Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

Abstract

cIn Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.T he most distinguishing feature of Gram-negative bacteria is their cell envelope, which is comprised of both an inner and an outer membrane bilayer. The outer membrane has a unique composition of lipoproteins, ␤-barrel proteins, lipopolysaccharides, and phospholipids. Lipoproteins, membrane proteins that are covalently modified with lipids, are involved in a variety of integral cellular functions, such as the synthesis and maintenance of the cell surface and the transport of substrates (reviewed in reference 1). Lipoproteins are synthesized as precursors in the cytoplasm. Upon transit across the inner membrane by either the Sec or Tat machinery, the export signal peptide is cleaved, and attached to its amino terminus is a lipid moiety. This lipid serves as a membrane anchor for the lipoprotein. Some lipoproteins remain in the outer leaflet of the inner membrane, while others must cross the hydrophilic periplasmic space to the outer membrane. This sorting of lipoproteins to the outer membrane is achieved by the Lol system, which consists of five proteins (Fig. 1) (reviewed in reference 1). In this process, lipoproteins destined for the outer membrane are released from the inner membrane by the LolCDE complex, an inner membrane ABC transporter. LolCDE transfers the lipoproteins to LolA, a diffusible periplasmic chaperone (2, 3). LolA then transfers the lipoprotein to LolB, the outer membrane lipoprotein receptor, which incorporates these lipoproteins into the inner leaflet of the outer membrane (1, 4). This is in contrast to Grampositive bacteria, which have a single membrane bilayer; therefore, localization of lipoproteins to the cell surface requires only export through the cytoplasmic membrane and acylation (5).Infections c...

Novel Antibacterial Targets and Compounds Revealed by a High-Throughput Cell Wall Reporter Assay

“…There was no change in susceptibility of the lpp mutant to compounds that do not inhibit lipoprotein transport to the outer membrane, as expected (Table 4). We previously identified a structurally distinct inhibitor of the LolCDE complex (12), with mutations conferring resistance mapped to LolC (N265K) and LolE (L371P). These mutants were found to be resistant to compound 2 as well.…”

“…Inhibitors of outer membrane lipoprotein trafficking have been reported previously (12); however, to date, none of these molecules have been used therapeutically. Compound 2 lacks the physical properties and antibacterial spectrum required for clinical use, but it can be employed as a tool to aid in exploration of lipoprotein transport to the outer membrane, such as the physiological impact of disruption on outer membrane integrity.…”

“…The cells were converted into spheroplasts as described previously (26). The Lpp release assay was performed as described previously (12). Briefly, suspensions containing 2 ϫ 10 8 spheroplasts were incubated with or without 3.5 g His-tagged LolA in the presence of DMSO, 1.4 g globomycin, or compound 2 at 30°C for 1 min.…”

“…Sun et al showed that inhibitors of other steps of cell wall biogenesis are also capable of inducing AmpC ␤-lactamase production and that AmpC can be used as a reporter to detect cell wall-active compounds (10,11). In a previous report using a hypersensitive Escherichia coli strain, we identified a novel inhibitor of lipoprotein transport to the outer membrane (12). Here we describe the adaptation of the AmpC ␤-lactamase reporter system (11) for phenotypic screening in a high-throughput, 384-well format, which identified another novel inhibitor of lipoprotein trafficking as well as a novel inhibitor of lipopolysaccharide (LPS) synthesis.…”

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

“…43 In support of this, one group examining SMIs opted to use a strain of E. coli that has a shortened LPS, which renders it more permeable to SMIs. 44 This technique will be worth considering in future studies.…”