Creation of High-Resolution Models of Group A Streptococcus Plasminogen Binding Proteins and Exploring Their Role in Protein Surface Expression

Virulence factors play an important role in the infectiousness of group A streptococcus.(GAS) They play a significant role in immune evasion, cellular adhesion, and spread of group A streptococcus (GAS) within human hosts. To understand the role and impact of various virulence factors a variety of techniques need to be used. One of the most prevalent techniques to appear in the last decade is cryogenic electron microscopy (cryo-EM). One common motif of multiple virulence factors in GAS is the ability to bind human plasminogen. Two of the most significant hPg binders are plasminogen binding group A streptococcal M-like protein (PAM ) and streptococcal surface enolase (SEN).The goal of this study is to research the method in which primary hPg binding proteins are expressed on the surface and to use high level cryo-EM imaging to determine structural information on plasminogen binders to determine their method of interaction. These mutants revealed the existence of another protein LPXTGase that can cleave the LPXTG motif of PAM and showed an increased affinity for aromatic mutants of PAM. PAM and enolase are two of the most abundant proteins on the surface of GAS cells. It has been shown that PAM plays a role in allowing SEN to be present on the surface. Through scanning electron microscopy (SEM) it was shown that 100 nm lipid vesicles on the surface are responsible for protein expression on the surface of S+ GAS cells. These lipid vesicles contain their own mechanisms for protein expression and utilize PAM to integrate into the surface. To determine the method in which both SEN and PAM can interact with hPg on the surface cryo-EM imaging was used to create high resolution 3D structural models of PAM and enolase interacting with hPg on the surface of a cell membrane. Lipid vesicles and lentiviruses were utilized to get 3D maps and models that were analyzed to determine the most probable interactions with hPg.