Severe streptococcal infections account for more than 500,000 deaths globally per year. The rapid emergence of multi-drug resistant bacterial pathogens, and the lack of new antibiotics in pharmaceutical development, represents a huge public health problem. Streptokinase (SK), a major streptococcal virulence factor, targets human fibrinolysis by activating plasminogen (Pg) to the proteinase, plasmin (Pm). By mechanisms that are not understood, Pm degrades protective fibrin (Fbn) barriers generated by the host response. Along with another virulence factor, PAM, SK produces, by an unknown mechanism, Pm bound to the bacterial surface, allowing Pm-coated bacteria to spread through tissues. Background - We proposed a unified mechanism of SK-Pg activation initiated by SK binding and inducing conformational activation of the Pg zymogen by the NH2-terminal insertion mechanism. A second Pg molecule binds to the SK7Pg* catalytic complex as a specific substrate, which is cleaved to Pm in an initial, triggering catalytic cycle. Pg is displaced from SK7Pg* by the high affinity binding of Pm produced, yielding the SK7Pm catalytic complex that binds Pg as a substrate, initiating the second, propagation cycle that converts the remaining Pg to Pm. Broad Goals - Biochemical and biophysical studies of Pg activation by SK are proposed with the goal of understanding the molecular mechanism by which SK subverts human fibrinolysis to promote severe streptococcal diseases, with the ultimate purpose of developing new mechanism-based drugs. The investigation addresses hypotheses to fill major gaps in the understanding of the infection mechanisms, and evaluation of novel therapeutic approaches. Experimental approaches include enzyme kinetics, rapid-reaction kinetics, equilibrium binding, mutagenesis, and collaborative electron microscopy, crystallography, and in vivo models of infection.
Aim 1 : To define the role of [Glu]Pg conformational changes in its binding to SK in the catalytic and substrate modes, and the mechanism of substrate/product recognition for [Glu]Pg, [Lys]Pg, and Pm.
Aim 2 : To define in rapid-reaction kinetic studies the pathway of molecular events in the mechanism of SK-induced conformational activation of [Glu]Pg and [Lys]Pg, and the mechanism of [Glu]Pg/[Lys]Pg substrate recognition by the SK7Pg*/Pm catalytic complexes.
Aim 3 : To determine the role of inter-domain flexibility of SK bound to Pg and Pm in dictating SK function, and to evaluate antibodies against the flexible segments linking SK domains as inhibitors for potential use in combating streptococcal diseases.
Aim 4 : To elucidate the mechanisms by which Fbg, Fbn, and PAM regulate SK-initiated Pg activation that underlie their roles in invasive streptococcal infections, and to evaluate an anti-substrate Pg recognition antibody inhibitor.
Aim 5 : To define the mechanism of skizzle (SkzL) as a novel cofactor of tPA-catalyzed Pg activation, and to evaluate its potential role as pathogenicity factor in Streptococcus agalactiae infections.

Public Health Relevance

Severe streptococcal infections account for 500,000 deaths per year globally, and there are at least 18.1 million people affected. The rapid emergence of antibiotic-resistant bacteria and the lack of new antibiotics under development represent a huge public health problem. This project proposes to define the molecular mechanisms by which streptokinase, a streptococcal pathogenicity factor, subverts the human blood clot- dissolving system to propagate life-threatening infections, with the ultimate goal of developing new drugs for targeting streptokinase to combat these bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056181-17
Application #
8452725
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
1996-07-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
17
Fiscal Year
2013
Total Cost
$330,810
Indirect Cost
$118,752
Name
Vanderbilt University Medical Center
Department
Pathology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Verhamme, Ingrid M; Bock, Paul E (2014) Rapid binding of plasminogen to streptokinase in a catalytic complex reveals a three-step mechanism. J Biol Chem 289:28006-18
Nolan, Miranda; Bouldin, Samantha D; Bock, Paul E (2013) Full time course kinetics of the streptokinase-plasminogen activation pathway. J Biol Chem 288:29482-93
Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias et al. (2011) Engineering streptokinase for generation of active site-labeled plasminogen analogs. Anal Biochem 415:105-15
Wiles, Karen G; Panizzi, Peter; Kroh, Heather K et al. (2010) Skizzle is a novel plasminogen- and plasmin-binding protein from Streptococcus agalactiae that targets proteins of human fibrinolysis to promote plasmin generation. J Biol Chem 285:21153-64
Tharp, Anthony C; Laha, Malabika; Panizzi, Peter et al. (2009) Plasminogen substrate recognition by the streptokinase-plasminogen catalytic complex is facilitated by Arg253, Lys256, and Lys257 in the streptokinase beta-domain and kringle 5 of the substrate. J Biol Chem 284:19511-21
Verhamme, Ingrid M; Bock, Paul E (2008) Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation. J Biol Chem 283:26137-47
Panizzi, Peter; Boxrud, Paul D; Verhamme, Ingrid M et al. (2006) Binding of the COOH-terminal lysine residue of streptokinase to plasmin(ogen) kringles enhances formation of the streptokinase.plasmin(ogen) catalytic complexes. J Biol Chem 281:26774-8
Bean, Ronald R; Verhamme, Ingrid M; Bock, Paul E (2005) Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin. J Biol Chem 280:7504-10
Panizzi, P; Friedrich, R; Fuentes-Prior, P et al. (2004) The staphylocoagulase family of zymogen activator and adhesion proteins. Cell Mol Life Sci 61:2793-8
Boxrud, Paul D; Bock, Paul E (2004) Coupling of conformational and proteolytic activation in the kinetic mechanism of plasminogen activation by streptokinase. J Biol Chem 279:36642-9

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