Structural and functional characterization of the teichoic acid flippase TacF from S. pneumoniae

Streptococcus pneumoniae, a Gram-positive bacterium, stands as a significant contributor to mortality among children under 5 years worldwide. Beyond causing community-acquired pneumonia, it can induce severe and fatal infections in adults, including meningitis and septicemia. Recognizing the escalating multi-drug resistance issue, the World Health Organization (WHO) incorporated S. pneumoniae into the list of 12 priority pathogens necessitating the development of novel antibiotics in 2017.
As a Gram-positive bacterium, S. pneumoniae possesses a robust cell wall primarily composed of peptidoglycan and teichoic acids. Teichoic acids manifest as glycopolymers, classified as lipoteichoic acids (LTA) when bound to a lipidic carrier or wall teichoic acids (WTA) when covalently linked to peptidoglycan in the cell wall. In S. pneumoniae, teichoic acids comprise repeating units of 2-acetamido-4-amino-2,4,6-trideoxygalactose (AATGal), glucose (Glc), ribitol-5-phosphate (RboP), and two molecules of N-acetylglucosamine (GalNAc). Phosphorylcholine moieties, added by the LicC protein, adorn the GalNAc moieties.
Choline-binding proteins (CBP), featuring a choline-binding domain (CBD), interact with phosphorylcholine moieties in teichoic acids (TA) located either in the cell wall (WTA) or plasmatic membrane (LTA). These proteins play various roles, including immune system evasion, host cell recognition, and biofilm formation. Despite the diverse functions attributed to CBPs, the biosynthetic pathway of TA in S. pneumoniae remains incompletely understood, particularly regarding the translocation process towards the extracellular leaflet of the membrane.
TacF, a member of the prokaryotic polysaccharide transporter (PST) family within the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily of transporters, is the putative protein responsible for translocating TA. Its homology model suggests a protein folding similar to the lipid II transporter MurJ of the peptidoglycan biosynthetic pathway. However, the mechanism of lipid translocation in the MOP superfamily is poorly elucidated and there are no structures available of TA flippases for this family. In this study, we present, for the first time, the cryo-electron Microscopy (cryo-EM) structure of TacF at a resolution of 3.5Å in an inward-open conformation. Notable structural features, such as a hydrophobic groove, aromatic residues in the cavity, and ion binding sites, were identified and deemed crucial for the function of the protein.
Through a combination of functional assays in vitro, molecular dynamics (MD) simulations, and in vivo approaches, we propose the TA binding sites and the transport mechanism of TacF. A method for synthesizing and purifying NBD-labelled TA was developed for functional assays. These findings not only lay the foundation for understanding other MOP superfamily lipid transporters but also provide insights for targeted structure- and function-based drug design against S. pneumoniae.