Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun’s Lipoprotein

Abstract

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of the major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. Importance As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last fifty years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance; namely, the deletion of the major Gram-negative outer membrane lipoprotein, lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway, and potentially points to new antibacterial targets in this pathway.

A Biological Signature for the Inhibition of Outer Membrane Lipoprotein Biogenesis

“…A recent study described a macrocyclic peptide (G2824) that inhibits Lgt activity and is bactericidal to E. coli (70). Notably, Δ lpp did not confer resistance to G2824.…”

Bacterial Lipoprotein Posttranslational Modifications. New Insights and Opportunities for Antibiotic and Vaccine Development

“…This nucleophile attacks the lipid substrate at the ester bond between the phosphate and diacylglyceryl moiety generating pBLP and glycerol-1-phosphate as products. Of note is the recent observation that Lgt from E. coli can use PG as a substrate in which the terminal glycerol is racemic at C2 ( Diao et al, 2021 ). The enzyme can therefore produce both glycerol-1-phosphate and glycerol-3-phosphate as products.…”

“…The putative blocking amine of the globomycin analog G0790 is shown in red. (B) Structure the macrocyclic peptide G2824 ( Diao et al, 2021 ), an inhibitor of Lgt.…”

“…Attention has recently turned to Lgt, the first enzyme in the canonical BLP posttranslational processing pathway, as a target for the development of antibiotics ( Diao et al, 2021 ). Considerable progress has already been made based on a screen of a macrocyclic peptide library generated by the mRNA-based RaPID (random non-standard peptides integrated discovery) method.…”

“…To understand the mechanism of action of these cyclic peptide inhibitors, Diao et al (2021) examined the effect that a titratable, inducible Lgt protein depletion had on the morphology, growth and survival of E. coli . Reducing Lgt to 75% of wild-type levels was enough to kill the cells.…”