Mechanistic basis of teichoic acid transport by a gatekeeper flippase

The cell wall is a complex structure that protects bacteria from environmental threats. Phosphocholine-containing teichoic acids are key cell wall biopolymers critical for host colonization, immune evasion, competence, and persistence in <i>Streptococcus pneumoniae</i> . The flippase TacF, a member of the multidrug/oligosaccharide-lipid/polysaccharide (MOP) superfamily, monitors the phosphocholine content of teichoic acids during transport, yet the underlying mechanism of this process remains unknown. We present a cryo-EM structure of <i>S. pneumoniae</i> TacF in lipid nanodiscs. <i>In vivo</i> complementation assays and molecular dynamics simulations reveal key residues involved in teichoic acid recognition and transport, while coevolutionary and conservation analyses delineate common mechanistic elements among MOP flippases, indicating a shared mechanism for polyprenyl-diphosphate-linked oligosaccharide lipid transport. Our findings provide mechanistic insights into an essential flippase involved in <i>S. pneumoniae</i> pathogenesis and a potential drug target.