The rapid emergence of antibiotic resistance, coupled with a decline in antibiotic discovery, poses a major threat to global public health and underscores the urgent need for the identification and characterization of new antibiotic targets and mechanisms. The lipid II flippase MurJ, an essential membrane transporter that flips the peptidoglycan precursor lipid II across the cytoplasmic membrane, represents a validated yet unexploited antibiotic target. MurJ has been repeatedly targeted by both bacteriophage-encoded lysis proteins and by small-molecule screening efforts. Distinct single gene lysis protein (Sgls) from small lytic phages have evolved independently to inhibit MurJ, suggesting that MurJ represents a key step for killing bacteria and an evolutionarily validated target. Structural and mechanistic understanding of MurJ has largely been derived from diderm bacteria. However, small molecule compounds that target MurJ have been identified specifically in monoderm pathogens, and the corresponding monoderm MurJ homologs remain uncharacterized. Despite strong validation of MurJ as a potential antibiotic target, the inhibition mechanisms are unknown, limiting efforts to translate this key step into effective therapeutic strategies. In this thesis, we define the inhibition mechanisms of MurJ by Sgls using structural approaches. We show that distinct Sgls converge on a common interface on MurJ and trap MurJ in a periplasm-open conformation. We further identified and structurally characterized a novel MurJ-targeting Sgl from the predicted phage Changjiang3, supporting convergent evolution toward a shared inhibition mechanism. In addition, we determined structures of monoderm MurJ in multiple conformational states, revealing both conserved and divergent features for the transport mechanism across monoderms and diderms. Together, these findings highlight new opportunities for targeting MurJ for the development of novel antimicrobials
Yancheng Li (Mon,) studied this question.