Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in Streptococcus pyogenes

Abstract

The proteolytic activity of human plasmin (hPm) is utilized by various cells to provide a surface protease that increases the potential of cells to migrate and disseminate. Skin-trophic Pattern D strains of Streptococcus pyogenes (GAS), e.g., GAS isolate AP53, contain a surface M-protein (PAM) that directly and strongly interacts (Kd ~ 1 nM) with human host plasminogen (hPg), after which it is activated to hPm by a specific coinherited bacterial activator, streptokinase (SK2b), or by host activators. Another ubiquitous class of hPg binding proteins on GAS cells includes “moonlighting” proteins, such as the glycolytic enzyme, enolase (Sen). However, the importance of Sen in hPg acquisition, especially when PAM is present, has not been fully developed. Sen forms a complex with hPg on different surfaces, but not in solution. Isogenic AP53 cells with a targeted deletion of PAM do not bind hPg, but the surface expression of Sen is also greatly diminished upon deletion of the PAM gene, thus confounding this approach for defining the role of Sen. However, cells with point deletions in PAM that negate hPg binding, but fully express PAM and Sen, show that hPg binds weakly to Sen on GAS cells. Despite this, Sen does not stimulate hPg activation by SK2b, but does stimulate tissue-type plasminogen activator-catalyzed activation of hPg. These data demonstrate that PAM plays the dominant role as a functional hPg receptor in GAS cells that also contain surface enolase.

A high-resolution single-particle cryo-EM hydrated structure ofStreptococcus pyogenesenolase offers insights into its function as a plasminogen receptor

“…This is also the case for all of the Sen variants constructed in this study. However, Sen does interact with hPg when Sen adheres to surfaces as shown by similar experiments (9,20,(28)(29)(30)(31)(32)(33).…”

“…We tested these concepts in this study. Sen in solution does not interact with solution phase hPg; however, surface-bound Sen, whether adsorbed to SPR chips, microtiter plates, GAS cell surfaces, or phospholipid vesicular surfaces, interacted with hPg with a Kd of ~100-200 nM (20).…”

“…The interaction of Sen variants with DOPG phospholipid (PL) vesicles was investigated essentially as described earlier (20). In summary, 0.1 mM PL vesicles pre-blocked with 2.5% BSA/PBS, pH 7.4, for 2 hr, were incubated with 0 or 10 M WT-Sen or Sen variants for 1 hr at ambient temperature.…”

“…We have previously shown that Sen interacts with DOPG vesicles and that the DOPG-bound Sen enhanced activation of hPg by (tissue-type plasminogen activator) tPA (20). DOPG provides a more biologically relevant system in which to study this interaction with surface-adsorbed Sen.…”

Functional and Transcriptome Analysis of Streptococcus pyogenes Virulence on Loss of Its Secreted Esterase

“…These virulence factors probably contribute directly or indirectly to the invasion process of actin rearrangement or endocytic pathways, which ultimately lead to the internalization of GAS [18][19][20][21]. M protein is a major surface virulence factor that binds to a wide range of host cells and proteins, including plasminogen, IgA, IgG, factor H, and C4b-binding protein [22,23]. It can efficiently promote uptake of streptococci by both human endothelial and epithelial cells.…”