Sequence Diversity in Streptokinase and M-Proteins, Their Co-Inheritance and Functional Relationships in Group A Streptococcal Virulence

Group A Streptococcus (GAS) is a strict human pathogen which causes both trivial and lethal infections. Although antibiotics are effective drugs against GAS diseases, GAS infections have undergone resurgence since the 1980s. To perpetuate in its host and cause disease, GAS employs several virulence factors that assist GAS in adherence, invasion, immune evasion, and colonization in distal tissues within the host, e.g., M or M-like proteins, hyaluronic acid capsule, fibronectin binding protein, streptokinase, streptolysins, and streptococcal pyogenic exotoxins. Of these factors, only M or M-like proteins and streptokinase (SK) are the focus of these studies. These virulence factors are used by GAS to interact with key components of the host fibrinolytic system and generate plasmin (hPm). The enzymatic activity of hPm is used by GAS to degrade basement membrane matrix and invade through tissues barriers. M or M-like proteins are membrane-anchored proteins, where they bind human plasminogen (hPg) and hPm. The GAS-bound or free hPg is subsequently activated to plasmin by GAS-secreted SK and generates a proteolytic surface on GAS.

SK is a three-domain protein, containing α, β, and γ modules. Phylogenetic analysis of variable β-domain sequences allows GAS to be grouped into cluster 1 and cluster 2 SK. Based on these same β-domain sequences, SK is subdivided into subclusters 2a and 2b. Cluster 2b strains are non-coincidentally associated with skin tropism and express an M-like protein called Plasminogen-binding group A streptococcal M-like protein (PAM) on their surface, e.g., strain AP53. Whereas, SK2a strains are associated with the respiratory tract infections and express a M protein, designated M1, on their surface, e.g., strain SF370. PAM binds to human hPg/hPm directly, while M1 binds to hPg/hPm indirectly through interaction with human fibrinogen (Fg). Subsequently, GAS-secreted SK non-proteolytically activates hPg to hPm. Due to different modes of substrate (hPg) recognition and activation by SK2a and SK2b strains, it was anticipated that there would be differences in virulence. To test this, AP53 strains were generated with a targeted replacement of its SK2b by SK2a and PAM by M1 from the SF370 strain of GAS. Additional strains were generated in which B1B2 repeats of M1 in the AP53/pam-to-M1 strain were replaced by a1a2 repeats of PAM so that it can bind hPg and hPm directly. The virulence of these strains was analyzed in a humanized hPg mouse model. The results indicated that SK2a in the background of a SK2b GAS strain is as virulent as its native SK2b containing strain. Additionally, to probe more fully the nature of the SK-hPm complexes and their roles in dictating virulence, we demonstrated that the SK2a-hPm activator complex is much more resistant to inactivation by host α2-antiplasmin, than the SK2b-hPm complex. These studies demonstrate that GAS virulence in different bacterial strains can be dictated by differences in the hPg activation properties of SK2a and SK2b.

Due to the importance of PAM as a virulence determinant in GAS, the binding interactions between PAM and hPg were characterized. Our results demonstrate for the first time that VEK38, a peptide within PAM representing the hPg binding domain harbors two binding sites for the kringle 2 domain of hPg which specifically binds to PAM. Therefore, to understand the mechanism of PAM-hPg interactions, we sought to determine the solution structure of this complex by NMR. Our studies revealed that subsequent to kringle 2 binding, VEK38 undergoes a conformational transition from a predominantly random coil to an end-to-end helix conformation. Completion of these studies will reveal a mechanism for binding of PAM and hPg which will facilitate the targeting these virulence factors of GAS in disease management.