ABSTRACT Human plasminogen (hPg)-binding M-protein (PAM), a major virulence factor of Pattern D Streptococcus pyogenes (GAS), is the primary receptor responsible for binding and activating hPg. PAM is covalently bound to the cell wall (CW) through cell membrane (CM)-resident sortase A (SrtA)-catalyzed cleavage of the PAM-proximal C-terminal LPST ↓ -GEAA motif present immediately upstream of its transmembrane domain (TMD), and subsequent transpeptidation to the CW. These steps expose the N-terminus of PAM to the extracellular milieu (EM) to interact with PAM ligands, e . g ., hPg. Previously, we found that inactivation of SrtA showed little reduction in functional binding of PAM to hPg, indicating that PAM retained in the cell membrane (CM) by the TMD nonetheless exposed its N-terminus to the EM. In the current study, we assessed the effects of mutating the Thr 4 (P1) residue of the SrtA-cleavage site in PAM (Thr 355 in PAM) to delay PAM in the CM in the presence of SrtA. Using rSrtA in vitro , LPS Y GEAA and LPS W GEAA peptides were shown to have low activities, while LPS T GEAA had the highest activity. Isolated CM fractions of AP53/ΔSrtA cells showed that LPS Y GEAA and LPS W GEAA peptides were cleaved at substantially faster rates than LPS T GEAA, even in CMs with an AP53/ΔSrtA/PAM[T 355 Y] double mutation, but the transpeptidation step did not occur. These results implicate another CM-resident enzyme that cleaves LPS Y GEAA and LPS W GEAA motifs, most likely LPXTGase, but cannot catalyze the transpeptidation step. We conclude that the natural P1 (Thr) of the SrtA cleavage site has evolved to dampen PAM from nonfunctional cleavage by LPXTGase. IMPORTANCE We show in this study that functional cleavage of the sortase A (SrtA) cleavage signal for M-protein, LPST*GEAA, in the Gram+ cell membrane, which allows transpeptidation of M-protein to the cell wall, as opposed to non-functional cleavage by the highly active cell membrane nonribosomal enzyme, LPXTGase, at the downstream G-residue, is highly dependent on the presence of T at position 4. From our studies, we conclude that Streptococcus pyogenes has evolved in a manner that maximized T at this position so that SrtA preferentially cleaved the sorting signal in order that the virulence factor, M-protein, was stabilized on the cell surface through covalent attachment to the cell wall.

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