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 Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σ^E stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli 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_Q258K), which confers high-level resistance to G0507 in vivo 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.

Keywords: LolCDE; antibiotic discovery; lipoprotein trafficking; outer membrane biogenesis; phenotypic assay; stress response.

FIG 1

Novel compound inhibits the growth of E. coli and induces the σ^E stress response. (A) Chemical structure of G0507. (B and C) Induction of the σ^E stress response by G0507. The σ^E reporter strain (MG1655 Δlac attB::rpoH_P3-lacZ tolC::aph; GNEID1222) was incubated with G0507 ranging from 0 to 100 μM for 4 h (B) or with 3.1 μM G0507 for 0 to 4 h (C). β-Galactosidase activity (measured by Beta-Glo assay reagent) was normalized for cell numbers by dividing activity by the BacTiter-Glo activity, yielding normalized β-galactosidase activity. σ^E induction equals normalized β-galactosidase activity in the presence of compound divided by normalized β-galactosidase activity in the presence of DMSO. The dotted line shows the level of σ^E activity in the absence of G0507. Error bars represent the standard deviations of triplicate samples.

FIG 2

Mutations conferring resistance to G0507 localized to the LolCDE ABC transporter. Topology models for LolC and LolE are based on Yasuda et al. (44). Mutations identified by whole-genome sequencing are highlighted in bold, while the remaining mutations were identified by sequencing the lolCDE operon of additional resistant mutants.

FIG 3

G0507 causes morphological changes in cells similar to those with treatment with globomycin. MG1655 imp4213 cells in the exponential phase of growth were treated with G0507 and globomycin at 4× MIC for 2 h. Cells were harvested and prepared for confocal microscopy. Cells were stained with the membrane dye Nile Red (red) to visualize membranes and the DNA-intercalating agent DAPI (blue) to label DNA. Images were acquired using confocal fluorescence microscopy at ×100 magnification. Bar, 1 μm.

FIG 4

Loss of Lpp confers resistance to G0507. The schematic represents 24 individual resistance mutants selected against 4× MIC G0507. Nineteen resistant mutants had mutations in lolC, lolD, or lolE. The five remaining mutants with wild-type lolCDE genes had mutations upstream of or in the lpp gene, identified by whole-genome sequencing. Immunoblotting with Lpp-specific polyclonal antibodies shows that four mutants lost expression of Lpp (inset).

FIG 5

Accumulation of Lpp in the inner membrane of cells treated with G0507. Total membranes from untreated cells (A) and cells treated with G0507 (B) were separated by floatation sucrose density gradient centrifugation, followed by fractionation into 20 fractions from the top (35% sucrose) to the bottom (58% sucrose) of the gradient. Each fraction was subjected to SDS-PAGE and immunoblotting using polyclonal antibodies against BamA, MsbA, and Lpp, as indicated. MsbA is a control protein for inner membrane localization, and BamA is a control protein for outer membrane localization.

FIG 6

Accumulated Lpp in the inner membrane of G0507-treated cells is fully processed. (A) Schematic depicting the lipoprotein modification and transport pathway showing each step of the process and the expected mass of Lpp intermediates. (B) Lpp_ΔK58-6×His was expressed in a strain lacking endogenous Lpp (GNEID4177) and treated with 4× MIC of G0507 or globomycin for 1 h. Total cell lysates were subsequently separated by SDS-PAGE and immunoblotted with Lpp-specific polyclonal antibodies.

FIG 7

G0507 stimulates ATPase activity in wild-type LolCDE but not the mutant ABC transporter. The graph shows the changes in the ATPase activity (see Materials and Methods for details of the ATPase assay) of LolCDE_WT and LolC_Q258KDE in the presence of 0.8 μM and 3.2 μM G0507. Error bars represent standard deviations of duplicate samples from three independent experiments. In all experiments, LolCDE_WT and LolC_Q258KDE proteins were used at 10 nM.

FIG 8

Analogs of G0507 gain wild-type activity. Structures and MIC values (μg/ml) of G0507 and its analogs are shown. MICs were determined against E. coli wild-type MG1655 and imp4213 and ΔtolC strains, Gram-positive S. aureus USA300, and OM-permeabilized E. aerogenes strain ATCC 13048 and K. pneumonia strain ATCC 43816. Permeabilization of OM by 4 mM EDTA was confirmed by increased susceptibility to vancomycin (MICs of 25 μg/ml for E. aerogenes ATCC 13048 and 12.5 μg/ml for K. pneumonia strain ATCC 43816), which is inactive against OM-intact Gram-negative bacteria. Red circles show the area of the molecule where modifications are tolerated, whereas blue circles show areas where modifications lead to loss of activity against E. coli. MICs were determined by microdilution.