Biology subjects

Readnour, B. M.

Publications and source records attributed to Readnour, B. M..

2 recordsLinked to original sources

bioRxiv2023

A high-resolution cryo-EM structure of a bacterial M-protein reveals a compact structure that diverges from related M-proteins

The surface of Streptococcus pyogenes (GAS) is studded with virulence determinants, with the most abundant being the characteristic M-protein used to serotype various strains of the bacterium. There are >250 strains of GAS serotypically distinguished by their M-proteins. Major pathogenic mechanisms of GAS require that this microorganism hijacks host components for survival, many of which are involved in hemostasis. One of these processes involves the binding of human host plasminogen (hPg) to an abundant GAS M-protein receptor (PAM). When bound to PAM, hPg is readily activated to the serine protease plasmin (hPm) by bacterial and host hPg activators, and cell-bound hPm is protected from inactivation by its natural inhibitors. This stabilizes a potent protease on GAS cells which aids in their survival and dissemination. Highly evolutionary domain-related M-proteins are assumed to form long alpha-helical projections, without tertiary structure, although no M-protein complete structure has been determined. Here, we employed cryogenic electron microscopy to solve such a structure anchored to a lentivirus particle membrane. Contrary to the belief in this field that M-proteins are extended long tropomyosin-like coils, we show that PAM folds through intra- and inter-domain interactions to a much more globular form on the cell surface. The nature of the folding and the many interactions involved in forming the PAM tertiary structure are summarized herein. SignificanceWe provide a unique approach to solve high-resolution structures of Streptococcus pyogenes (GAS) M-proteins, abundant virulence determinants on the GAS surface. Because of their unusual nature, no full high-resolution structure of any M-protein has been determined, especially when membrane-bound. Herein, we provide a unique general methodology for solving these structures by engineering a M-protein to be anchored to a lentivirus particle membrane for effective use in cryo-EM. Using this approach, we provide the first structure of a complete bacterial M-protein and show, that this M-protein is a monomeric globular structure on the cell surface, and not a dimeric coiled-coil, as generally believed. Thus, individual M-proteins may adopt structures that have evolved to accommodate their major host binding partner.

biochemistry↗

bioRxiv2021

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

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{downarrow}-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 Thr4 (P1) residue of the SrtA-cleavage site in PAM (Thr355 in PAM) to delay PAM in the CM in the presence of SrtA. Using rSrtA in vitro, LPSYGEAA and LPSWGEAA peptides were shown to have low activities, while LPSTGEAA had the highest activity. Isolated CM fractions of AP53/{Delta}SrtA cells showed that LPSYGEAA and LPSWGEAA peptides were cleaved at substantially faster rates than LPSTGEAA, even in CMs with an AP53/{Delta}SrtA/PAM[T355Y] double mutation, but the transpeptidation step did not occur. These results implicate another CM-resident enzyme that cleaves LPSYGEAA and LPSWGEAA 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. IMPORTANCEWe 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.

microbiology↗