Marta Wojnowska

The M protein is an essential virulence factor of Streptococcus pyogenes , or group A streptococcus (GAS), one of the most common and dangerous human pathogens. Molecular and functional characterization of M protein variants and their interactions with host components is crucial for understanding streptococcal pathogenesis and vaccine development. The M3 protein is produced by the prevalent emm 3 GAS serotype, which is frequently associated with severe invasive diseases. Here, we structurally and biochemically characterize the interaction of M3 with human collagens. High-resolution structures of the N-terminal M3 domain in the free state as well as bound to a collagen peptide derived from the Collagen Ligands Collection reveal a novel T-shaped protein fold that presents binding sites complementing the characteristic topology of collagen triple helices. The structure of the M3/collagen peptide complex explains how emm 3 GAS and related streptococci, such as Streptococcus dysgalactiae subsp. equisimilis , can target collagens to enable colonization of various tissues. In line with this, we demonstrate that the M3/collagen interaction promotes enhanced biofilm formation of emm 3 GAS in an emm type-specific manner, which can be inhibited with the recombinant N-terminal M3 domain. Further, emm 3 GAS are shown to colocalize with collagen in tissue biopsies from patients with necrotizing soft tissue infections, where GAS biofilms are common. This observation is reproduced in infected organotypic skin models. Together, these data provide detailed molecular insights into an important streptococcal virulence mechanism with implications for the understanding of invasive infections, strategies for treating biofilm and M-protein-based vaccine design.

TonB-dependent uptake systems of Gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrient acquisition, primarily siderophore-bound iron, to cross the highly impermeable outer membrane (OM). Remarkably, the ferredoxin uptake system (Fus) of Pectobacteriaceae , a group of soft rot-inducing plant pathogens, imports an entire folded host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homologue, plays two roles in facilitating ferredoxin import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the OM receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. In this work, we describe structures of FusB-ferredoxin and homodimeric FusB complexes and determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. We postulate that under resting conditions FusB exists a homodimer, stabilised by an intermolecular R241-D322 salt bridge. The homodimer dissociates when the “FusB-box” of FusA outcompetes one protomer, and FusA D53 displaces FusB D322. Upon ferredoxin binding, FusB undergoes a structural rearrangement, expanding its β-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces the receptor (FusA) from FusB with Arg241 forming an intramolecular salt bridge with Asp322 to stabilise the newly formed β-hairpin of FusB. We propose a mechanistic model for ferredoxin import in which FusB Arg241 acts as a molecular switch, and two distinct regions function as interaction hotspots.