A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria

Abstract

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of growth followed by selection of compounds that induced the extracytoplasmic σ stress response. Mutations in ,, and conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolC), which confers high-level resistance to G0507 but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

Insights into bacterial lipoprotein trafficking from a structure of LolA bound to the LolC periplasmic domain

“…In MacB, the stalk structure is intimately connected with mechanotransmission, suggesting that G0507 and C2 exert their effects by interfering with analogous movements necessary for coupling LolCDE's cytoplasmic ATPase activity with the lipoprotein transfer reaction. Consistent with this "mechanotransmission uncoupling" as a hypothesis for the action of these inhibitors, G0507 is known to stimulate ATPase activity of LolCDE while inhibiting the release of lipoproteins from the inner membrane (19). Because C2 also inhibits lipoprotein release, we tested its effect on LolCDE ATPase activity and found that, like G0507, C2 causes an increase in the rate of hydrolysis (SI Appendix, Fig.…”

“…LolA has been docked according to LolA-LolC crystallographic data (6F3Z). Positions at which mutations confer resistance to both compound 2 (21) and G0507 (19) inhibitors are shown mapped to the ATP-bound state.…”

“…Uncoupling. Homology models of LolCDE and LolA-LolCDE facilitate physical mapping of LolCDE mutations reported to provide resistance to two antimicrobial compounds: pyrrolopyrimidinedione G0507 (19) and pyridineimidazole compound 2 (21) (hereafter, C2). G0507 and C2 are both purported inhibitors of LolCDE with potent antibacterial activity against E. coli strains lacking the tripartite efflux pump component, TolC.…”

“…Mislocalization of outer-membrane lipoproteins on the inner membrane results in cell death (12,13), and proteins responsible for lipoylation and trafficking of outer-membrane lipoproteins are essential for bacterial viability (14)(15)(16)(17)(18). The relative accessibility of proteins involved in lipoprotein maturation and trafficking, combined with their essential functions, have made these systems attractive targets for developing new antimicrobial agents (19)(20)(21).…”

Lipid trafficking across the Gram-negative cell envelope

“…The OM is also essential for bacterial growth and therefore is a great molecular target for novel antibiotic intervention. To this end, small molecule inhibitors against the Lol (108,109) and MsbA/Lpt pathways (105,110) have already been identified. Continued efforts in deciphering PL-trafficking processes will ultimately yield new targets that can be exploited for combating Gram-negative bacterial pathogens.…”