. The finding that virulence was not fully attenuated in the humanised plasminogen uPA−/− background is not unexpected, given the well established role for streptokinase in GAS virulence, and the presence of the other major host plasminogen activator tPA in this model. However, GAS virulence was further attenuated following deletion of ska, providing further evidence of a role for co-operative plasminogen activation in GAS invasive disease. The contribution of streptokinase to plasminogen-dependant GAS virulence has been well documented[9], and data presented here clearly show that in the absence of host activators, streptokinase is an absolute requirement for cell surface plasmin acquisition by GAS strain 5448. However, previous studies suggest that even in the absence of streptokinase, GAS are able to acquire cell surface plasmin activity[9],[10]. Following deletion of ska from the chromosome, GAS strain 5448 acquired cell surface plasmin activity in both human plasma and in the presence of plasminogen and uPA. This study therefore demonstrates that streptokinase is not essential for sequestration of plasmin by GAS. The ability of GAS to access a source of plasmin in the absence of streptokinase may have relevance at stages of infection where streptokinase expression is downregulated, or when streptokinase is degraded by other GAS virulence factors such as SpeB. The GAS protease SpeB has been shown to degrade key mediators of plasminogen acquisition, including streptokinase, but not receptors for plasmin and plasminogen such as the streptococcal enolase[8]. uPA mediated plasmin acquisition may therefore be critical during early infection when SpeB is abundant[11], providing a source of plasmin for recruitment to the cell surface. uPA can be detected in normal human plasma and is expressed at the leading edge of keratinocytes and macrophages during wound healing[19],[30]. Additionally, uPA stored in intracellular vesicles in neutrophils is released into the extracellular space following activation[31]. The abundance of migrating and inflammatory cells during GAS infection therefore represents a significant source of uPA that can contribute to plasmin acquisition by GAS. Furthermore, receptor bound plasmin is resistant to α2-antiplasmin inhibition, enhancing the activation of receptor bound pro-uPA[32]. Plasmin bound to GAS cell surface receptors may therefore further amplify uPA mediated proteolysis. The finding in this study that GAS are able to enhance uPA generation in plasma supports a scenario in which plasmin localised to the GAS surface enhances activation of pro-uPA to uPA, effectively creating an activation cascade leading to enhanced proteolysis[18]. Upregulation of uPA in bacterial meningitis is associated with poor patient outcome and breaching of the blood cerebrospinal fluid barrier[21], and uPA is upregulated in response to numerous bacterial infections, including bacterial sepsis[20],[33]. Whilst the ability of bacteria to degrade clots and ECM using uPA activated plasmin has been demonstrated repeatedly in vitro( reviewed in[1]), the contribution of uPA to GAS virulence has not been established in vivo. We now show for the first time, that uPA mediated plasminogen activation contributes to systemic GAS disease in vivo using a novel model of GAS infection AlbPLG1/uPA−/− mice. It has been suggested that targeting the nexus between bacteria and the fibrinolytic system to inhibit bacterial plasmin activation may provide therapeutic benefit[2],[3],[4],[5], and indeed, data presented here support this proposal. However, the development of potential therapeutics is currently hampered by our limited understanding of this process. Clearly, inhibitors targeting streptokinase mediated plasmin acquisition would not be sufficient to prevent bacterial plasmin accumulation by GAS. Understanding the mechanisms behind bacterial interaction with key components of the fibrinolytic system may therefore aid in the development of therapeutics to control GAS infection.
Subversion of the host fibrinolytic system by bacterial pathogens is recognised as a key process in severe disease initiation . Co-opting of plasmin by bacteria contributes to tissue destruction and bacterial dissemination , both hallmarks of invasive Group A streptococcal disease , and research aimed at therapeutic targeting of the nexus between group A streptococcus and the fibrinolytic system is increasing . The host plasminogen activator uPA is found at the surface of cells that contribute to epithelial and innate immune defense against bacterial infection , and may contribute to bacterial recruitment of plasmin , however , the role of uPA in group A streptococcal infection is not well characterised . Here , we describe for the first time the key role played by uPA in invasive group A streptococcal disease . The ability of this pathogen to cause severe infection , even in the absence of the bacterial plasminogen activator streptokinase , has significant implications for the development of therapeutics to control invasive bacterial infection .