Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in .

is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against , we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical isolates. Mutations leading to G5132 resistance in map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in .