OC 46.1 - Streptococcal Surface Enolase (SEN) is Overshadowed by Plasminogen-binding Group A Streptococcal M-protein (PAM) for Human Plasminogen Acquisition and Activation

Background: Human plasmin (hPm) proteolytic activity is utilized by various pathogenic bacteria, such as Group A Streptococcus (GAS), to increase dissemination and pathogenicity. GAS is a human-selective Gram-positive bacterium that utilizes hPm through the expression of surface human plasminogen (hPg) receptors. Once hPg is recruited to the cell surface, it is converted into active hPm. There are at least three hPg receptors on the GAS cell surface with different binding affinities. Plasminogen-binding Group A Streptococcal M-protein (PAM) is typically found on skin-tropic Pattern D GAS strains, such as AP53. PAM binds hPg with a very high affinity (Kd ~1 nM) and is able to stimulate hPg activation in the presence of GAS-secreted streptokinase (SK) and host hPg activators such as urokinase. Unlike PAM, streptococcal surface enolase (SEN) is found on the surface of most strains and its importance in hPg acquisition by skin-tropic GAS strains is unknown.

Aims: An investigation of SEN structure and the contribution of SEN in hPg binding and activation.

Methods: AUC was used to determine the protein size. SEN octameric structure was imaged using CryoEM. Binding affinity of SEN and hPg was determined by ELISA and SPR. Surface expression of SEN was determined using FACS and SEM.

Results: The data generated showed that the 47 kDa monomer unit of recombinant SEN oligomerizes to form highly stable octamer in solution. The octameric SEN forms a complex with hPg, characterized by a Kd of ~100 nM. Although SEN tightly binds hPg, it does not stimulate hPg activation by SK or urokinase. Whole GAS cell studies demonstrated that the surface expression of SEN is diminished upon deletion of the PAM gene. hPg binding to AP53 cells has a lower affinity for SEN compared to PAM.

Conclusion(s): This study strongly suggests that SEN plays a secondary role as a hPg receptor in skin-tropic GAS strains.