The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene

Abstract

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface. IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Evolution of Gram+ Streptococcus pyogenes has maximized efficiency of the Sortase A cleavage site

“…In this case, PAM is found noncovalently bound within the CM and also the EM where it is functional regarding hPg binding and activation. We hypothesized that PAM delayed in the CM nonetheless spans the CW and presents its N-terminus to the EM, where hPg interacts with the exposed A-domain and is activated by SK2b secreted by the cells (15). Also, the PAM cleaved from the CM and present in the CW is likely noncovalently bound to peptidoglycan and can also expose its A-domain for the same purpose.…”

“…The surface exposed PAMs were also functional in stimulating hPg activation by SK2b (Figure 7B). Whether the PAM extending from the CW and/or from the CM is the source of the surface-exposed PAM is not known at this stage, but a SrtA deficiency does not significantly affect the ability of PAM to function in binding and stimulating the activation of hPg in whole AP53 cells (15).…”

“…Since WT-PAM was selectively retained in the CM in cells in which SrtA was inactivated (15), we designed experiments to determine the cellular distribution of AP53/PAM[T 355 Y] and AP53/PAM[T 355 A], in comparison to WT-PAM, in cell lines with a targeted expression of these variants. We began by evaluating whether these SrtA-resistant PAMs were processed and secreted in the extracellular medium (EM) at mid-log phase cell growth.…”

Group A Streptococcus-Induced Activation of Human Plasminogen Is Required for Keratinocyte Wound Retraction and Rapid Clot Dissolution

“…The binding and activation of hPg by the GAS virulence factors, PAM and SK, respectively, has been implicated in promoting GAS dissemination. Once hPm is bound to the GAS surface, proteolysis of the ECM and alteration of the cellular tight junctions (TJ) can occur (39)(40)(41). To further investigate the roles of PAM and SK in creating a proteolytic environment to promote host invasion, we performed these same experiments using the isogenic GAS strains, AP53R + S − / PAM and AP53R + S − / SK, neither of which is capable of generating hPm.…”

3D-QSAR Studies of 1,2,4-Oxadiazole Derivatives as Sortase A Inhibitors

“…This enzyme speeds up two consecutive reactions: (a) transpeptidation and (b) thioesterification. SrtA is involved in the bacterial adhesion process and acts by attaching proteins holding LPXTG to lipid II [1][2][3][4][5][6][7][8][9][10][11]. SrtA inhibitors do not influence bacterial growth, but instead, they prevent the emergence of the virulence of pathogenic bacterial strains, thereby hindering infections produced by Staphylococcus aureus (S. aureus) or other bacteria of Gram-positive strain.…”